free full text journal articles: genetics and proteomics




Can Genetics Influence Eating Steak and Red Meat?
Meat Consumption, Genetics, and Cancer
How Long to Cook Steak on the Grill
Eating Steak and Red Meats May be Genetic

Recent Articles in Nucleic Acids Research

Duvick J, Fu A, Muppirala U, Sabharwal M, Wilkerson MD, Lawrence CJ, Lushbough C, Brendel V
PlantGDB: a resource for comparative plant genomics.
Nucleic Acids Res. 2007 Dec 6; .
PlantGDB ( is a genomics database encompassing sequence data for green plants (Viridiplantae). PlantGDB provides annotated transcript assemblies for >100 plant species, with transcripts mapped to their cognate genomic context where available, integrated with a variety of sequence analysis tools and web services. For 14 plant species with emerging or complete genome sequence, PlantGDB's genome browsers (xGDB) serve as a graphical interface for viewing, evaluating and annotating transcript and protein alignments to chromosome or bacterial artificial chromosome (BAC)-based genome assemblies. Annotation is facilitated by the integrated yrGATE module for community curation of gene models. Novel web services at PlantGDB include Tracembler, an iterative alignment tool that generates contigs from GenBank trace file data and BioExtract Server, a web-based server for executing custom sequence analysis workflows. PlantGDB also hosts a plant genomics research outreach portal (PGROP) that facilitates access to a large number of resources for research and training. [Abstract/Link to Full Text]

Leulliot N, Bohnsack MT, Graille M, Tollervey D, Tilbeurgh HV
The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases.
Nucleic Acids Res. 2007 Dec 6;
Emg1 was previously shown to be required for maturation of the 18S rRNA and biogenesis of the 40S ribosomal subunit. Here we report the determination of the crystal structure of Emg1 at 2 A resolution in complex with the methyl donor, S-adenosyl-methionine (SAM). This structure identifies Emg1 as a novel member of the alpha/beta knot fold methyltransferase (SPOUT) superfamily. In addition to the conserved SPOUT core, Emg1 has two unique domains that form an extended surface, which we predict to be involved in binding of RNA substrates. A point mutation within a basic patch on this surface almost completely abolished RNA binding in vitro. Three point mutations designed to disrupt the interaction of Emg1 with SAM each caused>100-fold reduction in SAM binding in vitro. Expression of only Emg1 with these mutations could support growth and apparently normal ribosome biogenesis in strains genetically depleted of Emg1. We conclude that the catalytic activity of Emg1 is not essential and that the presence of the protein is both necessary and sufficient for ribosome biogenesis. [Abstract/Link to Full Text]

Petersen J, Poulsen L, Petronis S, Birgens H, Dufva M
Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays.
Nucleic Acids Res. 2007 Dec 6;
DNA microarrays are generally operated at a single condition, which severely limits the freedom of designing probes for allele-specific hybridization assays. Here, we demonstrate a fluidic device for multi-stringency posthybridization washing of microarrays on microscope slides. This device is called a multi-thermal array washer (MTAW), and it has eight individually controlled heating zones, each of which corresponds to the location of a subarray on a slide. Allele-specific oligonucleotide probes for nine mutations in the beta-globin gene were spotted in eight identical subarrays at positions corresponding to the temperature zones of the MTAW. After hybridization with amplified patient material, the slides were mounted in the MTAW, and each subarray was exposed to different temperatures ranging from 22 to 40 degrees C. When processed in the MTAW, probes selected without considering melting temperature resulted in improved genotyping compared with probes selected according to theoretical melting temperature and run under one condition. In conclusion, the MTAW is a versatile tool that can facilitate screening of a large number of probes for genotyping assays and can also enhance the performance of diagnostic arrays. [Abstract/Link to Full Text]

Katahira J, Miki T, Takano K, Maruhashi M, Uchikawa M, Tachibana T, Yoneda Y
Nuclear RNA export factor 7 is localized in processing bodies and neuronal RNA granules through interactions with shuttling hnRNPs.
Nucleic Acids Res. 2007 Dec 5;
The nuclear RNA export factor (NXF) family proteins have been implicated in various aspects of post-transcriptional gene expression. This study shows that mouse NXF7 exhibits heterologous localization, i.e. NXF7 associates with translating ribosomes, stress granules (SGs) and processing bodies (P-bodies), the latter two of which are believed to be cytoplasmic sites of storage, degradation and/or sorting of mRNAs. By yeast two-hybrid screening, a series of heterogeneous nuclear ribonucleoproteins (hnRNPs) were identified as possible binding partners for NXF7. Among them, hnRNP A3, which is believed to be involved in translational control and/or cytoplasmic localization of certain mRNAs, formed a stable complex with NXF7 in vitro. Although hnRNP A3 was not associated with translating ribosomes, it was co-localized with NXF7 in P-bodies. After exposing to oxidative stress, NXF7 trans-localized to SGs, whereas hnRNP A3 did not. In differentiated neuroblastoma Neuro2a cells, NXF7 was co-localized with hnRNP A3 in cell body and neurites. The amino terminal half of NXF7, which was required for stable complex formation with hnRNP A3, coincided with the region required for localization in both P-bodies and neuronal RNA granules. These findings suggest that NXF7 plays a role in sorting, transport and/or storage of mRNAs through interactions with hnRNP A3. [Abstract/Link to Full Text]

Hoischen C, Bussiek M, Langowski J, Diekmann S
Escherichia coli low-copy-number plasmid R1 centromere parC forms a U-shaped complex with its binding protein ParR.
Nucleic Acids Res. 2007 Dec 3;
The Escherichia coli low-copy-number plasmid R1 contains a segregation machinery composed of parC, ParR and parM. The R1 centromere-like site parC contains two separate sets of repeats. By atomic force microscopy (AFM) we show here that ParR molecules bind to each of the 5-fold repeated iterons separately with the intervening sequence unbound by ParR. The two ParR protein complexes on parC do not complex with each other. ParR binds with a stoichiometry of about one ParR dimer per each single iteron. The measured DNA fragment lengths agreed with B-form DNA and each of the two parC 5-fold interon DNA stretches adopts a linear path in its complex with ParR. However, the overall parC/ParR complex with both iteron repeats bound by ParR forms an overall U-shaped structure: the DNA folds back on itself nearly completely, including an angle of approximately 150 degrees . Analysing linear DNA fragments, we never observed dimerized ParR complexes on one parC DNA molecule (intramolecular) nor a dimerization between ParR complexes bound to two different parC DNA molecules (intermolecular). This bacterial segrosome is compared to other bacterial segregation complexes. We speculate that partition complexes might have a similar overall structural organization and, at least in part, common functional properties. [Abstract/Link to Full Text]

Ruan J, Li H, Chen Z, Coghlan A, Coin LJ, Guo Y, Hériché JK, Hu Y, Kristiansen K, Li R, Liu T, Moses A, Qin J, Vang S, Vilella AJ, Ureta-Vidal A, Bolund L, Wang J, Durbin R
TreeFam: 2008 Update.
Nucleic Acids Res. 2007 Dec 1;
TreeFam ( was developed to provide curated phylogenetic trees for all animal gene families, as well as orthologue and paralogue assignments. Release 4.0 of TreeFam contains curated trees for 1314 families and automatically generated trees for another 14 351 families. We have expanded TreeFam to include 25 fully sequenced animal genomes, as well as four genomes from plant and fungal outgroup species. We have also introduced more accurate approaches for automatically grouping genes into families, for building phylogenetic trees, and for inferring orthologues and paralogues. The user interface for viewing phylogenetic trees and family information has been improved. Furthermore, a new perl API lets users easily extract data from the TreeFam mysql database. [Abstract/Link to Full Text]

Tang X, Swaminathan J, Gewirtz AM, Dmochowski IJ
Regulating gene expression in human leukemia cells using light-activated oligodeoxynucleotides.
Nucleic Acids Res. 2007 Dec 1;
Light-activated antisense oligodeoxynucleotides (asODNs) were developed to control the degradation of target mRNA in living cells by RNase H. A 20-mer asODN previously shown to target c-myb, a hematopoietic transcription factor, was covalently attached via a photocleavable linker (PL) to partially complementary 20-mer sense strands (sODNs). In the 'caged' state, the sODN blocked hybridization of the asODN to c-myb mRNA. Six asODN-PL-sODN conjugates, C1-C6, were synthesized. C5, with twelve complementary bases, gave the largest decrease in melting temperature (T(m)) upon UV irradiation (DeltaT(m) = -29 degrees C). The most thermally stable conjugate, C6 (T(m) = 84 degrees C), gave the lowest background RNase H activity, with just 8.6% degradation of an RNA 40-mer after 1 h incubation. In biochemical assays with C6, RNA digestion increased 10-fold 10 min after UV irradiation. Finally, phosphorothioated analogs S-C5 and S-C6 were synthesized to test activity in cultured K562 (human leukemia) cells. No knockdown of c-myb mRNA or protein was observed with intact S-C5 or S-C6, whereas more than half of c-myb mRNA was degraded 24 h after photoactivation. Two-fold photomodulation of c-MYB protein levels was also observed with S-C5. However, no photomodulation of c-MYB protein levels was observed with S-C6, perhaps due to the greater stability of this duplex. [Abstract/Link to Full Text]

Saiz L, Vilar JM
Ab initio thermodynamic modeling of distal multisite transcription regulation.
Nucleic Acids Res. 2007 Dec 1;
Transcription regulation typically involves the binding of proteins over long distances on multiple DNA sites that are brought close to each other by the formation of DNA loops. The inherent complexity of assembling regulatory complexes on looped DNA challenges the understanding of even the simplest genetic systems, including the prototypical lac operon. Here we implement a scalable approach based on thermodynamic molecular properties to model ab initio systems regulated through multiple DNA sites with looping. We show that this approach applied to the lac operon accurately predicts the system behavior for a wide range of cellular conditions, which include the transcription rate over five orders of magnitude as a function of the repressor concentration for wild type and all seven combinations of deletions of three operators, as well as the observed induction curves for cells with and without active catabolite activator protein. Our results provide new insights into the detailed functioning of the lac operon and reveal an efficient avenue to incorporate the required underlying molecular complexity into fully predictive models of gene regulation. [Abstract/Link to Full Text]

Wisniewski JR, Zougman A, Mann M
N{varepsilon}-Formylation of lysine is a widespread post-translational modification of nuclear proteins occurring at residues involved in regulation of chromatin function.
Nucleic Acids Res. 2007 Dec 1;
Post-translational modification of histones and other chromosomal proteins regulates chromatin conformation and gene activity. Methylation and acetylation of lysyl residues are among the most frequently described modifications in these proteins. Whereas these modifications have been studied in detail, very little is known about a recently discovered chemical modification, the N(epsilon)-lysine formylation, in histones and other nuclear proteins. Here we mapped, for the first time, the sites of lysine formylation in histones and several other nuclear proteins. We found that core and linker histones are formylated at multiple lysyl residues located both in the tails and globular domains of histones. In core histones, formylation was found at lysyl residues known to be involved in organization of nucleosomal particles that are frequently acetylated and methylated. In linker histones and high mobility group proteins, multiple formylation sites were mapped to residues with important role in DNA binding. N(epsilon)-lysine formylation in chromosomal proteins is relatively abundant, suggesting that it may interfere with epigenetic mechanisms governing chromatin function, which could lead to deregulation of the cell and disease. [Abstract/Link to Full Text]

Venditti V, Niccolai N, Butcher SE
Measuring the dynamic surface accessibility of RNA with the small paramagnetic molecule TEMPOL.
Nucleic Acids Res. 2007 Dec 1;
The surface accessibility of macromolecules plays a key role in modulating molecular recognition events. RNA is a complex and dynamic molecule involved in many aspects of gene expression. However, there are few experimental methods available to measure the accessible surface of RNA. Here, we investigate the accessible surface of RNA using NMR and the small paramagnetic molecule TEMPOL. We investigated two RNAs with known structures, one that is extremely stable and one that is dynamic. For helical regions, the TEMPOL probing data correlate well with the predicted RNA surface, and the method is able to distinguish subtle variations in atom depths, such as the relative accessibility of pyrimidine versus purine aromatic carbon atoms. Dynamic motions are also detected by TEMPOL probing, and the method accurately reports a previously characterized pH-dependent conformational transition involving formation of a protonated C-A pair and base flipping. Some loop regions are observed to exhibit anomalously high accessibility, reflective of motions that are not evident within the ensemble of NMR structures. We conclude that TEMPOL probing can provide valuable insights into the surface accessibility and dynamics of RNA, and can also be used as an independent means of validating RNA structure and dynamics in solution. [Abstract/Link to Full Text]

Pang CN, Lin K, Wouters MA, Heringa J, George RA
Identifying foldable regions in protein sequence from the hydrophobic signal.
Nucleic Acids Res. 2007 Dec 1;
Structural genomics initiatives aim to elucidate representative 3D structures for the majority of protein families over the next decade, but many obstacles must be overcome. The correct design of constructs is extremely important since many proteins will be too large or contain unstructured regions and will not be amenable to crystallization. It is therefore essential to identify regions in protein sequences that are likely to be suitable for structural study. Scooby-Domain is a fast and simple method to identify globular domains in protein sequences. Domains are compact units of protein structure and their correct delineation will aid structural elucidation through a divide-and-conquer approach. Scooby-Domain predictions are based on the observed lengths and hydrophobicities of domains from proteins with known tertiary structure. The prediction method employs an A*-search to identify sequence regions that form a globular structure and those that are unstructured. On a test set of 173 proteins with consensus CATH and SCOP domain definitions, Scooby-Domain has a sensitivity of 50% and an accuracy of 29%, which is better than current state-of-the-art methods. The method does not rely on homology searches and, therefore, can identify previously unknown domains. [Abstract/Link to Full Text]

Haque ME, Grasso D, Spremulli LL
The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3mt.
Nucleic Acids Res. 2007 Dec 1;
Mammalian mitochondrial initiation factor 3 (IF3(mt)) has a central region with homology to bacterial IF3. This homology region is preceded by an N-terminal extension and followed by a C-terminal extension. The role of these extensions on the binding of IF3(mt) to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The K(d) for the binding of IF3(mt) to 28S subunits is approximately 30 nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3(mt) has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (K(d) = 1.5 muM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (K(d) = 0.12-0.25 muM). IF3(mt) does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3(mt) is very effective in dissociating 55S ribosomes. Removal of the N-terminal extension has little effect on this activity. However, removal of the C-terminal extension leads to a complex dissociation pattern due to the high affinity of this derivative for 39S subunits. These data suggest that the extensions have evolved to ensure the proper dissociation of IF3(mt) from the 28S subunits upon 39S subunit joining. [Abstract/Link to Full Text]

Bauer M, Marschaus L, Reuff M, Besche V, Sartorius-Neef S, Pfeifer F
Overlapping activator sequences determined for two oppositely oriented promoters in halophilic Archaea.
Nucleic Acids Res. 2007 Dec 1;
Transcription of the genomic region involved in gas vesicle formation in Halobacterium salinarum (p-vac) and Haloferax mediterranei (mc-vac) is driven by two divergent promoters, P(A) and P(D), separated by only 35 nt. Both promoters are activated by the transcription activator GvpE which in the case of P(mcA) requires a 20-nt sequence (UAS) consisting of two conserved 8-nt sequence portions located upstream of BRE. Here, we determined the two UAS elements in the promoter region of p-vac by scanning mutageneses using constructs containing P(pD) (without P(pA)) fused to the bgaH reporter gene encoding an enzyme with beta-galactosidase activity, or the dual reporter construct pApD with P(pD) fused to bgaH and P(pA) to an altered version of gvpA. The two UAS elements found exhibited a similar extension and distance to BRE as previously determined for the UAS in P(mcA). Their distal 8-nt portions almost completely overlapped in the centre of P(pD)-P(pA), and mutations in this region negatively affected the GvpE-mediated activation of both promoters. Any alteration of the distance between BRE and UAS resulted in the loss of the GvpE activation, as did a complete substitution of the proximal 8-nt portion, underlining that a close location of UAS and BRE was very important. [Abstract/Link to Full Text]

Berglund AC, Sjölund E, Ostlund G, Sonnhammer EL
InParanoid 6: eukaryotic ortholog clusters with inparalogs.
Nucleic Acids Res. 2007 Dec 5;
The InParanoid eukaryotic ortholog database ( has been updated to version 6 and is now based on 35 species. We collected all available 'complete' eukaryotic proteomes and Escherichia coli, and calculated ortholog groups for all 595 species pairs using the InParanoid program. This resulted in 2 642 187 pairwise ortholog groups in total. The orthology-based species relations are presented in an orthophylogram. InParanoid clusters contain one or more orthologs from each of the two species. Multiple orthologs in the same species, i.e. inparalogs, result from gene duplications after the species divergence. A new InParanoid website has been developed which is optimized for speed both for users and for updating the system. The XML output format has been improved for efficient processing of the InParanoid ortholog clusters. [Abstract/Link to Full Text]

Birzele F, Csaba G, Zimmer R
Alternative splicing and protein structure evolution.
Nucleic Acids Res. 2007 Nov 30;
Alternative splicing is thought to be one of the major sources for functional diversity in higher eukaryotes. Interestingly, when mapping splicing events onto protein structures, about half of the events affect structured and even highly conserved regions i.e. are non-trivial on the structure level. This has led to the controversial hypothesis that such splice variants result in nonsense-mediated mRNA decay or non-functional, unstructured proteins, which do not contribute to the functional diversity of an organism. Here we show in a comprehensive study on alternative splicing that proteins appear to be much more tolerant to structural deletions, insertions and replacements than previously thought. We find literature evidence that such non-trivial splicing isoforms exhibit different functional properties compared to their native counterparts and allow for interesting regulatory patterns on the protein network level. We provide examples that splicing events may represent transitions between different folds in the protein sequence-structure space and explain these links by a common genetic mechanism. Taken together, those findings hint to a more prominent role of splicing in protein structure evolution and to a different view of phenotypic plasticity of protein structures. [Abstract/Link to Full Text]

Burhans WC, Weinberger M
DNA replication stress, genome instability and aging.
Nucleic Acids Res. 2007 Nov 30;
Genome instability is a fundamentally important component of aging in all eukaryotes. How age-related genome instability occurs remains unclear. The free radical theory of aging posits oxidative damage to DNA and other cellular constituents as a primary determinant of aging. More recent versions of this theory predict that mitochondria are a major source of reactive oxygen species (ROS) that cause oxidative damage. Although substantial support for the free radical theory exists, the results of some tests of this theory have been contradictory or inconclusive. Enhanced growth signaling also has been implicated in aging. Many efforts to understand the effects of growth signaling on aging have focused on inhibition of oxidative stress responses that impact oxidative damage. However, recent experiments in the model organism Saccharomyces cerevisiae (budding yeast) and in higher eukaryotes suggest that growth signaling also impacts aging and/or age-related diseases-including cancer and neurodegeneration-by inducing DNA replication stress, which causes DNA damage. Replication stress, which has not been broadly considered as a factor in aging, may be enhanced by ROS that signal growth. In this article, we review evidence that points to DNA replication stress and replication stress-induced genome instability as important factors in aging. [Abstract/Link to Full Text]

Benson ML, Smith RD, Khazanov NA, Dimcheff B, Beaver J, Dresslar P, Nerothin J, Carlson HA
Binding MOAD, a high-quality protein ligand database.
Nucleic Acids Res. 2007 Nov 30;
Binding MOAD (Mother of All Databases) is a database of 9836 protein-ligand crystal structures. All biologically relevant ligands are annotated, and experimental binding-affinity data is reported when available. Binding MOAD has almost doubled in size since it was originally introduced in 2004, demonstrating steady growth with each annual update. Several technologies, such as natural language processing, help drive this constant expansion. Along with increasing data, Binding MOAD has improved usability. The website now showcases a faster, more featured viewer to examine the protein-ligand structures. Ligands have additional chemical data, allowing for cheminformatics mining. Lastly, logins are no longer necessary, and Binding MOAD is freely available to all at [Abstract/Link to Full Text]

Bird C
Nucleic Acids Res. 2007;35(20): [Abstract/Link to Full Text]

Todd BA, Rau DC
Interplay of ion binding and attraction in DNA condensed by multivalent cations.
Nucleic Acids Res. 2007 Nov 29;
We have measured forces generated by multivalent cation-induced DNA condensation using single-molecule magnetic tweezers. In the presence of cobalt hexammine, spermidine, or spermine, stretched DNA exhibits an abrupt configurational change from extended to condensed. This occurs at a well-defined condensation force that is nearly equal to the condensation free energy per unit length. The multivalent cation concentration dependence for this condensation force gives the apparent number of multivalent cations that bind DNA upon condensation. The measurements show that the lower critical concentration for cobalt hexammine as compared to spermidine is due to a difference in ion binding, not a difference in the electrostatic energy of the condensed state as previously thought. We also show that the resolubilization of condensed DNA can be described using a traditional Manning-Oosawa cation adsorption model, provided that cation-anion pairing at high electrolyte concentrations is taken into account. Neither overcharging nor significant alterations in the condensed state are required to describe the resolubilization of condensed DNA. The same model also describes the spermidine(3+)/Na(+) phase diagram measured previously. [Abstract/Link to Full Text]

El-Shemerly M, Hess D, Pyakurel AK, Moselhy S, Ferrari S
ATR-dependent pathways control hEXO1 stability in response to stalled forks.
Nucleic Acids Res. 2007 Nov 29;
Nucleases play important roles in DNA synthesis, recombination and repair. We have previously shown that human exonuclease 1 (hEXO1) is phosphorylated in response to agents stalling DNA replication and that hEXO1 consequently undergoes ubiquitination and degradation in a proteasome-dependent manner. In the present study, we have addressed the identity of the pathway transducing stalled-replication signals to hEXO1. Using chemical inhibitors, RNA interference, ATM- and ATR-deficient cell lines we have concluded that hEXO1 phosphorylation is ATR-dependent. By means of mass spectrometry, we have identified the sites of phosphorylation in hEXO1 in undamaged cells and in cells treated with hydroxyurea (HU). hEXO1 is phosphorylated at nine basal sites and three additional sites are induced by HU treatment. Analysis of single- and multiple-point mutants revealed that mutation to Ala of the three HU-induced sites of phosphorylation partially rescued HU-dependent degradation of hEXO1 and additionally stabilized the protein in non-treated cells. We have raised an antibody to pS(714), an HU-induced site of the S/T-Q type, and we provide evidence that S(714) is phosphorylated upon HU but not IR treatment. The antibody may be a useful tool to monitor signal transduction events triggered by stalled DNA replication. [Abstract/Link to Full Text]

Draper WE, Hayden EJ, Lehman N
Mechanisms of covalent self-assembly of the Azoarcus ribozyme from four fragment oligonucleotides.
Nucleic Acids Res. 2007 Nov 29;
RNA oligomers of length 40-60 nt can self-assemble into covalent versions of the Azoarcus group I intron ribozyme. This process requires a series of recombination reactions in which the internal guide sequence of a nascent catalytic complex makes specific interactions with a complement triplet, CAU, in the oligomers. However, if the CAU were mutated, promiscuous self-assembly may be possible, lessening the dependence on a particular set of oligomer sequences. Here, we assayed whether oligomers containing mutations in the CAU triplet could still self-construct Azoarcus ribozymes. The mutations CAC, CAG, CUU and GAU all inhibited self-assembly to some degree, but did not block it completely in 100 mM MgCl(2). Oligomers containing the CAC mutation retained the most self-assembly activity, while those containing GAU retained the least, indicating that mutations more 5' in this triplet are the most deleterious. Self-assembly systems containing additional mutant locations were progressively less functional. Analyses of properly self-assembled ribozymes revealed that, of two recombination mechanisms possible for self-assembly, termed 'tF2' and 'R2F2', the simpler one-step 'tF2' mechanism is utilized when mutations exist. These data suggest that self-assembling systems are more facile than previously believed, and have relevance to the origin of complex ribozymes during the RNA World. [Abstract/Link to Full Text]

Nolan T, Cecere G, Mancone C, Alonzi T, Tripodi M, Catalanotto C, Cogoni C
The RNA-dependent RNA polymerase essential for post-transcriptional gene silencing in Neurospora crassa interacts with replication protein A.
Nucleic Acids Res. 2007 Nov 29;
Post-transcriptional gene silencing (PTGS) pathways play a role in genome defence and have been extensively studied, yet how repetitive elements in the genome are identified is still unclear. It has been suggested that they may produce aberrant transcripts (aRNA) that are converted by an RNA-dependent RNA polymerase (RdRP) into double-stranded RNA (dsRNA), the essential intermediate of PTGS. However, how RdRP enzymes recognize aberrant transcripts remains a key question. Here we show that in Neurospora crassa the RdRP QDE-1 interacts with Replication Protein A (RPA), part of the DNA replication machinery. We show that both QDE-1 and RPA are nuclear proteins and that QDE-1 is specifically recruited onto the repetitive transgenic loci. We speculate that this localization of QDE-1 could allow the in situ production of dsRNA using transgenic nascent transcripts as templates, as in other systems. Supporting a link between the two proteins, we found that the accumulation of short interfering RNAs (siRNAs), the hallmark of silencing, is dependent on an ongoing DNA synthesis. The interaction between QDE-1 and RPA is important since it should guide further studies aimed at understanding the specificity of the RdRP and it provides for the first time a potential link between a PTGS component and the DNA replication machinery. [Abstract/Link to Full Text]

Masson P, Leimgruber E, Creton S, Collart MA
The dual control of TFIIB recruitment by NC2 is gene specific.
Nucleic Acids Res. 2007 Nov 29;
Negative co-factor 2 (NC2) is a conserved eukaryotic complex composed of two subunits, NC2alpha (Drap1) and NC2beta (Dr1) that associate through a histone-fold motif. In this work, we generated mutants of NC2, characterized target genes for these mutants and studied the assembly of NC2 and general transcription factors on target promoters. We determined that the two NC2 subunits mostly function together to be recruited to DNA and regulate gene expression. We found that NC2 strongly controls promoter association of TFIIB, both negatively and positively. We could attribute the gene-specific repressor effect of NC2 on TFIIB to the C-terminal domain of NC2beta, and define that it requires ORF sequences of the target gene. In contrast, the positive function of NC2 on TFIIB targets is more general and requires adequate levels of the NC2 histone-fold heterodimer on promoters. Finally, we determined that NC2 becomes limiting for TATA-binding protein (TBP) association with a heat inducible promoter under heat stress. This study demonstrates an important positive role of NC2 for formation of the pre-initiation complex on promoters, under normal conditions through control of TFIIB, or upon activation by stress via control of TBP. [Abstract/Link to Full Text]

Wishart DS, Knox C, Guo AC, Cheng D, Shrivastava S, Tzur D, Gautam B, Hassanali M
DrugBank: a knowledgebase for drugs, drug actions and drug targets.
Nucleic Acids Res. 2007 Dec 11;
DrugBank is a richly annotated resource that combines detailed drug data with comprehensive drug target and drug action information. Since its first release in 2006, DrugBank has been widely used to facilitate in silico drug target discovery, drug design, drug docking or screening, drug metabolism prediction, drug interaction prediction and general pharmaceutical education. The latest version of DrugBank (release 2.0) has been expanded significantly over the previous release. With approximately 4900 drug entries, it now contains 60% more FDA-approved small molecule and biotech drugs including 10% more 'experimental' drugs. Significantly, more protein target data has also been added to the database, with the latest version of DrugBank containing three times as many non-redundant protein or drug target sequences as before (1565 versus 524). Each DrugCard entry now contains more than 100 data fields with half of the information being devoted to drug/chemical data and the other half devoted to pharmacological, pharmacogenomic and molecular biological data. A number of new data fields, including food-drug interactions, drug-drug interactions and experimental ADME data have been added in response to numerous user requests. DrugBank has also significantly improved the power and simplicity of its structure query and text query searches. DrugBank is available at [Abstract/Link to Full Text]

Pollard LM, Bourn RL, Bidichandani SI
Repair of DNA double-strand breaks within the (GAA*TTC)n sequence results in frequent deletion of the triplet-repeat sequence.
Nucleic Acids Res. 2007 Nov 27;
Friedreich ataxia is caused by an expanded (GAA*TTC)(n) sequence, which is unstable during intergenerational transmission and in most patient tissues, where it frequently undergoes large deletions. We investigated the effect of DSB repair on instability of the (GAA*TTC)(n) sequence. Linear plasmids were transformed into Escherichia coli so that each colony represented an individual DSB repair event. Repair of a DSB within the repeat resulted in a dramatic increase in deletions compared with circular templates, but DSB repair outside the repeat tract did not affect instability. Repair-mediated deletions were independent of the orientation and length of the repeat, the location of the break within the repeat or the RecA status of the strain. Repair at the center of the repeat resulted in deletion of approximately half of the repeat tract, and repair at an off-center location produced deletions that were equivalent in length to the shorter of the two repeats flanking the DSB. This is consistent with a single-strand annealing mechanism of DSB repair, and implicates erroneous DSB repair as a mechanism for genetic instability of the (GAA*TTC)(n) sequence. Our data contrast significantly with DSB repair within (CTG*CAG)(n) repeats, indicating that repair-mediated instability is dependent on the sequence of the triplet repeat. [Abstract/Link to Full Text]

Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, Chetvernin V, Church DM, Dicuccio M, Edgar R, Federhen S, Feolo M, Geer LY, Helmberg W, Kapustin Y, Khovayko O, Landsman D, Lipman DJ, Madden TL, Maglott DR, Miller V, Ostell J, Pruitt KD, Schuler GD, Shumway M, Sequeira E, Sherry ST, Sirotkin K, Souvorov A, Starchenko G, Tatusov RL, Tatusova TA, Wagner L, Yaschenko E
Database resources of the National Center for Biotechnology Information.
Nucleic Acids Res. 2007 Nov 27;
In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data available through NCBI's web site. NCBI resources include Entrez, the Entrez Programming Utilities, My NCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link, Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genome, Genome Project and related tools, the Trace, Assembly, and Short Read Archives, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups, Influenza Viral Resources, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus, Entrez Probe, GENSAT, Database of Genotype and Phenotype, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool and the PubChem suite of small molecule databases. Augmenting the web applications are custom implementations of the BLAST program optimized to search specialized data sets. These resources can be accessed through the NCBI home page at [Abstract/Link to Full Text]

Amen M, Espinoza HM, Cox C, Liang X, Wang J, Link TM, Brennan RG, Martin JF, Amendt BA
Chromatin-associated HMG-17 is a major regulator of homeodomain transcription factor activity modulated by Wnt/ -catenin signaling.
Nucleic Acids Res. 2007 Nov 27;
Homeodomain (HD) transcriptional activities are tightly regulated during embryogenesis and require protein interactions for their spatial and temporal activation. The chromatin-associated high mobility group protein (HMG-17) is associated with transcriptionally active chromatin, however its role in regulating gene expression is unclear. This report reveals a unique strategy in which, HMG-17 acts as a molecular switch regulating HD transcriptional activity. The switch utilizes the Wnt/beta-catenin signaling pathway and adds to the diverse functions of beta-catenin. A high-affinity HMG-17 interaction with the PITX2 HD protein inhibits PITX2 DNA-binding activity. The HMG-17/PITX2 inactive complex is concentrated to specific nuclear regions primed for active transcription. beta-Catenin forms a ternary complex with PITX2/HMG-17 to switch it from a repressor to an activator complex. Without beta-catenin, HMG-17 can physically remove PITX2 from DNA to inhibit its transcriptional activity. The PITX2/HMG-17 regulatory complex acts independently of promoter targets and is a general mechanism for the control of HD transcriptional activity. HMG-17 is developmentally regulated and its unique role during embryogenesis is revealed by the early embryonic lethality of HMG-17 homozygous mice. This mechanism provides a new role for canonical Wnt/beta-catenin signaling in regulating HD transcriptional activity during development using HMG-17 as a molecular switch. [Abstract/Link to Full Text]

Bardin C, Leroy JL
The formation pathway of tetramolecular G-quadruplexes.
Nucleic Acids Res. 2007 Nov 27;
Oligonucleotides containing guanosine stretches associate into tetrameric structures stabilized by monovalent ions. In order to describe the sequence of reactions leading to association of four identical strands, we measured by NMR the formation and dissociation rates of (TGnT)(4) quadruplexes (n = 3-6), their dissociation constants and the reaction orders for quadruplex formation. The quadruplex formation rates increase with the salt concentration but weakly depend on the nature (K(+), Na(+) or Li(+)) of the counter ions. The activation energies for quadruplex formation are negative. The quadruplex lifetimes strongly increase with the G-tract length and are much more longer in K(+) solution than in Na(+) or Li(+) solutions. The reaction order for quadruplex formation is 3 in 0.125 M KCl and 4 in LiCl solutions. The kinetics measurements suggest that quadruplex formation proceeds step by step via sequential strand association into duplex and triplex intermediate species. Triplex formation is rate limiting in 0.125 M KCl solution. In LiCl, each step of the association process depends on the strand concentration. Parallel reactions to formation of the fully matched canonical quadruplex may result in kinetically trapped mismatched quadruplexes making the canonical quadruplex practically inaccessible in particular at low temperature in KCl solution. [Abstract/Link to Full Text]

The Universal Protein Resource (UniProt).
Nucleic Acids Res. 2007 Nov 27;
The Universal Protein Resource (UniProt) provides a stable, comprehensive, freely accessible, central resource on protein sequences and functional annotation. The UniProt Consortium is a collaboration between the European Bioinformatics Institute (EBI), the Protein Information Resource (PIR) and the Swiss Institute of Bioinformatics (SIB). The core activities include manual curation of protein sequences assisted by computational analysis, sequence archiving, development of a user-friendly UniProt website, and the provision of additional value-added information through cross-references to other databases. UniProt is comprised of four major components, each optimized for different uses: the UniProt Knowledgebase, the UniProt Reference Clusters, the UniProt Archive and the UniProt Metagenomic and Environmental Sequences database. UniProt is updated and distributed every three weeks, and can be accessed online for searches or download at [Abstract/Link to Full Text]

Dönitz J, Goemann B, Lizé M, Michael H, Sasse N, Wingender E, Potapov AP
EndoNet: an information resource about regulatory networks of cell-to-cell communication.
Nucleic Acids Res. 2007 Nov 27;
EndoNet is an information resource about intercellular regulatory communication. It provides information about hormones, hormone receptors, the sources (i.e. cells, tissues and organs) where the hormones are synthesized and secreted, and where the respective receptors are expressed. The database focuses on the regulatory relations between them. An elementary communication is displayed as a causal link from a cell that secretes a particular hormone to those cells which express the corresponding hormone receptor and respond to the hormone. Whenever expression, synthesis and/or secretion of another hormone are part of this response, it renders the corresponding cell an internal node of the resulting network. This intercellular communication network coordinates the function of different organs. Therefore, the database covers the hierarchy of cellular organization of tissues and organs as it has been modeled in the Cytomer ontology, which has now been directly embedded into EndoNet. The user can query the database; the results can be used to visualize the intercellular information flow. A newly implemented hormone classification enables to browse the database and may be used as alternative entry point. EndoNet is accessible at: [Abstract/Link to Full Text]

Recent Articles in Genome Research

Roca X, Olson AJ, Rao AR, Enerly E, Kristensen VN, Břrresen-Dale AL, Andresen BS, Krainer AR, Sachidanandam R
Features of 5'-splice-site efficiency derived from disease-causing mutations and comparative genomics.
Genome Res. 2007 Nov 21;
Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonuria, can be caused by 5'-splice-site (5'ss) mutations that are not predicted to disrupt splicing, according to position weight matrices. By using comparative genomics, we identify pairwise dependencies between 5'ss nucleotides as a conserved feature of the entire set of 5'ss. These dependencies are also conserved in human-mouse pairs of orthologous 5'ss. Many disease-associated 5'ss mutations disrupt these dependencies, as can some human SNPs that appear to alter splicing. The consistency of the evidence signifies the relevance of this approach and suggests that 5'ss SNPs play a role in complex diseases. [Abstract/Link to Full Text]

Korshunova Y, Maloney RK, Lakey N, Citek RW, Bacher B, Budiman A, Ordway JM, McCombie WR, Leon J, Jeddeloh JA, McPherson JD
Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA.
Genome Res. 2007 Nov 21;
Cytosine-methylation changes are stable and thought to be among the earliest events in tumorigenesis. Theoretically, DNA carrying tumor-specifying methylation patterns escape the tumors and may be found circulating in the sera from cancer patients, thus providing the basis for development of noninvasive clinical tests for early cancer detection. Indeed, using methylation-specific PCR-based techniques, several groups reported the detection of tumor-associated methylated DNA in the sera from cancer patients with varying clinical success. However, by design, such analytical approaches allow assessment of the presence of molecules with only one methylation pattern, leaving the bigger picture unexplored. The limited knowledge about circulating DNA methylation patterns hinders the efficient development of clinical methylation tests and testing platforms. Here, we report the results of a comprehensive methylation pattern analysis from breast cancer clinical tissues and sera obtained using massively parallel bisulphite pyrosequencing. The four loci studied were recently discovered by our group, and demonstrated to be powerful epigenetic biomarkers of breast cancer. The detailed analysis of more than 700,000 DNA fragments derived from more than 50 individuals (cancer and cancer-free) revealed an unappreciated complexity of genomic cytosine-methylation patterns in both tissue derived and circulating DNAs. Both tumor and cancer-free tissues (as well as sera) contained molecules with nearly every conceivable cytosine-methylation pattern at each locus. Tumor samples displayed more variation in methylation level than normal samples. Importantly, by establishing the methylation landscape within circulating DNA, this study has better defined the development challenges facing DNA methylation-based cancer-detection tests. [Abstract/Link to Full Text]

Torres TT, Metta M, Ottenwälder B, Schlötterer C
Gene expression profiling by massively parallel sequencing.
Genome Res. 2007 Nov 21;
Massively parallel sequencing holds great promise for expression profiling, as it combines the high throughput of SAGE with the accuracy of EST sequencing. Nevertheless, until now only very limited information had been available on the suitability of the current technology to meet the requirements. Here, we evaluate the potential of 454 sequencing technology for expression profiling using Drosophila melanogaster. We show that short (< approximately 80 bp) and long (> approximately 300-400 bp) cDNA fragments are under-represented in 454 sequence reads. Nevertheless, sequencing of 3' cDNA fragments generated by nebulization could be used to overcome the length bias of the 454 sequencing technology. Gene expression measurements generated by restriction analysis and nebulization for fragments within the 80- to 300-bp range showed correlations similar to those reported for replicated microarray experiments (0.83-0.91); 97% of the cDNA fragments could be unambiguously mapped to the genomic DNA, demonstrating the advantage of longer sequence reads. Our analyses suggest that the 454 technology has a large potential for expression profiling, and the high mapping accuracy indicates that it should be possible to compare expression profiles across species. [Abstract/Link to Full Text]

Rasmussen MD, Kellis M
Accurate gene-tree reconstruction by learning gene- and species-specific substitution rates across multiple complete genomes.
Genome Res. 2007 Dec;17(12):1932-42.
Comparative genomics provides a general methodology for discovering functional DNA elements and understanding their evolution. The availability of many related genomes enables more powerful analyses, but requires rigorous phylogenetic methods to resolve orthologous genes and regions. Here, we use 12 recently sequenced Drosophila genomes and nine fungal genomes to address the problem of accurate gene-tree reconstruction across many complete genomes. We show that existing phylogenetic methods that treat each gene tree in isolation show large-scale inaccuracies, largely due to insufficient phylogenetic information in individual genes. However, we find that gene trees exhibit common properties that can be exploited for evolutionary studies and accurate phylogenetic reconstruction. Evolutionary rates can be decoupled into gene-specific and species-specific components, which can be learned across complete genomes. We develop a phylogenetic reconstruction methodology that exploits these properties and achieves significantly higher accuracy, addressing the species-level heterotachy and enabling studies of gene evolution in the context of species evolution. [Abstract/Link to Full Text]

Engström PG, Ho Sui SJ, Drivenes O, Becker TS, Lenhard B
Genomic regulatory blocks underlie extensive microsynteny conservation in insects.
Genome Res. 2007 Dec;17(12):1898-908.
Insect genomes contain larger blocks of conserved gene order (microsynteny) than would be expected under a random breakage model of chromosome evolution. We present evidence that microsynteny has been retained to keep large arrays of highly conserved noncoding elements (HCNEs) intact. These arrays span key developmental regulatory genes, forming genomic regulatory blocks (GRBs). We recently described GRBs in vertebrates, where most HCNEs function as enhancers and HCNE arrays specify complex expression programs of their target genes. Here we present a comparison of five Drosophila genomes showing that HCNE density peaks centrally in large synteny blocks containing multiple genes. Besides developmental regulators that are likely targets of HCNE enhancers, HCNE arrays often span unrelated neighboring genes. We describe differences in core promoters between the target genes and the unrelated genes that offer an explanation for the differences in their responsiveness to enhancers. We show examples of a striking correspondence between boundaries of synteny blocks, HCNE arrays, and Polycomb binding regions, confirming that the synteny blocks correspond to regulatory domains. Although few noncoding elements are highly conserved between Drosophila and the malaria mosquito Anopheles gambiae, we find that A. gambiae regions orthologous to Drosophila GRBs contain an equivalent distribution of noncoding elements highly conserved in the yellow fever mosquito Aëdes aegypti and coincide with regions of ancient microsynteny between Drosophila and mosquitoes. The structural and functional equivalence between insect and vertebrate GRBs marks them as an ancient feature of metazoan genomes and as a key to future studies of development and gene regulation. [Abstract/Link to Full Text]

Heger A, Ponting CP
Evolutionary rate analyses of orthologs and paralogs from 12 Drosophila genomes.
Genome Res. 2007 Dec;17(12):1837-49.
The newly sequenced genome sequences of 11 Drosophila species provide the first opportunity to investigate variations in evolutionary rates across a clade of closely related species. Protein-coding genes were predicted using established Drosophila melanogaster genes as templates, with recovery rates ranging from 81%-97% depending on species divergence and on genome assembly quality. Orthology and paralogy assignments were shown to be self-consistent among the different Drosophila species and to be consistent with regions of conserved gene order (synteny blocks). Next, we investigated the rates of diversification among these species' gene repertoires with respect to amino acid substitutions and to gene duplications. Constraints on amino acid sequences appear to have been most pronounced on D. ananassae and least pronounced on D. simulans and D. erecta terminal lineages. Codons predicted to have been subject to positive selection were found to be significantly over-represented among genes with roles in immune response and RNA metabolism, with the latter category including each subunit of the Dicer-2/r2d2 heterodimer. The vast majority of gene duplications (96.5%) and synteny rearrangements were found to occur, as expected, within single Müller elements. We show that the rate of ancient gene duplications was relatively uniform. However, gene duplications in terminal lineages are strongly skewed toward very recent events, consistent with either a rapid-birth and rapid-death model or the presence of large proportions of copy number variable genes in these Drosophila populations. Duplications were significantly more frequent among trypsin-like proteases and DM8 putative lipid-binding domain proteins. [Abstract/Link to Full Text]

Villasante A, Abad JP, Planelló R, Méndez-Lago M, Celniker SE, de Pablos B
Drosophila telomeric retrotransposons derived from an ancestral element that was recruited to replace telomerase.
Genome Res. 2007 Dec;17(12):1909-18.
Drosophila telomeres do not have arrays of simple telomerase-generated G-rich repeats. Instead, Drosophila maintains its telomeres by occasional transposition of specific non-long terminal repeat (non-LTR) retrotransposons to chromosome ends. The genus Drosophila provides a superb model system for comparative telomere analysis. Here we present an evolutionary study of Drosophila telomeric elements to ascertain the significance of telomeric retrotransposons (TRs) in the maintenance of Drosophila telomeres. PCR and in silico surveys in the sibling species of Drosophila melanogaster and in more distantly related species show that multiple TRs maintain telomeres in Drosophila. In addition to TRs with two open reading frames (ORFs) capable of autonomous transposition, there are deleted telomeric retrotransposons that have lost their ORF2, which we refer to as half telomeric-retrotransposons (HTRs). The phylogenetic relationship among these telomeric elements is congruent with the phylogeny of the species, suggesting that they have been vertically inherited from a common ancestor. Our results suggest that an existing non-LTR retrotransposon was recruited to perform the cellular function of telomere maintenance. [Abstract/Link to Full Text]

Stage DE, Eickbush TH
Sequence variation within the rRNA gene loci of 12 Drosophila species.
Genome Res. 2007 Dec;17(12):1888-97.
Concerted evolution maintains at near identity the hundreds of tandemly arrayed ribosomal RNA (rRNA) genes and their spacers present in any eukaryote. Few comprehensive attempts have been made to directly measure the identity between the rDNA units. We used the original sequencing reads (trace archives) available through the whole-genome shotgun sequencing projects of 12 Drosophila species to locate the sequence variants within the 7.8-8.2 kb transcribed portions of the rDNA units. Three to 18 variants were identified in >3% of the total rDNA units from 11 species. Species where the rDNA units are present on multiple chromosomes exhibited only minor increases in sequence variation. Variants were 10-20 times more abundant in the noncoding compared with the coding regions of the rDNA unit. Within the coding regions, variants were three to eight times more abundant in the expansion compared with the conserved core regions. The distribution of variants was largely consistent with models of concerted evolution in which there is uniform recombination across the transcribed portion of the unit with the frequency of standing variants dependent upon the selection pressure to preserve that sequence. However, the 28S gene was found to contain fewer variants than the 18S gene despite evolving 2.5-fold faster. We postulate that the fewer variants in the 28S gene is due to localized gene conversion or DNA repair triggered by the activity of retrotransposable elements that are specialized for insertion into the 28S genes of these species. [Abstract/Link to Full Text]

Stark A, Kheradpour P, Parts L, Brennecke J, Hodges E, Hannon GJ, Kellis M
Systematic discovery and characterization of fly microRNAs using 12 Drosophila genomes.
Genome Res. 2007 Dec;17(12):1865-79.
MicroRNAs (miRNAs) are short regulatory RNAs that inhibit target genes by complementary binding in 3' untranslated regions (3' UTRs). They are one of the most abundant classes of regulators, targeting a large fraction of all genes, making their comprehensive study a requirement for understanding regulation and development. Here we use 12 Drosophila genomes to define structural and evolutionary signatures of miRNA hairpins, which we use for their de novo discovery. We predict >41 novel miRNA genes, which encompass many unique families, and 28 of which are validated experimentally. We also define signals for the precise start position of mature miRNAs, which suggest corrections of previously known miRNAs, often leading to drastic changes in their predicted target spectrum. We show that miRNA discovery power scales with the number and divergence of species compared, suggesting that such approaches can be successful in human as dozens of mammalian genomes become available. Interestingly, for some miRNAs sense and anti-sense hairpins score highly and mature miRNAs from both strands can indeed be found in vivo. Similarly, miRNAs with weak 5' end predictions show increased in vivo processing of multiple alternate 5' ends and have fewer predicted targets. Lastly, we show that several miRNA star sequences score highly and are likely functional. For mir-10 in particular, both arms show abundant processing, and both show highly conserved target sites in Hox genes, suggesting a possible cooperation of the two arms, and their role as a master Hox regulator. [Abstract/Link to Full Text]

Ruby JG, Stark A, Johnston WK, Kellis M, Bartel DP, Lai EC
Evolution, biogenesis, expression, and target predictions of a substantially expanded set of Drosophila microRNAs.
Genome Res. 2007 Dec;17(12):1850-64.
MicroRNA (miRNA) genes give rise to small regulatory RNAs in a wide variety of organisms. We used computational methods to predict miRNAs conserved among Drosophila species and large-scale sequencing of small RNAs from Drosophila melanogaster to experimentally confirm and complement these predictions. In addition to validating 20 of our top 45 predictions for novel miRNA loci, the large-scale sequencing identified many miRNAs that had not been predicted. In total, 59 novel genes were identified, increasing our tally of confirmed fly miRNAs to 148. The large-scale sequencing also refined the identities of previously known miRNAs and provided insights into their biogenesis and expression. Many miRNAs were expressed in particular developmental contexts, with a large cohort of miRNAs expressed primarily in imaginal discs. Conserved miRNAs typically were expressed more broadly and robustly than were nonconserved miRNAs, and those conserved miRNAs with more restricted expression tended to have fewer predicted targets than those expressed more broadly. Predicted targets for the expanded set of microRNAs substantially increased and revised the miRNA-target relationships that appear conserved among the fly species. Insights were also provided into miRNA gene evolution, including evidence for emergent regulatory function deriving from the opposite arm of the miRNA hairpin, exemplified by mir-10, and even the opposite strand of the DNA, exemplified by mir-iab-4. [Abstract/Link to Full Text]

Lin MF, Carlson JW, Crosby MA, Matthews BB, Yu C, Park S, Wan KH, Schroeder AJ, Gramates LS, St Pierre SE, Roark M, Wiley KL, Kulathinal RJ, Zhang P, Myrick KV, Antone JV, Celniker SE, Gelbart WM, Kellis M
Revisiting the protein-coding gene catalog of Drosophila melanogaster using 12 fly genomes.
Genome Res. 2007 Dec;17(12):1823-36.
The availability of sequenced genomes from 12 Drosophila species has enabled the use of comparative genomics for the systematic discovery of functional elements conserved within this genus. We have developed quantitative metrics for the evolutionary signatures specific to protein-coding regions and applied them genome-wide, resulting in 1193 candidate new protein-coding exons in the D. melanogaster genome. We have reviewed these predictions by manual curation and validated a subset by directed cDNA screening and sequencing, revealing both new genes and new alternative splice forms of known genes. We also used these evolutionary signatures to evaluate existing gene annotations, resulting in the validation of 87% of genes lacking descriptive names and identifying 414 poorly conserved genes that are likely to be spurious predictions, noncoding, or species-specific genes. Furthermore, our methods suggest a variety of refinements to hundreds of existing gene models, such as modifications to translation start codons and exon splice boundaries. Finally, we performed directed genome-wide searches for unusual protein-coding structures, discovering 149 possible examples of stop codon readthrough, 125 new candidate ORFs of polycistronic mRNAs, and several candidate translational frameshifts. These results affect >10% of annotated fly genes and demonstrate the power of comparative genomics to enhance our understanding of genome organization, even in a model organism as intensively studied as Drosophila melanogaster. [Abstract/Link to Full Text]

Bhutkar A, Russo SM, Smith TF, Gelbart WM
Genome-scale analysis of positionally relocated genes.
Genome Res. 2007 Dec;17(12):1880-7.
During evolution, genome reorganization includes large-scale events such as inversions, translocations, and segmental or even whole-genome duplications, as well as fine-scale events such as the relocation of individual genes. This latter category, which we will refer to as positionally relocated genes (PRGs), is the subject of this report. Assessment of the magnitude of such PRGs and of possible contributing mechanisms is aided by a comparative analysis of related genomes, where conserved chromosomal organization can aid in identifying genes that have acquired a new location in a lineage of these genomes. Here we utilize two methods to comprehensively identify relocated protein-coding genes in the recently sequenced genomes of 12 species of genus Drosophila. We use exceptions to the general rule of maintenance of chromosome arm (Muller element) association for most Drosophila genes to identify one major class of PRGs. We also identify a partially overlapping set of PRGs among "embedded genes," located within the extents of other surrounding genes. We provide evidence that PRG movements have at least two different origins: Some events occur via retrotransposition of processed RNAs and others via a DNA-based transposition mechanism. Overall, we identify several hundred PRGs that arose within a lineage of the genus Drosophila phylogeny and provide suggestive evidence that a few thousand such events have occurred within the radiation of the insect order Diptera, thereby illustrating the magnitude of the contribution of PRG movement to chromosomal reorganization during evolution. [Abstract/Link to Full Text]

Kheradpour P, Stark A, Roy S, Kellis M
Reliable prediction of regulator targets using 12 Drosophila genomes.
Genome Res. 2007 Dec;17(12):1919-31.
Gene expression is regulated pre- and post-transcriptionally via cis-regulatory DNA and RNA motifs. Identification of individual functional instances of such motifs in genome sequences is a major goal for inferring regulatory networks yet has been hampered due to the motifs' short lengths that lead to many chance matches and poor signal-to-noise ratios. In this paper, we develop a general methodology for the comparative identification of functional motif instances across many related species, using a phylogenetic framework that accounts for the evolutionary relationships between species, allows for motif movements, and is robust against missing data due to artifacts in sequencing, assembly, or alignment. We also provide a robust statistical framework for evaluating motif confidence, which enables us to translate evolutionary conservation into a confidence measure for each motif instance, correcting for varying motif length, composition, and background conservation of the target regions. We predict targets of fly transcription factors and miRNAs in alignments of 12 recently sequenced Drosophila species. When compared to extensive genome-wide experimental data, predicted targets are of high quality, matching and surpassing ChIP-chip microarrays and recovering miRNA targets with high sensitivity. The resulting regulatory network suggests significant redundancy between pre- and post-transcriptional regulation of gene expression. [Abstract/Link to Full Text]

Cornell MJ, Alam I, Soanes DM, Wong HM, Hedeler C, Paton NW, Rattray M, Hubbard SJ, Talbot NJ, Oliver SG
Comparative genome analysis across a kingdom of eukaryotic organisms: Specialization and diversification in the Fungi.
Genome Res. 2007 Dec;17(12):1809-22.
The recent proliferation of genome sequencing in diverse fungal species has provided the first opportunity for comparative genome analysis across a eukaryotic kingdom. Here, we report a comparative study of 34 complete fungal genome sequences, representing a broad diversity of Ascomycete, Basidiomycete, and Zygomycete species. We have clustered all predicted protein-encoding gene sequences from these species to provide a means of investigating gene innovations, gene family expansions, protein family diversification, and the conservation of essential gene functions-empirically determined in Saccharomyces cerevisiae-among the fungi. The results are presented with reference to a phylogeny of the 34 fungal species, based on 29 universally conserved protein-encoding gene sequences. We contrast this phylogeny with one based on gene presence and absence and show that, while the two phylogenies are largely in agreement, there are differences in the positioning of some species. We have investigated levels of gene duplication and demonstrate that this varies greatly between fungal species, although there are instances of coduplication in distantly related fungi. We have also investigated the extent of orthology for protein families and demonstrate unexpectedly high levels of diversity among genes involved in lipid metabolism. These analyses have been collated in the e-Fungi data warehouse, providing an online resource for comparative genomic analysis of the fungi. [Abstract/Link to Full Text]

Legendre M, Pochet N, Pak T, Verstrepen KJ
Sequence-based estimation of minisatellite and microsatellite repeat variability.
Genome Res. 2007 Dec;17(12):1787-96.
Variable tandem repeats are frequently used for genetic mapping, genotyping, and forensics studies. Moreover, variation in some repeats underlies rapidly evolving traits or certain diseases. However, mutation rates vary greatly from repeat to repeat, and as a consequence, not all tandem repeats are suitable genetic markers or interesting unstable genetic modules. We developed a model, "SERV," that predicts the variability of a broad range of tandem repeats in a wide range of organisms. The nonlinear model uses three basic characteristics of the repeat (number of repeated units, unit length, and purity) to produce a numeric "VARscore" that correlates with repeat variability. SERV was experimentally validated using a large set of different artificial repeats located in the Saccharomyces cerevisiae URA3 gene. Further in silico analysis shows that SERV outperforms existing models and accurately predicts repeat variability in bacteria and eukaryotes, including plants and humans. Using SERV, we demonstrate significant enrichment of variable repeats within human genes involved in transcriptional regulation, chromatin remodeling, morphogenesis, and neurogenesis. Moreover, SERV allows identification of known and candidate genes involved in repeat-based diseases. In addition, we demonstrate the use of SERV for the selection and comparison of suitable variable repeats for genotyping and forensic purposes. Our analysis indicates that tandem repeats used for genotyping should have a VARscore between 1 and 3. SERV is publicly available from [Abstract/Link to Full Text]

Green P
2x genomes--does depth matter?
Genome Res. 2007 Nov;17(11):1547-9. [Abstract/Link to Full Text]

Copley RR, Totrov M, Linnell J, Field S, Ragoussis J, Udalova IA
Functional conservation of Rel binding sites in drosophilid genomes.
Genome Res. 2007 Sep;17(9):1327-35.
Evolutionary constraints on gene regulatory elements are poorly understood: Little is known about how the strength of transcription factor binding correlates with DNA sequence conservation, and whether transcription factor binding sites can evolve rapidly while retaining their function. Here we use the model of the NFKB/Rel-dependent gene regulation in divergent Drosophila species to examine the hypothesis that the functional properties of authentic transcription factor binding sites are under stronger evolutionary constraints than the genomic background. Using molecular modeling we compare tertiary structures of the Drosophila Rel family proteins Dorsal, Dif, and Relish and demonstrate that their DNA-binding and protein dimerization domains undergo distinct rates of evolution. The accumulated amino acid changes, however, are unlikely to affect DNA sequence recognition and affinity. We employ our recently developed microarray-based experimental platform and principal coordinates statistical analysis to quantitatively and systematically profile DNA binding affinities of three Drosophila Rel proteins to 10,368 variants of the NFKB recognition sequences. We then correlate the evolutionary divergence of gene regulatory regions with differences in DNA binding affinities. Genome-wide analyses reveal a significant increase in the number of conserved Rel binding sites in promoters of developmental and immune genes. Significantly, the affinity of Rel proteins to these sites was higher than to less conserved sites and was maintained by the conservation of the DNA binding site sequence (static conservation) or in some cases despite significantly diverged sequences (dynamic conservation). We discuss how two types of conservation may contribute to the stabilization and optimization of a functional gene regulatory code in evolution. [Abstract/Link to Full Text]

Dreszer TR, Wall GD, Haussler D, Pollard KS
Biased clustered substitutions in the human genome: the footprints of male-driven biased gene conversion.
Genome Res. 2007 Oct;17(10):1420-30.
We examined fixed substitutions in the human lineage since divergence from the common ancestor with the chimpanzee, and determined what fraction are AT to GC (weak-to-strong). Substitutions that are densely clustered on the chromosomes show a remarkable excess of weak-to-strong "biased" substitutions. These unexpected biased clustered substitutions (UBCS) are common near the telomeres of all autosomes but not the sex chromosomes. Regions of extreme bias are enriched for genes. Human and chimp orthologous regions show a striking similarity in the shape and magnitude of their respective UBCS maps, suggesting a relatively stable force leads to clustered bias. The strong and stable signal near telomeres may have participated in the evolution of isochores. One exception to the UBCS pattern found in all autosomes is chromosome 2, which shows a UBCS peak midchromosome, mapping to the fusion site of two ancestral chromosomes. This provides evidence that the fusion occurred as recently as 740,000 years ago and no more than approximately 3 million years ago. No biased clustering was found in SNPs, suggesting that clusters of biased substitutions are selected from mutations. UBCS is strongly correlated with male (and not female) recombination rates, which explains the lack of UBCS signal on chromosome X. These observations support the hypothesis that biased gene conversion (BGC), specifically in the male germline, played a significant role in the evolution of the human genome. [Abstract/Link to Full Text]

Fiston-Lavier AS, Anxolabehere D, Quesneville H
A model of segmental duplication formation in Drosophila melanogaster.
Genome Res. 2007 Oct;17(10):1458-70.
Segmental duplications (SDs) are low-copy repeats of DNA segments that have long been recognized to be involved in genome organization and evolution. But, to date, the mechanism of their formation remains obscure. We propose a model for SD formation that we name "duplication-dependent strand annealing" (DDSA). This model is a variant of the synthesis-dependent strand annealing (SDSA) model--a double-strand break (DSB) homologous repair model. DSB repair in Drosophila melanogaster genome usually occurs primarily through homologous repair, more preferentially through the SDSA model. The DDSA model predicts that after a DSB, the search for an ectopic homologous region--here a repeat--initiates the repair. As expected by the model, the analysis of SDs detected by a computational analysis of the D. melanogaster genome indicates a high enrichment in transposable elements at SD ends. It shows moreover a preferential location of SDs in heterochromatic regions. The model has the advantage of also predicting specific traces left during synthesis. The observed traces support the DDSA model as one model of formation of SDs in D. melanogaster genome. The analysis of these DDSA signatures suggests moreover a sequestration of the dissociated strand in the repair complex. [Abstract/Link to Full Text]

Becquet C, Przeworski M
A new approach to estimate parameters of speciation models with application to apes.
Genome Res. 2007 Oct;17(10):1505-19.
How populations diverge and give rise to distinct species remains a fundamental question in evolutionary biology, with important implications for a wide range of fields, from conservation genetics to human evolution. A promising approach is to estimate parameters of simple speciation models using polymorphism data from multiple loci. Existing methods, however, make a number of assumptions that severely limit their applicability, notably, no gene flow after the populations split and no intralocus recombination. To overcome these limitations, we developed a new Markov chain Monte Carlo method to estimate parameters of an isolation-migration model. The approach uses summaries of polymorphism data at multiple loci surveyed in a pair of diverging populations or closely related species and, importantly, allows for intralocus recombination. To illustrate its potential, we applied it to extensive polymorphism data from populations and species of apes, whose demographic histories are largely unknown. The isolation-migration model appears to provide a reasonable fit to the data. It suggests that the two chimpanzee species became reproductively isolated in allopatry approximately 850 Kya, while Western and Central chimpanzee populations split approximately 440 Kya but continued to exchange migrants. Similarly, Eastern and Western gorillas and Sumatran and Bornean orangutans appear to have experienced gene flow since their splits approximately 90 and over 250 Kya, respectively. [Abstract/Link to Full Text]

Gierman HJ, Indemans MH, Koster J, Goetze S, Seppen J, Geerts D, van Driel R, Versteeg R
Domain-wide regulation of gene expression in the human genome.
Genome Res. 2007 Sep;17(9):1286-95.
Transcription factor complexes bind to regulatory sequences of genes, providing a system of individual expression regulation. Targets of distinct transcription factors usually map throughout the genome, without clustering. Nevertheless, highly and weakly expressed genes do cluster in separate chromosomal domains with an average size of 80-90 genes. We therefore asked whether, besides transcription factors, an additional level of gene expression regulation exists that acts on chromosomal domains. Here we show that identical green fluorescent protein (GFP) reporter constructs integrated at 90 different chromosomal positions obtain expression levels that correspond to the activity of the domains of integration. These domains are up to 80 genes long and can exert an eightfold effect on the expression levels of integrated genes. 3D-FISH shows that active domains of integration have a more open chromatin structure than integration domains with weak activity. These results reveal a novel domain-wide regulatory mechanism that, together with transcription factors, exerts a dual control over gene transcription. [Abstract/Link to Full Text]

DeCaprio D, Vinson JP, Pearson MD, Montgomery P, Doherty M, Galagan JE
Conrad: gene prediction using conditional random fields.
Genome Res. 2007 Sep;17(9):1389-98.
We present Conrad, the first comparative gene predictor based on semi-Markov conditional random fields (SMCRFs). Unlike the best standalone gene predictors, which are based on generalized hidden Markov models (GHMMs) and trained by maximum likelihood, Conrad is discriminatively trained to maximize annotation accuracy. In addition, unlike the best annotation pipelines, which rely on heuristic and ad hoc decision rules to combine standalone gene predictors with additional information such as ESTs and protein homology, Conrad encodes all sources of information as features and treats all features equally in the training and inference algorithms. Conrad outperforms the best standalone gene predictors in cross-validation and whole chromosome testing on two fungi with vastly different gene structures. The performance improvement arises from the SMCRF's discriminative training methods and their ability to easily incorporate diverse types of information by encoding them as feature functions. On Cryptococcus neoformans, configuring Conrad to reproduce the predictions of a two-species phylo-GHMM closely matches the performance of Twinscan. Enabling discriminative training increases performance, and adding new feature functions further increases performance, achieving a level of accuracy that is unprecedented for this organism. Similar results are obtained on Aspergillus nidulans comparing Conrad versus Fgenesh. SMCRFs are a promising framework for gene prediction because of their highly modular nature, simplifying the process of designing and testing potential indicators of gene structure. Conrad's implementation of SMCRFs advances the state of the art in gene prediction in fungi and provides a robust platform for both current application and future research. [Abstract/Link to Full Text]

Nakatani Y, Takeda H, Kohara Y, Morishita S
Reconstruction of the vertebrate ancestral genome reveals dynamic genome reorganization in early vertebrates.
Genome Res. 2007 Sep;17(9):1254-65.
Although several vertebrate genomes have been sequenced, little is known about the genome evolution of early vertebrates and how large-scale genomic changes such as the two rounds of whole-genome duplications (2R WGD) affected evolutionary complexity and novelty in vertebrates. Reconstructing the ancestral vertebrate genome is highly nontrivial because of the difficulty in identifying traces originating from the 2R WGD. To resolve this problem, we developed a novel method capable of pinning down remains of the 2R WGD in the human and medaka fish genomes using invertebrate tunicate and sea urchin genes to define ohnologs, i.e., paralogs produced by the 2R WGD. We validated the reconstruction using the chicken genome, which was not considered in the reconstruction step, and observed that many ancestral proto-chromosomes were retained in the chicken genome and had one-to-one correspondence to chicken microchromosomes, thereby confirming the reconstructed ancestral genomes. Our reconstruction revealed a contrast between the slow karyotype evolution after the second WGD and the rapid, lineage-specific genome reorganizations that occurred in the ancestral lineages of major taxonomic groups such as teleost fishes, amphibians, reptiles, and marsupials. [Abstract/Link to Full Text]

Ogata H, Claverie JM
Unique genes in giant viruses: regular substitution pattern and anomalously short size.
Genome Res. 2007 Sep;17(9):1353-61.
Large DNA viruses, including giant mimivirus with a 1.2-Mb genome, exhibit numerous orphan genes possessing no database homologs or genes with homologs solely in close members of the same viral family. Due to their solitary nature, the functions and evolutionary origins of those genes remain obscure. We examined sequence features and evolutionary rates of viral family-specific genes in three nucleo-cytoplasmic large DNA virus (NCLDV) lineages. First, we showed that the proportion of family-specific genes does not correlate with sequence divergence rate. Second, position-dependent nucleotide statistics were similar between family-specific genes and the remaining genes in the genome. Third, we showed that the synonymous-to-nonsynonymous substitution ratios in those viruses are at levels comparable to those estimated for vertebrate proteomes. Thus, the vast majority of family-specific genes do not exhibit an accelerated evolutionary rate, and are thus likely to specify functional polypeptides. On the other hand, these family-specific proteins exhibit several distinct properties: (1) they are shorter, (2) they include a larger fraction of predicted transmembrane proteins, and (3) they are enriched in low-complexity sequences. These results suggest that family-specific genes do not correspond to recent horizontal gene transfer. We propose that their characteristic features are the consequences of the specific evolutionary forces shaping the viral gene repertoires in the context of their parasitic lifestyles. [Abstract/Link to Full Text]

Wong LH, Brettingham-Moore KH, Chan L, Quach JM, Anderson MA, Northrop EL, Hannan R, Saffery R, Shaw ML, Williams E, Choo KH
Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere.
Genome Res. 2007 Aug;17(8):1146-60.
The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric alpha-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I-dependent manner. The nucleolar targeting of CENPC1 and INCENP requires alpha-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following alpha-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds alpha-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and "RNA replenishment" experiments, we show that alpha-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. Our data suggest that centromere satellite RNA directly facilitates the accumulation and assembly of centromere-specific nucleoprotein components at the nucleolus and mitotic centromere, and that the sequestration of these components in the interphase nucleolus provides a regulatory mechanism for their timely release into the nucleoplasm for kinetochore assembly at the onset of mitosis. [Abstract/Link to Full Text]

Wang C, Mitsuya Y, Gharizadeh B, Ronaghi M, Shafer RW
Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance.
Genome Res. 2007 Aug;17(8):1195-201.
The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in > or =3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations. [Abstract/Link to Full Text]

Blankenberg D, Taylor J, Schenck I, He J, Zhang Y, Ghent M, Veeraraghavan N, Albert I, Miller W, Makova KD, Hardison RC, Nekrutenko A
A framework for collaborative analysis of ENCODE data: making large-scale analyses biologist-friendly.
Genome Res. 2007 Jun;17(6):960-4.
The standardization and sharing of data and tools are the biggest challenges of large collaborative projects such as the Encyclopedia of DNA Elements (ENCODE). Here we describe a compact Web application, Galaxy2(ENCODE), that effectively addresses these issues. It provides an intuitive interface for the deposition and access of data, and features a vast number of analysis tools including operations on genomic intervals, utilities for manipulation of multiple sequence alignments, and molecular evolution algorithms. By providing a direct link between data and analysis tools, Galaxy2(ENCODE) allows addressing biological questions that are beyond the reach of existing software. We use Galaxy2(ENCODE) to show that the ENCODE regions contain >2000 unannotated transcripts under strong purifying selection that are likely functional. We also show that the ENCODE regions are representative of the entire genome by estimating the rate of nucleotide substitution and comparing it to published data. Although each of these analyses is complex, none takes more than 15 min from beginning to end. Finally, we demonstrate how new tools can be added to Galaxy2(ENCODE) with almost no effort. Every section of the manuscript is supplemented with QuickTime screencasts. Galaxy2(ENCODE) and the screencasts can be accessed at [Abstract/Link to Full Text]

Elnitski LL, Shah P, Moreland RT, Umayam L, Wolfsberg TG, Baxevanis AD
The ENCODEdb portal: simplified access to ENCODE Consortium data.
Genome Res. 2007 Jun;17(6):954-9.
The Encyclopedia of DNA Elements (ENCODE) project aims to identify and characterize all functional elements in a representative chromosomal sample comprising 1% of the human genome. Data generated by members of The ENCODE Project Consortium are housed in a number of public databases, such as the UCSC Genome Browser, NCBI's Gene Expression Omnibus (GEO), and EBI's ArrayExpress. As such, it is often difficult for biologists to gather all of the ENCODE data from a particular genomic region of interest and integrate them with relevant information found in other public databases. The ENCODEdb portal was developed to address this problem. ENCODEdb provides a unified, single point-of-access to data generated by the ENCODE Consortium, as well as to data from other source databases that lie within ENCODE regions; this provides the user a complete view of all known data in a particular region of interest. ENCODEdb Genomic Context searches allow for the retrieval of information on functional elements annotated within ENCODE regions, including mRNA, EST, and STS sequences; single nucleotide polymorphisms, and UniGene clusters. Information is also retrieved from GEO, OMIM, and major genome sequence browsers. ENCODEdb Consortium Data searches allow users to perform compound queries on array-based ENCODE data available both from GEO and from the UCSC Genome Browser. Results are retrieved from a specific genomic area of interest and can be further manipulated in a variety of contexts, including the UCSC Genome Browser and the Galaxy large-scale genome analysis platform. The ENCODEdb portal is freely accessible at [Abstract/Link to Full Text]

Greenbaum JA, Pang B, Tullius TD
Construction of a genome-scale structural map at single-nucleotide resolution.
Genome Res. 2007 Jun;17(6):947-53.
Few methods are available for mapping the local structure of DNA throughout a genome. The hydroxyl radical cleavage pattern is a measure of the local variation in solvent-accessible surface area of duplex DNA, and thus provides information on the local shape and structure of DNA. We report the construction of a relational database, ORChID (OH Radical Cleavage Intensity Database), that contains extensive hydroxyl radical cleavage data produced from two DNA libraries. We have used the ORChID database to develop a set of algorithms that are capable of predicting the hydroxyl radical cleavage pattern of a DNA sequence of essentially any length, to high accuracy. We have used the prediction algorithm to produce a structural map of the 30 Mb of the ENCODE regions of the human genome. [Abstract/Link to Full Text]

Greenbaum JA, Parker SC, Tullius TD
Detection of DNA structural motifs in functional genomic elements.
Genome Res. 2007 Jun;17(6):940-6.
The completion of the human genome project has fueled the search for regulatory elements by a variety of different approaches. Many successful analyses have focused on examining primary DNA sequence and/or chromatin structure. However, it has been difficult to detect common sequence motifs within the feature of chromatin structure most closely associated with regulatory elements, DNase I hypersensitive sites (DHSs). Considering just the nucleotide sequence and/or the chromatin structure of regulatory elements may neglect a critical feature of what is recognized by the regulatory machinery--DNA structure. We introduce a new computational method to detect common DNA structural motifs in a large collection of DHSs that are found in the ENCODE regions of the human genome. We show that DHSs have common DNA structural motifs that show no apparent sequence consensus. One such structural motif is much more highly enriched in experimentally identified DHSs that are in CpG islands and near transcription start sites (TSSs), compared to DHSs not in CpG islands and farther from TSSs, suggesting that DNA structural motifs may participate in the formation of functional regulatory elements. We propose that studies of the conservation of DNA structure, independent of sequence conservation, will provide new information about the link between the nucleotide sequence of a DNA molecule and its experimentally demonstrated function. [Abstract/Link to Full Text]

Recent Articles in Journal of Applied Genetics

Wiejacha K, Trzewik A, Orlikowski LB, Szkuta G, Orlikowska T
Genomic polymorphism of isolates of Phytophthora ramorum from Polish ornamental nurseries compared with other European and North American isolates.
J Appl Genet. 2007;48(4):413-9.
We undertook an analysis of the genomic relationships between 15 isolates of Phytophthora ramorum Werres, de Cock et Man in't Veld, obtained from symptomatic plants growing in Polish ornamental nurseries, and 2 representatives of the European population and 3 of the North American population. Dendrograms were generated by UPGMA based on 786 amplification products obtained in ISSR-PCR reactions. The representatives of the European population and 13 of the "Polish" isolates formed a common cluster. The other 2 "Polish" isolates, which were found in 1998, and the 3 American representatives formed 2 separate clusters. There was no observed link between genomic distance on the basis of polymorphism and the origin of the isolates from plant species. [Abstract/Link to Full Text]

Podgorska B, Pazdro K, Wegrzyn G
The use of the Vibrio harveyi luminescence mutagenicity assay as a rapid test for preliminary assessment of mutagenic pollution of marine sediments.
J Appl Genet. 2007;48(4):409-12.
Mutagenic pollution of the natural environment is currently one of the most serious environmental problems. It includes the pollution of marine sediments. Therefore, rapid detection of the presence of mutagens is an important issue. Recently, we have developed a novel microbiological assay for rapid assessment of mutagenicity of samples from the natural environment. This assay is based on bioluminescence of a mutant Vibrio harveyi strain, and was shown to be useful in testing samples of marine water and plant tissues. Here we demonstrate the usefulness of this assay in preliminary assessment of mutagenic pollution of marine sediments. Mutagenicity of environmental samples taken from the Baltic Sea, is documented and compared here with a commercially available standard sediment sample (IAEA 383), which contains known amounts of mutagenic compounds. The whole procedure, from obtaining a sample in the laboratory to getting final results, is very short (less than 4 h). [Abstract/Link to Full Text]

Duran-Gonzalez J, Gutierrez-Angulo M, Garcia-Cruz D, Ayala M, Padilla M, Davalos IP
A de novo interstitial 6q deletion in a boy with a split hand malformation.
J Appl Genet. 2007;48(4):405-407.
We report on a de novo interstitial deletion of (6)(q15q22.2) in a 5-year-old boy with developmental delay, microcephaly, facial dysmorphism, cryptorchidism, congenital heart defect, and split-hand malformation. Previous reports and this patient suggest that 6q21 may contain a gene or genes related either directly or indirectly to limb development. [Abstract/Link to Full Text]

Przybylski GK, Kreuzer KA, Siegert W, Schmidt CA
No recovery of T-cell receptor excision circles (TRECs) after non-myeloablative allogeneic hematopoietic stem cell transplantation is correlated with the onset of GvHD.
J Appl Genet. 2007;48(4):397-404.
Improper T-cell reconstitution with its consequences, graft-vs-host disease (GvHD) and outbreak of viral infections, is the major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). To determine the factors affecting reconstitution of naive T-cells after non-myeloablative HSCT (NM-HSCT), the T-cell receptor excision circle (TREC) content was measured on a weekly basis in 24 transplanted patients with various malignant diseases. We analysed correlations of the results with the development of GvHD. In addition, in 11 chronic myeloid leukaemia (CML) patients, we correlated TREC and BCR-ABL transcript numbers. After HSCT, in most patients (22/24) TRECs became undetectable. In 12 patients, TRECs reappeared 3-4 months after HSCT, in 1 patient TRECs reappeared 5 months after HSCT, and in 11 patients TRECs remained negative for more than a year. All 11 patients who remained TREC-negative, developed acute GvHD grade 2-3, while only 6 out of 13 patients who recovered TRECs developed GvHD. We show that after non-myeloablative HSCT, thymopoiesis takes place and is affected by GvHD. Our results indicate that no recovery of TRECs after NM-HSCT (which most likely reflect the expansion of host-reactive co-transplanted mature T-cells) correlates with the onset of GvHD. [Abstract/Link to Full Text]

Corona-Rivera A, Urbina-Cano P, Bobadilla-Morales L, Vargas-Lares J, Ramirez-Herrera M, Mendoza-Magaua M, Troyo-Sanroman R, Diaz-Esquivel P, Corona-Rivera J
Protective in vivo effect of curcumin on copper genotoxicity evaluated by comet and micronucleus assays.
J Appl Genet. 2007;48(4):389-396.
Curcumin is a phytochemical with antiinflammatory, antioxidant and anticarcinogenic activities. Apparently, curcumin is not genotoxic in vivo, but in vitro copper and curcumin interactions induce genetic damage. The aim of this study was to test if in vivo copper excess induces DNA damage measured by comet and micronucleus assays in the presence of curcumin. We tested 0.2&percnt; curcumin in Balb-C mice at normal (13 ppm) and high (65, 130 and 390 ppm) copper ion concentrations. The comet and micronucleus assays were performed 48 hr after chemical application. Comet tail length in animals treated with 0.2&percnt; curcumin was not significantly different from the control. Animals exposed to copper cations (up to 390 ppm) exhibited higher oxidative DNA damage. Curcumin reduced the DNA damage induced by 390 ppm copper. We observed statistically significant increase in damage in individuals exposed to 390 ppm copper versus the control or curcumin groups, which was lowered by the presence of curcumin. Qualitative data on comets evidenced that cells from individuals exposed to 390 ppm copper had longer tails (categories 3 and 4) than in 390 ppm copper + curcumin. A statistically significant increase in frequency of micronucleated erythrocytes (MNE/10000TE) was observed only in 390 ppm copper versus the control and curcumin alone. Also cytotoxicity measured as the frequency of polychromatic erythrocytes (PE/1000TE) was attributable to 390 ppm copper. The lowest cytotoxic effect observed was attributed to curcumin. In vivo exposure to 0.2&percnt; curcumin for 48 hr did not cause genomic damage, while 390 ppm copper was genotoxic, but DNA damage induced by 390 ppm copper was diminished by curcumin. Curcumin seems to exert a genoprotective effect against DNA damage induced by high concentrations of copper cations. The comet and micronucleus assays prove to be suitable tools to detect DNA damage by copper in the presence of curcumin. [Abstract/Link to Full Text]

Jakobkiewicz-Banecka J, Wegrzyn A, Wegrzyn G
Substrate deprivation therapy: a new hope for patients suffering from neuronopathic forms of inherited lysosomal storage diseases.
J Appl Genet. 2007;48(4):383-8.
Lysosomal storage diseases are a group of disorders caused by defects in enzymes responsible for degradation of particular compounds in lysosomes. In most cases, these diseases are fatal, and until recently no treatment was available. Introduction of enzyme replacement therapy was a breakthrough in the treatment of some of the diseases. However, while this therapy is effective in reduction of many somatic symptoms, its efficacy in the treatment of the central nervous system is negligible, if any, mainly because of problems with crossing the blood-brain-barrier by intravenously administered enzyme molecules. On the other hand, there are many lysosomal storage diseases in which the central nervous system is affected. Results of very recent studies indicate that in at least some cases, another type of therapy, called substrate deprivation therapy (or substrate reduction therapy) may be effective in the treatment of neuronopathic forms of lysosomal storage diseases. This therapy, based on inhibition of synthesis of the compounds that cannot be degraded in cells of the patients, has been shown to be effective in several animal models of various diseases, and recent reports demonstrate its efficacy in the treatment of patients suffering from Niemann-Pick C disease and Sanfilippo disease. [Abstract/Link to Full Text]

Danielak-Czech B, Slota E
A new case of reciprocal translocation t(10;13)(q16;q21) diagnosed in an AI boar.
J Appl Genet. 2007;48(4):379-388.
A new case of reciprocal translocation t(10;13)(q16;q21) was detected in a hybrid boar (Large White &times; Pietrain &times; Duroc &times; Hampshire) from an artificial insemination (AI) station. Altogether, 258 sires of 4 pure breeds as well as hybrid lines and crossbreeds were investigated. The diagnosis was based on classical cytogenetic examination following the standard protocols of lymphocyte cultures, Giemsa staining and G-, C- and Ag-I banding techniques. The population screening performed was an initial part of a long-term karyotype control system of boars kept at AI stations, which was started by the National Research Institute of Animal Production in Poland in 2007. [Abstract/Link to Full Text]

Czarnik U, Grzybowski G, Kaminski S, Prusak B, Zabolewicz T
Effectiveness of a program aimed at the elimination of BLAD-carrier bulls from Polish Holstein-Friesian cattle.
J Appl Genet. 2007;48(4):375-7.
The molecular basis of BLAD is the D128G mutation of the gene coding for the CD18 subunit of beta-2 integrin. This mutation is lethal, since homozygous (BL/BL) animals die before they reach sexual maturity. In the 1990s, BLAD was the most widespread genetic disease in HF cattle worldwide. The aim of the present study was to determine the frequency of BLAD carriers among 4645 young breeding bulls in Poland in 1995-2006. The frequency of carriers of the mutated allele showed a clear decreasing trend. The highest frequency (7.9%) was recorded while implementing the BLAD control program (1995-1997). Regular monitoring has enabled a great reduction of this threat to the tested population. Today only sporadic cases of BL/TL heterozygotes are reported (ca. 0.8% in 2004-2006). [Abstract/Link to Full Text]

Song CY, Gao B, Teng SH, Wang XY, Xie F, Chen GH, Wang ZY, Jing RB, Mao JD
Polymorphisms in intron 1 of the porcine POU1F1 gene.
J Appl Genet. 2007;48(4):371-4.
This study was conducted to detect polymorphisms in intron 1 of porcine POU1F1 (POU domain, class 1, transcription factor 1, Pit1, renamed as POU1F1) by comparative sequencing. Within the intron, 23 sites of variation were identified, including 16 single-nucleotide substitutions, 4 single-nucleotide indels, 2 short (3-bp and 17-bp), and one long (313-bp) indels. Several important regulatory motifs were found within the 313-bp indel by in silico analysis. The 313-bp indel was next genotyped in 11 Chinese native pig breeds and 4 western meat-type pig breeds. The appearance of genotypes varied between breeds: among Chinese native breeds, no AA and AB genotypes were found in Tibetan, Lingao, Min, Rongchang, and Songliao Black pigs, no AA genotype was found in Fenjing and Leping Spotted pigs, whereas in Pietrain and Landrace there were no BB genotypes, and all 19 Duroc pigs were AA homozygotes. The western meat-type pigs had high A allele frequencies and the Chinese pigs had more B alleles, except Jianquhai pigs. A positive association of the AA genotype with birth weight was observed in a commercial pig line. This paper demonstrated that the genetic variation in intron 1 of the pig POU1F1 gene was high and these polymorphisms may provide useful makers for QTL analysis. [Abstract/Link to Full Text]

Zhang J, Xiong Y, Zuo B, Lei M, Jiang S, Li F, Zheng R, Li J, Xu D
Quantitative trait loci for carcass traits on pig chromosomes 4, 6, 7, 8 and 13.
J Appl Genet. 2007;48(4):363-369.
For 22 carcass traits, we identified 16 QTLs (based on data for pig resource population no. 214, including 180 F2 hybrids of 3 Yorkshire boars and 8 Meishan sows) and mapped them with the use of 39 microsatellite marker loci on chromosomes 4, 6, 7, 8 and 13. Five QTLs were highly significant (P &le; 0.01 at chromosome level): for skin weight (on chromosome 7 at SW1856 and on chromosome 13 at SW1495), skin percentage (on chromosome 7 between SW2155 and SW1856 and on chromosome 13 between SW1495 and SW520), and ratio of leg and butt to carcass (on chromosome 4 at SW1996). The remaining 11 QTLs were significant (P &le; 0.05 at chromosome level): for backfat thickness at shoulder, loin eye width, loin eye height, fat meat weight, lean meat weight, skin weight, bone weight, skin percentage, fat meat percentage, and ratio of lean meat to fat meat. The proportion of phenotypic variance explained by these QTLs ranged from 0.06&percnt; (QTL for loin eye width on chromosome 8 between SW1037 and SW1953) to 18.04&percnt; (QTL for ratio of lean meat to fat meat on chromosome 7 between SW252 and SW581). Seven of the QTLs reported here are novel. [Abstract/Link to Full Text]

Kontek R, Osiecka R, Kontek B
Clastogenic and mitodepressive effects of the insecticide dichlorvos on root meristems of Vicia faba.
J Appl Genet. 2007;48(4):359-61.
Plant bioassays are an important and integral part of the test battery used in detecting genotoxic/carcinogenic contamination in the environment. Highly sensitive biomonitoring of plant models have been developed, which enables the detection of hazards arising from pesticides, insecticides, industrial contamination, heavy metals and radiation. Root tips of Vicia faba ssp. minor were treated with 1-60 mM of the organophosphorus insecticide dichlorvos (DDVP) for 2 h, followed by a 20-h recovery period. Maleic acid hydrazide (MH) was used as a positive control for the mitotic index, micronucleus and chromosomal aberration assays performed on the Vicia model system. All treatments with DDVP significantly decreased the mitotic activity and increased the frequency of chromosomal aberrations at the metaphase. The frequency of micronuclei was significantly increased at DDVP concentrations starting from 10 mM. The results demonstrate clastogenic and mitodepressive effects of DDVP on Vicia faba cells. [Abstract/Link to Full Text]

Salmanowicz BP, Dylewicz M
Identification and characterization of high-molecular-weight glutenin genes in Polish triticale cultivars by PCR-based DNA markers.
J Appl Genet. 2007;48(4):347-57.
Molecular markers were used to identify the allele/gene composition of complex loci Glu-A1 and Glu-B1 of high-molecular-weight (HMW) glutenin subunits in triticale cultivars. Forty-six Polish cultivars of both winter and spring triticale were analysed with 7 PCR-based markers. Amplified DNA fragments of HMW glutenin Glu-1 genes were separated by agarose slab-gel electrophoresis. Differences between all 3 alleles at the locus Glu-A1 [Glu-A1a (encoding Ax1), 1b (Ax2*), and 1c (AxNull)], 4 alleles at Glu-B1-1 [Glu-B1-1a (Bx7), 1b (Bx7*), 1d (Bx6), 1ac (Bx6.8)], and 5 alleles at Glu-B1-2 [Glu-B1-2a (By8), 2b (By9), 2o (By8*), 2s (By18*), and 2z (By20*)] were revealed. In total, 16 allele combinations were observed. Molecular markers are particularly helpful in distinguishing the wheat Glu-A1a and Glu-A1b alleles from the rye Glu-R1a and Glu-R1b alleles in triticale genotypes, respectively, as well as subunits Bx7 from Bx7* and By8 from By8*, which could not be distinguished by SDS-PAGE. Novel glutenin subunits By18* and By20* (unique to triticale) were identified. HMW glutenin subunit combinations of Polish triticale cultivars, earlier identified by SDS-PAGE analyses, were verified by PCR-based DNA markers. Rapid identification of wheat Glu-1 alleles by molecular markers can be an efficient alternative to the standard separation procedure for early selection of useful triticale genotypes with good bread-making quality. [Abstract/Link to Full Text]

Ram S, Thiruvengadam V, Vinod K
Genetic diversity among cultivars, landraces and wild relatives of rice as revealed by microsatellite markers.
J Appl Genet. 2007;48(4):337-345.
Genetic diversity among 35 rice accessions, which included 19 landraces, 9 cultivars and 7 wild relatives, was investigated by using microsatellite (SSR) markers distributed across the rice genome. The mean number of alleles per locus was 4.86, showing 95.2&percnt; polymorphism and an average polymorphism information content of 0.707. Cluster analysis based on microsatellite allelic diversity clearly demarcated the landraces, cultivars and wild relatives into different groups. The allelic richness computed for the clusters indicated that genetic diversity was the highest among wild relatives (0.436), followed by landraces (0.356), and the lowest for cultivars. Allelic variability among the SSR markers was high enough to categorize cultivars, landraces and wild relatives of the rice germplasm, and to catalogue the genetic variability observed for future use. The results also suggested the necessity to introgress genes from landraces and wild relatives into cultivars, for cultivar improvement. [Abstract/Link to Full Text]

Ngezahayo F, Dong Y, Liu B
Somaclonal variation at the nucleotide sequence level in rice (Oryza sativa L.) as revealed by RAPD and ISSR markers, and by pairwise sequence analysis.
J Appl Genet. 2007;48(4):329-336.
The nature of somaclonal variation at the nucleotide sequence level was studied in rice cv. Nipponbare. First, we investigated genomic variations by using 2 molecular marker systems: RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This was followed by sequencing of selected bands that represented genomic variations, and pairwise sequence analysis taking advantage of the whole genome sequence of rice. In addition, transpositional activity of the active MITE, mPing, was analysed by locus-specific PCR amplifications. The 2-year-old calli and their regenerated plants, analysed with 24 RAPD and 20 ISSR primers, showed moderate levels of genomic variation (20.83&percnt; and 17.04&percnt;, respectively). To test whether DNA methylation plays a role in somaclonal variation, the calli were treated with 5-azacytidine, a chemical agent that reduces cytosine methylation by blocking the activity of DNA methyltransferase. Though dwarfism occurred in regenerants from treated calli (a hallmark of the drug treatment), there was only a slight increase in the frequency of somaclonal variation detected in the treated calli and their regenerated plants relative to untreated controls. The transposon mPing also remained immobile in both treated and untreated calli. Nevertheless, dendrograms constructed according to the Jaccard coefficient calculated by UPGMA of the ISSR bands revealed that the 5-azacytidine-treated and untreated somaclones were grouped into 2 distinct clusters, suggesting a possible indirect effect of the treatment on the genomic changes, depending on the marker used. Sequence analysis indicated a low level of variation (0.31&percnt;), with single-base-pair substitutions predominating. [Abstract/Link to Full Text]

Kuczynska A, Surma M, Adamski T
Methods to predict transgressive segregation in barley and other self-pollinated crops.
J Appl Genet. 2007;48(4):321-8.
Most of agronomically important characters are biometric traits. An improvement of these traits in cultivated plants by deriving segregants superior to parents, which could be developed as cultivars, is a main goal in breeding of self-pollinated crops. Two problems need to be solved: when will the progeny be better than its parents and how can a genetic potential of a given pair of parental genotypes be predicted? In this paper, transgressive segregation in homozygous barley populations is shortly reviewed. Various approaches to choosing parental forms are shown, and a theoretical method for predicting the frequency of transgressive segregants in a homozygous population is presented. Additionally, relationships between parental diversity estimated with molecular markers and the progeny performance are discussed. Although the prediction of transgressive segregation is still a problem, it seems promising to apply an approach measuring the performance of the parental genotypes and estimating their genetic distance by molecular markers. [Abstract/Link to Full Text]

Ratajczak MZ, Zuba-Surma EK, Machalinski B, Kucia M
Bone-marrow-derived stem cells - our key to longevity?
J Appl Genet. 2007;48(4):307-19.
Bone marrow (BM) was for many years primarily regarded as the source of hematopoietic stem cells. In this review we discuss current views of the BM stem cell compartment and present data showing that BM contains not only hematopoietic but also heterogeneous non-hematopoietic stem cells. It is likely that similar or overlapping populations of primitive non-hematopoietic stem cells in BM were detected by different investigators using different experimental strategies and hence were assigned different names (e.g., mesenchymal stem cells, multipotent adult progenitor cells, or marrow-isolated adult multilineage inducible cells). However, the search still continues for true pluripotent stem cells in adult BM, which would fulfill the required criteria (e.g. complementation of blastocyst development). Recently our group has identified in BM a population of very small embryonic-like stem cells (VSELs), which express several markers characteristic for pluripotent stem cells and are found during early embryogenesis in the epiblast of the cylinder-stage embryo. [Abstract/Link to Full Text]

Kozubek E, Irzykowski W, Lehmann P
Genetic and molecular variability of a Turnip mosaic virus population from horseradish (Cochlearia armoracia L.).
J Appl Genet. 2007;48(3):295-306.
Variability and genetic structure of a novel Turnip mosaic virus (TuMV) population from horseradish (Cochlearia armoracia L.) were examined. Over 60 horseradish plants were tested to identify a total of 28 TuMV isolates, constituting the Cochlearia ARmoracia (CAR) TuMV population. Two subgroups of the CAR TuMV isolates could be distinguished: subgroup N did not infect oilseed rape (Brassica napus var. oleifera) cv. Westar plants, while subgroup A infected these plants systemically. Two types of infection of oilseed rape plants were induced by inoculation with the CAR TuMV isolates: systemic mosaic infection and systemic necrotic lesions. The complete sequences of isolates CAR37 (subgroup N) and CAR37A (subgroup A) were determined and compared. The sequences of HC-Pro and CP genes of CAR37 and CAR37A and other isolates of TuMV from other countries were compared to provide some insight into their relatedness. CAR37A, initially regarded as a variant, proved to be very different from CAR37. Re-sequencing after repeated passages confirmed the genetic stability of both isolates. [Abstract/Link to Full Text]

Szalewska-Palasz A, Wegrzyn G, Wegrzyn A
Mechanisms of physiological regulation of RNA synthesis in bacteria: new discoveries breaking old schemes.
J Appl Genet. 2007;48(3):281-94.
Although in bacterial cells all genes are transcribed by RNA polymerase, there are 2 additional enzymes capable of catalyzing RNA synthesis: poly(A) polymerase I, which adds poly(A) residues to transcripts, and primase, which produces primers for DNA replication. Mechanisms of actions of these 3 RNA-synthesizing enzymes were investigated for many years, and schemes of their regulations have been proposed and generally accepted. Nevertheless, recent discoveries indicated that apart from well-understood mechanisms, there are additional regulatory processes, beyond the established schemes, which allow bacterial cells to respond to changing environmental and physiological conditions. These newly discovered mechanisms, which are discussed in this review, include: (i) specific regulation of gene expression by RNA polyadenylation, (ii) control of DNA replication by interactions of the starvation alarmones, guanosine pentaphosphate and guanosine tetraphosphate, (p)ppGpp, with DnaG primase, (iii) a role for the DksA protein in ppGpp-mediated regulation of transcription, (iv) allosteric modulation of the RNA polymerase catalytic reaction by specific inhibitors of transcription, rifamycins, (v) stimulation of transcription initiation by proteins binding downstream of the promoter sequences, and (vi) promoter-dependent control of transcription antitermination efficiency. [Abstract/Link to Full Text]

Cimbalistiene L, Lehnert W, Huoponen K, Kucinskas V
First reported case of lysinuric protein intolerance (LPI) in Lithuania, confirmed biochemically and by DNA analysis.
J Appl Genet. 2007;48(3):277-80.
We report on an 18-year-old Lithuanian girl with hepatosplenomegaly noticed at birth, which progressed thereafter. The patient had to wait about 17 years for an accurate diagnosis and appropriate therapy. Lactase deficiency, congenital cataract of the right eye, and osteoporosis were observed. Episodes of drowsiness were caused by intake of high-protein food. Laboratory findings included slight hyperammonaemia, high plasma Citr, Ala, Gly, Glu, Ser levels, as well as citrullinuria, lysinuria, glutaminuria, alaninuria, argininuria, prolinuria, hydroxyprolinuria, ornithinuria, and orotic aciduria. Aversion to high-protein diet strongly suggested a disorder resulting in hyperammonaemia. Citrullinaemia was suspected. Subsequently the diagnosis of LPI was made on the basis of biochemical and clinical features. Molecular genetic testing revealed a mutation in the SLC7A7 gene, confirming the diagnosis. [Abstract/Link to Full Text]

Szczerkowska-Dobosz A, Niespodziana K, Reba?a K, Garstecka J, Lange M, Bara?ska-Rybak W
Lack of association of HLA-C alleles with late-onset psoriasis in the northern Polish population.
J Appl Genet. 2007;48(3):273-5.
The HLA (human leukocyte antigen) Cw*06 allele demonstrates the strongest association with susceptibility to early-onset psoriasis in most populations. Recent data have indicated that late-onset psoriasis (LOP) demonstrates only a weak association with Cw*0602, and suggest that this type of psoriasis may represent a distinct subtype of the disease. The aim of this study was to compare the frequency of human leukocyte antigen C (HLA-C) alleles in patients with LOP and in healthy subjects within the same ethnic group in northern Poland. HLA-C alleles of 89 patients with psoriasis with onset at the age of 40 y or later and 80 control subjects were determined by a polymerase chain reaction (PCR), low-resolution method. The results showed that the Cw*05 allele was detected less frequently in patients with LOP than in control subjects, but this failed to retain significance after correction for multiple comparisons. There were no differences in the frequency of other HLA-C alleles between the patients and the control group. Our results confirm no association between HLA-C alleles and LOP in the northern Polish population. The lack of this association supports the hypothesis about different genetic backgrounds of early- and late-onset psoriasis. [Abstract/Link to Full Text]

Ahmad I, Hunter RE, Flax JD, Snyder EY, Erickson RP
Neural stem cell implantation extends life in Niemann-Pick C1 mice.
J Appl Genet. 2007;48(3):269-72.
In order to evaluate the phenotypic effects of implanted neural stem cells (NSCs) in the mouse model of Niemann-Pick C (NPC) disease, we injected a well-characterized clone of murine NSCs into the cerebella of neonatal Npc1(-/-) and control mice. The implanted cells survived and were abundant in some regions of the cerebellum. Life span was lengthened in NPC mice with the implanted NSCs. However, the rate of weight gain and subsequent weight loss, resulting from neurodegeneration, was not significantly different from un-injected controls. Ataxia was measured by Rota-Rod performance. The overall rate of decline in time on the Rota-Rod was not significantly slowed down. Thus, in this small group of NPC mice, a single administration in the neonatal period of the NSCs (which were not engineered to over-express the missing gene and not directed into the parenchyma) was only partially therapeutic. [Abstract/Link to Full Text]

Vázquez-Cárdenas A, Vásquez-Velásquez AI, Barros-Núńez P, Mantilla-Capacho J, Rocchi M, Rivera H
Familial whole-arm translocations (1;19), (9;13), and (12;21): a review of 101 constitutional exchanges.
J Appl Genet. 2007;48(3):261-8.
We report here on 3 familial whole-arm translocations (WATs), namely the 8th instance of t(1;19)(p10;q10) and 2 novel exchanges: t(9;13)(p10;q10) and t(12;21)(p10;q10). The exchanges (1;19) and (12;21) were ascertained through a balanced carrier, whereas the t(9;13) was first diagnosed in a boy with a trisomy 9p syndrome and der(9p13p). Results of FISH analyses with the appropriate ?-satellite probes were as follows. Family 1, t(1;19): the D1Z5 probe gave a strong signal on both the normal chromosome 1 and the der(1q19p) as well as a weak signal on the der(1p19q). Family 2, t(9;13): the centromere-9 alphoid and D13Z1/D21Z1 probes under standard stringency gave no signal on the der(9p13p) in both the proband and a carrier brother, whereas the der(9q13q) was labelled only with the centromere-9 alphoid repeat in the latter; yet, this probe under low stringency revealed a residual amount of alphoid DNA on the der(9p13p) in the carrier. Family 3, t(12;21): the D12Z3 probe gave a signal on the normal chromosome 12 and the der(12p21q), whereas the D13Z1/D21Z1 repeat labelled the der(12q21p), the normal chromosome 21, and both chromosomes 13. Out of 101 WATs compiled here, 73 are distinct exchanges, including 32 instances between chromosomes with common alphoid repeats. Moreover, 7/9 of recurrent WATs involved chromosomes from the same alphoid family. Thus constitutional WATs appear to recur more frequently than other reciprocal exchanges, often involve chromosomes with common alphoid repeats, and can mostly be accounted for the great homology in alphoid DNA that favours mispairing and illegitimate nonhomologous recombination. [Abstract/Link to Full Text]

Szwaczkowski T, Wezyk S, Stanis?awska-Barczak E, Badowski J, Bieli?ska H, Wolc A
Genetic variability of body weight in two goose strains under long-term selection.
J Appl Genet. 2007;48(3):253-60.
Body weight is one of the most important traits in any genetic improvement program in geese for at least 2 reasons. First, measurements of the trait are very easy. Second, body weight is correlated with a number of other meat performance traits. However, the genetic background of body weight shows considerable complexity. Three genetic models (with direct, maternal genetic and permanent maternal environmental effects) were employed in this study. Records of 3076 individuals of maternal strain W11 and 2656 individuals of paternal strain W33 over 6 consecutive generations, kept in the pedigree farm of Ko?uda Wielka, were analysed. Body weight (in kilograms) was measured in weeks 8 (BW8) and 11 (BW11). The inbreeding levels in both populations were relatively low (0.14% and 0.02% for W11 and W33, respectively), therefore these effects were not included in the linear models to estimate genetic parameters. Three fixed effects (hatch period, sex and year) were included in each linear model. Two criteria (AIC, BIC) were used to check the goodness of fit of the models. The computations were performed by WOMBAT software. In general, the genetic parameter estimates varied across the traits, models and strains studied. Direct additive heritability estimates ranged from 0.0001 (for BW11 of W33) to 0.55 (for BW11 of W33). Maternal and total heritabilities were also variable. Estimates of ratios of direct-maternal effect covariance in phenotypic variance were both positive and negative, but they were negligible, whereas ratios of the permanent environmental maternal variance to phenotypic variance were close to zero. Both of the applied criteria of model adequacy indicate that the model with maternal genetic and environmental effects should be considered as optimal. Genetic trends were close to zero. It seems that they were influenced by long-term selection. Similar tendencies have been observed for phenotypic trends, as well. [Abstract/Link to Full Text]

Ru?? A, Kami?ski S
Prevalence of complex vertebral malformation carriers among Polish Holstein-Friesian bulls.
J Appl Genet. 2007;48(3):247-52.
An increasing number of Holstein calf births exhibiting vertebral deformations has been detected in Denmark since 1999 by a program monitoring the incidence of genetic diseases. Pedigree analysis demonstrated that the affected calves originated from a family afflicted by an autosomally recessively inherited complex vertebral malformation (CVM) syndrome. To determine the actual carrier frequency of the CVM-determining mutation in a population of Polish Holstein-Friesian (=Polish Black-and-White) cattle, we examined 202 proven bulls (active in 2001-2005) used by 4 domestic artificial insemination companies and 403 unproven bulls (under evaluation for breeding value). Out of the 605 bulls examined, 150 T/G heterozygotes were diagnosed, including 118 that were sons of known CVM carriers. Identification of a gene polymorphism in a bovine solute carrier family 35 member 3, termed SLC35A3, was conducted with the use of a new PCR-SSCP method (polymerase chain reaction - single stranded conformation polymorphism), which - due to its ease of use and high reliability - can be applied in widespread screening programs aimed at reducing the incidence of the CVM defect. [Abstract/Link to Full Text]

Stranzinger GF, Steiger D, Kneubuhler J, Hagger C
Y chromosome polymorphism in various breeds of cattle (Bos taurus) in Switzerland.
J Appl Genet. 2007;48(3):241-5.
The evolutionary development of mammals involves mutations and fixations of chromosomal types. The Y chromosome polymorphism in cattle is important for the breeding strategy, since chromosomal incompatibilities in crossings result in fertility problems. In bulls of various breeds in Switzerland, data on chromosome status have been collected for over 20 years. Data from 7 years were analysed in this study through chromosome measurements and their normalization. Some highly significant differences were found between the 7 groups of breeds, especially between Holsteins and the original Swiss breeds Braunvieh and Simmental. Fleckvieh (purebred or crossbred) did not differ significantly from Black or Red Holsteins. The results were discussed with respect to fertility problems. The observed Y chromosome polymorphism should be taken into account in breeding, and research in this field should be continued. [Abstract/Link to Full Text]

De Felice B, Ciarmiello LF, Wilson RR, Conicella C
Molecular analysis of a novel tandemly organized repetitive DNA sequence in Citrus limon (L.) Burm.
J Appl Genet. 2007;48(3):233-9.
Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and molecular analysis of a novel tandemly organized repetitive DNA sequence from the genome of Citrus limon. Digestion of C. limon DNA with Hinf I produced a prominent fragment of approximately 300 bp. Southern blotting revealed a ladder composed of DNA fragments that were multimers of the 300-bp Hinf I band. Thus, Hinf I digestion revealed a novel satellite, which we have called the C. limon satellite DNA 300 (CL300). Sequence analysis shows significant homology between a portion of the CL300 monomer and the transposase box of an En/Spm-like element. The CL300 satellite was also detected in grapefruit, sour orange, trifoliate orange and kumquat. These results suggest that the CL300 repeat is an ancient satellite, and we propose that a significant portion originated by amplification of a genomic region containing the En/Spm-like transposase element. [Abstract/Link to Full Text]

Carlos de Oliveira A, Bastianel M, Cristofani-Yaly M, Morais do Amaral A, Machado MA
Development of genetic maps of the citrus varieties 'Murcott' tangor and 'Pera' sweet orange by using fluorescent AFLP markers.
J Appl Genet. 2007;48(3):219-31.
The progeny of 87 BC(1) hybrids of 'Murcott' tangor and 'Pera' sweet orange, genotyped with fluorescent amplified fragment length polymorphism (fAFLP) markers, was used for the construction of genetic maps for both citrus varieties. Mapping strategies, considering the progeny as a result of backcrossing and cross-pollination, were exploited in Mapmaker 2.0 (LOD score >or= 3.0 and <or= 0.40) and JoinMap 3.0 software (LOD score >or= 3.0 and theta <or = 0.25), respectively. Genetic map distances (in cM) between the linked fAFLPs were estimated, in both packages, by the Kosambi's function. Maps of both parents were constructed in Mapmaker with 121 of the 202 fAFLP markers showing 1:1 Mendelian segregation rates ('Murcott' map: 65 fAFLPs, average distance between them 29.5 cM, divided into 9 linkage groups (LGs), total size 1651.47 cM; 'Pera' map: 55 fAFLPs, average distance between them 31.9 cM, divided into 5 LGs, total size 1596.2 cM). The second 'Murcott' map, constructed through linkage analysis of 347 fAFLP markers with 3:1 or 1:1 segregation rates by using JoinMap, resulted in the linkage of 227 markers with an average distance of 4.25 cM among them, divided into 9 LGs of 845 cM. fAFLP loci showing distorted segregation and/or clustered were observed in different LGs of the maps generated by all the software. The use of the 'Murcott' tangor and 'Pera' sweet orange genetic maps in research on identification of citrus QRLs (quantitative resistance loci) to Xylella fastidiosa and QTLs (quantitative trait loci) related to the productivity and quality of the juice, respectively, is discussed. [Abstract/Link to Full Text]

Masoj? P, Banek-Tabor A, Milczarski P, Twardowska M
QTLs for resistance to preharvest sprouting in rye (Secale cereale L.).
J Appl Genet. 2007;48(3):211-7.
Grain quality of rye is often negatively affected by sprouting - a complex trait with a poorly understood genetic background and strong interaction with weather conditions. The aim of this report was to detect the main quantitative trait loci (QTLs) underlying preharvest sprouting resistance in rye, measured as a percentage of sprouted kernels after spraying spikes with water for 7 days. Simple and composite interval mapping, carried out in 3 environments on 94 F3 and F4 families of the cross between sprouting-susceptible (541) and sprouting-resistant (Ot1-3) inbred lines, revealed 5 QTLs located on chromosome arms 1RL, 2RL, 5RL, 6RL and 7RL. The significance of these QTLs was additionally proved by disruptive selection carried out on 5000 F2 plants of the 541 x Ot1-3 cross and continued to the F5 generation of recombinant inbred lines (RIL), which strongly affected allele frequencies at linked marker loci. Resistance to preharvest sprouting showed dominant inheritance except for QPhs.uas-7R.1 (recessive) and QPhs.uas-1R.1 (additive). Results of the present study suggest that introgression of 4-5 QTLs, identified in line Ot1-3, should substantially reduce sprouting risk in rye varieties. [Abstract/Link to Full Text]

Pathan AK, Park RF, Wellings CR, Bariana HS
The expression and genetics of resistance to stripe (yellow) rust in three European and four New Zealand wheat cultivars.
J Appl Genet. 2007;48(3):199-210.
Adult plant resistance (APR) to stripe rust in three European (Pegaso, Victo and Aztec) and four New Zealand cultivars (Weka, Kopara, Kokart and Takahe) was characterised using hybrid analysis and tests of allelism. In agreement with earlier work, the APR in most of these cultivars appeared to be controlled by two or more genes with additive effects. It was suggested that heavy selection pressure should be avoided in early generations in breeding programs utilising APR, because lines in which APR genes are heterozygous may display lower levels of resistance due to the incompletely dominant and interactive nature of many APRs. Such lines are capable of generating more resistant progenies following selfing. It was also demonstrated that it is possible to misclassify F2 plants as susceptible if APR genes are in a heterozygous condition, especially in the case of gene(s) conferring intermediate levels of resistance. The presence of a common APR gene in Kopara and Takahe, and perhaps Weka, was suggested because all shared a common parent in their pedigree and no susceptible plants were observed in F2 populations derived from intercrossing them. The difficulties inherent in conducting genetic studies on APRs and the need for large population sizes for such studies were emphasised. [Abstract/Link to Full Text]

Kami?ski S, Ciesli?ska A, Kostyra E
Polymorphism of bovine beta-casein and its potential effect on human health.
J Appl Genet. 2007;48(3):189-98.
Proteins in bovine milk are a common source of bioactive peptides. The peptides are released by the digestion of caseins and whey proteins. In vitro the bioactive peptide beta-casomorphin 7 (BCM-7) is yielded by the successive gastrointestinal proteolytic digestion of bovine beta-casein variants A1 and B, but this was not seen in variant A2. In hydrolysed milk with variant A1 of beta-casein, BCM-7 level is 4-fold higher than in A2 milk. Variants A1 and A2 of beta-casein are common among many dairy cattle breeds. A1 is the most frequent in Holstein-Friesian (0.310-0.660), Ayrshire (0.432-0.720) and Red (0.710) cattle. In contrast, a high frequency of A2 is observed in Guernsey (0.880-0.970) and Jersey (0.490-0.721) cattle. BCM-7 may play a role in the aetiology of human diseases. Epidemiological evidence from New Zealand claims that consumption of beta-casein A1 is associated with higher national mortality rates from ischaemic heart disease. It seems that the populations that consume milk containing high levels of beta-casein A2 have a lower incidence of cardiovascular disease and type 1 diabetes. BCM-7 has also been suggested as a possible cause of sudden infant death syndrome. In addition, neurological disorders, such as autism and schizophrenia, seem to be associated with milk consumption and a higher level of BCM-7. Therefore, careful attention should be paid to that protein polymorphism, and deeper research is needed to verify the range and nature of its interactions with the human gastrointestinal tract and whole organism. [Abstract/Link to Full Text]

Recent Articles in Genetics and Molecular Research

Freitas JS, Silva EM, Rossi A
Identification of nutrient-dependent changes in extracellular pH and acid phosphatase secretion in Aspergillus nidulans.
Genet Mol Res. 2007;6(3):721-9.
The present study was designed to identify nutrient-dependent changes in extracellular pH and acid phosphatase secretion in the biA1 palC4 mutant strain of Aspergillus nidulans. The palC4 mutant was selected as lacking alkaline phosphatase, but having substantially increased acid phosphatase activity when grown on solid minimal medium under phosphate starvation, pH 6.5. Gene palC was identified as a putative member of a conserved signaling cascade involved in ambient alkaline sensing whose sole function is to promote the proteolytic activation of PacC at alkaline pH. We showed that both poor growth and conidiation of the palC4 mutant strain on solid medium, alkaline pH, were relative to its hypersensitivity to Tris (hydroxymethyl) aminomethane buffer. Also, the secretion of acid phosphatase was repressed when both the wild-type and palC4 mutant strains were grown in low-phosphate yeast extract liquid medium, pH 5.0, indicating that the secretion of this enzyme is not necessary to regenerate inorganic phosphate from the organic phosphate pool present in yeast extract. [Abstract/Link to Full Text]

Morielle-Souza A, Azeredo-Oliveira MT
Differential characterization of holocentric chromosomes in triatomines (Heteroptera, Triatominae) using different staining techniques and fluorescent in situ hybridization.
Genet Mol Res. 2007;6(3):713-20.
A comparative study of holocentric chromosomes in the triatomine species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans was carried out in order to characterize heterochromatin, rDNA active sites and nucleolar proteins. Cytological preparations of seminiferous tubules were stained by silver impregnation, C banding, fluorochromes cma3/da and dapi/da, and fluorescent in situ hybridization (FISH) with Drosophila melanogaster 28S rDNA probe. Our results showed interesting aspects of the organization of chromatin and chromosomes in the meiotic cells of these insects. In R. pallescens, sex chromosomes (X, Y) were distinct from autosomes, when submitted to silver impregnation, C banding, CMA3 staining, and FISH, confirming that these chromosomes bear nucleolar organizer regions (NORs). In P. megistus, two of the three sex chromosomes were CMA3/DAPI-; at early meiotic prophase and at diakinesis, silver impregnation corresponded with FISH signals, indicating that in this species, two chromosomes (probably a sex chromosome and an autosome) bear NORs. In T. infestans, silver nitrate and FISH also stained corresponding areas on meiotic chromosomes. Our data suggest that in triatomines, in general, the number and location of NORs are species-specific. These regions may be considered important chromosome markers for comparative studies to improve the understanding of evolutionary mechanisms in these hematophagous insects. [Abstract/Link to Full Text]

Vargas SM, Torres GA, Sobrinho FS, Pereira AV, Davide LC
Karyotypic studies of Cratylia argentea (Desv.) O. Kuntze and C. mollis Mart. ex Benth. (Fabaceae - Papilionoideae).
Genet Mol Res. 2007;6(3):707-12.
Cratylia argentea and C. mollis (Fabaceae-Papilionoideae) are legume shrubs native to the Cerrado and Caatinga, respectively. Both species show great resistance to drought and high nutritive value, which makes them a valuable forage resource in tropical regions. Cytogenetic studies were carried out on accessions of C. argentea and C. mollis from Germplasm Banks of Embrapa Gado de Leite (Juiz de Fora, MG) and Embrapa Semi-Arido (Petrolina, PE), respectively. Root tips were treated with 3 mM 8-hydroxyquinoline and slides were made using the air-dry technique. Karyotype description for each accession took into account the following features: chromosome number; total length, relative length and arm ratio of each chromosome; haploid set length, and degree of asymmetry. Mitotic metaphases in both species showed 2n = 22 chromosomes, where this is the first report of diploid number for C. mollis. Chromosome length was also quite similar for the two species, ranging from 5.08 to 2.50 microm in C. argentea and 5.12 to 2.51 microm in C. mollis, with haploid sets of equal size, measuring 38.10 and 37.85 microm, respectively. However, they did not show the same karyotypic formula, which was 5 m + 4 sm + 2 st for C. argentea and 7 m + 2 sm + 2 st for C. mollis. This indicates the occurrence of rearrangements within chromosomes I and VI. Both karyotypes showed a tendency for asymmetry. [Abstract/Link to Full Text]

Grisi MC, Blair MW, Gepts P, Brondani C, Pereira PA, Brondani RP
Genetic mapping of a new set of microsatellite markers in a reference common bean (Phaseolus vulgaris) population BAT93 x Jalo EEP558.
Genet Mol Res. 2007;6(3):691-706.
The present study describes a new set of 61 polymorphic microsatellite markers for beans and the construction of a genetic map using the BAT93 x Jalo EEP558 (BJ) population for the purpose of developing a reference linkage map for common bean (Phaseolus vulgaris). The main objectives were to integrate new microsatellites on the existing framework map of the BJ population, and to develop the first linkage map for the BJ population based exclusively on microsatellites. Of the total of 264 microsatellites evaluated for polymorphism, 42.8% showed polymorphism between the genitors. An integrated map was created totaling 199 mapped markers in 13 linkage groups, with an observed length of 1358 cM and a mean distance between markers of 7.23 cM. For the map constructed exclusively with microsatellites, 106 markers were placed in 12 groups with a total length of 606.8 cM and average distance of 6.8 cM. Linkage group designation and marker order for BM microsatellites generally agreed with previous mapping, while the new microsatellites were well distributed across the genome, corroborating the utility of the BJ population for a reference map. The extensive use of the microsatellites and the availability of a reference map can help in the development of other genetic maps for common bean through the transfer of information of marker order and linkage, which will allow comparative analysis and map integration, especially for future quantitative trait loci and association mapping studies. [Abstract/Link to Full Text]

Van Vleck LD
Computing numerator relationships between any pair of animals.
Genet Mol Res. 2007;6(3):685-90.
We describe a simple method to compute the numerator relationship between any or all pairs of animals in the numerator relationship matrix. The method depends on output of the MTDFNRM program from the MTDFREML set of programs. An option of the MTDFNRM program creates a file that includes the inbreeding coefficient for each animal. The method also makes use of how the inbreeding coefficient is traditionally calculated: one-half of the relationship between the animal's parents. To obtain the numerator relationship between any pair of animals, the original pedigree file is augmented with a dummy animal with an identification number (ID) greater than for any animal in the original pedigree file. The ID of the pair of animals for which the relationship is wanted is included as parents. MTDFNRM is then run with the option to create a file of ordered and original IDs for animals and their parents along with the inbreeding coefficients. We then multiply the inbreeding coefficient for a dummy animal by two to obtain the numerator relationship between the two animals designated as parents. [Abstract/Link to Full Text]

Freitas FO, Moretzsohn MC, Valls JF
Genetic variability of Brazilian Indian landraces of Arachis hypogaea L.
Genet Mol Res. 2007;6(3):675-84.
The Kayabi Indians who inhabit the Xingu Indigenous Park, located in West Central Brazil, have grown and managed peanuts for a long time. A great number of landraces are being maintained by these tribes and some of this germplasm has morphological traits that exceed the variation described in the taxonomic literature. Here, we analyzed the genetic variability of these landraces using a set of microsatellite markers. The analysis showed that, in general, the indigenous samples grouped according to the villages where they were collected. The microsatellite markers used in the present study detected high levels of genetic variation. Similarity groups, genetically distant from each other, were formed, allowing a more efficient use of the existing genetic variability. The present study also showed that these materials can extend the genetic variability available for peanut-breeding programs. Additionally, the microsatellite markers revealed a large dissimilarity among germplasm accessions representing Arachis hypogaea varieties so far included in the same subspecies fastigiata (aequatoriana + peruviana vs fastigiata + vulgaris), a subject that deserves further investigation. Finally, the Xingu Indigenous Park proved to be an important center of diversity for peanut. [Abstract/Link to Full Text]

El Dib RP, Pastores GM
Laronidase for treating mucopolysaccharidosis type I.
Genet Mol Res. 2007;6(3):667-74.
Mucopolysaccharidoses are a group of inherited metabolic diseases caused by the absence or deficiency of the lysosomal enzymes that are needed for breaking down glycosaminoglycans (GAGs). Over time, GAGs collect in cells, blood and connective tissues, and increased amounts are excreted in the urine. The result is permanent and includes progressive cell damage that affects the individual's appearance, physical abilities, organ and system functioning and, in certain cases, mental development. Enzyme replacement therapies are currently in use or are being tested for at least three different subtypes (I, II and VI). The aim of the present study was to evaluate the effectiveness and safety of laronidase for treating mucopolysaccharidosis type I. A systematic review of the literature was conducted. A computerized electronic search was then conducted using the CENTRAL, Pubmed, EMBASE, and LILACS databases, to identify any randomized controlled trials. The last date of the search was June 2006. There was no possibility of combining the results, because only one study was included. In the pivotal placebo-controlled trial conducted over a 26-week period, there was a reduction in the urinary excretion of GAGs among treated patients. Regarding adverse events, there were no laronidase-related serious adverse events or deaths. Laronidase seems to be a promising agent for treating mucopolysaccharidosis type I, as shown by the reduction in the urinary excretion of GAGs and the associated improvements in vital capacity and in the performance of defined physical tasks. [Abstract/Link to Full Text]

Oliveira CG, Martinez RA, Gaiotto FA
DNA extraction from bristles and quills of Chaetomys subspinosus (Rodentia: Erethizontidae) using a novel protocol.
Genet Mol Res. 2007;6(3):657-66.
DNA extraction protocols are as varied as DNA sources. When it comes to endangered species, it is especially important to pay attention to all details that ensure the completion of the study goals and effectiveness in attaining useful data for conservation. Chaetomys subspinosus (Rodentia: Erethizontidae) is a secretive arboreal porcupine endemic to certain ecosystems of the Brazilian Atlantic Forest. A multidisciplinary study (including genetic data) was performed to create a management plan for the conservation of this species. Individuals from natural populations of the states of Bahia, Espírito Santo and Sergipe were sampled. To obtain a reliable and abundant amount of starting material, non-destructive methods were tested, extracting DNA from the bristles and quills that comprise most of this animal's hide. This method has also been innovative in adapting a DNA extraction protocol traditionally used for plants. Digestion using proteinase K was followed by protein precipitation with CTAB, a chloroform-isoamyl alcohol cleaning and DNA precipitation with isopropyl alcohol. This protocol supplies good-quality DNA for genetic analysis with molecular markers based on PCR. [Abstract/Link to Full Text]

Mizoguchi SM, Portela-Castro AL, Martins-Santos IC
Cytogenetic characterization of Crenicichla (Pisces, Perciformes, Cichlidae) of the Iguaçu River.
Genet Mol Res. 2007;6(3):650-6.
Three populations of the genus Crenicichla, namely Crenicichla iguassuensis, Crenicichla sp 1 and Crenicichla sp 2, from the Iguaçu River, were analyzed cytogenetically, and their nucleolus organizer regions, constitutive heterochromatin distribution and chromomycin A3 markings were studied. Karyotype analyses showed a diploid number of 48 chromosomes, made up of 2 metacentric pairs, 2 submetacentric pairs, 7 subtelocentric pairs, and 13 acrocentric pairs for the three Crenicichla species and no sexual chromosome differentiation. Nucleolus organizer regions showed strong interstitial marking on the first chromosome pair, coincident with a constriction presented by Giemsa and positive marking by chromomycin. Although constitutive heterochromatin patterns were also similar, with pericentromeric markings, small differences in the three species could be observed. Crenicichla sp 2 presented some chromosomes with bitelomeric markings absent in Crenicichla iguassuensis and Crenicichla sp 1. [Abstract/Link to Full Text]

Rieger TT, Oliveira-Silva SV, Pachęco IA, Chagas BS, Santos JF
Localization of HSP single-copy genes by inexpensive, permanent non-fluorescent in situ hybridization on meiotic chromosomes of the grasshopper Schistocerca pallens (Acrididae).
Genet Mol Res. 2007;6(3):643-9.
There have been many studies on Schistocerca gregaria and Locusta migratoria, which are important grasshopper pests in many parts of the world. However, the main pest grasshopper species in Brazil, S. pallens, Rhammatocerus schistocercoides and Stiphra robusta, are very poorly characterized genetically. We adapted a permanent in situ hybridization method to extend the genetic characterization of S. pallens by mapping the single-copy genes Hsp70, Hsp83, Hsp27, and Ubi on meiotic chromosomes. Hsp70 was mapped on the L2 chromosome, in which 82% of the signals were observed. Hsp83 was mapped on a medium-sized chromosome, on which 81% of the signals were observed, tentatively identified as M7. The hybridization signals for the Hsp27 gene were detected on the L1 chromosome at a frequency of 58%. The main hybridization site of the Ubi probe was on the L2 chromosome, with 73% of the signals. All mapped genes also presented secondary hybridization signals, always at frequencies below 30%. These are the first single-copy genes mapped for S. pallens and also for the Acrididae family. Since the Acrididae generally present very similar karyotypes, these data are useful as new landmarks for chromosome identification and as a tool for phylogenetic studies on the genus Schistocerca and for comparison with other insects. [Abstract/Link to Full Text]

Castro-Prado MA, Querol CB, Sant'Anna JR, Miyamoto CT, Franco CC, Mangolin CA, Machado MF
Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean.
Genet Mol Res. 2007;6(3):634-42.
The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum. [Abstract/Link to Full Text]

Moraes VP, Cereali SS, Froehlich O, Dias AL
Cytogenetic characterization of Rhamdia quelen (Siluriformes, Heptapteridae) from the Bodoquena Plateau, Mato Grosso do Sul, Brazil.
Genet Mol Res. 2007;6(3):627-33.
We made a cytogenetic study of the fish Rhamdia quelen collected from the Bodoquena Plateau, an isolated national park region in Mato Grosso do Sul State, Brazil. The diploid number was 2n = 58, with 36 metacentric + 16 submetacentric + 6 subtelocentric chromosomes. We found one to three B chromosomes, which were metacentric and submetacentric and of medium size, showing both intra- and interindividual variation. The nucleolus organizer region (NOR) was located in the terminal region of the short arm of submetacentric pair 20. Staining with CMA3 fluorochrome revealed the NOR location, while there was no evidence of fluorescent staining with DAPI. C banding revealed heterochromatin mainly in the terminal regions of the chromosome arms, including the NOR pair. In addition, metacentric pair 2 showed three heterochromatic blocks in the terminal portions and in the pericentromeric region. The B chromosomes appeared euchromatic. The CB + CMA3 staining combination demonstrated only one chromosome pair with fluorescence, probably the NOR-bearing one, while CB + DAPI gave various fluorescent signals, including metacentric pair 2, indicating that these heterochromatic regions are AT-rich in this population of R. quelen. The R. quelen population in this isolated region of Brazil is chromosomally distinct from that of other populations that have been studied. [Abstract/Link to Full Text]

Rao VB, Kerketta L, Korgaonkar S, Ghosh K
Differentiation of Nijmegen breakage syndrome from Fanconi anemia.
Genet Mol Res. 2007;6(3):622-6.
Nijmegen breakage syndrome (NBS) is a rare auto-somal recessive condition with chromosomal instability. Clinical and biological overlap between Fanconi anemia and ataxia telangiectasia has been reported. We report two cases of NBS born to consanguineous parents. Case one had NBS and Falconi anemia clinical features but relatively little chromosome breakage. The second case had mild NBS features, while cytogenetic evaluation with mitomycin C induction showed chromosome damage. Chromosomal analysis of bone marrow cells revealed tetraploidy, which indicates progression towards leukemia. On the basis of clinical and cytogenetic evaluation, these two cases were confirmed as NBS. However, detailed molecular studies are essential for accurate diagnosis and management of this disease. [Abstract/Link to Full Text]

Adamowski EV, Boldrini KR, Pagliarini MS, do Valle CB
Abnormal cytokinesis in microsporogenesis of Brachiaria humidicola (Poaceae: Paniceae).
Genet Mol Res. 2007;6(3):616-21.
Microsporogenesis was evaluated in the Brachiaria humidicola collection of the Embrapa Beef Cattle Center, represented by 60 accessions. One accession (H121) presented an abnormal pattern of cytokinesis that had never been reported in this genus. Among 900 meiocytes analyzed in the first division, 10.7% underwent precocious and multiple cytokinesis in metaphase I, fractionating the genome and the cytoplasm into two or more parts. The expected cytokinesis after telophase I did not occur. The abnormal meiocytes from the first division entered the second division but the second cytokinesis after telophase II was also abnormal. Among the 857 meiocytes analyzed in the second division, 10.9% presented abnormal, incomplete or total absence of cytokinesis. Dyads and binucleated microspores were recorded among the meiotic products. The use of this accession in the Embrapa breeding program is compromised. [Abstract/Link to Full Text]

Camargo OA, Souza EA, Mendes-Costa MC, Santos JB, Soares MA
Identification of Glomerella cingulata f. sp phaseoli recombinants by RAPD markers.
Genet Mol Res. 2007;6(3):607-15.
We examined the capacity of strains of Glomerella cingulata f. sp phaseoli fungus (Colletotrichum lindemuthianum sexual stage) to form recombinants, using random amplified polymorphic DNA (RAPD). Crosses of all possible combinations between strains 40, 42, 20, 21, 22, 23, 24, 25, and 26 were made on Petri dishes using M3 culture medium. The 42 x 21 cross produced the largest number of perithecia and five asci; the respective ascospores were isolated. RAPD analysis was performed on the parents and descendants. The 62 polymorphic RAPD bands obtained were used to assess the genetic similarity using the method of Sorence and Dice and clustering analysis in the form of a dendrogram by the UPGMA method. The RAPD markers allowed identification of recombinants from the cross between strains 42 and 21 of G. cingulata f. sp phaseoli and 40 ascospores presented 63 and 49% genetic similarity with parents 2 (strain 42) and 1 (strain 21), respectively. [Abstract/Link to Full Text]

Chen CY, Johnson RK, Newman S, Van Vleck LD
A general review of competition genetic effects with an emphasis on swine breeding.
Genet Mol Res. 2007;6(3):594-606.
A review of previous studies is presented on estimates of genetic parameters and responses to selection with traditional breeding approaches, on correlations between agonistic behavior and growth performance, and on theoretical frameworks for selection incorporating interactions among individuals and on practical methods for incorporating competition effects in breeding programs. [Abstract/Link to Full Text]

Kehdy FS, Cerqueira EM, Bonjardim MB, Camelo RM, Castro MC
Study of the cytogenetic effects of occupational exposure to pesticides on sanitation workers in Belo Horizonte, Brazil.
Genet Mol Res. 2007;6(3):581-93.
Sanitation workers handling pesticides in the control of disease vectors constitute an occupationally exposed population to genotoxic substances. The aim of the present study was to investigate the relation between the occupational exposure to various pesticides and the presence of cytogenetic damage. Fifty-nine men were selected (29 sanitation workers and 30 control individuals) with ages varying between 18-57 years who lived and worked in the same area in Belo Horizonte (Brazil). The following parameters were determined for all individuals using the cytokinesis-block micronucleus (MN) assay in peripheral blood lymphocytes: MN/1000 binucleated cells (BC), BC with MN (BCMN)/1000 BC, nucleoplasmic bridges (NB)/1000 BC, apoptotic and necrotic cells/500 cells and nuclear division index. The analysis of covariance showed significantly higher (p < 0.05) mean frequencies of MN (15.81 +/- 1.31 vs 4.71 +/- 0.42), BCMN (15.10 +/- 1.22 vs 4.62 +/- 0.44), NB (4.59 +/- 0.76 vs 1.00 +/- 0.34), and necrotic cells (12.07 +/- 1.45 vs 5.17 +/- 0.70) in the exposed group when compared to the control group. There was no significant difference in the apoptotic cell frequency between the two groups, while the nuclear division index was significantly lower (1.49 +/- 0.02 vs 1.61 +/- 0.02) in the control group. Neither the time of exposure nor the smoking or alcohol drinking habit influenced the cytogenetic parameters examined. According to these results, occupational exposure to pesticides induced genotoxic and cytotoxic effects in sanitation workers. [Abstract/Link to Full Text]

Sena DC, Molina WF
Robertsonian rearrangements and pericentric inversions in Scaridae fish (Perciformes).
Genet Mol Res. 2007;6(3):575-80.
The parrotfishes (family Scaridae) are comprised of the subfamilies Sparisomatinae and Scarinae. They are important agents of marine bioerosion, which rework the substrate with their beaklike jaws. Despite their importance, there are no published cytogenetic data on this group. We made cytogenetic analyses of Sparisoma axillare (Sparisomatinae) and Scarus coelestinus (Scarinae) from the Brazilian coast. Differentiation in the diploid number in S. axillare compared to the basal karyotype of the Perciformes apparently occurred due to a Robertsonian fusion, combined with pericentric inversions. S. coelestinus presented a conserved diploid number, but showed considerable structural karyotypic changes, resulting mainly from pericentric inversions. The Ag-NOR sites were unique and located on the short arm of the 1st subtelocentric pair in both species (possibly homeologous), corresponding to the 11th pair in S. axillare and the 9th pair in S. coelestinus. The constitutive heterochromatin is reduced in these species and is distributed in centromeric and pericentromeric regions in most of the chromosomes. The low fundamental number compared to the Scarus genus suggests a more basal condition for Sparisoma. The chromosome formula in S. coelestinus was more diversified, deriving from large-scale pericentric inversions. Karyotypic evolution patterns observed for these representatives of the Sparisomatinae and Scarinae subfamilies, added to new data from a larger number of species, would allow us to determine if there is a tendency among the Sparisomatinae for centric fusion events. [Abstract/Link to Full Text]

Oliveira-Martins CR, Grisolia CK
Determination of micronucleus frequency by acridine orange fluorescent staining in peripheral blood reticulocytes of mice treated topically with different lubricant oils and cyclophosphamide.
Genet Mol Res. 2007;6(3):566-74.
To ascertain whether used and re-refined lubricant oil absorbed through the skin can produce a genotoxic effect or cytotoxicity in mouse bone marrow cells, we examined the induction of micronucleated erythrocytes of peripheral blood after cutaneous application. Both re-refined and used lubricant oils showed a weak but significant induction of micronucleated polychromatic erythrocytes compared with control, while virgin oil did not show micronucleus induction. Cyclophosphamide (CP) was used not only as positive control but also to compare the sensitivity between intraperitoneal and dermal routes of administration of the test compounds in mice. The efficacy of intraperitoneal injection of CP is well known. On the other hand, dermal exposure is not so common and when CP was diluted in glycerin statistically significant values (P = 0.0036) of micronuclei were also found. Topically applied lubricant oils (virgin, re-refined and used) have the capacity to interfere with mouse bone marrow hematopoiesis evidenced by a statistically significant decrease in the proportion of polychromatic erythrocytes in the peripheral blood. Physical and chemical analysis revealed that used oil is more viscous than other lubricants, suggesting the presence of insoluble compounds, oxidized products and water as well as aromatic hydrocarbons. Used oil differs from other lubricant oils in metal and polyaromatic hydrocarbon content. Re-refined oil revealed a neutral value typical of pure mineral oil. This assay is an important tool to evaluate environmental pollutants that cause genotoxicity and/or cytotoxicity through skin exposure. [Abstract/Link to Full Text]

Targa AC, César AC, Cury PM, Silva AE
Apoptosis in different gastric lesions and gastric cancer: relationship with Helicobacter pylori, overexpression of p53 and aneuploidy.
Genet Mol Res. 2007;6(3):554-65.
Apoptosis has an essential function in maintaining the integrity of the gastrointestinal mucosa. Its deregulation is associated with the occurrence of lesions such as in atrophic gastritis, peptic ulcers, intestinal metaplasia, and stomach tumorigenesis. Thus, the aim of the present study was to investigate the frequency of apoptotic cells (apoptotic index, AI) by using two different immunohistochemical techniques, TUNEL and anti-activated caspase-3 antibody (CPP32), in gastric dyspepsia [chronic gastritis (CG, N = 34), chronic atrophic gastritis (CAG, N = 11), gastric ulcer (GU, N = 17), and intestinal metaplasia (IM, N = 15)], normal gastric mucosae (NM, N = 8), and gastric adenocarcinoma (GC, N = 12). The relationship was investigated between the AI and Helicobacter pylori infection, diagnosed by PCR, overexpression of p53 protein determined by immunohistochemistry, and aneuploidy by fluorescence in situ hybridization, as performed by our laboratory in previous studies. No significant differences were observed in AI between the different groups, whether by the TUNEL technique (F = 1.60; p = 0.1670) or by CPP32 antibody (F = 1.70; p = 0.1420). Nonetheless, CAG and CG groups had AI statistically higher than those of normal mucosae. These two groups (CAG and CG) also showed a higher frequency of apoptosis-positive cases (TUNEL+ or CPP32+). Generally, there was no correlation between the AI detected by the TUNEL and CPP32 techniques in the groups studied, except in the GC group (r = 0.70). Moreover, there was no significant association between apoptosis and H. pylori infection, overexpression of p53 protein and aneuploidy, but the H. pylori-positive cases only of GU (p = 0.0233) and IM (p = 0.0253) groups displayed a statistically higher AI compared to H. pylori-negative NM, when the CPP32 antibody technique was used. Thus, CG and CAG have increased apoptosis, which may occur independent of an association with H. pylori infection, aneuploidy and overexpression of p53 protein. [Abstract/Link to Full Text]

Juchum FS, Leal JB, Santos LM, Almeida MP, Ahnert D, Corręa RX
Evaluation of genetic diversity in a natural rosewood population (Dalbergia nigra Vell. Allemăo ex Benth.) using RAPD markers.
Genet Mol Res. 2007;6(3):543-53.
Dalbergia nigra (rosewood) is a long-lived leguminous species, which is endemic to the Brazilian Atlantic forest. Because of the high economic value of its wood, this species has been over-explored in recent years. Currently, rosewood is included in the IUCN Red List as vulnerable. We examined the genetic diversity of 87 specimens of D. nigra sampled from a continuous forest in the Veracel Reserve and Brazilwood Ecological Station, Porto Seguro, Bahia state, with random amplified polymorphic DNA markers. Grouping analyses were done using unweighted pair group method with arithmetic averages. Using the 16 most informative primers, 112 markers were obtained; 39% (44 bands) were polymorphic. A genetic similarity matrix was made based on the polymorphic bands. The dispersion graph and dendrogram analyses showed three distinct sub-populations. The degree of polymorphism was high, near that of other populations of similar species; however, it was considered low for the conservation of this species. [Abstract/Link to Full Text]

Mimbacas A, Trujillo J, Gascue C, Javiel G, Cardoso H
Prevalence of vitamin D receptor gene polymorphism in a Uruguayan population and its relation to type 1 diabetes mellitus.
Genet Mol Res. 2007;6(3):534-42.
Vitamin D has important immuno-modulatory properties and it influences insulin secretion. It acts through a vitamin D receptor (VDR), for which several gene polymorphisms have been described. The Uruguayan population presents several epidemiological characteristics that make it different from that of other counties, including other Latin-American countries. It went through miscegenation processes, with a tri-hybrid European, Amerindian and African origin, with no contribution from isolated Amerindian communities. Such differences have important consequences for the relationship between frequencies of several genes in the general population and their association with the diabetes mellitus. We examined the prevalence of VDR gene polymorphisms in the general population and their relation to type 1 diabetes in a parent-case design. One hundred unrelated individuals from the general population and 45 parent-patient triads with a child affected with type 1 diabetes were genotyped for FokI, BsmI and TaqI VDR gene polymorphisms by RFLP-PCR. We used a transmission disequilibrium test to assess preferential transmission of parents to affected offspring. The prevalence of the three VDR polymorphisms was: allele F = 48%, B = 35%, T = 64%. The f, b, T alleles and heterozygous genotypes were found at a high frequency in this population. Among 36 informative heterozygous parental genotypes, 30 transmitted the F allele (probability of transmission = 83%). The other two polymorphisms did not show significant transmission. We suggest that FokI polymorphism indicates susceptibility to type 1 diabetes mellitus in the Uruguayan population. [Abstract/Link to Full Text]

Li J, Guo M
Evolutionary tree reconstruction using structural expectation maximization and homotopy.
Genet Mol Res. 2007;6(3):522-33.
The evolutionary tree reconstruction algorithm called SEMPHY using structural expectation maximization (SEM) is an efficient approach but has local optimality problem. To improve SEMPHY, a new algorithm named HSEMPHY based on the homotopy continuation principle is proposed in the present study for reconstructing evolutionary trees. The HSEMPHY algorithm computes the condition probability of hidden variables in the structural through maximum entropy principle. It can reduce the influence of the initial value of the final resolution by simulating the process of the homotopy principle and by introducing the homotopy parameter beta. HSEMPHY is tested on real datasets and simulated dataset to compare with SEMPHY and the two most popular reconstruction approaches PHYML and RAXML. Experimental results show that HSEMPHY is at least as good as PHYML and RAXML and is very robust to poor starting trees. [Abstract/Link to Full Text]

Lima-Bittencourt CI, Cursino L, Gonçalves-Dornelas H, Pontes DS, Nardi RM, Callisto M, Chartone-Souza E, Nascimento AM
Multiple antimicrobial resistance in Enterobacteriaceae isolates from pristine freshwater.
Genet Mol Res. 2007;6(3):510-21.
A freshwater enterobacterial population (N = 111) was studied for antimicrobial and mercury resistance patterns, and for its possible association with biotic and abiotic factors in that environment. Conventional biochemical tests identified Klebsiella sp, Morganella sp, Serratia sp, Escherichia sp, Enterobacter sp, Edwarsiella sp, Proteus sp, Citrobacter sp, Providencia sp, and Kluyvera sp. There was no correlation between antimicrobial resistance patterns of isolates and bacterial genera, but resistance patterns varied among water samples and between seasons. Resistance to multiple antimicrobials was common (61%). The percentage of bacteria resistant to at least one antimicrobial differed between the rainy (100%) and dry seasons (89%). Resistance to beta-lactams and chloramphenicol was the most frequent and resistance to amikacin, gentamicin and kanamycin was less frequent. The main water variables examined (abiotic factors pH and temperature; biotic factor chlorophyll a concentration) did not influence antimicrobial resistance. Significant impact on freshwater enterobacteria, as evidenced by antimicrobial-multiple resistance and by the presence of bla(TEM) gene, may point to the fact that it has an important role in horizontal spread of resistance. [Abstract/Link to Full Text]

Santi-Rampazzo AP, Nishiyama PB, Ferreira PE, Martins-Santos IC
Cytogenetic analysis and description of the sexual chromosome determination system ZZ/ZW of species of the fish genus Serrapinnus (Characidae, Cheirodontinae).
Genet Mol Res. 2007;6(3):504-9.
Four populations of Serrapinnus notomelas and one population of Serrapinnus sp.1, both belonging to the subfamily Cheirodontinae, were analyzed by Giemsa and silver nitrate impregnation techniques. We found 2n = 52 chromosomes for all populations, with interspecific differences in the karyotype formula; S. notomelas showed 16 m + 22 sm + 10 st + 4a, with fundamental number (FN) = 100 for males, and 16 m + 23 sm + 10 st + 3a, with FN = 101 for females. Serrapinnus sp.1 had 8m + 16 sm + 4 st + 24 a, with FN = 80 for males, and 8m + 15 sm + 4 st + 25 a, with FN = 79 for females. The difference in FN for the two sexes is due to a pair of heteromorphic chromosomes in the females of both species, which characterizes a ZZ/ZW-type mechanism of chromosome sexual determination. Interspecies differences were also found in nucleolus organizer regions (NORs). A simple NOR system was detected in three of four S. notomelas populations, while Serrapinnus sp.1 had two chromosome pairs with NOR. Although S. notomelas and Serrapinnus sp.1 have the same diploid number, differences in the karyotype structure indicate that these are different species. Apparently there was pericentric inversion during the karyotype evolution of these species. [Abstract/Link to Full Text]

Amorim MR, Vargas FR, Llerena JC, Pombo-de-Oliveira MS
DNA extraction from fixed cytogenetic cell suspensions.
Genet Mol Res. 2007;6(3):500-3.
We developed a procedure for DNA extraction from small volumes of fixed cell suspensions previously prepared for conventional cytogenetic analysis. Good quality DNA was isolated with a fast and simple protocol using DNAzol reagent. This provided suitable DNA for various types of molecular analyses, including polymerase chain reaction, restriction fragment length polymorphism, denaturing high-performance liquid chromatography, and direct sequencing. This technique provides sufficient material for such test, which are important for diagnosis of neoplastic diseases in pediatric patients. [Abstract/Link to Full Text]

Antonialli WF, Lima SM, Andrade LH, Súarez YR
Comparative study of the cuticular hydrocarbon in queens, workers and males of Ectatomma vizottoi (Hymenoptera, Formicidae) by Fourier transform-infrared photoacoustic spectroscopy.
Genet Mol Res. 2007;6(3):492-9.
Fourier transform-infrared photoacoustic spectroscopy was applied for the first time, to our knowledge, to distinguish different castes of an ant species. The method was applied directly to the abdomen of queens, workers and males of Ectatomma vizottoi ants, without any special sample preparation. The absorption bands of secondary amide and hydrocarbons were identified; using these as variables in a canonical discriminant analysis we found significant differences between the castes. Queens have a greater hydrocarbon content than do workers and males, which is related to their function in the colony. This technique can be used to analyze and distinguish small chemical differences in biological systems, even in opaque samples. [Abstract/Link to Full Text]

Sarmiento RM, Garcia JP
Estimation of genetic parameters and variance components for growth traits in Romosinuano cattle in the Colombian humid tropics.
Genet Mol Res. 2007;6(3):482-91.
(Co)variance components and genetic parameters were estimated for body weights of a Romosinuano herd located in Sinú Valley, Cordoba, Colombia. Restricted maximum likelihood methods were used with a univariate animal model for birth weight, weaning weight (270 days), 16-month weight (480 days), weaning daily gain, and post-weaning daily gain. Models included random animal direct and maternal genetic effects, maternal permanent environmental effect (c2), and sex-year-month of birth and age of dam, as fixed effects. Estimates of direct effect for birth weight, weaning weight, 480-day weight, weaning daily gain, and post-weaning daily gain were: 0.25 +/- 0.0001, 0.34 +/- 0.063, 0.33 +/- 0.066, 0.32 +/- 0.062, and 0.17 +/- 0.052, respectively. Estimates of direct maternal genetic effects were low and ranged from 0.06 +/- 0.003 for birth weight to 0.20 +/- 0.054 for weaning daily gain. The genetic correlations between direct and maternal genetic effects were negative and low for 480-day weight (-0.05 +/- 0.219) and showed values of -0.37 +/- 0.007, -0.34 +/- 0.133, -0.33 +/- 0.135, and -0.38 +/- 0.232 for birth, weaning weight, weaning, and post-weaning daily gain, respectively. Permanent environmental maternal effects were not significant; the highest values were found for weaning weight, and weaning daily gain (0.086 +/- 0.031 and 0.078 +/- 0.031, respectively). We conclude that direct and maternal effects should be included in a selection program for all of these traits, and also that selection of weaning weights would be the most productive way to improve performance in Romosinuano cattle. [Abstract/Link to Full Text]

Iqbal A, Khan AS, Khan IA, Awan FS, Ahmad A, Khan AA
Study of genetic divergence among wheat genotypes through random amplified polymorphic DNA.
Genet Mol Res. 2007;6(3):476-81.
The degree of genetic divergence was estimated in seven wheat genotypes, six exotic genotypes and one local variety, through random amplified polymorphic DNA methodology. A total of 112 DNA fragments were generated by the 15 random primers, with an average of about 7.4 bands per primer. Among the 112, 50 fragments showed polymorphism among the seven wheat genotypes. Nei and Li's similarity matrix ranged from 86.2 to 93.0%, which indicated a narrow genetic base among the genotypes. The maximum similarity, 93.0%, was observed between 12WLRG/1-12 and WL-43. The local variety, Chenab-70, showed the lowest similarity with the exotic types. We conclude that random amplified polymorphic DNA analysis can be used for the characterization and grouping of wheat genotypes; these results will be helpful in our wheat breeding program. [Abstract/Link to Full Text]

Kedar PS, Nampoothiri S, Sreedhar S, Ghosh K, Shimizu K, Kanno H, Colah RB
First-trimester prenatal diagnosis of pyruvate kinase deficiency in an Indian family with the pyruvate kinase-Amish mutation.
Genet Mol Res. 2007;6(2):470-5.
Pyruvate kinase (PK) deficiency is a rare red cell glycolytic enzymopathy. The purpose of the present investigation was to offer prenatal diagnosis for PK deficiency to a couple who had a previous child with severe enzyme deficiency and congenital non-spherocytic hemolytic anemia. PK deficiency was identified in the family by assaying the enzyme activity in red cells. Chorionic villus sampling was performed in an 11-week gestation and the mutation was located in exon 10 of the PKLR gene characterized by polymerase chain reaction and using restriction endonuclease digestion with the MspI enzyme, which was confirmed by DNA sequencing on the ABI 310 DNA sequencer. Both the parents were heterozygous for the 1436G-->A [479 Arg-->His] mutation in exon 10 and the proband was homozygous for this mutation. The fetus was also heterozygous for this mutation and the pregnancy was continued. Prenatal diagnosis allowed the parents with a severely affected child with PK deficiency to have the reproductive choice of having the fetus tested in a subsequent pregnancy. [Abstract/Link to Full Text]

Recent Articles in BMC Genetics

Egito AA, Paiva SR, Albuquerque MD, Mariante AS, Almeida LD, Castro SR, Grattapaglia D
Microsatellite based genetic diversity and relationships among ten Creole and commercial cattle breeds raised in Brazil.
BMC Genet. 2007 Dec 7;8(1):83.
ABSTRACT: BACKGROUND: Brazil holds the largest commercial cattle populations worldwide. Local cattle breeds can be classified according to their origin, as exotic or Creole. Exotic breeds imported in the last 100 years, both zebuine and taurine, currently make up the bulk of the intensively managed populations. Locally adapted Creole breeds, originated from cattle introduced by the European conquerors derive from natural selection and events of breed admixture. While historical knowledge exists on the Brazilian Creole breeds very little is known on their genetic composition. The objective of this study was to assess the levels of genetic diversity, phylogenetic relationships and patterns of taurine/zebuine admixture among ten cattle breeds raised in Brazil. RESULTS: Significant reduction of heterozygosity exists due both to within-population inbreeding and to breed differentiation in both subspecies (taurine and zebuine). For taurine breeds the number of markers that contribute to breed differentiation is larger than for zebuine. A consistently similar number of alleles was seen in both subspecies for all microsatellites. Four Creole breeds were the most genetically diverse followed by the zebuine breeds, the two specialized taurine breeds and the Creole Caracu. Pairwise genetic differentiation were all significant indicating that all breeds can be considered as genetically independent entities. A STRUCTURE based diagram indicated introgression of indicine genes in the local Creole breeds and suggested that occasional Creole introgression can be detected in some Zebuine animals. CONCLUSIONS: This study reports on a comprehensive study of the genetic structure and diversity of cattle breeds in Brazil. A significant amount of genetic variation is maintained in the local cattle populations. The genetic data show that Brazilian Creole breeds constitute an important and diverse reservoir of genetic diversity for bovine breeding and conservation. The genetic data was able to shed light on a number of issues related to the local breeds origin and structure. The Brazilian Creole breeds are all important and viable targets for conservation for they display peculiar traits both phenotypic and of cultural and historical nature that deserve conservation efforts. [Abstract/Link to Full Text]

Zhu C, Zhang YM
An EM algorithm for mapping segregation distortion loci.
BMC Genet. 2007 Nov 29;8(1):82.
ABSTRACT: BACKGROUND: Chromosomal region that causes distorted segregation ratios may be referred to as segregation distortion locus (SDL). The distortion is caused either by differential representation of SDL genotypes in gametes before fertilization or by viability differences of SDL genotypes after fertilization but before genotype scoring. In both cases, observable phenotypes are distorted for marker loci in the chromosomal region close to the SDL. Under the quantitative genetics model for viability selection by proposing a continuous liability controlling the viability of individual, the simplex algorithm has been used to search for the solution in SDL mapping. However, they did not consider the effects of SDL on the construction of linkage map. RESULTS: We proposed a multipoint maximum-likelihood method to estimate the position and the effects of SDL under the liability model together with both selection coefficients of marker genotypes and recombination fractions. The method was implemented via an expectation and maximization (EM) algorithm. The superiority of the method proposed under the liability model over the previous methods was verified by a series of Monte Carlo simulation experiments, together with a working example derived from the MAPMAKER/QTL software. CONCLUSIONS: Our results suggest that the proposed method can serve as a powerful alternative to existing methods for SDL mapping. Under the liability model, the new method can simultaneously estimate the position and the effects of SDL as well as the recombination fractions between adjacent markers, and also be used to probe into the genetic mechanism for the bias of uncorrected map distance and to elucidate the relationship between the viability selection and genetic linkage. [Abstract/Link to Full Text]

Seo BY, Park EW, Ahn SJ, Lee SH, Kim JH, Im HT, Lee JH, Cho IC, Kong IK, Jeon JT
An accurate method for quantifying and analyzing copy number variation in porcine KIT by an oligonucleotide ligation assay.
BMC Genet. 2007 Nov 23;8(1):81.
ABSTRACT: BACKGROUND: Aside from single nucleotide polymorphisms, copy number variations (CNVs) are the most important factors in susceptibility to genetic disorders because they affect expression levels of genes. In previous studies, pyrosequencing, mini-sequencing, real-time PCR, invader assays and other techniques have been used to detect CNVs. However, the higher the copy number in a genome, the more difficult it is to resolve the copies, so a more accurate method for measuring CNVs and assigning genotype is needed. RESULTS: PCR followed by a quantitative oligonucleotide ligation assay (qOLA) was developed for quantifying CNVs. The accuracy and precision of the assay were evaluated for porcine KIT, which was selected as a model locus. Overall, the root mean squares of bias and standard deviation of qOLA were 2.09 and 0.45, respectively. These values are less than half of those in the published pyrosequencing assay for analyzing CNV in porcine KIT. Using a combined method of qOLA and another pyrosequencing for quantitative analysis of KIT copies with spliced forms, we confirmed the segregation of KIT alleles in 145 F1 animals with pedigree information and verified the correct assignment of genotypes. In a diagnostic test on 100 randomly sampled commercial pigs, there was perfect agreement between the genotypes obtained by grouping observations on a scatter plot and by clustering using the nearest centroid sorting method implemented in PROC FASTCLUS of the SAS package. In a test on 159 Large White pigs, there were only two discrepancies between genotypes assigned by the two clustering methods (98.7% agreement), confirming that the quantitative ligation assay established here makes genotyping possible through the accurate measurement of high KIT copy numbers (>4 per diploid genome). Moreover, the assay is sensitive enough for use on DNA from hair follicles, indicating that DNA from various sources could be used. CONCLUSIONS: We have established a high resolution quantification method using an oligonucleotide ligation assay to measure CNVs, and verified the reliability of genotype assignment for random animal samples using the nearest centroid sorting method. This new method will make it more practical to determine KIT CNV and to genotype the complicated Dominant White/KIT locus in pigs. This procedure could have wide applications for studying gene or segment CNVs in other species. [Abstract/Link to Full Text]

Kijas JW, McCulloch R, Hocking Edwards JE, Oddy VH, Lee SH, van der Werf J
Evidence for multiple alleles effecting muscling and fatness at the Ovine GDF8 locus.
BMC Genet. 2007 Nov 8;8(1):80.
ABSTRACT: BACKGROUND: The current investigation surveyed genetic polymorphism at the ovine GDF8 locus and determined its contribution to variation in muscling and fatness in sheep. RESULTS: Re-sequencing 2988 bp from a panel of 15 sires revealed a total of six SNP, none of which were located within exons of the gene. One of the identified SNP, g+6723G>A, is known to increase muscularity within the Belgian Texel. A genetic survey of 326 animals revealed that the mutation is near fixation within Australian Texels and present in additional breeds including White Suffolk, Poll Dorset and Lincoln. Using a resource population comprising 15 sires and 1191 half-sib progeny with genotypic data, the effect of this and other SNP was tested against a set of 50 traits describing growth, muscling, fatness, yield, meat and eating quality. The loss of function allele (g+6723A) showed significant effects on slaughter measurements of muscling and fatness. No effect was detected on objectively assessed meat quality however evidence was found for an association between g+6723G>A, decreased intramuscular fat and reduced eating quality. Haplotype analysis using flanking microsatellites was performed to search for evidence of currently unidentified mutations which might affect production traits. Four haplotypes were identified that do not carry g+6723A but which showed significant associations with muscling and fatness. CONCLUSIONS: The finding that g+6723G>A is present within Australian sheep facilitated an independent evaluation into its phenotypic consequence. Testing was conducted using a separate genetic background and animals raised in different environments to the Belgian Texel in which it was first identified. The observation that the direction and size of effects for g+6723A is approximately consistent represented a robust validation of the effects of the mutation. Based on observed allele frequencies within breeds, selection for g+6723A will have the largest impact within the White Suffolk. GDF8 may harbour additional mutations which serve to influence economically important traits in sheep. [Abstract/Link to Full Text]

Woo JG, Sun G, Haverbusch M, Indugula S, Martin LJ, Broderick JP, Deka R, Woo D
Quality assessment of buccal versus blood genomic DNA using the Affymetrix 500K GeneChip.
BMC Genet. 2007 Nov 8;8(1):79.
ABSTRACT: BACKGROUND: With the advent of genome-wide genotyping, the utility of stored buccal brushes for DNA extraction and genotyping has been questioned. We sought to describe the genomic DNA yield and concordance between stored buccal brushes and blood samples from the same individuals in the context of Affymetrix 500K Human GeneChip genotyping. RESULTS: Buccal cytobrushes stored for ~7 years at -80 degrees C prior to extraction yielded sufficient double stranded DNA (dsDNA) to be successfully genotyped on the Affymetrix ~262K NspI chip, with yields between 536 and 1047 ng dsDNA. Using the BRLMM algorithm, genotyping call rates for blood samples averaged 98.4%, and for buccal samples averaged 97.8%. Matched blood samples exhibited 99.2% concordance, while matched blood and buccal samples exhibited 98.8% concordance. CONCLUSIONS: Buccal cytobrushes stored long-term result in sufficient dsDNA concentrations to achieve high genotyping call rates and concordance with stored blood samples in the context of Affymetrix 500K SNP genotyping. Thus, given high-quality collection and storage protocols, it is possible to use stored buccal cytobrush samples for genome-wide association studies. [Abstract/Link to Full Text]

Mersch B, Sela N, Ast G, Suhai S, Hotz-Wagenblatt A
SERpredict: Detection of tissue- or tumor-specific isoforms generated through exonization of transposable elements.
BMC Genet. 2007 Nov 6;8(1):78.
ABSTRACT: BACKGROUND: Transposable elements (TEs) are known to affect transcriptomes, because either new exons are generated from intronic transposable elements (this is called exonization), or the element inserts into the exon, leading to a new transcript. Several examples in the literature show that isoforms generated by an exonization are specific to a certain tissue (for example the heart muscle) or inflict a disease. Thus, exonizations can have negative effects for the transcriptome of an organism. RESULTS: As we aimed at detecting other tissue- or tumor-specific isoforms in human and mouse genomes which were generated through exonization of a transposable element, we designed the automated analysis pipeline SERpredict (SER = Specific Exonized Retroelement) making use of Bayesian Statistics. With this pipeline, we found several genes in which a transposable element formed a tissue or tumor-specific isoform. CONCLUSIONS: Our results show that SERpredict produces relevant results, demonstrating the importance of transposable elements in shaping both the human and the mouse transcriptomes. The effect of transposable elements on the human transcriptome is several times higher than the effect on the mouse transcriptome, due to the contribution of the primate-specific Alu elements. [Abstract/Link to Full Text]

Nguyen TT, Genini S, Bui LC, Voegeli P, Stranzinger G, Renard JP, Maillard JC, Nguyen BX
Genomic conservation of cattle microsatellite loci in wild gaur (Bos gaurus) and current genetic status of this species in Vietnam.
BMC Genet. 2007 Nov 6;8(1):77.
ABSTRACT: BACKGROUND: The wild gaur (Bos gaurus) is an endangered wild cattle species. In Vietnam, the total number of wild gaur is estimated at a maximum of 500 individuals. Inbreeding and genetic drift are current relevant threats to this small population size. Therefore, information about the genetic status of the Vietnamese wild gaur population is essential to develop strategies for conservation and effective long-term management for this species. In the present study, we performed cross-species amplification of 130 bovine microsatellite markers, in order to evaluate the applicability and conservation of cattle microsatellite loci in the wild gaur genome. The genetic diversity of Vietnamese wild gaur was also investigated, based on data collected from 117 successfully amplified loci. RESULTS: One hundred-thirty cattle microsatellite markers were tested on a panel of 11 animals. Efficient amplifications were observed for 117 markers (90%) with a total of 264 alleles, and of these, 68 (58.1%) gave polymorphic band patterns. The number of alleles per locus among the polymorphic markers ranged from two to six. Thirteen loci (BM1314, BM2304, BM6017, BMC2228, BMS332, BMS911, CSSM023, ETH123, HAUT14, HEL11, HEL5, ILSTS005 and INRA189) distributed on nine different cattle chromosomes failed to amplify wild gaur genomic DNA. Three cattle Y-chromosome specific microsatellite markers (INRA124, INRA126 and BM861) were also highly specific in wild gaur, only displaying an amplification product in the males. Genotype data collected from the 117 successfully amplified microsatellites were used to assess the genetic diversity of this species in Vietnam. Polymorphic Information Content (PIC) values varied between 0.083 and 0.767 with a mean of 0.252 while observed heterozygosities (Ho) ranged from 0.091 to 0.909 (mean of 0.269). Nei's unbiased mean heterozygosity and the mean allele number across loci were 0.298 and 2.2, respectively. CONCLUSIONS: Extensive conservation of cattle microsatellite loci in the wild gaur genome, as shown by our results, indicated a high applicability of bovine microsatellites for genetic characterization and population genetic studies of this species. Moreover, the low genetic diversity observed in Vietnamese wild gaur further underlines the necessity of specific strategies and appropriate management plans to preserve this endangered species from extinction. [Abstract/Link to Full Text]

El Mokhtari NE, Ott SJ, Nebel A, Schaefer A, Rosenstiel P, Foerster M, Nothnagel M, Simon R, Schreiber S
Role of NOD2/CARD15 in coronary heart disease.
BMC Genet. 2007 Nov 2;8(1):76.
ABSTRACT: BACKGROUND: Bacterial DNA has been repeatedly detected in atheromatous lesions of coronary heart disease (CHD) patients. Phylogenetic signatures in the atheroma lesions that are similar to those of bacterial biofilms on human barrier organs, including the respiratory or gastrointestinal tract, raise the question of a defective barrier function in CHD. NOD2 plays a major role in defense against bacterial invasion. Genetic variation in the CARD15 gene, which encodes NOD2, was previously shown to result in a barrier defect that causes chronic inflammatory disorders (e.g. Crohn disease). In the present study, we investigated the possible involvement of NOD2/CARD15 in the pathology of CHD by i) analyzing the local expression of NOD2 in atherectomy versus healthy tissue (n=5 each) using histochemical immunofluorescence and ii) by testing the three major functional CARD15 variants (R702W, G908R and 1007fs) for association with early-onset CHD in 900 German patients and 632 healthy controls. RESULTS: In atherectomy tissue of CHD patients, NOD2 was detected in inflammatory cells at the luminal sides of the lesions. However, the allele and genotype frequencies of the three major CARD15 polymorphisms did not differ between CHD patients and controls. CONCLUSION: In conclusion, the NOD2 up-regulation in atheroma lesions indicates an involvement of this protein in the pathology of CHD. Although NOD2 could be important in local immune response mechanisms, none of the analyzed CARD15 variants seem to play a significant role in the etiology of CHD. [Abstract/Link to Full Text]

Feng BJ, Goldgar DE, Corbex M
Trend-TDT - a transmission/disequilibrium based association test on functional mini/microsatellites.
BMC Genet. 2007 Nov 1;8(1):75.
ABSTRACT: BACKGROUND: Minisatellites and microsatellites are associated with human disease, not only as markers of risk but also involved directly in disease pathogenesis. They may play significant roles in replication, repair and mutation of DNA, regulation of gene transcription and protein structure alteration. Phenotypes can thus be affected by mini/microsatellites in a manner proportional to the length of the allele. Here we propose a new method to assess the linear trend toward transmission of shorter or longer alleles from heterozygote parents to affected child. RESULTS: This test (trend-TDT) performs better than other TDT (Transmission/Disequilibrium Test) type tests, such as TDTmax and TDTL/S, under most marker-disease association models. CONCLUSIONS: The trend-TDT test is a more powerful association test when there is a biological basis to suspect a relationship between allele length and disease risk. [Abstract/Link to Full Text]

McKay SD, Schnabel RD, Murdoch BM, Matukumalli LK, Aerts J, Coppieters W, Crews D, Dias Neto E, Gill CA, Gao C, Mannen H, Stothard P, Wang Z, Van Tassell CP, Williams JL, Taylor JF, Moore SS
Whole genome linkage disequilibrium maps in cattle.
BMC Genet. 2007 Oct 25;8(1):74.
ABSTRACT: BACKGROUND: Bovine whole genome linkage disequilibrium maps were constructed for eight breeds of cattle. These data provide fundamental information concerning bovine genome organization which will allow the design of studies to associate genetic variation with economically important traits and also provides background information concerning of the extent of long range linkage disequilibrium in cattle. RESULTS: Linkage disequilibrium was assessed using r2 among all pairs of syntenic markers within eight breeds of cattle from the Bos taurus and Bos indicus subspecies. Bos taurus breeds included Angus, Charolais, Dutch Black and White Dairy, Holstein, Japanese Black and Limousin while Bos indicus breeds included Brahman and Nelore. Approximately 2670 markers spanning the entire bovine autosomal genome were used to estimate pairwise r2 values. We found that the extent of linkage disequilibrium is no more than 0.5 Mb in these eight breeds of cattle. CONCLUSIONS: Linkage disequilibrium in cattle has previously been reported to extend several tens of centimorgans. Our results, based on a much larger sample of marker loci and across eight breeds of cattle indicate that in cattle linkage disequilibrium persists over much more limited distances. Our findings suggest that 30,000-50,000 loci will be needed to conduct whole genome association studies in cattle. [Abstract/Link to Full Text]

Lopez-Orduna E, Cruz M, Garcia-Mena J
The transcription of MGAT4A glycosyl transferase is increased in white cells of peripheral blood of Type 2 Diabetes patients.
BMC Genet. 2007 Oct 22;8(1):73.
ABSTRACT: BACKGROUND: Human glycosylase IV is involved in GLUT2 transporter regulation in pancreatic beta cells. A KO of this gene along with a high fat diet in a mice model has been associated with the development of type 2 diabetes (T2D). The aims of this study were to measure and compare the MGAT4A mRNA levels in white blood cells (WBC) from T2D subjects and healthy subjects (T2NB), and to measure the half-life of the MGAT4A mRNA. RESULTS: We studied a sample of 73 individuals, 40 T2D subjects and 33 T2NB subjects. Anthropometrical and biochemical profiles were registered. The MGAT4A mRNA levels in WBC and the transcript half-life in Jurkat T cells were determined by Real-Time PCR. A blood differential cell counting was made for each individual. Cell counting showed T2D subjects exhibited an increased number of WBC compared to T2NB subjects (P = 0.0001). Biochemical parameters such as fasting glucose (P = 0.0001), and triglycerides (P = 0.002) were statistically significant. T2D subjects had 4.2-fold more MGAT4A transcript compared to T2NB subjects (P = 0.002). The MGAT4A mRNA had a half-life of 2.04 h in Jurkat T cells. CONCLUSION: The results of this work suggest that in T2D subjects, high levels of glucose and triglycerides are accompanied by an increase on MGAT4A mRNA levels and WBC count; condition that suggests a pro-inflammatory state due to a chronic metabolic stress. [Abstract/Link to Full Text]

Royer B, Soares DC, Barlow PN, Bontrop RE, Roll P, Robaglia-Schlupp A, Blancher A, Levasseur A, Cau P, Pontarotti P, Szepetowski P
Molecular evolution of the human SRPX2 gene that causes brain disorders of the Rolandic and Sylvian speech areas.
BMC Genet. 2007 Oct 18;8(1):72.
ABSTRACT: BACKGROUND: The X-linked SRPX2 gene encodes a Sushi Repeat-containing Protein of unknown function and is mutated in two disorders of the Rolandic/Sylvian speech areas. Since it is linked to defects in the functioning and the development of brain areas for speech production, SRPX2 may thus have participated in the adaptive organization of such brain regions. To address this issue, we have examined the recent molecular evolution of the SRPX2 gene. RESULTS: The complete coding region was sequenced in 24 human X chromosomes from worldwide populations and in six representative nonhuman primate species. One single, fixed amino acid change (R75K) has been specifically incorporated in human SRPX2 since the human-chimpanzee split. The R75K substitution occurred in the first sushi domain of SRPX2, only three amino acid residues away from a previously reported disease-causing mutation (Y72S). Three-dimensional structural modeling of the first sushi domain revealed that Y72 and K75 are both situated in the hypervariable loop that is usually implicated in protein-protein interactions. The side-chain of residue 75 is exposed, and is located within an unusual and SRPX-specific protruding extension to the hypervariable loop. The analysis of non-synonymous/synonymous substitution rate (Ka/Ks) ratio in primates was performed in order to test for positive selection during recent evolution. Using the branch models, the Ka/Ks ratio for the human branch was significantly different (p = 0.027) from that of the other branches. In contrast, the branch-site tests did not reach significance. Genetic analysis was also performed by sequencing 9,908 kilobases (kb) of intronic SRPX2 sequences. Despite low nucleotide diversity, neither the HKA (Hudson-Kreitman-Aguade) test nor the Tajima's D test reached significance. CONCLUSION: The R75K human-specific variation occurred in an important functional loop of the first sushi domain of SRPX2, indicating that this evolutionary mutation may have functional importance; however, positive selection for R75K could not be demonstrated. Nevertheless, our data contribute to the first understanding of molecular evolution of the human SPRX2 gene. Further experiments are now required in order to evaluate the possible consequences of R75K on SRPX2 interactions and functioning. [Abstract/Link to Full Text]

Hirunsatit R, Ilomaki R, Malison R, Rasanen P, Ilomaki E, Kranzler HR, Kosten T, Sughondhabirom A, Thavichachart N, Tangwongchai S, Listman J, Mutirangura A, Gelernter J, Lappalainen J
Sequence variation and linkage disequilibrium in the GABA transporter-1 gene (SLC6A1) in five populations: implications for pharmacogenetic research.
BMC Genet. 2007 Oct 17;8(1):71.
ABSTRACT: BACKGROUND: GABA transporter-1 (GAT-1; genetic locus SLC6A1) is emerging as a novel target for treatment of neuropsychiatric disorders. To understand how population differences might influence strategies for pharmacogenetic studies, we identified patterns of genetic variation and linkage disequilibrium (LD) in SLC6A1 in five populations representing three continental groups. RESULTS: We resequenced 12.4 kb of SLC6A1, including the promoters, exons and flanking intronic regions in African-American, Thai, Hmong, Finnish, and European-American subjects (total n=40). LD in SLC6A1 was examined by genotyping 16 SNPs in larger samples. Sixty-three variants were identified through resequencing. Common population-specific variants were found in African-Americans, including a novel 21-bp promoter region variable number tandem repeat (VNTR), but no such variants were found in any of the other populations studied. Low levels of LD and the absence of major LD blocks were characteristic of all five populations. African-Americans had the highest genetic diversity. European-Americans and Finns did not differ in genetic diversity or LD patterns. Although the Hmong had the highest level of LD, our results suggest that a strategy based on the use of tag SNPs would not translate to a major improvement in genotyping efficiency. CONCLUSION: Owing to the low level of LD and presence of recombination hotspots, SLC6A1 may be an example of a problematic gene for association and haplotype tagging-based genetic studies. The 21-bp promoter region VNTR polymorphism is a putatively functional candidate allele for studies focusing on variation in GAT-1 function in the African-American population. [Abstract/Link to Full Text]

Tan Q, Christiansen L, Brasch-Andersen C, Zhao JH, Li S, Kruse TA, Christensen K
Retrospective analysis of main and interaction effects in genetic association studies of human complex traits.
BMC Genet. 2007;870.
BACKGROUND: The etiology of multifactorial human diseases involves complex interactions between numerous environmental factors and alleles of many genes. Efficient statistical tools are demanded in identifying the genetic and environmental variants that affect the risk of disease development. This paper introduces a retrospective polytomous logistic regression model to measure both the main and interaction effects in genetic association studies of human discrete and continuous complex traits. In this model, combinations of genotypes at two interacting loci or of environmental exposure and genotypes at one locus are treated as nominal outcomes of which the proportions are modeled as a function of the disease trait assigning both main and interaction effects and with no assumption of normality in the trait distribution. Performance of our method in detecting interaction effect is compared with that of the case-only model. RESULTS: Results from our simulation study indicate that our retrospective model exhibits high power in capturing even relatively small effect with reasonable sample sizes. Application of our method to data from an association study on the catalase -262C/T promoter polymorphism and aging phenotypes detected significant main and interaction effects for age-group and allele T on individual's cognitive functioning and produced consistent results in estimating the interaction effect as compared with the popular case-only model. CONCLUSION: The retrospective polytomous logistic regression model can be used as a convenient tool for assessing both main and interaction effects in genetic association studies of human multifactorial diseases involving genetic and non-genetic factors as well as categorical or continuous traits. [Abstract/Link to Full Text]

Meyers SN, Rodriguez-Zas SL, Beever JE
Fine-mapping of a QTL influencing pork tenderness on porcine chromosome 2.
BMC Genet. 2007 Oct 12;8(1):69.
ABSTRACT: BACKGROUND: In a previous study, a quantitative trait locus (QTL) exhibiting large effects on both Instron shear force and taste panel tenderness was detected within the Illinois Meat Quality Pedigree (IMQP). This QTL mapped to the q arm of porcine chromosome 2 (SSC2q). Comparative analysis of SSC2q indicates that it is orthologous to a segment of human chromosome 5 (HSA5) containing a strong positional candidate gene, calpastatin (CAST). CAST polymorphisms have recently been shown to be associated with meat quality characteristics; however, the possible involvement of other genes and/or molecular variation in this region cannot be excluded, thus requiring fine-mapping of the QTL. RESULTS: Recent advances in porcine genome resources, including high-resolution radiation hybrid and bacterial artificial chromosome (BAC) physical maps, were utilized for development of novel informative markers. Marker density in the ~30-Mb region surrounding the most likely QTL position was increased by addition of eighteen new microsatellite markers, including nine publicly-available and nine novel markers. Two newly-developed markers were derived from a porcine BAC clone containing the CAST gene. Refinement of the QTL position was achieved through linkage and haplotype analyses. Within-family linkage analyses revealed at least two families segregating for a highly-significant QTL in strong positional agreement with CAST markers. A combined analysis of these two families yielded QTL intervals of 36 cM and 7 cM for Instron shear force and taste panel tenderness, respectively, while haplotype analyses suggested further refinement to a 1.8 cM interval containing CAST markers. The presence of additional tenderness QTL on SSC2q was also suggested. CONCLUSIONS: These results reinforce CAST as a strong positional candidate. Further analysis of CAST molecular variation within the IMQP F1 boars should enhance understanding of the molecular basis of pork tenderness, and thus allow for genetic improvement of pork products. Furthermore, additional resources have been generated for the targeted investigation of other putative QTL on SSC2q, which may lead to further advancements in pork quality. [Abstract/Link to Full Text]

Pereira LH, Pineda MA, Rowe WH, Fonseca LR, Greene MH, Offit K, Ellis NA, Zhang J, Collins A, Struewing JP
The BRCA1 Ashkenazi founder mutations occur on common haplotypes and are not highly correlated with anonymous single nucleotide polymorphisms likely to be used in genome-wide case-control association studies.
BMC Genet. 2007;868.
BACKGROUND: We studied linkage disequilibrium (LD) patterns at the BRCA1 locus, a susceptibility gene for breast and ovarian cancer, using a dense set of 114 single nucleotide polymorphisms in 5 population groups. We focused on Ashkenazi Jews in whom there are known founder mutations, to address the question of whether we would have been able to identify the 185delAG mutation in a case-control association study (should one have been done) using anonymous genetic markers. This mutation is present in approximately 1% of the general Ashkenazi population and 4% of Ashkenazi breast cancer cases. We evaluated LD using pairwise and haplotype-based methods, and assessed correlation of SNPs with the founder mutations using Pearson's correlation coefficient. RESULTS: BRCA1 is characterized by very high linkage disequilibrium in all populations spanning several hundred kilobases. Overall, haplotype blocks and pair-wise LD bins were highly correlated, with lower LD in African versus non-African populations. The 185delAG and 5382insC founder mutations occur on the two most common haplotypes among Ashkenazim. Because these mutations are rare, even though they are in strong LD with many other SNPs in the region as measured by D-prime, there were no strong associations when assessed by Pearson's correlation coefficient, r (maximum of 0.04 for the 185delAG). CONCLUSION: Since the required sample size is related to the inverse of r, this suggests that it would have been difficult to map BRCA1 in an Ashkenazi case-unrelated control association study using anonymous markers that were linked to the founder mutations. [Abstract/Link to Full Text]

Curtis D
Extended homozygosity is not usually due to cytogenetic abnormality.
BMC Genet. 2007;867.
BACKGROUND: Previous studies have reported frequent stretches of homozygosity in human subjects but have failed to clarify whether these are due to cytogenetic abnormalities or to autozygosity. METHODS: Trios which had been typed for closely spaced SNPs spanning the genome were studied. Stretches of extended homozygosity were identified in the child members, as were occasions on which the child had been genotyped as not inheriting one parental allele. The number of times such transmission errors occurred within regions of extended homozygosity was compared with the chance expectation. RESULTS: Transmission errors occurred more rarely in regions of extended homozygosity than would be expected by chance. DISCUSSION: Regions of extended homozygosity are not generally due to cytogenetic abnormalities such as uniparental isodisomy. They reflect the Mendelian inheritance of haplotypes from a common ancestor. This may have implications for mapping disease genes. [Abstract/Link to Full Text]

McArdle PF, Dytch H, O'Connell JR, Shuldiner AR, Mitchell BD, Abney M
Homozygosity by descent mapping of blood pressure in the Old Order Amish: evidence for sex specific genetic architecture.
BMC Genet. 2007;866.
BACKGROUND: High blood pressure is a well established risk factor for morbidity and mortality acting through heart disease, stroke and cardiovascular disease. Genome wide scans have linked regions of nearly every human chromosome to blood pressure related traits. We have capitalized on beneficial qualities of the Old Order Amish of Lancaster, PA, a closed founder population with a relatively small number of founders, to perform a genome wide homozygosity by descent mapping scan. Each individual in the study has a non zero probability of consanguinity. Systolic and diastolic blood pressures are shown to have appreciable dominance variance components. RESULTS: Areas of two chromosomes were identified as suggestive of linkage to SBP and 5 areas to DBP in either the overall or sex specific analyses. The strongest evidence for linkage in the overall sample was to Chromosome 18q12 (LOD = 2.6 DBP). Sex specific analyses identified a linkage on Chromosome 4p12-14 (LOD in men only = 3.4 SBP). At Chromosome 2q32-33, an area where we previously reported significant evidence for linkage to DBP using a conventional identity by descent approach, the LOD was 1.4; however an appreciable sex effect was observed with men accounting for most of the linkage (LOD in men only = 2.6). CONCLUSION: These results add evidence to a sex specific genetic architecture to blood pressure related traits, particularly in regions of linkage on chromosome 2, 4 and 18. [Abstract/Link to Full Text]

Zhang Z, Zhang S, Sha Q
A multi-marker test based on family data in genome-wide association study.
BMC Genet. 2007;865.
BACKGROUND: Complex diseases are believed to be the results of many genes and environmental factors. Hence, multi-marker methods that can use the information of markers from different genes are appropriate for mapping complex disease genes. There already have been several multi-marker methods proposed for case-control studies. In this article, we propose a multi-marker test called a Multi-marker Pedigree Disequilibrium Test (MPDT) to analyze family data from genome-wide association studies. If the parental phenotypes are available, we also propose a two-stage test in which a genomic screening test is used to select SNPs, and then the MPDT is used to test the association of the selected SNPs. RESULTS: We use simulation studies to evaluate the performance of the MPDT and the two-stage approach. The results show that the MPDT constantly outperforms the single marker transmission/disequilibrium test (TDT) 1. Comparing the power of the two-stage approach with that of the one-stage approach, which approach is more powerful depends on the value of the prevalence; when the prevalence is no less than 10%, the two-stage approach may be more powerful than the one-stage approach. Otherwise, the one-stage approach is more powerful. CONCLUSION: The proposed MPDT, is more powerful than the single marker TDT. When the parental phenotypes are available and the prevalence is no less than 10%, the proposed two-stage approach is more powerful than the one-stage approach. [Abstract/Link to Full Text]

Burdzinski C, Wendell DL
Mapping the anthocyaninless (anl) locus in rapid-cycling Brassica rapa (RBr) to linkage group R9.
BMC Genet. 2007;864.
BACKGROUND: Anthocyanins are flavonoid pigments that are responsible for purple coloration in the stems and leaves of a variety of plant species. Anthocyaninless (anl) mutants of Brassica rapa fail to produce anthocyanin pigments. In rapid-cycling Brassica rapa, also known as Wisconsin Fast Plants, the anthocyaninless trait, also called non-purple stem, is widely used as a model recessive trait for teaching genetics. Although anthocyanin genes have been mapped in other plants such as Arabidopsis thaliana, the anl locus has not been mapped in any Brassica species. RESULTS: We tested primer pairs known to amplify microsatellites in Brassicas and identified 37 that amplified a product in rapid-cycling Brassica rapa. We then developed three-generation pedigrees to assess linkage between the microsatellite markers and anl. 22 of the markers that we tested were polymorphic in our crosses. Based on 177 F2 offspring, we identified three markers linked to anl with LOD scores >or= 5.0, forming a linkage group spanning 46.9 cM. Because one of these markers has been assigned to a known B. rapa linkage group, we can now assign the anl locus to B. rapa linkage group R9. CONCLUSION: This study is the first to identify the chromosomal location of an anthocyanin pigment gene among the Brassicas. It also connects a classical mutant frequently used in genetics education with molecular markers and a known chromosomal location. [Abstract/Link to Full Text]

El Galta R, Uitte de Willige S, de Visser MC, Helmer Q, Hsu L, Houwing-Duistermaat JJ
Generalizing Terwilliger's likelihood approach: a new score statistic to test for genetic association.
BMC Genet. 2007 Sep 24;8(1):63.
ABSTRACT: BACKGROUND: In this paper, we propose a one degree of freedom test for association between a candidate gene and a binary trait. This method is a generalization of Terwilliger's likelihood ratio statistic and is especially powerful for the situation of one associated haplotype. As an alternative to the likelihood ratio statistic, we derive a score statistic, which has a tractable expression. For haplotype analysis, we assume that phase is known. RESULTS: By means of a simulation study, we compare the performance of the score statistic to Pearson's chi-square statistic and the likelihood ratio statistic proposed by Terwilliger. We illustrate the method on three candidate genes studied in the Leiden Thrombophilia Study. CONCLUSIONS: We conclude that the statistic follows a chi square distribution under the null hypothesis and that the score statistic is more powerful than Terwilliger's likelihood ratio statistic when the associated haplotype has frequency between 0.1 and 0.4 and has a small impact on the studied disorder. With regard to Pearson's chi-square statistic, the score statistic has more power when the associated haplotype has frequency above 0.2 and the number of variants is above five. [Abstract/Link to Full Text]

Kuehn C, Edel C, Weikard R, Thaller G
Dominance and parent-of-origin effects of coding and non-coding alleles at the acylCoA-diacylglycerol-acyltransferase (DGAT1) gene on milk production traits in German Holstein cows.
BMC Genet. 2007;862.
BACKGROUND: Substantial gene substitution effects on milk production traits have formerly been reported for alleles at the K232A and the promoter VNTR loci in the bovine acylCoA-diacylglycerol-acyltransferase 1 (DGAT1) gene by using data sets including sires with accumulated phenotypic observations of daughters (breeding values, daughter yield deviations). However, these data sets prevented analyses with respect to dominance or parent-of-origin effects, although an increasing number of reports in the literature outlined the relevance of non-additive gene effects on quantitative traits. RESULTS: Based on a data set comprising German Holstein cows with direct trait measurements, we first confirmed the previously reported association of DGAT1 promoter VNTR alleles with milk production traits. We detected a dominant mode of effects for the DGAT1 K232A and promoter VNTR alleles. Namely, the contrasts between the effects of heterozygous individuals at the DGAT1 loci differed significantly from the midpoint between the effects for the two homozygous genotypes for several milk production traits, thus indicating the presence of dominance. Furthermore, we identified differences in the magnitude of effects between paternally and maternally inherited DGAT1 promoter VNTR - K232A haplotypes indicating parent-of-origin effects on milk production traits. CONCLUSION: Non-additive effects like those identified at the bovine DGAT1 locus have to be accounted for in more specific QTL detection models as well as in marker assisted selection schemes. The DGAT1 alleles in cattle will be a useful model for further investigations on the biological background of non-additive effects in mammals due to the magnitude and consistency of their effects on milk production traits. [Abstract/Link to Full Text]

Coker JA, DasSarma S
Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA.
BMC Genet. 2007;861.
BACKGROUND: Archaea are prokaryotic organisms with simplified versions of eukaryotic transcription systems. Genes coding for the general transcription factors TBP and TFB are present in multiple copies in several Archaea, including Halobacterium sp. NRC-1. Multiple TBP and TFBs have been proposed to participate in transcription of genes via recognition and recruitment of RNA polymerase to different classes of promoters. RESULTS: We attempted to knock out all six TBP and seven TFB genes in Halobacterium sp. NRC-1 using the ura3-based gene deletion system. Knockouts were obtained for six out of thirteen genes, tbpCDF and tfbACG, indicating that they are not essential for cell viability under standard conditions. Screening of a population of 1,000 candidate mutants showed that genes which did not yield mutants contained less that 0.1% knockouts, strongly suggesting that they are essential. The transcriptomes of two mutants, Delta tbpD and DeltatfbA, were compared to the parental strain and showed coordinate down regulation of many genes. Over 500 out of 2,677 total genes were regulated in the Delta tbpD and DeltatfbA mutants with 363 regulated in both, indicating that over 10% of genes in both strains require the action of both TbpD and TfbA for normal transcription. Culturing studies on the Delta tbpD and DeltatfbA mutant strains showed them to grow more slowly than the wild-type at an elevated temperature, 49 degrees C, and they showed reduced viability at 56 degrees C, suggesting TbpD and TfbA are involved in the heat shock response. Alignment of TBP and TFB protein sequences suggested the expansion of the TBP gene family, especially in Halobacterium sp. NRC-1, and TFB gene family in representatives of five different genera of haloarchaea in which genome sequences are available. CONCLUSION: Six of thirteen TBP and TFB genes of Halobacterium sp. NRC-1 are non-essential under standard growth conditions. TbpD and TfbA coordinate the expression of over 10% of the genes in the NRC-1 genome. The Delta tbpD and DeltatfbA mutant strains are temperature sensitive, possibly as a result of down regulation of heat shock genes. Sequence alignments suggest the existence of several families of TBP and TFB transcription factors in Halobacterium which may function in transcription of different classes of genes. [Abstract/Link to Full Text]

Wu J, Province MA, Coon H, Hunt SC, Eckfeldt JH, Arnett DK, Heiss G, Lewis CE, Ellison RC, Rao DC, Rice T, Kraja AT
An investigation of the effects of lipid-lowering medications: genome-wide linkage analysis of lipids in the HyperGEN study.
BMC Genet. 2007;860.
BACKGROUND: Use of anti-hyperlipidemic medications compromises genetic analysis because of altered lipid profiles. We propose an empirical method to adjust lipid levels for medication effects so that the adjusted lipid values substitute the unmedicated lipid values in the genetic analysis. RESULTS: Published clinical trials were reviewed for HMG-CoA reductase inhibitors and fibric acid derivatives as mono-drug therapy. HMG-CoA reductase inhibitors showed similar effects in African Americans (AA) and non-African Americans (non-AA) for lowering total cholesterol (TC, -50.7 mg/dl), LDL cholesterol (LDL-C, -48.1 mg/dl), and triglycerides (TG, -19.7 mg/dl). Their effect on increasing HDL cholesterol (HDL-C) in AA (+0.4 mg/dl) was lower than in Non-AA (+2.3 mg/dl). The effects of fibric acid derivatives were estimated as -46.1 mg/dl for TC, -40.1 mg/dl for LDL-C, and +5.9 mg/dl for HDL-C in non-AA. The corresponding effects in AA were less extreme (-20.1 mg/dl, -11.4 mg/dl, and +3.1 mg/dl). Similar effect for TG (59.0 mg/dl) was shown in AA and non-AA. The above estimated effects were applied to a multipoint variance components linkage analysis on the lipid levels in 2,403 Whites and 2,214 AA in the HyperGEN study. The familial effects did vary depending on whether the lipids were adjusted for medication use. For example, the heritabilities increased after medication adjustment for TC and LDL-C, but did not change significantly for HDL-C and TG. CONCLUSION: Ethnicity-specific medication adjustments using our empirical method can be employed in epidemiological and genetic analysis of lipids. [Abstract/Link to Full Text]

Bhave SV, Hornbaker C, Phang TL, Saba L, Lapadat R, Kechris K, Gaydos J, McGoldrick D, Dolbey A, Leach S, Soriano B, Ellington A, Ellington E, Jones K, Mangion J, Belknap JK, Williams RW, Hunter LE, Hoffman PL, Tabakoff B
The PhenoGen informatics website: tools for analyses of complex traits.
BMC Genet. 2007;859.
BACKGROUND: With the advent of "omics" (e.g. genomics, transcriptomics, proteomics and phenomics), studies can produce enormous amounts of data. Managing this diverse data and integrating with other biological data are major challenges for the bioinformatics community. Comprehensive new tools are needed to store, integrate and analyze the data efficiently. DESCRIPTION: The PhenoGen Informatics website is a comprehensive toolbox for storing, analyzing and integrating microarray data and related genotype and phenotype data. The site is particularly suited for combining QTL and microarray data to search for "candidate" genes contributing to complex traits. In addition, the site allows, if desired by the investigators, sharing of the data. Investigators can conduct "in-silico" microarray experiments using their own and/or "shared" data. CONCLUSION: The PhenoGen website provides access to tools that can be used for high-throughput data storage, analyses and interpretation of the results. Some of the advantages of the architecture of the website are that, in the future, the present set of tools can be adapted for the analyses of any type of high-throughput "omics" data, and that access to new tools, available in the public domain or developed at PhenoGen, can be easily provided. [Abstract/Link to Full Text]

Klein RJ
Power analysis for genome-wide association studies.
BMC Genet. 2007;858.
BACKGROUND: Genome-wide association studies are a promising new tool for deciphering the genetics of complex diseases. To choose the proper sample size and genotyping platform for such studies, power calculations that take into account genetic model, tag SNP selection, and the population of interest are required. RESULTS: The power of genome-wide association studies can be computed using a set of tag SNPs and a large number of genotyped SNPs in a representative population, such as available through the HapMap project. As expected, power increases with increasing sample size and effect size. Power also depends on the tag SNPs selected. In some cases, more power is obtained by genotyping more individuals at fewer SNPs than fewer individuals at more SNPs. CONCLUSION: Genome-wide association studies should be designed thoughtfully, with the choice of genotyping platform and sample size being determined from careful power calculations. [Abstract/Link to Full Text]

Gutierrez A, Sommer RJ
Functional diversification of the nematode mbd2/3 gene between Pristionchus pacificus and Caenorhabditis elegans.
BMC Genet. 2007;857.
BACKGROUND: Several members of the Methyl-Binding Domain protein family link DNA methylation with chromatin remodeling complexes in vertebrates. Amongst the four classes of MBD proteins, MBD2/3 is the most highly conserved and widespread in metazoans. We have previously reported that an mbd2/3 like gene (mbd-2) is encoded in the genomes of the nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae. RNAi knock-down of mbd-2 in the two Caenorhabditis species results in varying percentages of lethality. RESULTS: Here, we report that a general feature of nematode MBD2/3 proteins seems to be the lack of a bona fide methyl-binding domain. We isolated a null allele of mbd-2 in P. pacificus and show that Ppa-mbd-2 mutants are viable, fertile and display a fully penetrant egg laying defect. This egg laying defect is partially rescued by treatment with acetylcholine or nicotine suggesting a specific function of this protein in vulval neurons. Using Yeast-two-hybrid screens, Ppa-MBD-2 was found to associate with microtubule interacting and vesicle transfer proteins. CONCLUSION: These results imply that MBD2/3 proteins in nematodes are more variable than their relatives in insects and vertebrates both in structure and function. Moreover, nematode MBD2/3 proteins assume functions independent of DNA methylation ranging from the indispensable to the non-essential. [Abstract/Link to Full Text]

Gutiérrez-Gil B, Wiener P, Williams JL
Genetic effects on coat colour in cattle: dilution of eumelanin and phaeomelanin pigments in an F2-Backcross Charolais x Holstein population.
BMC Genet. 2007;856.
BACKGROUND: In cattle, the gene coding for the melanocortin receptor 1 (MC1R) is known to be the main regulator of the switch between the two coat colour pigments: eumelanin (black pigment) and phaeomelanin (red pigment). Some breeds, such as Charolais and Simmental, exhibit a lightening of the original pigment over the whole body. The dilution mutation in Charolais (Dc) is responsible for the white coat colour of this breed. Using an F2-Backcross Charolais x Holstein population which includes animals with both pigment backgrounds, we present a linkage mapping study of the Charolais dilution locus. RESULTS: A Charolais x Holstein crossbred population was investigated for genetic effects on coat colour dilution. Three different traits representing the dilution of the phaeomelanin, eumelanin, and non-pigment-specific dilution were defined. Highly significant genome-wide associations were detected on chromosome 5 for the three traits analysed in the marker interval [ETH10-DIK5248]. The SILV gene was examined as the strongest positional and functional candidate gene. A previously reported non-synonymous mutation in exon 1 of this gene, SILV c.64A>G, was associated with the coat colour dilution phenotype in this resource population. Although some discrepancies were identified between this mutation and the dilution phenotype, no convincing recombination events were found between the SILV c.64A>G mutation and the Dc locus. Further analysis identified a region on chromosome 28 influencing the variation in pigment intensity for a given coat colour category. CONCLUSION: The present study has identified a region on bovine chromosome 5 that harbours the major locus responsible for the dilution of the eumelanin and phaeomelanin seen in Charolais crossbred cattle. In this study, no convincing evidence was found to exclude SILV c.64A>G as the causative mutation for the Charolais dilution phenotype, although other genetic effects may influence the coat colour variation in the population studied. A region on chromosome 28 influences the intensity of pigment within coat colour categories, and therefore may include a modifier of the Dc locus. A candidate gene for this effect, LYST, was identified. [Abstract/Link to Full Text]

Sanchez MP, Iannuccelli N, Basso B, Bidanel JP, Billon Y, Gandemer G, Gilbert H, Larzul C, Legault C, Riquet J, Milan D, Le Roy P
Identification of QTL with effects on intramuscular fat content and fatty acid composition in a Duroc x Large White cross.
BMC Genet. 2007;855.
BACKGROUND: Improving pork quality can be done by increasing intramuscular fat (IMF) content. This trait is influenced by quantitative trait loci (QTL) sought out in different pig populations. Considering the high IMF content observed in the Duroc pig, it was appealing to determine whether favourable alleles at a major gene or QTL could be found. The detection was performed in an experimental F2 Duroc x Large White population first by segregation analysis, then by QTL mapping using additional molecular information. RESULTS: Segregation analysis provided evidence for a major gene, with a recessive Duroc allele increasing IMF by 1.8% in Duroc homozygous pigs. However, results depended on whether data were normalised or not. After Box-Cox transformation, likelihood ratio was indeed 12 times lower and no longer significant. The QTL detection results were partly consistent with the segregation analysis. Three QTL significant at the chromosome wide level were evidenced. Two QTL, located on chromosomes 13 and 15, showed a high IMF Duroc recessive allele with an overall effect slightly lower than that expected from segregation analysis (+0.4 g/100 g muscle). The third QTL was located on chromosome 1, with a dominant Large White allele inducing high IMF content (+0.5 g/100 g muscle). Additional QTL were detected for muscular fatty acid composition. CONCLUSION: The study presented results from two complementary approaches, a segregation analysis and a QTL detection, to seek out genes involved in the higher IMF content observed in the Duroc population. Discrepancies between both methods might be partially explained by the existence of at least two QTL with similar characteristics located on two different chromosomes for which different boars were heterozygous. The favourable and dominant allele detected in the Large White population was unexpected. Obviously, in both populations, the favourable alleles inducing high IMF content were not fixed and improving IMF by fixing favourable alleles using markers can then be applied both in Duroc and LW populations. With QTL affecting fatty acid composition, combining an increase of IMF content enhancing monounsaturated fatty acid percentage would be of great interest. [Abstract/Link to Full Text]

Ekstrřm PO, Bjřrheim J, Thilly WG
Technology to accelerate pangenomic scanning for unknown point mutations in exonic sequences: cycling temperature capillary electrophoresis (CTCE).
BMC Genet. 2007;854.
BACKGROUND: Rapid means to discover and enumerate unknown mutations in the exons of human genes on a pangenomic scale are needed to discover the genes carrying inherited risk for common diseases or the genes in which somatic mutations are required for clonal diseases such as atherosclerosis and cancers. The method of constant denaturing capillary electrophoresis (CDCE) permitted sensitive detection and enumeration of unknown point mutations but labor-intensive optimization procedures for each exonic sequence made it impractical for application at a pangenomic scale. RESULTS: A variant denaturing capillary electrophoresis protocol, cycling temperature capillary electrophoresis (CTCE), has eliminated the need for the laboratory optimization of separation conditions for each target sequence. Here are reported the separation of wild type mutant homoduplexes from wild type/mutant heteroduplexes for 27 randomly chosen target sequences without any laboratory optimization steps. Calculation of the equilibrium melting map of each target sequence attached to a high melting domain (clamp) was sufficient to design the analyte sequence and predict the expected degree of resolution. CONCLUSION: CTCE provides practical means for economical pangenomic detection and enumeration of point mutations in large-scale human case/control cohort studies. We estimate that the combined reagent, instrumentation and labor costs for scanning the approximately 250,000 exons and splice sites of the approximately 25,000 human protein-coding genes using automated CTCE instruments in 100 case cohorts of 10,000 individuals each are now less than U.S. $500 million, less than U.S. $500 per person. [Abstract/Link to Full Text]

Recent Articles in BMC Genomics

Monier A, Claverie JM, Ogata H
Horizontal gene transfer and nucleotide compositional anomaly in large DNA viruses.
BMC Genomics. 2007 Dec 10;8(1):456.
ABSTRACT: BACKGROUND: DNA viruses have a wide range of genome sizes (5 kb up to 1.2 Mb, compared to 0.16 Mb to 1.5 Mb for obligate parasitic bacteria) that do not correlate with their virulence or the taxonomic distribution of their hosts. The reasons for such large variation are unclear. According to the traditional view of viruses as gifted "gene pickpockets", large viral genome sizes could originate from numerous gene acquisitions from their hosts. We investigated this hypothesis by studying 67 large DNA viruses with genome sizes larger than 150 kb, including the recently characterized giant mimivirus. Given that horizontally transferred DNA often have anomalous nucleotide compositions differing from the rest of the genome, we conducted a detailed analysis of the inter- and intra-genome compositional properties of these viruses. We then interpreted their compositional heterogeneity in terms of possible causes, including strand asymmetry, gene function/expression, and horizontal transfer. RESULTS: We first show that the global nucleotide composition and nucleotide word usage of viral genomes are species-specific and distinct from those of their hosts. Next, we identified compositionally anomalous (cA) genes in viral genomes, using a method based on Bayesian inference. The proportion of cA genes is highly variable across viruses and does not exhibit a significant correlation with genome size. The vast majority of the cA genes were of unknown function, lacking homologs in the databases. For genes with known homologs, we found a substantial enrichment of cA genes in specific functional classes for some of the viruses. No significant association was found between cA genes and compositional strand asymmetry. A possible exogenous origin for a small fraction of the cA genes could be confirmed by phylogenetic reconstruction. CONCLUSION: At odds with the traditional dogma, our results argue against frequent genetic transfers to large DNA viruses from their modern hosts. The large genome sizes of these viruses are not simply explained by an increased propensity to acquire foreign genes. This study also confirms that the anomalous nucleotide compositions of the cA genes is sometimes linked to particular biological functions or expression patterns, possibly leading to an overestimation of recent horizontal gene transfers. [Abstract/Link to Full Text]

Xu Y, Ikegami M, Wang Y, Matsuzaki Y, Whitsett JA
Gene expression and biological processes influenced by deletion of Stat3 in pulmonary type II epithelial cells.
BMC Genomics. 2007 Dec 10;8(1):455.
ABSTRACT: BACKGROUND: The signal transducer and activator of transcription 3 (STAT3) mediates gene expression in response to numerous growth factors and cytokines, playing an important role in many cellular processes. To better understand the molecular mechanisms by which Stat3 influences gene expression in the lung, the effect of pulmonary epithelial cell specific deletion of Stat3 on genome wide mRNA expression profiling was assessed. Differentially expressed genes were identified from Affymetrix Murine GeneChips analysis and subjected to gene ontology classification, promoter analysis, pathway mapping and literature mining. RESULTS: Total of 791 mRNAs were significantly increased and 314 mRNAs were decreased in response to the deletion of Stat3 in the lung. STAT is the most enriched cis-elements in the promoter regions of those differentially expressed genes. Deletion of Stat3 induced genes influencing protein metabolism, transport, chemotaxis and apoptosis and decreased the expression of genes mediating lipid synthesis and metabolism. Expression of Srebf1 and 2, genes encoding key regulators of fatty acid and steroid biosynthesis, was decreased in type II cells from the Stat3 deletion mice, consistent with the observation that lung surfactant phospholipid content was decreased. Stat3 influenced both pro- and anti-apoptotic pathways that determine cell death or survival. Akt, a potential transcriptional target of Stat3, was identified as an important participant in Stat3 mediated pathways including Jak-Stat signaling, apoptosis, Mapk signaling, cholesterol and fatty acid biosynthesis. CONCLUSIONS: Deletion of Stat3 from type II epithelial cells altered the expression of genes regulating diverse cellular processes, including cell growth, apoptosis and lipid metabolism. Pathway analysis indicates that STAT3 regulates cellular homeostasis through a complex regulatory network that likely enhances alveolar epithelial cell survival and surfactant/lipid synthesis, necessary for the protection of the lung during injury. [Abstract/Link to Full Text]

Baker DA, Meadows LA, Wang J, Dow JA, Russell S
Variable sexually dimorphic gene-expression in laboratory strains of Drosophila melanogaster.
BMC Genomics. 2007 Dec 10;8(1):454.
ABSTRACT: BACKGROUND: Wild-type laboratory strains of model organisms are typically kept in isolation for many years, with the action of genetic drift and selection on mutational variation causing lineages to diverge with time. Natural populations from which such strains are established, show that gender-specific interactions in particular drive many aspects of sequence level and transcriptional level variation. Here, our goal was to identify genes that display transcriptional variation between laboratory strains of Drosophila melanogaster, and to explore evidence of gender-biased interactions underlying that variability. RESULTS: Transcriptional variation among the laboratory genotypes studied occurs more frequently in males than in females. Qualitative differences are also apparent to suggest that genes within particular functional classes disproportionately display variation in gene expression. Our analysis indicates that genes with reproductive functions are most often divergent between genotypes in both sexes, however a large proportion of female variation can also be attributed to genes without expression in the ovaries. CONCLUSIONS: The present study clearly shows that transcriptional variation between common laboratory strains of Drosophila can differ dramatically due to sexual dimorphism. Much of this variation reflects sex-specific challenges associated with divergent physiological trade-offs, morphology and regulatory pathways operating within males and females. [Abstract/Link to Full Text]

Moral R, Wang R, Russo IH, Mailo DA, Balogh GA, Lamartininere CA, Russo J
The plasticizer butyl benzyl phthalate induces genomic changes in rat mammary gland after neonatal/prepubertal exposure.
BMC Genomics. 2007 Dec 6;8(1):453.
ABSTRACT: BACKGROUND: Phthalate esters like n-butyl benzyl phthalate (BBP) are widely used plasticizers. BBP has shown endocrine-disrupting properties, thus having a potential effect on hormone-sensitive tissues. The aim of this study is to determine the effect of neonatal/prepubertal exposure (post-natal days 2-20) to BBP on maturation parameters and on the morphology, proliferative index and genomic signature of the rat mammary gland at different ages of development (21, 35, 50 and 100 days). RESULTS: Here we show that exposure to BBP increased the uterine weight/body weight ratio at 21 days and decreased the body weight at time of vaginal opening. BBP did not induce significant changes on the morphology of the mammary gland, but increased proliferative index in terminal end buds at 35 days and in lobules 1 at several ages. Moreover, BBP had an effect on the genomic profile of the mammary gland mainly at the end of the exposure (21 days), becoming less prominent thereafter. By this age a significant number of genes related to proliferation and differentiation, communication and signal transduction were up-regulated in the glands of the exposed animals. CONCLUSIONS: These results suggest that BBP has an effect in the gene expression profile of the mammary gland. [Abstract/Link to Full Text]

Delahaye NF, Coltel N, Puthier D, Barbier M, Benech P, Joly F, Iraqi FA, Grau GE, Nguyen C, Rihet P
Gene expression analysis reveals early changes in several molecular pathways in cerebral malaria-susceptible mice versus cerebral malaria-resistant mice.
BMC Genomics. 2007 Dec 6;8(1):452.
ABSTRACT: BACKGROUND: : Microarray analyses allow the identification and assessment of molecular signatures in whole tissues undergoing pathological processes. To better understand cerebral malaria pathogenesis, we investigated intra-cerebral gene-expression profiles in well-defined genetically cerebral malaria-resistant (CM-R) and CM-susceptible (CM-S) mice, upon infection by Plasmodium berghei ANKA (PbA). We investigated mouse transcriptional responses at early and late stages of infection by use of cDNA microarrays. RESULTS: : Through a rigorous statistical approach with multiple testing corrections, we showed that PbA significantly altered brain gene expression in CM-R (BALB/c), and in CM-S (CBA/J and C57BL/6) mice, and that 327 genes discriminated between early and late infection stages, between mouse strains, and between CM-R and CM-S mice. We further identified 104, 56, 84 genes with significant differential expression between CM-R and CM-S mice on days 2, 5, and 7 respectively. The analysis of their functional annotation indicates that genes involved in metabolic energy pathways, the inflammatory response, and the neuroprotection/neurotoxicity balance play a major role in cerebral malaria pathogenesis. In addition, our data suggest that cerebral malaria and Alzheimer's disease may share some common mechanisms of pathogenesis, as illustrated by the accumulation of beta-amyloid proteins in brains of CM-S mice, but not of CM-R mice. CONCLUSION: Our microarray analysis highlighted marked changes in several molecular pathways in CM-S compared to CM-R mice, particularly at early stages of infection. This study revealed some promising areas for exploration that may both provide new insight into the knowledge of CM pathogenesis and the development of novel therapeutic strategies. [Abstract/Link to Full Text]

Aguilar R, Das S, Dong Y, Dimopoulos G
Continuous exposure to Plasmodium results in decreased susceptibility and transcriptomic divergence of the Anopheles gambiae immune system.
BMC Genomics. 2007 Dec 5;8(1):451.
ABSTRACT: BACKGROUND: Plasmodium infection has been shown to compromise the fitness of the mosquito vector, reducing its fecundity and longevity. However, from an evolutionary perspective, the impact of Plasmodium infection as a selective pressure on the mosquito is largely unknown. RESULTS: In the present study we have addressed the effect of a continuous Plasmodium berghei infection on the resistance to infection and global gene expression in Anopheles gambiae. Exposure of A. gambiae to P. berghei-infected blood and infection for 16 generations resulted in a decreased susceptibility to infection, altered constitutive expression levels for approximately 2.4% of the mosquito's total transcriptome and a lower basal level of immune genes expression, including several anti-Plasmodium factors. The infection-responsiveness for several anti-Plasmodium defense genes was elevated in the P. berghei exposed mosquito colonies. CONCLUSION: Our study establishes the existence of a selective pressure exerted by the parasite P. berghei on the malaria vector A. gambiae, which results in a decreased permissiveness to infection and changes in the mosquito transcriptome regulation that suggest a decreased constitutive immune gene activity but a more potent immune response upon Plasmodium challenge. [Abstract/Link to Full Text]

Ramel F, Sulmon C, Cabello-Hurtado F, Taconnat L, Martin-Magniette ML, Renou JP, Elamrani A, Couee I, Gouesbet G
Genome-wide interacting effects of sucrose and herbicide-mediated stress in Arabidopsis thaliana: novel insights into atrazine toxicity and sucrose-induced tolerance.
BMC Genomics. 2007 Dec 5;8(1):450.
ABSTRACT: BACKGROUND: Soluble sugars, which play a central role in plant structure and metabolism, are also involved in the responses to a number of stresses, and act as metabolite signalling molecules that activate specific or hormone-crosstalk transduction pathways. The different roles of exogenous sucrose in the tolerance of Arabidopsis thaliana plantlets to the herbicide atrazine and oxidative stress were studied by a transcriptomic approach using CATMA arrays. RESULTS: Parallel situations of xenobiotic stress and sucrose-induced tolerance in the presence of atrazine, of sucrose, and of sucrose plus atrazine were compared. These approaches revealed that atrazine affected gene expression and therefore seedling physiology at a much larger scale than previously described, with potential impairment of protein translation and of reactive-oxygen-species (ROS) defence mechanisms. Correlatively, sucrose-induced protection against atrazine injury was associated with important modifications of gene expression related to ROS defence mechanisms and repair mechanisms. These protection-related changes of gene expression did not result only from the effects of sucrose itself, but from combined effects of sucrose and atrazine, thus strongly suggesting important interactions of sucrose and xenobiotic signalling or of sucrose and ROS signalling. CONCLUSIONS: These interactions resulted in characteristic differential expression of gene families such as ascorbate peroxidases, glutathione-S-transferases and cytochrome P450s, and in the early induction of an original set of transcription factors. These genes used as molecular markers will eventually be of great importance in the context of xenobiotic tolerance and phytoremediation. [Abstract/Link to Full Text]

Schuster A, Kubicek CP, Friedl MA, Druzhinina IS, Schmoll M
Impact of light on Hypocrea jecorina and the multiple cellular roles of ENVOY in this process.
BMC Genomics. 2007 Dec 4;8(1):449.
ABSTRACT: BACKGROUND: In fungi, light is primarily known to influence general morphogenesis and both sexual and asexual sporulation. In order to expand the knowledge on the effect of light in fungi and to determine the role of the light regulatory protein ENVOY in the implementation of this effect, we performed a global screen for genes, which are specifically effected by light in the fungus Hypocrea jecorina (anamorph Trichoderma reesei) using Rapid Subtraction Hybridization (RaSH). Based on these data, we analyzed whether these genes are influenced by ENVOY and if overexpression of ENVOY in darkness would be sufficient to execute its function. RESULTS: The cellular functions of the detected light responsive genes comprised a variety of roles in transcription, translation, signal transduction, metabolism, and transport. Their response to light with respect to the involvement of ENVOY could be classified as follows: (i) ENVOY-mediated upregulation by light; (ii) ENVOY-independent upregulation by light; (iii) ENVOY-antagonized upregulation by light; ENVOY-dependent repression by light; (iv) ENVOY-independent repression by light; and (v) both positive and negative regulation by ENVOY of genes not responsive to light in the wild-type. ENVOY was found to be crucial for normal growth in light on various carbon sources and is not able to execute its regulatory function if overexpressed in the darkness. CONCLUSIONS: The different responses indicate that light impacts fungi like H. jecorina at several cellular processes, and that it has both positive and negative effects. The data also emphasize that ENVOY has an apparently more widespread cellular role in this process than only in modulating the response to light. [Abstract/Link to Full Text]

Nanjo T, Sakurai T, Totoki Y, Toyoda A, Nishiguchi M, Kado T, Igasaki T, Futamura N, Seki M, Sakaki Y, Shinozaki K, Shinohara K
Functional annotation of 19,841 Populus nigra full-length enriched cDNA clones.
BMC Genomics. 2007 Dec 3;8(1):448.
ABSTRACT: BACKGROUND: Populus is one of favorable model plants because of its small genome. Structural genomics of Populus has reached a breakpoint as nucleotides of the entire genome have been determined. Reaching the post genome era, functional genomics of Populus is getting more important for well-comprehended plant science. Development of bioresorce serving functional genomics is making rapid progress. Huge efforts have achieved deposits of expressed sequence tags (ESTs) in various plant species consequently accelerating functional analysis of genes. ESTs from full-length cDNA clones are especially powerful for accurate molecular annotation. We promoted collection and annotation of the ESTs from Populus full-length enriched cDNA clones as part of functional genomics of tree species. RESULTS: We have been collecting the full-length enriched cDNA of the female poplar (Populus nigra var. italica) for years. By sequencing P. nigra full-length (PnFL) cDNA libraries, we generated about 116,000 5'-end or 3'-end ESTs corresponding to 19,841 nonredundant PnFL clones. Population of PnFL cDNA clones represents 44% of the predicted genes in the Populus genome. CONCLUSION: Our resource of P. nigra full-length enriched clones is expected to provide valuable tools to gain further insight into genome annotation and functional genomics in Populus. [Abstract/Link to Full Text]

Ruzanov P, Jones SJ, Riddle DL
Discovery of novel alternatively spliced C. elegans transcripts by computational analysis of SAGE data.
BMC Genomics. 2007 Nov 30;8(1):447.
ABSTRACT: BACKGROUND: Alternative RNA splicing allows cells to produce multiple protein isoforms from one gene. These isoforms may have specialized functions, and may be tissue- or stage-specific. Our aim was to use computational analysis of SAGE and genomic data to predict alternatively spliced transcripts expressed in C. elegans. RESULTS: We predicted novel alternatively spliced variants and confirmed five of eighteen candidates selected for experimental validation by RT-PCR tests and DNA sequencing. CONCLUSIONS: We show that SAGE data can be efficiently used to discover alternative mRNA isoforms, including those with skipped exons or retained introns. Our results also imply that C. elegans may produce a larger number of alternatively spliced transcripts than initially estimated. [Abstract/Link to Full Text]

Smiraglia DJ, Kazhiyur-Mannar R, Oakes CC, Wu YZ, Liang P, Ansari T, Su J, Rush LJ, Smith LT, Yu L, Liu C, Dai Z, Chen SS, Wang SH, Costello J, Ioshikhes I, Dawson DW, Hong JS, Teitell MA, Szafranek A, Camoriano M, Song F, Elliott R, Held W, Trasler JM, Plass C, Wenger R
Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations.
BMC Genomics. 2007 Nov 30;8(1):446.
ABSTRACT: BACKGROUND: Restriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning". RESULTS: We report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation. CONCLUSIONS: The new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation. [Abstract/Link to Full Text]

Norden-Krichmar TM, Holtz J, Pasquinelli AE, Gaasterland T
Computational prediction and experimental validation of Ciona intestinalis microRNA genes.
BMC Genomics. 2007 Nov 29;8(1):445.
ABSTRACT: BACKGROUND: This study reports the first collection of validated microRNA genes in the sea squirt, Ciona intestinalis. MicroRNAs are processed from hairpin precursors to ~22 nucleotide RNAs that base pair to target mRNAs and inhibit expression. As a member of the subphylum Urochordata (Tunicata) whose larval form has a notochord, the sea squirt is situated at the emergence of vertebrates, and therefore may provide information about the evolution of molecular regulators of early development. RESULTS: In this study, computational methods were used to predict 14 microRNA gene families in Ciona intestinalis. The microRNA prediction algorithm utilizes configurable microRNA sequence conservation and stem-loop specificity parameters, grouping by miRNA family, and phylogenetic conservation to the related species, Ciona savignyi. The expression for 8, out of 9 attempted, of the putative microRNAs in the adult tissue of Ciona intestinalis was validated by Northern blot analyses. Additionally, a target prediction algorithm was implemented, which identified a high confidence list of 240 potential target genes. Over half of the predicted targets can be grouped into the gene ontology categories of metabolism, transport, regulation of transcription, and cell signaling. CONCLUSIONS: The computational techniques implemented in this study can be applied to other organisms and serve to increase the understanding of the origins of non-coding RNAs, embryological and cellular developmental pathways, and the mechanisms for microRNA-controlled gene regulatory networks. [Abstract/Link to Full Text]

Moura GR, Lousado JP, Pinheiro M, Carreto L, Silva RM, Oliveira JL, Santos MA
Codon-triplet context unveils unique features of the Candida albicans protein coding genome.
BMC Genomics. 2007 Nov 29;8(1):444.
ABSTRACT: BACKGROUND: The evolutionary forces that determine the arrangement of synonymous codons within open reading frames and fine tune mRNA translation efficiency are not yet understood. In order to tackle this question we have carried out a large scale study of codon-triplet contexts in 11 fungal species to unravel associations or relationships between codons present at the ribosome A-, P- and E-sites during each decoding cycle. RESULTS: Our analysis unveiled high bias within the context of codon-triplets, in particular strong preference for triplets of identical codons. We have also identified a surprisingly large number of codon-triplet combinations that vanished from fungal ORFeomes. Candida albicans exacerbated these features, showed an unbalanced tRNA population for decoding its pool of codons and used near-cognate decoding for a large set of codons, suggesting that unique evolutionary forces shaped the evolution of its ORFeome. CONCLUSIONS: We have developed bioinformatics tools for large-scale analysis of codon-triplet contexts. These algorithms identified codon-triplets context biases, allowed for large scale comparative codon-triplet analysis, and identified rules governing codon-triplet context. They could also detect alterations to the standard genetic code. [Abstract/Link to Full Text]

Madrigal I, Rodriguez-Revenga L, Armengol L, Gonzalez E, Rodriguez B, Badenas C, Sanchez A, Martinez F, Guitart M, Fernandez I, Arranz JA, Tejada MI, Perez-Jurado LA, Estivill X, Mila M
X-chromosome tiling path array detection of copy number variants in patients with chromosome X-linked mental retardation.
BMC Genomics. 2007 Nov 29;8(1):443.
ABSTRACT: BACKGROUND: Aproximately 5-10% of cases of mental retardation in males are due to copy number variations (CNV) on the X chromosome. Novel technologies, such as array comparative genomic hybridization (aCGH), may help to uncover cryptic rearrangements in X-linked mental retardation (XLMR) patients. We have constructed an X-chromosome tiling path array using bacterial artificial chromosomes (BACs) and validated it using samples with cytogenetically defined copy number changes. We have studied 54 patients with idiopathic mental retardation and 20 controls subjects. RESULTS: Known genomic aberrations were reliably detected on the array and eight novel submicroscopic imbalances, likely causative for the mental retardation (MR) phenotype, were detected. Putatively pathogenic rearrangements included three deletions and five duplications (ranging between 82 kb to one Mb), all but two affecting genes previously known to be responsible for XLMR. Additionally, we describe different CNV regions with significant different frequencies in XLMR and control subjects (44% vs. 20%). CONCLUSIONS: This tiling path array of the human X chromosome has proven successful for the detection and characterization of known rearrangements and novel CNVs in XLMR patients. [Abstract/Link to Full Text]

Lee CN, Hu RM, Chow TY, Lin JW, Chen HY, Tseng YH, Weng SF
Comparison of Genomes of Three Xanthomonas oryzae Bacteriophages.
BMC Genomics. 2007 Nov 29;8(1):442.
ABSTRACT: BACKGROUND: Xp10 and OP1 are phages of Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight in rice plants, which were isolated in 1967 in Taiwan and in 1954 in Japan, respectively. We recently isolated the Xoo phage Xop411. RESULTS: The linear Xop411 genome (44,520 bp, 58 ORFs) sequenced here is 147 bp longer than that of Xp10 (60 ORFs) and 735 bp longer than that of OP1 (59 ORFs). The G+C contents of OP1 (51%) and Xop411 and Xp10 (52% each) are less than that of the host (65%). The 9-bp 3'-overhangs (5'-GGACAGTCT-3') in Xop411 and Xp10 are absent from OP1. More of the deduced Xop411 proteins share higher degrees of identity with Xp10 than with OP1 proteins, while the right end of the genomes of Xp10 and OP1, containing all predicted promoters, share stronger homology. Xop411, Xp10, and OP1 contain 8, 7, and 6 freestanding HNH endonuclease genes, respectively. These genes can be classified into five groups depending on their possession of the HNH domain (HNN or HNH type) and/or AP2 domain in intact or truncated forms. While the HNN-AP2 type endonuclease genes dispersed in the genome, the HNH type endonuclease genes, each with a unique copy, were located within the same genome context. Mass spectrometry and N-terminal sequencing showed nine Xop411 coat proteins, among which three were identified, six were assigned as coat proteins (4) and conserved phage proteins (2) in Xp10. The major coat protein, in which only the N-terminal methionine is removed, appears to exist in oligomeric forms containing 2 to 6 subunits. The three phages exhibit different patterns of domain duplication in the N-terminus of the tail fiber, which are involved in determination of the host range. Many short repeated sequences are present in and around the duplicated domains. CONCLUSIONS: Geographical separation may have confined lateral gene transfer among the Xoo phages. The HNN-AP2 type endonucleases were more likely to transfer their genes randomly in the genome and may degenerate after successful transmission. Some repeated sequences may be involved in duplication/loss of the domains in the tail fiber genes. [Abstract/Link to Full Text]

Lee AP, Yang Y, Brenner S, Venkatesh B
TFCONES: A database of vertebrate transcription factor-encoding genes and their associated conserved noncoding elements.
BMC Genomics. 2007 Nov 29;8(1):441.
ABSTRACT: BACKGROUND: Transcription factors (TFs) regulate gene transcription and play pivotal roles in various biological processes such as development, cell cycle progression, cell differentiation and tumor suppression. Identifying cis-regulatory elements associated with TF-encoding genes is a crucial step in understanding gene regulatory networks. To this end, we have used a comparative genomics approach to identify putative cis-regulatory elements associated with TF-encoding genes in vertebrates. Description: We have created a database named TFCONES (Transcription Factor Genes & Associated COnserved Noncoding ElementS) ( which contains all human, mouse and fugu TF-encoding genes and conserved noncoding elements (CNEs) associated with them. The CNEs were identified by gene-by-gene alignments of orthologous TF-encoding gene loci using MLAGAN. We also predicted putative transcription factor binding sites within the CNEs. A significant proportion of human-fugu CNEs contain experimentally defined binding sites for transcriptional activators and repressors, indicating that a majority of the CNEs may function as transcriptional regulatory elements. The TF-encoding genes that are involved in nervous system development are generally enriched for human-fugu CNEs. Users can retrieve TF-encoding genes and their associated CNEs by conducting a keyword search or by selecting a family of DNA-binding proteins. CONCLUSIONS: The conserved noncoding elements identified in TFCONES represent a catalog of highly prioritized putative cis-regulatory elements of TF-encoding genes and are candidates for functional assay. [Abstract/Link to Full Text]

Lee YK, Chew A, Fitzsimon L, Thomas R, Greenhalgh D, Cho K
Genome-wide changes in expression profile of murine endogenous retroviruses (MuERVs) in distant organs after burn injury.
BMC Genomics. 2007 Nov 28;8(1):440.
ABSTRACT: BACKGROUND: Previous studies have shown that burn-elicited stress signals alter expression of certain murine endogenous retroviruses (MuERVs) in distant organs of mice. These findings suggest that MuERVs may participate in a network of pathophysiologic events during post-burn systemic response. To gain a better understanding of the biological roles of MuERVs in post-burn systemic response, we examined the genome-wide changes in the MuERV expression profiles in distant organs and the biological properties of the putative-burn related MuERVs were characterized. RESULTS: Female C57BL/6J mice were subjected to an approximately 18 % total body surface area flame burn and tissues (liver, lung, and kidney) were harvested at 3 hours and 24 hours after injury. The changes in the MuERV expression profiles in these tissues were examined by RT-PCR using a primer set flanking the non-ecotropic MuERV U3 promoter region within the 3 long terminal repeat. There were differential changes in the expression profiles of MuERV U3 regions after injury in all three tissues examined. Subsequently, a total of 31 unique U3 promoter sequences were identified from the tissues of both burn and no burn mice. An analysis of viral tropisms revealed that putative MuERVs harboring these U3 promoter sequences were presumed to be either xenotropic or polytropic. Some putative transcription regulatory elements were present predominantly in U3 promoter sequences isolated from burn and no burn mice, respectively. In addition, in silico mapping using these U3 sequences as a probe against the mouse genome database identified 59 putative MuERVs. The biological properties (coding potentials for retroviral polypeptides, primer binding sites, tropisms, branching ages, recombination events, and neighboring host genes) of each putative MuERV were characterized. In particular, 16 putative MuERVs identified in this study retained intact coding potentials for all three retroviral polypeptides (gag, pol, and env). None of the putative MuERVs identified in this study were mapped to the coding sequences of host genes. CONCLUSION: In this study, we identified and characterized putative MuERVs whose expression might be altered in response to burn-elicited systemic stress signals. Further investigation is needed to understand the role of these MuERVs in post-burn systemic pathogenesis, in particular, via characterization of their interaction with host genes, MuERV gene products, and viral activities. [Abstract/Link to Full Text]

Chelala C, Hahn SA, Whiteman HJ, Barry S, Hariharan D, Radon TP, Lemoine NR, Crnogorac-Jurcevic T
Pancreatic Expression database: a generic model for the organization, integration and mining of complex cancer datasets.
BMC Genomics. 2007 Nov 28;8(1):439.
ABSTRACT: BACKGROUND: Pancreatic cancer is the 5th leading cause of cancer death in both males and females. In recent years, a wealth of gene and protein expression studies have been published broadening our understanding of pancreatic cancer biology. Due to the explosive growth in publicly available data from multiple different sources it is becoming increasingly difficult for individual researchers to integrate these into their current research programmes. The Pancreatic Expression database, a generic web-based system, is aiming to close this gap by providing the research community with an open access tool, not only to mine currently available pancreatic cancer data sets but also to include their own data in the database. Description Currently, the database holds 32 datasets comprising 7636 gene expression measurements extracted from 20 different published gene or protein expression studies from various pancreatic cancer types, pancreatic precursor lesions (PanINs) and chronic pancreatitis. The pancreatic data are stored in a data management system based on the BioMart technology alongside the human genome gene and protein annotations, sequence, homologue, SNP and antibody data. Interrogation of the database can be achieved through both a web-based query interface and through web services using combined criteria from pancreatic (disease stages, regulation, differential expression, expression, platform technology, publication) and/or public data (antibodies, genomic region, gene-related accessions, ontology, expression patterns, multi-species comparisons, protein data, SNPs). Thus, our database enables connections between otherwise disparate data sources and allows relatively simple navigation between all data types and annotations. CONCLUSIONS: The database structure and content provides a powerful and high-speed data-mining tool for cancer research. It can be used for target discovery i.e. of biomarkers from body fluids, identification and analysis of genes associated with the progression of cancer, cross-platform meta-analysis, SNP selection for pancreatic cancer association studies, cancer gene promoter analysis as well as mining cancer ontology information. The data model is generic and can be easily extended and applied to other types of cancer. The database is available online with no restrictions for the scientific community at [Abstract/Link to Full Text]

Rescan PY, Montfort J, Ralliere C, Le Cam A, Esquerre D, Hugot K
Dynamic gene expression in fish muscle during recovery growth induced by a fasting-refeeding schedule.
BMC Genomics. 2007 Nov 28;8(1):438.
ABSTRACT: BACKGROUND: Recovery growth is a phase of rapid growth that is triggered by adequate refeeding of animals following a period of weight loss caused by starvation. In this study, to obtain more information on the system-wide integration of recovery growth in muscle, we undertook a time-course analysis of transcript expression in trout subjected to a food deprivation-refeeding sequence. For this purpose complex targets produced from muscle of trout fasted for one month and from muscle of trout fasted for one month and then refed for 4, 7, 11 and 36 days were hybridized to cDNA microarrays containing 9023 clones. RESULTS: Significance analysis of microarrays (SAM) and temporal expression profiling led to the segregation of differentially expressed genes into four major clusters. One cluster comprising 1020 genes with high expression in muscle from fasted animals included a large set of genes involved in protein catabolism. A second cluster that included approximately 550 genes with transient induction 4 to 11 days post-refeeding was dominated by genes involved in transcription, ribosomal biogenesis, translation, chaperone activity, mitochondrial production of ATP and cell division. A third cluster that contained 480 genes that were up-regulated 7 to 36 days post-refeeding was enriched with genes involved in reticulum and Golgi dynamics and with genes indicative of myofiber and muscle remodelling such as genes encoding sarcomeric proteins and matrix compounds. Finally, a fourth cluster of 200 genes overexpressed only in 36-day refed trout muscle contained genes with function in carbohydrate metabolism and lipid biosynthesis. Remarkably, among the genes induced during muscle recovery were several transcriptional regulators which might be important for the gene-specific transcriptional adaptations that underlie muscle recovery. CONCLUSIONS: Our study is the first demonstration of a coordinated expression of functionally related genes during muscle recovery growth. Furthermore, the generation of a useful database of novel genes associated with muscle recovery growth will allow further investigations on particular genes, pathways or cellular process involved in muscle growth and regeneration. [Abstract/Link to Full Text]

Schriek S, Rueckert C, Staiger D, Pistorius EK, Michel KP
Bioinformatic evaluation of L-arginine catabolic pathways in 24 cyanobacteria and transcriptional analysis of genes encoding enzymes of L-arginine catabolism in the cyanobacterium Synechocystis sp. PCC 6803.
BMC Genomics. 2007 Nov 28;8(1):437.
ABSTRACT: BACKGROUND: So far very limited knowledge exists on L-arginine catabolism in cyanobacteria, although six major L-arginine-degrading pathways have been described for prokaryotes. Thus, we have performed a bioinformatic analysis of possible L-arginine-degrading pathways in cyanobacteria. Further, we chose Synechocystis sp. PCC 6803 for a more detailed bioinformatic analysis and for validation of the bioinformatic predictions on L arginine catabolism by a transcript analysis. RESULTS: We have evaluated 24 cyanobacterial genomes of freshwater or marine strains for the presence of putative L-arginine-degrading enzymes. We identified an L-arginine decarboxylase pathway in all 24 strains. In addition, cyanobacteria have one or two further pathways representing either an arginase pathway or L-arginine deiminase pathway or an L arginine oxidase/dehydrogenase pathway. An L-arginine amidinotransferase pathway as a major L-arginine-degrading pathway is not likely but can not be entirely excluded. A rather unusual finding was that the cyanobacterial L-arginine deiminases are substantially larger than the enzymes in non-photosynthetic bacteria and that they are membrane-bound. A more detailed bioinformatic analysis of Synechocystis sp. PCC 6803 revealed that three different L arginine-degrading pathways may in principle be functional in this cyanobacterium: these are (i) an L arginine decarboxylase pathway, (ii) an L-arginine deiminase pathway, and (iii) an L arginine oxidase/dehydrogenase pathway. A transcript analysis of cells grown either with nitrate or L-arginine as sole N-source and with an illumination of 50 umol photons m-2 s-1 showed that the transcripts for the first enzyme(s) of all three pathways are present, but that the transcript levels for the L-arginine deiminase and the L-arginine oxidase/dehydrogenase were substantially higher than that of the three isoenzymes of L arginine decarboxylase. CONCLUSION: The evaluation of 24 cyanobacterial genomes revealed that five different L arginine-degrading pathways are present in the investigated cyanobacterial species. In Synechocystis sp. PCC 6803 an L-arginine deiminase pathway and an L arginine oxidase/dehydrogenase pathway represent the major pathways, while the L-arginine decarboxylase pathway most likely only functions in polyamine biosynthesis. The transcripts encoding the enzymes of the two major pathways are constitutively expressed with the exception of the transcript for the carbamate kinase, which is substantially up-regulated in cells grown with L-arginine. [Abstract/Link to Full Text]

Srivastava J, Premi S, Kumar S, Parwez I, Ali S
Characterization of Smoc-1 uncovers two transcript variants showing differential tissue and age specific expression in Bubalus bubalis.
BMC Genomics. 2007 Nov 28;8(1):436.
ABSTRACT: BACKGROUND: Secreted modular calcium binding protein-1 (Smoc-1) belongs to the BM-40 family which has been implicated with tissue remodeling, angiogenesis and bone mineralization. Besides its anticipated role in embryogenesis, Smoc-1 has been characterized only in a few mammalian species. We made use of the consensus sequence (5' CACCTCTCCAC CTGCC 3') of 33.15 repeat loci to explore the buffalo transcriptome and uncovered the Smoc-1 transcript tagged with this repeat. The main objective of this study was to gain an insight into its structural and functional organization, and expressional status of Smoc-1 in water buffalo, Bubalus bubalis. RESULTS: We cloned and characterized the buffalo Smoc-1, including its copy number status, in-vitro protein expression, tissue & age specific transcription/translation, chromosomal mapping and localization to the basement membrane zone. Buffalo Smoc-1 was found to encode a secreted matricellular glycoprotein containing two EF-hand calcium binding motifs homologous to that of BM-40/SPARC family. In buffalo, this single copy gene consisted of 12 exons and was mapped onto the acrocentric chromosome 11. Though this gene was found to be evolutionarily conserved, the buffalo Smoc-1 showed conspicuous nucleotide/amino acid changes altering its secondary structure compared to that in other mammals. In silico analysis of the Smoc-1 proposed its glycoprotein nature with a calcium dependent conformation. Further, we unveiled two transcript variants of this gene, varying in their 3'UTR lengths but both coding for identical protein(s). Smoc-1 evinced highest expression of both the variants in liver and modest to negligible in other tissues. The relative expression of variant-02 was markedly higher compared to that of variant-01 in all the tissues examined. Moreover, expression of Smoc-1, though modest during the early ages, was conspicuously enhanced after 1 year and remained consistently higher during the entire life span of buffalo with gradual increment in expression of variant-02. Immunohistochemically, Smoc-1 was localized in the basement membrane zones and extracellular matrices of various tissues. CONCLUSION: These data added to our understandings about the tissue, age and species specific functions of the Smoc-1. It also enabled us to demonstrate varying expression of the two transcript variants of Smoc-1 amongst different somatic tissues/gonads and ages, in spite of their identical coding frames. Pursuance of these variants for their roles in various disease phenotypes such as hepatocellular carcinoma and angiogenesis is envisaged to establish broader biological significance of this gene. [Abstract/Link to Full Text]

Priya B, Premanandh J, Dhanalakshmi RT, Seethalakshmi T, Uma L, Prabaharan D, Subramanian G
Comparative analysis of cyanobacterial superoxide dismutases to discriminate canonical forms.
BMC Genomics. 2007 Nov 27;8(1):435.
ABSTRACT: BACKGROUND: Superoxide dismutases (SOD) are ubiquitous metalloenzymes that catalyze the disproportion of superoxide to peroxide and molecular oxygen through alternate oxidation and reduction of their metal ions. In general, SODs are classified into four forms by their catalytic metals namely; FeSOD, MnSOD, Cu/ZnSOD and NiSOD. In addition, a cambialistic form that uses Fe/Mn in its active site also exists. Cyanobacteria, the oxygen evolving photosynthetic prokaryotes, produce reactive oxygen species that can damage cellular components leading to cell death. Thus, the co-evolution of an antioxidant system was necessary for the survival of photosynthetic organisms with SOD as the initial enzyme evolved to alleviate the toxic effect. Cyanobacteria represent the first oxygenic photoautotrophs and their SOD sequences available in the databases lack clear annotation. Hence, the present study focuses on structure and sequence pattern of subsets of cyanobacterial superoxide dismutases. Result The sequence conservation and structural analysis of Fe (Thermosynechococcus elongatus BP1) and MnSOD (Anabaena sp. PCC7120) reveal the sharing of N and C terminal domains. At the C terminal domain, the metal binding motif in cyanoprokaryotes is DVWEHAYY while it is D-X-[WF]-E-H-[STA]-[FY]-[FY] in other pro- and eukaryotes. The cyanobacterial FeSOD differs from MnSOD at least in three ways viz. (i) FeSOD has a metal specific signature F184X3A188Q189.......T280......F/Y303 while, in Mn it is R184X3G188G189.......G280......W303, (ii) aspartate ligand forms a hydrogen bond from the active site with the outer sphere residue of W243 in Fe where as it is Q262 in MnSOD; and (iii) two unique lysine residues at positions 201 and 255 with a photosynthetic role, found only in FeSOD. Further, most of the cyanobacterial Mn metalloforms have a specific transmembrane hydrophobic pocket that distinguishes FeSOD from Mn isoform. Cyanobacterial Cu/ZnSOD has a copper domain and two different signatures G-F-H-[ILV]-H-x-[NGT]-[GPDA]-[SQK]-C and G-[GA]-G-G-[AEG]-R-[FIL]-[AG]-C-G, while Ni isoform has an nickel containing SOD domain containing a Ni-hook HCDGPCVYDPA. CONCLUSION: The present analysis unravels the ambiguity among cyanobacterial SOD isoforms. NiSOD is the only SOD found in lower forms; whereas, Fe and Mn occupy the higher orders of cyanobacteria. In conclusion, cyanobacteria harbor either Ni alone or a combination of Fe and Ni or Fe and Mn as their catalytic active metal while Cu/Zn is rare. [Abstract/Link to Full Text]

Brenchley R, Tariq H, McElhinney H, Szoor B, Huxley-Jones J, Stevens R, Matthews K, Tabernero L
The TriTryp Phosphatome: analysis of the protein phosphatase catalytic domains.
BMC Genomics. 2007 Nov 26;8(1):434.
ABSTRACT: BACKGROUND: The genomes of the three parasitic protozoa Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the main subject of this study. These parasites are responsible for devastating human diseases known as Chagas disease, African sleeping sickness and cutaneous Leishmaniasis, respectively, that affect millions of people in the developing world. The prevalence of these neglected diseases results from a combination of poverty, inadequate prevention and difficult treatment. Protein phosphorylation is an important mechanism of controlling the development of these kinetoplastids. With the aim to further our knowledge of the biology of these organisms we present a characterisation of the phosphatase complement (phosphatome) of the three parasites. RESULTS: An ontology-based scan of the three genomes was used to identify 86 phosphatase catalytic domains in T. cruzi, 78 in T. brucei, and 88 in L. major. We found interesting differences with other eukaryotic genomes, such as the low proportion of tyrosine phosphatases and the expansion of the serine/threonine phosphatase family. Additionally, a large number of atypical protein phosphatases were identified in these species, representing more than one third of the total phosphatase complement. Most of the atypical phosphatases belong to the dual-specificity phosphatase (DSP) family and show considerable divergence from classic DSPs in both the domain organisation and sequence features. CONCLUSIONS: The analysis of the phosphatome of the three kinetoplastids indicates that they possess orthologues to many of the phosphatases reported in other eukaryotes, including humans. However, novel domain architectures and unusual combinations of accessory domains, suggest distinct functional roles for several of the kinetoplastid phosphatases, which await further experimental exploration. These distinct traits may be exploited in the selection of suitable new targets for drug development to prevent transmission and spread of the diseases, taking advantage of the already extensive knowledge on protein phosphatase inhibitors. [Abstract/Link to Full Text]

Zhang J, He Q, Liu QY, Guo W, Deng XM, Zhang WW, Hu XX, Li N
Differential gene expression profile in pig adipose tissue treated with/without clenbuterol.
BMC Genomics. 2007 Nov 26;8(1):433.
ABSTRACT: BACKGROUND: Clenbuterol, a beta-agonist, can dramatically reduce pig adipose accumulation at high dosages. However, it has been banned in pig production because people who eat pig products treated with clenbuterol can be poisoned by the clenbuterol residues. To understand the molecular mechanism for this fat reduction, cDNA microarray, real-time PCR, two-dimensional electrophoresis and mass spectra were used to study the differential gene expression profiles of pig adipose tissues treated with/without clenbuterol. The objective of this research is to identify novel genes and physiological pathways that potentially facilitate clenbuterol induced reduction of adipose accumulation. RESULTS: Clenbuterol was found to improve the lean meat percentage about 10 percent (P< .05). The adipose cells became smaller and the muscle fibers became thicker with the administration of clenbuterol. The mRNA abundance levels of 82 genes (ESTs) were found to be statistically differentially expressed based on the Student t-test (P<.05) in the microarray analyses which contained 3358 genes (ESTs). These 82 genes (ESTs) were divided into four groups according to their Gene Ontology Biological Process descriptions. 16 genes were cellular metabolism related genes (including five related to lipid metabolism such as apolipoprotein D and apolipoprotein R), 10 were signal transduction related genes, 45 were expressed sequence tags (ESTs) and 11 others were of various categories. Eleven of the 82 genes (ESTs) were chosen for real-time PCR analysis, with eight genes showing similar induction magnitude as that seen in the microarray data. Apolipoprotein R was also found to be up-regulated by the proteomic analysis. CONCLUSIONS: Pig fat accumulation was reduced dramatically with clenbuterol treatment. Histological sections and global evaluation of gene expression after administration of clenbuterol in pigs identified profound changes in adipose cells. With clenbuterol stimulation, adipose cell volumes decreased and their gene expression profile changed, which indicate some metabolism processes have been also altered. Although the biological functions of the differentially expressed genes are not completely known, higher expressions of these molecules in adipose tissue might contribute to the reduction of fat accumulation. Among these genes, five lipid metabolism related genes were of special interest for further study, including apoD and apoR. The apoR expression was increased at both the RNA and protein levels. The apoR may be one of the critical molecules through which clenbuterol reduces fat accumulation. [Abstract/Link to Full Text]

Jackson AP
Evolutionary consequences of a large duplication event in Trypanosoma brucei: Chromosomes 4 and 8 are partial duplicons.
BMC Genomics. 2007 Nov 23;8(1):432.
ABSTRACT: BACKGROUND: Gene order along the genome sequence of the human parasite Trypanosoma brucei provides evidence for a 0.5Mb duplication, comprising the 3' regions of chromosomes 4 and 8. Here, the principal aim was to examine the contribution made by this duplication event to the T. brucei genome sequence, emphasising the consequences for gene content and the evolutionary change subsequently experienced by paralogous gene copies. The duplicated region may be browsed online at RESULTS: Comparisons of trypanosomatid genomes demonstrated widespread gene loss from each duplicon, but also showed that 47% of duplicated genes were retained on both chromosomes as paralogous loci. Secreted and surface-expressed genes were over-represented among retained paralogs, reflecting a bias towards important factors at the host-parasite interface, and consistent with a dosage-balance hypothesis. Genetic divergence in both coding and regulatory regions of retained paralogs was bimodal, with a deficit in moderately divergent paralogs; in particular, non-coding sequences were either conserved or entirely remodelled. The conserved paralogs included examples of remarkable sequence conservation, but also considerable divergence of both coding and regulatory regions. Sequence divergence typically displayed strong negative selection; but several features, such as asymmetric evolutionary rates, positively-selected codons and other non-neutral substitutions, suggested that divergence of some paralogs was driven by functional change. The absence of orthologs to retained paralogs in T. congolense indicated that the duplication event was specific to T. brucei. CONCLUSIONS: The duplication of this chromosomal region doubled the dosage of many genes. Rather than creating 'more of the same', these results show that paralogs were structurally modified according to various evolutionary trajectories. The retention of paralogs, and subsequent elaboration of both their primary structures and regulatory regions, strongly suggests that this duplication was a seminal development, stimulating functional innovation and fundamentally altering the genetic repertoire of T. brucei relative to other trypanosomatids. [Abstract/Link to Full Text]

Irsigler AS, Costa MD, Zhang P, Braga PA, Dewey RE, Boston RS, Fontes EP
Expression profiling on soybean leaves reveals integration of ER- and osmotic-stress pathways.
BMC Genomics. 2007 Nov 23;8(1):431.
ABSTRACT: BACKGROUND: Despite the potential of the endoplasmic reticulum (ER) stress response to accommodate adaptive pathways, its integration with other environmental-induced responses is poorly understood in plants. We have previously demonstrated that the ER-stress sensor binding protein (BiP) from soybean exhibits an unusual response to drought. The members of the soybean BiP gene family are differentially regulated by osmotic stress and soybean BiP confers tolerance to drought. While these results may reflect crosstalk between the osmotic and ER-stress signaling pathways, the lack of mutants, transcriptional response profiles to stresses and genome sequence information of this relevant crop has limited our attempts to identify integrated networks between osmotic and ER stress-induced adaptive responses. As a fundamental step towards this goal, we performed global expression profiling on soybean leaves exposed to polyethylene glycol treatment (osmotic stress) or to ER stress inducers. RESULTS: The up-regulated stress-specific changes unmasked the major branches of the ER-stress response, which include enhancing protein folding and degradation in the ER, as well as specific osmotically regulated changes linked to cellular responses induced by dehydration. However, a small proportion (5.5%) of total up-regulated genes represented a shared response that seemed to integrate the two signaling pathways. These co-regulated genes were considered downstream targets based on similar induction kinetics and a synergistic response to the combination of osmotic- and ER-stress-inducing treatments. Genes in this integrated pathway with the strongest synergistic induction encoded proteins with diverse roles, such as plant-specific development and cell death (DCD) domain-containing proteins, an ubiquitin-associated (UBA) protein homolog and NAC domain-containing proteins. This integrated pathway diverged further from characterized specific branches of ER-stress as downstream targets were inversely regulated by osmotic stress. CONCLUSIONS: The present ER-stress- and osmotic-stress-induced transcriptional studies demonstrate a clear predominance of stimulus-specific positive changes over shared responses on soybean leaves. This scenario indicates that PEG-induced cellular dehydration and ER stress elicited very different up-regulated responses within a 10-h stress treatment regime. In addition to identifying ER-stress and osmotic-stress-specific responses in soybean (Glycine max), our global expression-profiling analyses provided a list of candidate regulatory components, which may integrate the osmotic-stress and ER-stress signaling pathways in plants. [Abstract/Link to Full Text]

Saxena V, Orgill D, Kohane I
A set of genes previously implicated in the hypoxia response might be an important modulator in the rat ear tissue response to mechanical stretch.
BMC Genomics. 2007 Nov 23;8(1):430.
ABSTRACT: BACKGROUND: Wounds are increasingly important in our aging societies. Pathologies such as diabetes predispose patients to chronic wounds that can cause pain, infection, and amputation. The vacuum assisted closure device shows remarkable outcomes in wound healing. Its mechanism of action is unclear despite several hypotheses advanced. We previously hypothesized that micromechanical forces can heal wounds. To understand better the biological response of soft tissue to forces, rat ears in vivo were stretched and their gene expression patterns over time obtained. The absolute enrichment (AE) algorithm that obtains a combined up and down regulated picture of the expression analysis was implemented. RESULTS: With the use of AE, the hypoxia gene set was the most important at a highly significant level. A co-expression network analysis showed that important co-regulated members of the hypoxia pathway include a glucose transporter (slc2a8), heme oxygenase, and nitric oxide synthase2 among others. CONCLUSION: It appears that the hypoxia pathway may be an important modulator of response of soft tissue to forces. This finding gives us insights not only into the underlying biology, but also into clinical interventions that could be designed to mimic within wounded tissue the effects of forces without all the negative effects that forces themselves create. [Abstract/Link to Full Text]

Deluc LG, Grimplet J, Wheatley MD, Tillett RL, Quilici DR, Osborne C, Schooley DA, Schlauch KA, Cushman JC, Cramer GR
Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development.
BMC Genomics. 2007 Nov 22;8(1):429.
ABSTRACT: BACKGROUND: Grape berry development is a dynamic process that involves a complex series of molecular genetic and biochemical changes divided into three major phases. During initial berry growth (Phase I), berry size increases along a sigmoidal growth curve due to cell division and subsequent cell expansion, and organic acids (mainly malate and tartrate), tannins, and hydroxycinnamates accumulate to peak levels. The second major phase (Phase II) is defined as a lag phase in which cell expansion ceases and sugars begin to accumulate. Veraison (the onset of ripening) marks the beginning of the third major phase (Phase III) in which berries undergo a second period of sigmoidal growth due to additional mesocarp cell expansion, accumulation of anthocyanin pigments for berry color, accumulation of volatile compounds for aroma, softening, peak accumulation of sugars (mainly glucose and fructose), and a decline in organic acid accumulation. In order to understand the transcriptional network responsible for controlling berry development, mRNA expression profiling was conducted on berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip(R) Vitis oligonucleotide microarray ver. 1.0 spanning seven stages of berry development from small pea size berries (E-L stages 31 to 33 as defined by the modified E-L system), through veraison (E-L stages 34 and 35), to mature berries (E-L stages 36 and 38). Selected metabolites were profiled in parallel with mRNA expression profiling to understand the effect of transcriptional regulatory processes on specific metabolite production that ultimately influence the organoleptic properties of wine. RESULTS: Over the course of berry development whole fruit tissues were found to express an average of 74.5% of probes represented on the Vitis microarray. Approximately 60% of these transcripts exhibited significant differential expression between at least two out of the seven stages of berry development with more than 28% of transcripts (4,151 Unigenes) showing pronounced ([greater than or equal to] 2 fold) differences in mRNA expression illustrating the dynamic nature of the developmental process. Grouping 4,151 Unigenes having at least two-fold differential expression between two developmental phases revealed twenty well-correlated expression profile groups of interest. Expression profile patterns included those with declining or increasing mRNA expression over the course of berry development as well as transient peak or trough patterns across various developmental stages as defined by the modified E-L system. These detailed surveys revealed the expression patterns for genes that play key functional roles in phytohormone biosynthesis and response, calcium sequestration, transport and signaling, cell wall metabolism mediating expansion, ripening, and softening, flavonoid metabolism, transport, organic and amino acid metabolism, hexose sugar and triose phosphate metabolism and transport, and starch metabolism. In particular, mRNA expression patterns of transcription factor, abscisic acid (ABA) biosynthesis, and calcium signaling genes identified candidate factors likely to participate in the progression of key developmental events such as veraison and potential candidate genes associated with such processes as auxin partitioning within berry cells, aroma compound production, and flavonoid pathway regulation and sequestration of flavonoid compounds. Finally, analysis of sugar metabolism gene expression patterns indicated the existence of an alternative pathway for glucose and triose phosphate production that is invoked from veraison to mature berries. CONCLUSIONS: These results reveal the first high-resolution picture of the transcriptome dynamics that occur during seven stages of grape berry development. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern berry development in a widely grown cultivar of wine grape. More importantly, this analysis identified a set of previously unknown genes potentially involved in critical steps associated with fruit development that can now be subjected to functional testing. [Abstract/Link to Full Text]

Pilati S, Perazzolli M, Malossini A, Cestaro A, Dematte L, Fontana P, Dal Ri A, Viola R, Velasco R, Moser C
Genome-wide transcriptional analysis of grapevine berry ripening reveals a set of genes similarly modulated during three seasons and the occurrence of an oxidative burst at veraison.
BMC Genomics. 2007 Nov 22;8(1):428.
ABSTRACT: BACKGROUND: Grapevine (Vitis species) is among the most important fruit crops in terms of cultivated area and economic impact. Despite this relevance, little is known about the transcriptional changes and the regulatory circuits underlying the biochemical and physical changes occurring during berry development. RESULTS: Fruit ripening in the non-climacteric crop species Vitis vinifera L. has been investigated at the transcriptional level by the use of the Affymetrix Vitis GeneChip which contains approximately 14,500 unigenes. Gene expression data obtained from berries sampled before and after veraison in three growing years, were analyzed to identify genes specifically involved in fruit ripening and to investigate seasonal influences on the process. From these analyses a core set of 1477 genes was found which was similarly modulated in all seasons. We were able to separate ripening specific isoforms within gene families and to identify ripening related genes which appeared strongly regulated also by the seasonal weather conditions. Transcripts annotation by Gene Ontology vocabulary revealed five overrepresented functional categories of which cell wall organization and biogenesis, carbohydrate and secondary metabolisms and stress response were specifically induced during the ripening phase, while photosynthesis was strongly repressed. About 19% of the core gene set was characterized by genes involved in regulatory processes, such as transcription factors and transcripts related to hormonal metabolism and signal transduction. Auxin, ethylene and light emerged as the main stimuli influencing berry development. In addition, an oxidative burst, previously not detected in grapevine, characterized by rapid accumulation of H2O2 starting from veraison and by the modulation of many ROS scavenging enzymes, was observed. CONCLUSIONS: The time-course gene expression analysis of grapevine berry development has identified the occurrence of two well distinct phases along the process. The pre-veraison phase represents a reprogramming stage of the cellular metabolism, characterized by the expression of numerous genes involved in hormonal signalling and transcriptional regulation. The post-veraison phase is characterized by the onset of a ripening-specialized metabolism responsible for the phenotypic traits of the ripe berry. Between the two phases, at veraison, an oxidative burst and the concurrent modulation of the anti-oxidative enzymatic network was observed. The large number of regulatory genes we have identified represents a powerful new resource for dissecting the mechanisms of fruit ripening control in non-climacteric plants. [Abstract/Link to Full Text]

Macas J, Neumann P, Navratilova A
Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula.
BMC Genomics. 2007 Nov 21;8(1):427.
ABSTRACT: BACKGROUND: Extraordinary size variation of higher plant nuclear genomes is in large part caused by differences in accumulation of repetitive DNA. This makes repetitive DNA of great interest for studying the molecular mechanisms shaping architecture and function of complex plant genomes. However, due to methodological constraints of conventional cloning and sequencing, a global description of repeat composition is available for only a very limited number of higher plants. In order to provide further data required for investigating evolutionary patterns of repeated DNA within and between species, we used a novel approach based on massive parallel sequencing which allowed a comprehensive repeat characterization in our model species, garden pea (Pisum sativum). RESULTS: Analysis of 33.3 Mb sequence data resulted in quantification and partial sequence reconstruction of major repeat families occurring in the pea genome with at least thousands of copies. Our results showed that the pea genome is dominated by LTR-retrotransposons, estimated at 140,000 copies /1C. Ty3/gypsy elements are less diverse and accumulated to higher copy numbers than Ty1/copia. This is in part due to a large population of Ogre-like retrotransposons which alone make up over 20% of the genome. In addition to numerous types of mobile elements, we have discovered a set of novel satellite repeats and two additional variants of telomeric sequences. Comparative genome analysis revealed that there are only a few repeat sequences conserved between pea and soybean genomes. On the other hand, all major families of pea mobile elements are well represented in M. truncatula. CONCLUSIONS: We have demonstrated that even in a species with a relatively large genome like pea, where a single 454-sequencing run provided only 0.77% coverage, the generated sequences were sufficient to reconstruct and analyze major repeat families corresponding to a total of 35-48% of the genome. These data provide a starting point for further investigations of legume plant genomes based on their global comparative analysis and for the development of more sophisticated approaches for data mining. [Abstract/Link to Full Text]

Recent Articles in BMC Medical Genetics

Grewal RP, Dutra AV, Liao YC, Juo SH, Papamitsakis NI
The intron 4c allele of the NOS3 gene is associated with ischemic stroke in African Americans.
BMC Med Genet. 2007 Dec 10;8(1):76.
ABSTRACT: BACKGROUND: Ischemic stroke is the most common cause of disability in North America and in addition to the generally accepted risk factors, there is increasing evidence for the potential pathophysiological role of genes. One of these genes, the endothelial nitric oxide synthase gene (NOS3) has been reported as a genetic risk factor for ischemic stroke. To independently confirm and extend the results of these previous reports, we investigated this gene as a risk factor for stroke in an ethnically diverse study population. METHODS: Using the TOAST classification, we characterized and studied 377 patients with ischemic stroke. We genotyped two common variants in the NOS3 gene, the intron 4 insertion/deletion and an exonic single nucleotide polymorphism (SNP), G894T, in these patients and compared them with 502 controls. Chi-square or Fisher's exact tests were used to examine allele effects on stroke and stroke subtypes. Logistic regression analysis was used to adjust for confounding covariate effects. RESULTS: All genotypes are in Hardy-Weinberg equilibrium except for intron 4c, which is overrepresented in ischemic stroke patients. In pooled analysis of all patients, intron 4c, but not intron 4a, intron 4b or G894T alleles are associated with stroke (p< 0.01). In subgroup analysis by race, the intron 4c allele is most strongly associated with large artery ischemic stroke in African Americans (p<0.01). CONCLUSIONS: We are unable to confirm previous reports of an association of the intron 4a or the G894T alleles with ischemic stroke. However, although limited by a relatively small sample size, our study suggests a potentially important role of the intron 4c allele as a genetic marker of ischemic stroke in African Americans. [Abstract/Link to Full Text]

Nunez C, Oliver J, Mendoza JL, Gomez-Garcia M, Taxonera C, Gomez LM, Lopez-Nevot MA, G de la Concha E, Urcelay E, Martinez A, Martin J
CD209 in inflammatory bowel disease: a case-control study in the Spanish population.
BMC Med Genet. 2007 Dec 10;8(1):75.
ABSTRACT: BACKGROUND: the etiology of Ulcerative Colitis (UC) and Crohn's Disease (CD), considered together as Inflammatory Bowel Diseases (IBD), involves environmental and genetic factors. Although some genes are already known, the genetics underlying these diseases is complex and new candidates are continuously emerging. The CD209 gene is located in a region linked previously to IBD and a CD209 functional polymorphism (rs4804803) has been associated to other inflammatory conditions. Our aim was to study the potential involvement of this CD209 variant in IBD susceptibility. METHODS: we performed a case-control study with 515 CD patients, 497 UC patients and 731 healthy controls, all of them white Spaniards. Samples were typed for the CD209 single nucleotide polymorphism (SNP) rs4804803 by TaqMan technology. Frequency comparisons were performed using chi squared tests. RESULTS: no association between CD209 and UC or CD was observed initially. However, stratification of UC patients by HLA-DR3 status, a strong protective allele, showed that carriage of the CD209_G allele could increase susceptibility in the subgroup of HLA-DR3-positive individuals (p=0.03 OR=1.77 95% CI 1.04-3.02, vs. controls). CONCLUSIONS: a functional variant in the CD209 gene, rs4804803, does not seem to be influencing Crohn's disease susceptibility. However, it could be involved in the etiology or pathology of Ulcerative Colitis in HLA-DR3-positive individuals but further studies are necessary. [Abstract/Link to Full Text]

Philippi A, Tores F, Carayol J, Rousseau F, Letexier M, Roschmann E, Lindenbaum P, Benajjou A, Fontaine K, Vazart C, Gesnouin P, Brooks P, Hager J
Association of autism with polymorphisms in the paired-like homeodomain transcription factor 1 (PITX1) on chromosome 5q31: a candidate gene analysis.
BMC Med Genet. 2007 Dec 6;8(1):74.
ABSTRACT: BACKGROUND: Autism is a complex, heterogeneous, behaviorally-defined disorder characterized by disruptions of the nervous system and of other systems such as the pituitary-hypothalamic axis. In a previous genome wide screen, we reported linkage of autism with a 1.2 Megabase interval on chromosome 5q31. For the current study, we hypothesized that 3 of the genes in this region could be involved in the development of autism: 1) paired-like homeodomain transcription factor 1 (PITX1), which is a key regulator of hormones within the pituitary-hypothalamic axis, 2) neurogenin 1, a transcription factor involved in neurogenesis, and 3) histone family member Y (H2AFY), which is involved in X-chromosome inactivation in females and could explain the 4:1 male:female gender distortion present in autism. METHODS: A total of 276 families from the Autism Genetic Resource Exchange (AGRE) repository composed of 1086 individuals including 530 affected children were included in the study. Single nucleotide polymorphisms tagging the three candidate genes were genotyped on the initial linkage sample of 116 families. A second step of analysis was performed using tightly linked SNPs covering the PITX1 gene. Association was evaluated using the FBAT software version 1.7.3 for single SNP analysis and the HBAT command from the same package for haplotype analysis respectively. RESULTS: Association between SNPs and autism was only detected for PITX1. Haplotype analysis within PITX1 showed evidence for overtransmission of the A-C haplotype of markers rs11959298 - rs6596189 (p = 0.0004). Individuals homozygous or heterozygous for the A-C haplotype risk allele were 2.54 and 1.59 fold more likely to be autistic than individuals who were not carrying the allele, respectively. CONCLUSIONS: Strong and consistent association was observed between a 2 SNPs within PITX1 and autism. Our data suggest that PITX1, a key regulator of hormones within the pituitary-hypothalamic axis, may be implicated in the etiology of autism. [Abstract/Link to Full Text]

Nilsson M, Dahlman I, Jiao H, Gustafsson JA, Arner P, Dahlman-Wright K
Impact of estrogen receptor gene polymorphisms and mRNA levels on obesity and lipolysis - a cohort study.
BMC Med Genet. 2007 Dec 4;8(1):73.
ABSTRACT: BACKGROUND: The estrogen receptors alpha and beta (ESR1, ESR2) have been implicated in adiposity, lipid metabolism and feeding behaviour. In this report we analyse ESR1 and ESR2 gene single nucleotide polymorphisms (SNPs) for association with obesity. We also relate adipose tissue ESR1 mRNA levels and ESR1 SNPs to adipocyte lipolysis and lipogenesis phenotypes. METHODS: 23 ESR1 and 11 ESR2 tag-SNPs, covering most of the common haplotype variation in each gene according to HAPMAP data, were analysed by Chi2 for association with obesity in a cohort comprising 705 adults with severe obesity and 402 lean individuals. Results were replicated in a cohort comprising 837 obese and 613 lean subjects. About 80% of both cohorts comprised women, and 20% men. Adipose tissue ESR1 mRNA was quantified in 122 women and related to lipolysis and lipogenesis by multiple regression. ESR1 SNPs were analysed for association with adipocyte lipolysis and lipogenesis phenotypes in 204 obese women by simple regression. RESULTS: No ESR1 SNP was associated with obesity. Five ESR2 SNPs displayed nominal significant allelic association with obesity in women and one in men. The two ESR2 SNPs associated with obesity with nominal P value < 0.01 were genotyped in a second cohort where no association with obesity was observed. There was an inverse correlation between ESR1 mRNA levels in abdominal subcutaneous (sc) adipose tissue and basal lipolysis, as well as responsiveness to adrenoceptor agonists independent of age and BMI (P value 0.009-0.045). ESR1 rs532010 was associated with lipolytic sensitivity to noradrenaline (nominal P value 0.012), and ESR1 rs1884051 with responsiveness to the non-selective beta-adrenoceptor agonist isoprenaline (nominal P value 0.05). These associations became non-significant after Bonferroni correction. CONCLUSION: ESR1 gene alleles are unlikely to be a major cause of obesity in women. A minor importance of ESR2 on severe obesity cannot be excluded. The inverse correlation between ESR1 mRNA levels and lipolytic responsiveness to adrenoceptor agonists implies that low adipose tissue ESR1 levels attenuate catecholamine resistance in sc fat cells of obese women hereby contributing to loss of sc and gain of visceral fat. There is no evidence for a genetic impact of ESR1 on lipolysis or lipogenesis. [Abstract/Link to Full Text]

Lee SA, Haiman CA, Burtt NP, Pooler LC, Cheng I, Kolonel LN, Pike MC, Altshuler D, Hirschhorn JN, Henderson BE, Stram DO
A comprehensive analysis of common genetic variation in prolactin (PRL) and PRL receptor (PRLR) genes in relation to plasma prolactin levels and breast cancer risk: the Multiethnic Cohort.
BMC Med Genet. 2007 Dec 1;8(1):72.
ABSTRACT: BACKGROUND: Studies in animals and humans clearly indicate a role for prolactin (PRL) in breast epithelial proliferation, differentiation, and tumorigenesis. Prospective epidemiological studies have also shown that women with higher circulating PRL levels have an increase in risk of breast cancer, suggesting that variability in PRL may also be important in determining a woman's risk. METHODS: We evaluated genetic variation in the PRL and PRL receptor (PRLR) genes as predictors of plasma PRL levels and breast cancer risk among African-American, Native Hawaiian, Japanese-American, Latina, and White women in the Multiethnic Cohort Study (MEC). We selected single nucleotide polymorphisms (SNPs) from both the public (dbSNP) and private (Celera) databases to construct high density SNP maps that included up to 20 kilobases (kb) upstream of the transcription initiation site and 10 kb downstream of the last exon of each gene, for a total coverage of 59 kb in PRL and 210 kb in PRLR. We genotyped 80 SNPs in PRL and 173 SNPs in PRLR in a multiethnic panel of 349 unaffected subjects to characterize linkage disequilibrium (LD) and haplotype patterns. We sequenced the coding regions of PRL and PRLR in 95 advanced breast cancer cases (19 of each racial/ethnic group) to uncover putative functional variation. A total of 33 and 60 haplotype "tag" SNPs (tagSNPs) that allowed for high predictability (Rh2 >= 0.70) of the common haplotypes in PRL and PRLR, respectively, were then genotyped in a multiethnic breast cancer case-control study of 1,615 invasive breast cancer cases and 1,962 controls in the MEC. We also assessed the association of common genetic variation with circulating PRL levels in 362 postmenopausal controls without a history of hormone therapy use at blood draw. Because of the large number of comparisons being performed we used a relatively stringent type I error criteria (p < 0.0005) for evaluating the significance of any single association to correct for performing approximately 100 independent tests, close to the number of tagSNPs genotyped for both genes. RESULTS: We observed no significant associations between PRL and PRLR haplotypes or individual SNPs in relation to breast cancer risk. A nominally significant association was noted between prolactin levels and a tagSNP (tagSNP 44, rs2244502) in intron 1 of PRL. This SNP showed approximately a 50% increase in levels between minor allele homozygotes vs. major allele homozygotes. However, this association was not significant (p = 0.002) using our type I error criteria to correct for multiple testing, nor was this SNP associated with breast cancer risk (p = 0.58). CONCLUSION: In this comprehensive analysis covering 59 kb of the PRL locus and 210 kb of the PRLR locus, we found no significant association between common variation in these candidate genes and breast cancer risk or plasma PRL levels. The LD characterization of PRL and PRLR in this multiethnic population provide a framework for studying these genes in relation to other disease outcomes that have been associated with PRL, as well as for larger studies of plasma PRL levels. [Abstract/Link to Full Text]

Cooper JD, Smyth DJ, Bailey R, Payne F, Downes K, Godfrey LM, Masters J, Zeitels LR, Vella A, Walker NM, Todd JA
The candidate genes TAF5L, TCF7, PDCD1, IL6 and ICAM1 cannot be excluded from having effects in type 1 diabetes.
BMC Med Genet. 2007 Nov 28;8(1):71.
ABSTRACT: BACKGROUND: As genes associated with immune-mediated diseases have an increased prior probability of being associated with other immune-mediated diseases, we tested three such genes, IL23R, IRF5 and CD40, for an associated with type 1 diabetes. In addition, we tested seven genes, TAF5L, PDCD1, TCF7, IL12B, IL6, ICAM1 and TBX21, with published marginal or inconsistent evidence of an association with type 1 diabetes. METHODS: We genotyped reported polymorphisms of the ten genes, nonsynonymous SNPs (nsSNPs) and, for the IL12B and IL6 regions, tag SNPs in up to 7,888 case, 8,858 control and 3,142 parent-child trio samples. In addition, we analysed data from the Wellcome Trust Case Control Consortium genome-wide association study to determine whether there was any further evidence of an association in each gene region. RESULTS: We found some evidence of associations between type 1 diabetes and TAF5L, PDCD1, TCF7 and IL6 (ORs = 1.05-1.13; P = 0.0291 - 4.16x10-4). No evidence of an association was obtained for IL12B, IRF5, IL23R, ICAM1, TBX21 and CD40, although there was some evidence of an association (OR = 1.10; P = 0.0257) from the genome-wide association study for ICAM1 region. CONCLUSIONS: We failed to exclude the possibility of some effect in type 1 diabetes for TAF5L, PDCD1, TCF7, IL6 and ICAM1. Additional studies, of these and other candidate genes, employing much larger sample sizes and analysis of additional polymorphisms in each gene and its flanking region will be required to ascertain their contributions to type 1 diabetes susceptibility. [Abstract/Link to Full Text]

Kim KS, Kim GS, Hwang JY, Lee HJ, Park MH, Kim KJ, Jung J, Cha HS, Shin HD, Kang JH, Park EK, Kim TH, Hong JM, Koh JM, Oh B, Kimm K, Kim SY, Lee JY
Single nucleotide polymorphisms in bone turnover-related genes in Koreans: Ethnic differences in linkage disequilibrium and haplotype.
BMC Med Genet. 2007 Nov 26;8(1):70.
ABSTRACT: BACKGROUND: Osteoporosis is defined as the loss of bone mineral density that leads to bone fragility with aging. Population-based case-control studies have identified polymorphisms in many candidate genes that have been associated with bone mass maintenance or osteoporotic fracture. To investigate single nucleotide polymorphisms (SNPs) that are associated with osteoporosis, we examined the genetic variation among Koreans by analyzing 81 genes according to bone formation and resorption during bone remodeling. METHODS: We resequenced all the exons, splice junctions and promoter regions of candidate osteoporosis genes using 24 unrelated Korean individuals. Using the common SNPs from our study and the HapMap database, a statistical analysis of deviation in heterozygosity was performed. RESULTS: We identified 942 variants, including 888 SNPs, 43 insertion/deletion polymorphisms, and 11 microsatellite markers. Of the SNPs, 557 (63%) had been previously identified and 331 (37%) were newly discovered in the Korean population. When those SNPs in the Korean population were compared with those in HapMap database, 1% (or less) of SNPs in the Japanese and Chinese subpopulations and 20% of those in Caucasian and African subpopulations were significantly deviated from the Hardy-Weinberg expectations. In addition, an analysis of the genetic diversity showed that there were no significant differences among Korean, Han Chinese and Japanese populations, but African and Caucasian populations were significantly differentiated in selected genes. Nevertheless, in the detailed analysis of genetic properties, the LD and Haplotype block patterns among the five sub-populations were substantially different from one another. CONCLUSIONS: Through the resequencing of 81 osteoporosis candidate genes, 118 unknown SNPs with a minor allele frequency (MAF) >0.05 were discovered in the Korean population. In addition, using the common SNPs between our study and HapMap, an analysis of genetic diversity and deviation in heterozygosity was performed and the polymorphisms of the above genes among the five populations were substantially differentiated from one another. Further studies of osteoporosis could utilize the polymorphisms identified in our data since they may have important implications for the selection of highly informative SNPs for future association studies. [Abstract/Link to Full Text]

Cukjati M, Vaupotic T, Rupreht R, Curin Serbec V
Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay.
BMC Med Genet. 2007 Nov 23;8(1):69.
ABSTRACT: BACKGROUND: Hereditary hemochromatosis (HH) is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for HFE mutations screening based on TaqMan technology and to determine the frequencies of HFE mutations in the Slovenian population. METHODS: Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of HFE mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP)-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing. RESULTS: The genotyping assay of the H63D, S65C and C282Y mutations in the HFE gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous HFE genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI) 11.5 - 14.2%), 1.8% (95% CI 1.4 - 2.5%) and 3.6% (95% CI 3.0 - 4.5%), respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions. CONCLUSIONS: The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for routine screening and diagnostic procedures. The genotype frequencies in the Slovenian population agree with those reported for the Central European populations although some deviations where observed in comparison with other populations of Slavic origin. Regional distribution of the mutations should be considered when planning population screening. [Abstract/Link to Full Text]

Buxbaum JD, Cai G, Nygren G, Chaste P, Delorme R, Goldsmith J, Rastam M, Silverman JM, Hollander E, Gillberg C, Leboyer M, Betancur C
Mutation analysis of the NSD1 gene in patients with autism spectrum disorders and macrocephaly.
BMC Med Genet. 2007 Nov 14;8(1):68.
ABSTRACT: BACKGROUND: Sotos syndrome is an overgrowth syndrome characterized by macrocephaly, advanced bone age, characteristic facial features, and learning disabilities, caused by mutations or deletions of the NSD1 gene, located at 5q35. Sotos syndrome has been described in a number of patients with autism spectrum disorders, suggesting that NSD1 could be involved in other cases of autism and macrocephaly. METHODS: We screened the NSD1 gene for mutations and deletions in 88 patients with autism spectrum disorders and macrocephaly (head circumference 2 standard deviations or more above the mean). Mutation analysis was performed by direct sequencing of all exons and flanking regions. Dosage analysis of NSD1 was carried out using multiplex ligation-dependent probe amplification. RESULTS: We identified three missense variants (R604L, S822C and E1499G) in one patient each, but none is within a functional domain. In addition, segregation analysis showed that all variants were inherited from healthy parents and in two cases were also present in unaffected siblings, indicating that they are probably nonpathogenic. No partial or whole gene deletions/duplications were observed. CONCLUSIONS: Our findings suggest that Sotos syndrome is a rare cause of autism spectrum disorders and that screening for NSD1 mutations and deletions in patients with autism and macrocephaly is not warranted in the absence of other features of Sotos syndrome. [Abstract/Link to Full Text]

Bohlega S, Al-Shubili A, Edris A, Alreshaid A, Alkhairallah T, Alsous MW, Farah S, Abu-Amero K
CADASIL in Arabs: clinical and genetic findings.
BMC Med Genet. 2007 Nov 9;8(1):67.
ABSTRACT: BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is increasingly recognized as an inherited arterial disease leading to a step-wise decline and eventually to dementia. CADASIL is caused by mutations in NOTCH3 epidermal growth factor-like repeat that maps to chromosome 19. CADASIL cases have been identified in most countries of Western and Central Europe, the Americas, Japan, Australia, the Caribbean, South America, Tanzania, Turkey, South Africa and Southeast Asia, but not in Arabs. METHODS: We studied three families from Saudi Arabia (Family A), Kuwait (Family B) and Yemen (Family C) with 19 individuals affected by CADASIL. RESULTS: The mean age of onset was 31 +/- 6 and the clinical presentation included stroke in 68%, subcortical dementia in 17% and asymptomatic leukoariosis detected by MRI in 15%. Migraine and depression were frequently associated, 38% and 68% respectively. The mean age of death was 56 +/- 11. All NOTCH3 exons were screened for mutations, which revealed the presence of previously reported mutations c.406 C to T (p.Arg110Cys) in two families (family A&B) and c.475 C to T (p.Arg133Cys) mutation in family C. CONCLUSION: CADASIL occurs in Arabs, with clinical phenotype and genotype similar to that in other ethnic groups. [Abstract/Link to Full Text]

Gosso FM, de Geus EJ, Polderman TJ, Boomsma DI, Posthuma D, Heutink P
Exploring the functional role of the CHRM2 gene in human cognition: results from a dense genotyping and brain expression study.
BMC Med Genet. 2007 Nov 8;8(1):66.
ABSTRACT: BACKGROUND: The CHRM2 gene, located on the long arm of chromosome 7 (7q31-35), is involved in neuronal excitability, synaptic plasticity and feedback regulation of acetylcholine release, and has been implicated in higher cognitive processing. The aim of this study is the identification of functional (non)coding variants underlying cognitive phenotypic variation. METHODS: We previously reported an association between polymorphisms in the 5'UTR regions of the CHRM2 gene and intelligence. However, no functional variants within this area have currently been identified. In order to identify the relevant functional variant(s), we conducted a denser coverage of SNPs, using two independent Dutch cohorts, consisting of a children's sample (N = 371 ss; mean age 12.4) and an adult sample (N= 391 ss; mean age 37.6). For all individuals standardized intelligence measures were available. Subsequently, we investigated genotype-dependent CHRM2 gene expression levels in the brain, to explore putative enhancer/inhibition activity exerted by variants within the muscarinic acetylcholinergic receptor. RESULTS: Using a test of within-family association two of the previously reported variants - rs2061174, and rs324650 - were again strongly associated with intelligence (P< 0.01). A new SNP (rs2350780) showed a trend towards significance. SNP rs324650, is located within a short interspersed repeat (SINE). Although the function of short interspersed repeats remains contentious, recent research revealed potential functionality of SINE repeats in a gene-regulatory context. Gene-expression levels in post-mortem brain material, however were not dependent on rs324650 genotype. CONCLUSION: Using a denser coverage of SNPs in the CHRM2 gene, we confirmed the 5'UTR regions to be most interesting in the context of intelligence, and ruled out other regions of this gene. Although no correlation between genomic variants and gene expression was found, it would be interesting to examine allele-specific effects on CHRM2 transcripts expression in much more detail, for example in relation to transcripts specific halve-life and their relation to LTP and memory. [Abstract/Link to Full Text]

Beffagna G, De Bortoli M, Nava A, Salamon M, Lorenzon A, Zaccolo M, Mancuso L, Sigalotti L, Bauce B, Occhi G, Basso C, Lanfranchi G, Towbin JA, Thiene G, Danieli GA, Rampazzo A
Missense mutations in Desmocollin-2 N-terminus, associated with arrhythmogenic right ventricular cardiomyopathy, affect intracellular localization of desmocollin-2 in vitro.
BMC Med Genet. 2007 Oct 26;8(1):65.
ABSTRACT: BACKGROUND: Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC), an autosomal dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement. We screened 54 ARVC probands for mutations in desmocollin-2 (DSC2), the only desmocollin isoform expressed in cardiac tissue. METHODS: Mutation screening was performed by denaturing high-performance liquid chromatography and direct sequencing. To evaluate the pathogenic potentials of the DSC2 mutations detected in patients affected with ARVC, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to obtain a fusion protein with green fluorescence protein (GFP); constructs were transfected in neonatal rat cardiomyocytes and in HL-1 cells. RESULTS: We identified two heterozygous mutations (c.304G>A (p.E102K) and c.1034T>C (p.I345T)) in two probands and in four family members. The two mutations p.E102K and p.I345T map to the N-terminal region, relevant to adhesive interactions. In vitro functional studies demonstrated that, unlike wild-type DSC2, the two N-terminal mutants are predominantly localised in the cytoplasm. CONCLUSIONS: The two missense mutations in the N-terminal domain affect the normal localisation of DSC2, thus suggesting the potential pathogenic effect of the reported mutations. Identification of additional DSC2 mutations associated with ARVC may result in increased diagnostic accuracy with implications for genetic counseling. [Abstract/Link to Full Text]

Vasan RS, Larson MG, Aragam J, Wang TJ, Mitchell GF, Kathiresan S, Newton-Cheh C, Vita JA, Keyes MJ, O'Donnell CJ, Levy D, Benjamin EJ
Genome-wide association of echocardiographic dimensions, brachial artery endothelial function and treadmill exercise responses in the Framingham Heart Study.
BMC Med Genet. 2007;8 Suppl 1S2.
BACKGROUND: Echocardiographic left ventricular (LV) measurements, exercise responses to standardized treadmill test (ETT) and brachial artery (BA) vascular function are heritable traits that are associated with cardiovascular disease risk. We conducted a genome-wide association study (GWAS) in the community-based Framingham Heart Study. METHODS: We estimated multivariable-adjusted residuals for quantitative echocardiography, ETT and BA function traits. Echocardiography residuals were averaged across 4 examinations and included LV mass, diastolic and systolic dimensions, wall thickness, fractional shortening, left atrial and aortic root size. ETT measures (single exam) included systolic blood pressure and heart rate responses during exercise stage 2, and at 3 minutes post-exercise. BA measures (single exam) included vessel diameter, flow-mediated dilation (FMD), and baseline and hyperemic flow responses. Generalized estimating equations (GEE), family-based association tests (FBAT) and variance-components linkage were used to relate multivariable-adjusted trait residuals to 70,987 SNPs (Human 100K GeneChip, Affymetrix) restricted to autosomal SNPs with minor allele frequency > or =0.10, genotype call rate > or =0.80, and Hardy-Weinberg equilibrium p > or = 0.001. RESULTS: We summarize results from 17 traits in up to 1238 related middle-aged to elderly men and women. Results of all association and linkage analyses are web-posted at webcite. We confirmed modest-to-strong heritabilities (estimates 0.30-0.52) for several Echo, ETT and BA function traits. Overall, p < 10(-5) in either GEE or FBAT models were observed for 21 SNPs (nine for echocardiography, eleven for ETT and one for BA function). The top SNPs associated were (GEE results): LV diastolic dimension, rs1379659 (SLIT2, p = 1.17*10(-7)); LV systolic dimension, rs10504543 (KCNB2, p = 5.18*10(-6)); LV mass, rs10498091 (p = 5.68*10(-6)); Left atrial size, rs1935881 (FAM5C, p = 6.56*10(-6)); exercise heart rate, rs6847149 (NOLA1, p = 2.74*10(-6)); exercise systolic blood pressure, rs2553268 (WRN, p = 6.3*10(-6)); BA baseline flow, rs3814219 (OBFC1, 9.48*10(-7)), and FMD, rs4148686 (CFTR, p = 1.13*10(-5)). Several SNPs are reasonable biological candidates, with some being related to multiple traits suggesting pleiotropy. The peak LOD score was for LV mass (4.38; chromosome 5); the 1.5 LOD support interval included NRG2. CONCLUSION: In hypothesis-generating GWAS of echocardiography, ETT and BA vascular function in a moderate-sized community-based sample, we identified several SNPs that are candidates for replication attempts and we provide a web-based GWAS resource for the research community. [Abstract/Link to Full Text]

Chandler RJ, Sloan J, Fu H, Tsai M, Stabler S, Allen R, Kaestner KH, Kazazian HH, Venditti CP
Metabolic phenotype of methylmalonic acidemia in mice and humans: the role of skeletal muscle.
BMC Med Genet. 2007 Oct 15;8(1):64.
ABSTRACT: BACKGROUND: Mutations in methylmalonyl-CoA mutase cause methylmalonic acidemia, a common organic aciduria. Current treatment regimens rely on dietary management and, in severely affected patients, liver or combined liver-kidney transplantation. For undetermined reasons, transplantation does not correct the biochemical phenotype. METHODS: To study the metabolic disturbances seen in this disorder, we have created a murine model with a null allele at the methylmalonyl-CoA mutase locus and correlated the results observed in the knock-out mice to patient data. To gain insight into the origin and magnitude of methylmalonic acid (MMA) production in humans with methylmalonyl-CoA mutase deficiency, we evaluated two methylmalonic acidemia patients who had received different variants of combined liver-kidney transplants, one with a complete liver replacement-kidney transplant and the other with an auxiliary liver graft-kidney transplant, and compared their metabolite production to four untransplanted patients with intact renal function. RESULTS: Enzymatic, Western and Northern analyses demonstrated that the targeted allele was null and correctable by lentiviral complementation. Metabolite studies defined the magnitude and tempo of plasma MMA concentrations in the mice. Before a fatal metabolic crisis developed in the first 24-48 hours, the methylmalonic acid (MMA) content per gram wet-weight was massively elevated in the skeletal muscle as well as the kidneys, liver and brain. Near the end of life, extreme elevations in tissue MMA were present primarily in the liver. The transplant patients studied when well and on dietary therapy, displayed massive elevations of MMA in the plasma and urine, comparable to the levels seen in the untransplanted patients with similar enzymatic phenotypes and dietary regimens. CONCLUSIONS: The combined observations from the murine metabolite studies and patient investigations indicate that during homeostasis, a large portion of circulating MMA has an extra-heptorenal origin and likely derives from the skeletal muscle. Our studies suggest that modulating skeletal muscle metabolism may represent a strategy to increase metabolic capacity in methylmalonic acidemia as well as other organic acidurias. This mouse model will be useful for further investigations exploring disease mechanisms and therapeutic interventions in methylmalonic acidemia, a devastating disorder of intermediary metabolism. [Abstract/Link to Full Text]

Gottlieb DJ, O'Connor GT, Wilk JB
Genome-wide association of sleep and circadian phenotypes.
BMC Med Genet. 2007;8 Suppl 1S9.
BACKGROUND: Numerous studies suggest genetic influences on sleepiness and circadian rhythms. The Sleep Heart Health Study collected questionnaire data on sleep habits and sleepiness from 2848 Framingham Heart Study Offspring Cohort participants. More than 700 participants were genotyped using the Affymetrix 100K SNP GeneChip, providing a unique opportunity to assess genetic linkage and association of these traits. METHODS: Sleepiness (defined as the Epworth Sleepiness Scale score), usual bedtime and usual sleep duration were assessed by self-completion questionnaire. Standardized residual measures adjusted for age, sex and BMI were analyzed. Multipoint variance components linkage analysis was performed. Association of SNPs to sleep phenotypes was analyzed with both population-based and family-based association tests, with analysis limited to 70,987 autosomal SNPs with minor allele frequency > or =10%, call rate > or =80%, and no significant deviation from Hardy-Weinberg equilibrium (p > or = 0.001). RESULTS: Heritability of sleepiness was 0.29, bedtime 0.22, and sleep duration 0.17. Both genotype and sleep phenotype data were available for 749 subjects. Linkage analysis revealed five linkage peaks of LOD >2: four to usual bedtime, one to sleep duration. These peaks include several candidate sleep-related genes, including CSNK2A2, encoding a known component of the circadian molecular clock, and PROK2, encoding a putative transmitter of the behavioral circadian rhythm from the suprachiasmatic nucleus. Association tests identified an association of usual bedtime with a non-synonymous coding SNP in NPSR1 that has been shown to encode a gain of function mutation of the neuropeptide S receptor, whose endogenous ligand is a potent promoter of wakefulness. Each copy of the minor allele of this SNP was associated with a 15 minute later mean bedtime. The lowest p value was for association of sleepiness with a SNP located in an intron of PDE4D, which encodes a cAMP-specific phosphodiesterase widely expressed in human brain. Full association results are posted at webcite. CONCLUSION: This analysis confirms prior reports of significant heritability of sleepiness, usual bedtime, and usual sleep duration. Several genetic loci with suggestive linkage to these traits are identified, including linkage peaks containing circadian clock-related genes. Association tests identify NPSR1 and PDE4D as possible mediators of bedtime and sleepiness. [Abstract/Link to Full Text]

Wilk JB, Walter RE, Laramie JM, Gottlieb DJ, O'Connor GT
Framingham Heart Study genome-wide association: results for pulmonary function measures.
BMC Med Genet. 2007;8 Suppl 1S8.
BACKGROUND: Pulmonary function measures obtained by spirometry are used to diagnose chronic obstructive pulmonary disease (COPD) and are highly heritable. We conducted genome-wide association (GWA) analyses (Affymetrix 100K SNP GeneChip) for measures of lung function in the Framingham Heart Study. METHODS: Ten spirometry phenotypes including percent of predicted measures, mean spirometry measures over two examinations, and rates of change based on forced expiratory volume in one second (FEV1), forced vital capacity (FVC), forced expiratory flow from the 25th to 75th percentile (FEF25-75), the FEV1/FVC ratio, and the FEF25-75/FVC ratio were examined. Percent predicted phenotypes were created using each participant's latest exam with spirometry. Predicted lung function was estimated using models defined in the set of healthy never-smokers, and standardized residuals of percent predicted measures were created adjusting for smoking status, pack-years, and body mass index (BMI). All modeling was performed stratified by sex and cohort. Mean spirometry phenotypes were created using data from two examinations and adjusting for age, BMI, height, smoking and pack-years. Change in pulmonary function over time was studied using two to four examinations with spirometry to calculate slopes, which were then adjusted for age, height, smoking and pack-years. RESULTS: Analyses were restricted to 70,987 autosomal SNPs with minor allele frequency > or = 10%, genotype call rate > or = 80%, and Hardy-Weinberg equilibrium p-value > or = 0.001. A SNP in the interleukin 6 receptor (IL6R) on chromosome 1 was among the best results for percent predicted FEF25-75. A non-synonymous coding SNP in glutathione S-transferase omega 2 (GSTO2) on chromosome 10 had top-ranked results studying the mean FEV1 and FVC measurements from two examinations. SNPs nearby the SOD3 and vitamin D binding protein genes, candidate genes for COPD, exhibited association to percent predicted phenotypes. CONCLUSION: GSTO2 and IL6R are credible candidate genes for association to pulmonary function identified by GWA. These and other observed associations warrant replication studies. This resource of GWA results for pulmonary function measures is publicly available at webcite. [Abstract/Link to Full Text]

Newton-Cheh C, Guo CY, Wang TJ, O'donnell CJ, Levy D, Larson MG
Genome-wide association study of electrocardiographic and heart rate variability traits: the Framingham Heart Study.
BMC Med Genet. 2007;8 Suppl 1S7.
BACKGROUND: Heritable electrocardiographic (ECG) and heart rate variability (HRV) measures, reflecting pacemaking, conduction, repolarization and autonomic function in the heart have been associated with risks for cardiac arrhythmias. Whereas several rare monogenic conditions with extreme phenotypes have been noted, few common genetic factors contributing to interindividual variability in ECG and HRV measures have been identified. We report the results of a community-based genomewide association study of six ECG and HRV intermediate traits. METHODS: Genotyping using Affymetrix 100K GeneChip was conducted on 1345 related Framingham Heart Study Original and Offspring cohort participants. We analyzed 1175 Original and Offspring participants with ECG data (mean age 52 years, 52% women) and 548 Offspring participants with HRV data (mean age 48 years, 51% women), in relation to 70,987 SNPs with minor allele frequency > or = 0.10, call rate > or = 80%, Hardy-Weinberg p-value > or = 0.001. We used generalized estimating equations to test association of SNP alleles with multivariable-adjusted residuals for QT, RR, and PR intervals, the ratio of low frequency to high frequency power (LF/HFP), total power (TP) and the standard deviation of normal RR intervals (SDNN). RESULTS: Associations at p < 10(-3) were found for 117 (QT), 105 (RR), 111 (PR), 102 (LF/HF), 121 (TP), and 102 (SDNN) SNPs. Several common variants in NOS1AP (4 SNPs with p-values < 10(-3); lowest p-value, rs6683968, p = 1 x 10(-4)) were associated with adjusted QT residuals, consistent with our previously reported finding for NOS1AP in an unrelated sample of FHS Offspring and other cohorts. All results are publicly available at NCBI's dbGaP at webcite. CONCLUSION: In the community-based Framingham Heart Study none of the ECG and HRV results individually attained genomewide significance. However, the presence of bona fide QT-associated SNPs among the top 117 results for QT duration supports the importance of efforts to validate top results from the reported scans. Finding genetic variants associated with ECG and HRV quantitative traits may identify novel genes and pathways implicated in arrhythmogenesis and allow for improved recognition of individuals at high risk for arrhythmias in the general population. [Abstract/Link to Full Text]

Murabito JM, Rosenberg CL, Finger D, Kreger BE, Levy D, Splansky GL, Antman K, Hwang SJ
A genome-wide association study of breast and prostate cancer in the NHLBI's Framingham Heart Study.
BMC Med Genet. 2007;8 Suppl 1S6.
BACKGROUND: Breast and prostate cancer are two commonly diagnosed cancers in the United States. Prior work suggests that cancer causing genes and cancer susceptibility genes can be identified. METHODS: We conducted a genome-wide association study (Affymetrix 100K SNP GeneChip) of cancer in the community-based Framingham Heart Study. We report on 2 cancer traits--prostate cancer and breast cancer--in up to 1335 participants from 330 families (54% women, mean entry age 33 years). Multivariable-adjusted residuals, computed using Cox proportional hazards models, were tested for association with qualifying SNPs (70, 987 autosomal SNPs with genotypic call rate > or =80%, minor allele frequency > or =10%, Hardy-Weinberg test p > or = 0.001) using generalized estimating equations (GEE) models and family based association tests (FBAT). RESULTS: There were 58 women with breast cancer and 59 men with prostate cancer. No SNP associations attained genome-wide significance. The top SNP associations in GEE models for each trait were as follows: breast cancer, rs2075555, p = 8.0 x 10(-8) in COL1A1; and prostate cancer, rs9311171, p = 1.75 x 10(-6) in CTDSPL. In analysis of selected candidate cancer susceptibility genes, two MSR1 SNPs (rs9325782, GEE p = 0.008 and rs2410373, FBAT p = 0.021) were associated with prostate cancer and three ERBB4 SNPs (rs905883 GEE p = 0.0002, rs7564590 GEE p = 0.003, rs7558615 GEE p = 0.0078) were associated with breast cancer. The previously reported risk SNP for prostate cancer, rs1447295, was not included on the 100K chip. Results of cancer phenotype-genotype associations for all autosomal SNPs are web posted at webcite. CONCLUSION: Although no association attained genome-wide significance, several interesting associations emerged for breast and prostate cancer. These findings can serve as a resource for replication in other populations to identify novel biologic pathways contributing to cancer susceptibility. [Abstract/Link to Full Text]

Larson MG, Atwood LD, Benjamin EJ, Cupples LA, D'Agostino RB, Fox CS, Govindaraju DR, Guo CY, Heard-Costa NL, Hwang SJ, Murabito JM, Newton-Cheh C, O'Donnell CJ, Seshadri S, Vasan RS, Wang TJ, Wolf PA, Levy D
Framingham Heart Study 100K project: genome-wide associations for cardiovascular disease outcomes.
BMC Med Genet. 2007;8 Suppl 1S5.
BACKGROUND: Cardiovascular disease (CVD) and its most common manifestations--including coronary heart disease (CHD), stroke, heart failure (HF), and atrial fibrillation (AF)--are major causes of morbidity and mortality. In many industrialized countries, cardiovascular disease (CVD) claims more lives each year than any other disease. Heart disease and stroke are the first and third leading causes of death in the United States. Prior investigations have reported several single gene variants associated with CHD, stroke, HF, and AF. We report a community-based genome-wide association study of major CVD outcomes. METHODS: In 1345 Framingham Heart Study participants from the largest 310 pedigrees (54% women, mean age 33 years at entry), we analyzed associations of 70,987 qualifying SNPs (Affymetrix 100K GeneChip) to four major CVD outcomes: major atherosclerotic CVD (n = 142; myocardial infarction, stroke, CHD death), major CHD (n = 118; myocardial infarction, CHD death), AF (n = 151), and HF (n = 73). Participants free of the condition at entry were included in proportional hazards models. We analyzed model-based deviance residuals using generalized estimating equations to test associations between SNP genotypes and traits in additive genetic models restricted to autosomal SNPs with minor allele frequency > or =0.10, genotype call rate > or =0.80, and Hardy-Weinberg equilibrium p-value > or = 0.001. RESULTS: Six associations yielded p < 10(-5). The lowest p-values for each CVD trait were as follows: major CVD, rs499818, p = 6.6 x 10(-6); major CHD, rs2549513, p = 9.7 x 10(-6); AF, rs958546, p = 4.8 x 10(-6); HF: rs740363, p = 8.8 x 10(-6). Of note, we found associations of a 13 Kb region on chromosome 9p21 with major CVD (p 1.7-1.9 x 10(-5)) and major CHD (p 2.5-3.5 x 10(-4)) that confirm associations with CHD in two recently reported genome-wide association studies. Also, rs10501920 in CNTN5 was associated with AF (p = 9.4 x 10(-6)) and HF (p = 1.2 x 10(-4)). Complete results for these phenotypes can be found at the dbgap website webcite. CONCLUSION: No association attained genome-wide significance, but several intriguing findings emerged. Notably, we replicated associations of chromosome 9p21 with major CVD. Additional studies are needed to validate these results. Finding genetic variants associated with CVD may point to novel disease pathways and identify potential targeted preventive therapies. [Abstract/Link to Full Text]

O'Donnell CJ, Cupples LA, D'Agostino RB, Fox CS, Hoffmann U, Hwang SJ, Ingellson E, Liu C, Murabito JM, Polak JF, Wolf PA, Demissie S
Genome-wide association study for subclinical atherosclerosis in major arterial territories in the NHLBI's Framingham Heart Study.
BMC Med Genet. 2007;8 Suppl 1S4.
INTRODUCTION: Subclinical atherosclerosis (SCA) measures in multiple arterial beds are heritable phenotypes that are associated with increased incidence of cardiovascular disease. We conducted a genome-wide association study (GWAS) for SCA measurements in the community-based Framingham Heart Study. METHODS: Over 100,000 single nucleotide polymorphisms (SNPs) were genotyped (Human 100K GeneChip, Affymetrix) in 1345 subjects from 310 families. We calculated sex-specific age-adjusted and multivariable-adjusted residuals in subjects tested for quantitative SCA phenotypes, including ankle-brachial index, coronary artery calcification and abdominal aortic calcification using multi-detector computed tomography, and carotid intimal medial thickness (IMT) using carotid ultrasonography. We evaluated associations of these phenotypes with 70,987 autosomal SNPs with minor allele frequency > or = 0.10, call rate > or = 80%, and Hardy-Weinberg p-value > or = 0.001 in samples ranging from 673 to 984 subjects, using linear regression with generalized estimating equations (GEE) methodology and family-based association testing (FBAT). Variance components LOD scores were also calculated. RESULTS: There was no association result meeting criteria for genome-wide significance, but our methods identified 11 SNPs with p < 10(-5) by GEE and five SNPs with p < 10(-5) by FBAT for multivariable-adjusted phenotypes. Among the associated variants were SNPs in or near genes that may be considered candidates for further study, such as rs1376877 (GEE p < 0.000001, located in ABI2) for maximum internal carotid artery IMT and rs4814615 (FBAT p = 0.000003, located in PCSK2) for maximum common carotid artery IMT. Modest significant associations were noted with various SCA phenotypes for variants in previously reported atherosclerosis candidate genes, including NOS3 and ESR1. Associations were also noted of a region on chromosome 9p21 with CAC phenotypes that confirm associations with coronary heart disease and CAC in two recently reported genome-wide association studies. In linkage analyses, several regions of genome-wide linkage were noted, confirming previously reported linkage of internal carotid artery IMT on chromosome 12. All GEE, FBAT and linkage results are provided as an open-access results resource at webcite. CONCLUSION: The results from this GWAS generate hypotheses regarding several SNPs that may be associated with SCA phenotypes in multiple arterial beds. Given the number of tests conducted, subsequent independent replication in a staged approach is essential to identify genetic variants that may be implicated in atherosclerosis. [Abstract/Link to Full Text]

Levy D, Larson MG, Benjamin EJ, Newton-Cheh C, Wang TJ, Hwang SJ, Vasan RS, Mitchell GF
Framingham Heart Study 100K Project: genome-wide associations for blood pressure and arterial stiffness.
BMC Med Genet. 2007;8 Suppl 1S3.
BACKGROUND: About one quarter of adults are hypertensive and high blood pressure carries increased risk for heart disease, stroke, kidney disease and death. Increased arterial stiffness is a key factor in the pathogenesis of systolic hypertension and cardiovascular disease. Substantial heritability of blood-pressure (BP) and arterial-stiffness suggests important genetic contributions. METHODS: In Framingham Heart Study families, we analyzed genome-wide SNP (Affymetrix 100K GeneChip) associations with systolic (SBP) and diastolic (DBP) BP at a single examination in 1971-1975 (n = 1260), at a recent examination in 1998-2001 (n = 1233), and long-term averaged SBP and DBP from 1971-2001 (n = 1327, mean age 52 years, 54% women) and with arterial stiffness measured by arterial tonometry (carotid-femoral and carotid-brachial pulse wave velocity, forward and reflected pressure wave amplitude, and mean arterial pressure; 1998-2001, n = 644). In primary analyses we used generalized estimating equations in models for an additive genetic effect to test associations between SNPs and phenotypes of interest using multivariable-adjusted residuals. A total of 70,987 autosomal SNPs with minor allele frequency > or = 0.10, genotype call rate > or = 0.80, and Hardy-Weinberg equilibrium p > or = 0.001 were analyzed. We also tested for association of 69 SNPs in six renin-angiotensin-aldosterone pathway genes with BP and arterial stiffness phenotypes as part of a candidate gene search. RESULTS: In the primary analyses, none of the associations attained genome-wide significance. For the six BP phenotypes, seven SNPs yielded p values < 10(-5). The lowest p-values for SBP and DBP respectively were rs10493340 (p = 1.7 x 10(-6)) and rs1963982 (p = 3.3 x 10(-6)). For the five tonometry phenotypes, five SNPs had p values < 10(-5); lowest p-values were for reflected wave (rs6063312, p = 2.1 x 10(-6)) and carotid-brachial pulse wave velocity (rs770189, p = 2.5 x 10(-6)) in MEF2C, a regulator of cardiac morphogenesis. We found only weak association of SNPs in the renin-angiotensin-aldosterone pathway with BP or arterial stiffness. CONCLUSION: These results of genome-wide association testing for blood pressure and arterial stiffness phenotypes in an unselected community-based sample of adults may aid in the identification of the genetic basis of hypertension and arterial disease, help identify high risk individuals, and guide novel therapies for hypertension. Additional studies are needed to replicate any associations identified in these analyses. [Abstract/Link to Full Text]

Fox CS, Heard-Costa N, Cupples LA, Dupuis J, Vasan RS, Atwood LD
Genome-wide association to body mass index and waist circumference: the Framingham Heart Study 100K project.
BMC Med Genet. 2007;8 Suppl 1S18.
BACKGROUND: Obesity is related to multiple cardiovascular disease (CVD) risk factors as well as CVD and has a strong familial component. We tested for association between SNPs on the Affymetrix 100K SNP GeneChip and measures of adiposity in the Framingham Heart Study. METHODS: A total of 1341 Framingham Heart Study participants in 310 families genotyped with the Affymetrix 100K SNP GeneChip had adiposity traits measured over 30 years of follow up. Body mass index (BMI), waist circumference (WC), weight change, height, and radiographic measures of adiposity (subcutaneous adipose tissue, visceral adipose tissue, waist circumference, sagittal height) were measured at multiple examination cycles. Multivariable-adjusted residuals, adjusting for age, age-squared, sex, smoking, and menopausal status, were evaluated in association with the genotype data using additive Generalized Estimating Equations (GEE) and Family Based Association Test (FBAT) models. We prioritized mean BMI over offspring examinations (1-7) and cohort examinations (10, 16, 18, 20, 22, 24, 26) and mean WC over offspring examinations (4-7) for presentation. We evaluated associations with 70,987 SNPs on autosomes with minor allele frequencies of at least 0.10, Hardy-Weinberg equilibrium p > or = 0.001, and call rates of at least 80%. RESULTS: The top SNPs to be associated with mean BMI and mean WC by GEE were rs110683 (p-value 1.22*10(-7)) and rs4471028 (p-values 1.96*10(-7)). Please see webcite for the complete set of results. We were able to validate SNPs in known genes that have been related to BMI or other adiposity traits, including the ESR1 Xba1 SNP, PPARG, and ADIPOQ. CONCLUSION: Adiposity traits are associated with SNPs on the Affymetrix 100K SNP GeneChip. Replication of these initial findings is necessary. These data will serve as a resource for replication as more genes become identified with BMI and WC. [Abstract/Link to Full Text]

Kathiresan S, Manning AK, Demissie S, D'Agostino RB, Surti A, Guiducci C, Gianniny L, Burtt NP, Melander O, Orho-Melander M, Arnett DK, Peloso GM, Ordovas JM, Cupples LA
A genome-wide association study for blood lipid phenotypes in the Framingham Heart Study.
BMC Med Genet. 2007;8 Suppl 1S17.
BACKGROUND: Blood lipid levels including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) are highly heritable. Genome-wide association is a promising approach to map genetic loci related to these heritable phenotypes. METHODS: In 1087 Framingham Heart Study Offspring cohort participants (mean age 47 years, 52% women), we conducted genome-wide analyses (Affymetrix 100K GeneChip) for fasting blood lipid traits. Total cholesterol, HDL-C, and TG were measured by standard enzymatic methods and LDL-C was calculated using the Friedewald formula. The long-term averages of up to seven measurements of LDL-C, HDL-C, and TG over a approximately 30 year span were the primary phenotypes. We used generalized estimating equations (GEE), family-based association tests (FBAT) and variance components linkage to investigate the relationships between SNPs (on autosomes, with minor allele frequency > or =10%, genotypic call rate > or =80%, and Hardy-Weinberg equilibrium p > or = 0.001) and multivariable-adjusted residuals. We pursued a three-stage replication strategy of the GEE association results with 287 SNPs (P < 0.001 in Stage I) tested in Stage II (n approximately 1450 individuals) and 40 SNPs (P < 0.001 in joint analysis of Stages I and II) tested in Stage III (n approximately 6650 individuals). RESULTS: Long-term averages of LDL-C, HDL-C, and TG were highly heritable (h2 = 0.66, 0.69, 0.58, respectively; each P < 0.0001). Of 70,987 tests for each of the phenotypes, two SNPs had p < 10(-5) in GEE results for LDL-C, four for HDL-C, and one for TG. For each multivariable-adjusted phenotype, the number of SNPs with association p < 10(-4) ranged from 13 to 18 and with p < 10(-3), from 94 to 149. Some results confirmed previously reported associations with candidate genes including variation in the lipoprotein lipase gene (LPL) and HDL-C and TG (rs7007797; P = 0.0005 for HDL-C and 0.002 for TG). The full set of GEE, FBAT and linkage results are posted at the database of Genotype and Phenotype (dbGaP). After three stages of replication, there was no convincing statistical evidence for association (i.e., combined P < 10(-5) across all three stages) between any of the tested SNPs and lipid phenotypes. CONCLUSION: Using a 100K genome-wide scan, we have generated a set of putative associations for common sequence variants and lipid phenotypes. Validation of selected hypotheses in additional samples did not identify any new loci underlying variability in blood lipids. Lack of replication may be due to inadequate statistical power to detect modest quantitative trait locus effects (i.e., <1% of trait variance explained) or reduced genomic coverage of the 100K array. GWAS in FHS using a denser genome-wide genotyping platform and a better-powered replication strategy may identify novel loci underlying blood lipids. [Abstract/Link to Full Text]

Meigs JB, Manning AK, Fox CS, Florez JC, Liu C, Cupples LA, Dupuis J
Genome-wide association with diabetes-related traits in the Framingham Heart Study.
BMC Med Genet. 2007;8 Suppl 1S16.
BACKGROUND: Susceptibility to type 2 diabetes may be conferred by genetic variants having modest effects on risk. Genome-wide fixed marker arrays offer a novel approach to detect these variants. METHODS: We used the Affymetrix 100K SNP array in 1,087 Framingham Offspring Study family members to examine genetic associations with three diabetes-related quantitative glucose traits (fasting plasma glucose (FPG), hemoglobin A1c, 28-yr time-averaged FPG (tFPG)), three insulin traits (fasting insulin, HOMA-insulin resistance, and 0-120 min insulin sensitivity index); and with risk for diabetes. We used additive generalized estimating equations (GEE) and family-based association test (FBAT) models to test associations of SNP genotypes with sex-age-age2-adjusted residual trait values, and Cox survival models to test incident diabetes. RESULTS: We found 415 SNPs associated (at p < 0.001) with at least one of the six quantitative traits in GEE, 242 in FBAT (18 overlapped with GEE for 639 non-overlapping SNPs), and 128 associated with incident diabetes (31 overlapped with the 639) giving 736 non-overlapping SNPs. Of these 736 SNPs, 439 were within 60 kb of a known gene. Additionally, 53 SNPs (of which 42 had r2 < 0.80 with each other) had p < 0.01 for incident diabetes AND (all 3 glucose traits OR all 3 insulin traits, OR 2 glucose traits and 2 insulin traits); of these, 36 overlapped with the 736 other SNPs. Of 100K SNPs, one (rs7100927) was in moderate LD (r2 = 0.50) with TCF7L2 (rs7903146), and was associated with risk of diabetes (Cox p-value 0.007, additive hazard ratio for diabetes = 1.56) and with tFPG (GEE p-value 0.03). There were no common (MAF > 1%) 100K SNPs in LD (r2 > 0.05) with ABCC8 A1369S (rs757110), KCNJ11 E23K (rs5219), or SNPs in CAPN10 or HNFa. PPARG P12A (rs1801282) was not significantly associated with diabetes or related traits. CONCLUSION: Framingham 100K SNP data is a resource for association tests of known and novel genes with diabetes and related traits posted at webcite. Framingham 100K data replicate the TCF7L2 association with diabetes. [Abstract/Link to Full Text]

Seshadri S, DeStefano AL, Au R, Massaro JM, Beiser AS, Kelly-Hayes M, Kase CS, D'Agostino RB, Decarli C, Atwood LD, Wolf PA
Genetic correlates of brain aging on MRI and cognitive test measures: a genome-wide association and linkage analysis in the Framingham Study.
BMC Med Genet. 2007;8 Suppl 1S15.
BACKGROUND: Brain magnetic resonance imaging (MRI) and cognitive tests can identify heritable endophenotypes associated with an increased risk of developing stroke, dementia and Alzheimer's disease (AD). We conducted a genome-wide association (GWA) and linkage analysis exploring the genetic basis of these endophenotypes in a community-based sample. METHODS: A total of 705 stroke- and dementia-free Framingham participants (age 62 +9 yrs, 50% male) who underwent volumetric brain MRI and cognitive testing (1999-2002) were genotyped. We used linear models adjusting for first degree relationships via generalized estimating equations (GEE) and family based association tests (FBAT) in additive models to relate qualifying single nucleotide polymorphisms (SNPs, 70,987 autosomal on Affymetrix 100K Human Gene Chip with minor allele frequency > or = 0.10, genotypic call rate > or = 0.80, and Hardy-Weinberg equilibrium p-value > or = 0.001) to multivariable-adjusted residuals of 9 MRI measures including total cerebral brain (TCBV), lobar, ventricular and white matter hyperintensity (WMH) volumes, and 6 cognitive factors/tests assessing verbal and visuospatial memory, visual scanning and motor speed, reading, abstract reasoning and naming. We determined multipoint identity-by-descent utilizing 10,592 informative SNPs and 613 short tandem repeats and used variance component analyses to compute LOD scores. RESULTS: The strongest gene-phenotype association in FBAT analyses was between SORL1 (rs1131497; p = 3.2 x 10(-6)) and abstract reasoning, and in GEE analyses between CDH4 (rs1970546; p = 3.7 x 10(-8)) and TCBV. SORL1 plays a role in amyloid precursor protein processing and has been associated with the risk of AD. Among the 50 strongest associations (25 each by GEE and FBAT) were other biologically interesting genes. Polymorphisms within 28 of 163 candidate genes for stroke, AD and memory impairment were associated with the endophenotypes studied at p < 0.001. We confirmed our previously reported linkage of WMH on chromosome 4 and describe linkage of reading performance to a marker on chromosome 18 (GATA11A06), previously linked to dyslexia (LOD scores = 2.2 and 5.1). CONCLUSION: Our results suggest that genes associated with clinical neurological disease also have detectable effects on subclinical phenotypes. These hypothesis generating data illustrate the use of an unbiased approach to discover novel pathways that may be involved in brain aging, and could be used to replicate observations made in other studies. [Abstract/Link to Full Text]

Kiel DP, Demissie S, Dupuis J, Lunetta KL, Murabito JM, Karasik D
Genome-wide association with bone mass and geometry in the Framingham Heart Study.
BMC Med Genet. 2007;8 Suppl 1S14.
BACKGROUND: Osteoporosis is characterized by low bone mass and compromised bone structure, heritable traits that contribute to fracture risk. There have been no genome-wide association and linkage studies for these traits using high-density genotyping platforms. METHODS: We used the Affymetrix 100K SNP GeneChip marker set in the Framingham Heart Study (FHS) to examine genetic associations with ten primary quantitative traits: bone mineral density (BMD), calcaneal ultrasound, and geometric indices of the hip. To test associations with multivariable-adjusted residual trait values, we used additive generalized estimating equation (GEE) and family-based association tests (FBAT) models within each sex as well as sexes combined. We evaluated 70,987 autosomal SNPs with genotypic call rates > or =80%, HWE p > or = 0.001, and MAF > or =10% in up to 1141 phenotyped individuals (495 men and 646 women, mean age 62.5 yrs). Variance component linkage analysis was performed using 11,200 markers. RESULTS: Heritability estimates for all bone phenotypes were 30-66%. LOD scores > or =3.0 were found on chromosomes 15 (1.5 LOD confidence interval: 51,336,679-58,934,236 bp) and 22 (35,890,398-48,603,847 bp) for femoral shaft section modulus. The ten primary phenotypes had 12 associations with 100K SNPs in GEE models at p < 0.000001 and 2 associations in FBAT models at p < 0.000001. The 25 most significant p-values for GEE and FBAT were all less than 3.5 x 10(-6) and 2.5 x 10(-5), respectively. Of the 40 top SNPs with the greatest numbers of significantly associated BMD traits (including femoral neck, trochanter, and lumbar spine), one half to two-thirds were in or near genes that have not previously been studied for osteoporosis. Notably, pleiotropic associations between BMD and bone geometric traits were uncommon. Evidence for association (FBAT or GEE p < 0.05) was observed for several SNPs in candidate genes for osteoporosis, such as rs1801133 in MTHFR; rs1884052 and rs3778099 in ESR1; rs4988300 in LRP5; rs2189480 in VDR; rs2075555 in COLIA1; rs10519297 and rs2008691 in CYP19, as well as SNPs in PPARG (rs10510418 and rs2938392) and ANKH (rs2454873 and rs379016). All GEE, FBAT and linkage results are provided as an open-access results resource at webcite. CONCLUSION: The FHS 100K SNP project offers an unbiased genome-wide strategy to identify new candidate loci and to replicate previously suggested candidate genes for osteoporosis. [Abstract/Link to Full Text]

Lunetta KL, D'Agostino RB, Karasik D, Benjamin EJ, Guo CY, Govindaraju R, Kiel DP, Kelly-Hayes M, Massaro JM, Pencina MJ, Seshadri S, Murabito JM
Genetic correlates of longevity and selected age-related phenotypes: a genome-wide association study in the Framingham Study.
BMC Med Genet. 2007;8 Suppl 1S13.
BACKGROUND: Family studies and heritability estimates provide evidence for a genetic contribution to variation in the human life span. METHODS: We conducted a genome wide association study (Affymetrix 100K SNP GeneChip) for longevity-related traits in a community-based sample. We report on 5 longevity and aging traits in up to 1345 Framingham Study participants from 330 families. Multivariable-adjusted residuals were computed using appropriate models (Cox proportional hazards, logistic, or linear regression) and the residuals from these models were used to test for association with qualifying SNPs (70, 987 autosomal SNPs with genotypic call rate > or =80%, minor allele frequency > or =10%, Hardy-Weinberg test p > or = 0.001). RESULTS : In family-based association test (FBAT) models, 8 SNPs in two regions approximately 500 kb apart on chromosome 1 (physical positions 73,091,610 and 73, 527,652) were associated with age at death (p-value < 10(-5)). The two sets of SNPs were in high linkage disequilibrium (minimum r2 = 0.58). The top 30 SNPs for generalized estimating equation (GEE) tests of association with age at death included rs10507486 (p = 0.0001) and rs4943794 (p = 0.0002), SNPs intronic to FOXO1A, a gene implicated in lifespan extension in animal models. FBAT models identified 7 SNPs and GEE models identified 9 SNPs associated with both age at death and morbidity-free survival at age 65 including rs2374983 near PON1. In the analysis of selected candidate genes, SNP associations (FBAT or GEE p-value < 0.01) were identified for age at death in or near the following genes: FOXO1A, GAPDH, KL, LEPR, PON1, PSEN1, SOD2, and WRN. Top ranked SNP associations in the GEE model for age at natural menopause included rs6910534 (p = 0.00003) near FOXO3a and rs3751591 (p = 0.00006) in CYP19A1. Results of all longevity phenotype-genotype associations for all autosomal SNPs are web posted at webcite. CONCLUSION: Longevity and aging traits are associated with SNPs on the Affymetrix 100K GeneChip. None of the associations achieved genome-wide significance. These data generate hypotheses and serve as a resource for replication as more genes and biologic pathways are proposed as contributing to longevity and healthy aging. [Abstract/Link to Full Text]

Yang Q, Kathiresan S, Lin JP, Tofler GH, O'Donnell CJ
Genome-wide association and linkage analyses of hemostatic factors and hematological phenotypes in the Framingham Heart Study.
BMC Med Genet. 2007;8 Suppl 1S12.
BACKGROUND: Increased circulating levels of hemostatic factors as well as anemia have been associated with increased risk of cardiovascular disease (CVD). Known associations between hemostatic factors and sequence variants at genes encoding these factors explain only a small proportion of total phenotypic variation. We sought to confirm known putative loci and identify novel loci that may influence either trait in genome-wide association and linkage analyses using the Affymetrix GeneChip 100K single nucleotide polymorphism (SNP) set. METHODS: Plasma levels of circulating hemostatic factors (fibrinogen, factor VII, plasminogen activator inhibitor-1, von Willebrand factor, tissue plasminogen activator, D-dimer) and hematological phenotypes (platelet aggregation, viscosity, hemoglobin, red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin concentration) were obtained in approximately 1000 Framingham Heart Study (FHS) participants from 310 families. Population-based association analyses using the generalized estimating equations (GEE), family-based association test (FBAT), and multipoint variance components linkage analyses were performed on the multivariable adjusted residuals of hemostatic and hematological phenotypes. RESULTS: In association analysis, the lowest GEE p-value for hemostatic factors was p = 4.5*10(-16) for factor VII at SNP rs561241, a variant located near the F7 gene and in complete linkage disequilibrium (LD) (r2 = 1) with the Arg353Gln F7 SNP previously shown to account for 9% of total phenotypic variance. The lowest GEE p-value for hematological phenotypes was 7*10(-8) at SNP rs2412522 on chromosome 4 for mean corpuscular hemoglobin concentration. We presented top 25 most significant GEE results with p-values in the range of 10(-6) to 10(-5) for hemostatic or hematological phenotypes. In relating 100K SNPs to known candidate genes, we identified two SNPs (rs1582055, rs4897475) in erythrocyte membrane protein band 4.1-like 2 (EPB41L2) associated with hematological phenotypes (GEE p < 10(-3)). In linkage analyses, the highest linkage LOD score for hemostatic factors was 3.3 for factor VII on chromosome 10 around 15 Mb, and for hematological phenotypes, LOD 3.4 for hemoglobin on chromosome 4 around 55 Mb. All GEE and FBAT association and variance components linkage results can be found at webcite. CONCLUSION: Using genome-wide association methodology, we have successfully identified a SNP in complete LD with a sequence variant previously shown to be strongly associated with factor VII, providing proof of principle for this approach. Further study of additional strongly associated SNPs and linked regions may identify novel variants that influence the inter-individual variability in hemostatic factors and hematological phenotypes. [Abstract/Link to Full Text]

Benjamin EJ, Dupuis J, Larson MG, Lunetta KL, Booth SL, Govindaraju DR, Kathiresan S, Keaney JF, Keyes MJ, Lin JP, Meigs JB, Robins SJ, Rong J, Schnabel R, Vita JA, Wang TJ, Wilson PW, Wolf PA, Vasan RS
Genome-wide association with select biomarker traits in the Framingham Heart Study.
BMC Med Genet. 2007;8 Suppl 1S11.
BACKGROUND: Systemic biomarkers provide insights into disease pathogenesis, diagnosis, and risk stratification. Many systemic biomarker concentrations are heritable phenotypes. Genome-wide association studies (GWAS) provide mechanisms to investigate the genetic contributions to biomarker variability unconstrained by current knowledge of physiological relations. METHODS: We examined the association of Affymetrix 100K GeneChip single nucleotide polymorphisms (SNPs) to 22 systemic biomarker concentrations in 4 biological domains: inflammation/oxidative stress; natriuretic peptides; liver function; and vitamins. Related members of the Framingham Offspring cohort (n = 1012; mean age 59 +/- 10 years, 51% women) had both phenotype and genotype data (minimum-maximum per phenotype n = 507-1008). We used Generalized Estimating Equations (GEE), Family Based Association Tests (FBAT) and variance components linkage to relate SNPs to multivariable-adjusted biomarker residuals. Autosomal SNPs (n = 70,987) meeting the following criteria were studied: minor allele frequency > or = 10%, call rate > or = 80% and Hardy-Weinberg equilibrium p > or = 0.001. RESULTS: With GEE, 58 SNPs had p < 10(-6): the top SNPs were rs2494250 (p = 1.00*10(-14)) and rs4128725 (p = 3.68*10(-12)) for monocyte chemoattractant protein-1 (MCP1), and rs2794520 (p = 2.83*10(-8)) and rs2808629 (p = 3.19*10(-8)) for C-reactive protein (CRP) averaged from 3 examinations (over about 20 years). With FBAT, 11 SNPs had p < 10(-6): the top SNPs were the same for MCP1 (rs4128725, p = 3.28*10(-8), and rs2494250, p = 3.55*10(-8)), and also included B-type natriuretic peptide (rs437021, p = 1.01*10(-6)) and Vitamin K percent undercarboxylated osteocalcin (rs2052028, p = 1.07*10(-6)). The peak LOD (logarithm of the odds) scores were for MCP1 (4.38, chromosome 1) and CRP (3.28, chromosome 1; previously described) concentrations; of note the 1.5 support interval included the MCP1 and CRP SNPs reported above (GEE model). Previous candidate SNP associations with circulating CRP concentrations were replicated at p < 0.05; the SNPs rs2794520 and rs2808629 are in linkage disequilibrium with previously reported SNPs. GEE, FBAT and linkage results are posted at webcite. CONCLUSION: The Framingham GWAS represents a resource to describe potentially novel genetic influences on systemic biomarker variability. The newly described associations will need to be replicated in other studies. [Abstract/Link to Full Text]

Hwang SJ, Yang Q, Meigs JB, Pearce EN, Fox CS
A genome-wide association for kidney function and endocrine-related traits in the NHLBI's Framingham Heart Study.
BMC Med Genet. 2007;8 Suppl 1S10.
BACKGROUND: Glomerular filtration rate (GFR) and urinary albumin excretion (UAE) are markers of kidney function that are known to be heritable. Many endocrine conditions have strong familial components. We tested for association between the Affymetrix GeneChip Human Mapping 100K single nucleotide polymorphism (SNP) set and measures of kidney function and endocrine traits. METHODS: Genotype information on the Affymetrix GeneChip Human Mapping 100K SNP set was available on 1345 participants. Serum creatinine and cystatin-C (cysC; n = 981) were measured at the seventh examination cycle (1998-2001); GFR (n = 1010) was estimated via the Modification of Diet in Renal Disease (MDRD) equation; UAE was measured on spot urine samples during the sixth examination cycle (1995-1998) and was indexed to urinary creatinine (n = 822). Thyroid stimulating hormone (TSH) was measured at the third and fourth examination cycles (1981-1984; 1984-1987) and mean value of the measurements were used (n = 810). Age-sex-adjusted and multivariable-adjusted residuals for these measurements were used in association with genotype data using generalized estimating equations (GEE) and family-based association tests (FBAT) models. We presented the results for association tests using additive allele model. We evaluated associations with 70,987 SNPs on autosomes with minor allele frequencies of at least 0.10, Hardy-Weinberg Equilibrium p-value > or = 0.001, and call rates of at least 80%. RESULTS: The top SNPs associated with these traits using the GEE method were rs2839235 with GFR (p-value 1.6*10(-05)), rs1158167 with cysC (p-value 8.5*10(-09)), rs1712790 with UAE (p-value 1.9*10(-06)), and rs6977660 with TSH (p-value 3.7*10(-06)), respectively. The top SNPs associated with these traits using the FBAT method were rs6434804 with GFR(p-value 2.4*10(-5)), rs563754 with cysC (p-value 4.7*10(-5)), rs1243400 with UAE (p-value 4.8*10(-6)), and rs4128956 with TSH (p-value 3.6*10(-5)), respectively. Detailed association test results can be found at webcite. Four SNPs in or near the CST3 gene were highly associated with cysC levels (p-value 8.5*10(-09) to 0.007). CONCLUSION : Kidney function traits and TSH are associated with SNPs on the Affymetrix GeneChip Human Mapping 100K SNP set. These data will serve as a valuable resource for replication as more SNPs associated with kidney function and endocrine traits are identified. [Abstract/Link to Full Text]

Recent Articles in American Journal of Human Genetics

Tian C, Hinds DA, Shigeta R, Adler SG, Lee A, Pahl MV, Silva G, Belmont JW, Hanson RL, Knowler WC, Gregersen PK, Ballinger DG, Seldin MF
A genomewide single-nucleotide-polymorphism panel for Mexican American admixture mapping.
Am J Hum Genet. 2007 Jun;80(6):1014-23.
For admixture mapping studies in Mexican Americans (MAM), we define a genomewide single-nucleotide-polymorphism (SNP) panel that can distinguish between chromosomal segments of Amerindian (AMI) or European (EUR) ancestry. These studies used genotypes for >400,000 SNPs, defined in EUR and both Pima and Mayan AMI, to define a set of ancestry-informative markers (AIMs). The use of two AMI populations was necessary to remove a subset of SNPs that distinguished genotypes of only one AMI subgroup from EUR genotypes. The AIMs set contained 8,144 SNPs separated by a minimum of 50 kb with only three intermarker intervals >1 Mb and had EUR/AMI FST values >0.30 (mean FST = 0.48) and Mayan/Pima FST values <0.05 (mean FST < 0.01). Analysis of a subset of these SNP AIMs suggested that this panel may also distinguish ancestry between EUR and other disparate AMI groups, including Quechuan from South America. We show, using realistic simulation parameters that are based on our analyses of MAM genotyping results, that this panel of SNP AIMs provides good power for detecting disease-associated chromosomal segments for genes with modest ethnicity risk ratios. A reduced set of 5,287 SNP AIMs captured almost the same admixture mapping information, but smaller SNP sets showed substantial drop-off in admixture mapping information and power. The results will enable studies of type 2 diabetes, rheumatoid arthritis, and other diseases among which epidemiological studies suggest differences in the distribution of ancestry-associated susceptibility. [Abstract/Link to Full Text]

Boughman JA
ASHG Board emphasizes communication and involvement.
Am J Hum Genet. 2007 Jun;80(6):1195-6. [Abstract/Link to Full Text]

Risch NJ, Bressman SB, Senthil G, Ozelius LJ
Intragenic Cis and Trans modification of genetic susceptibility in DYT1 torsion dystonia.
Am J Hum Genet. 2007 Jun;80(6):1188-93.
A GAG deletion in the DYT1 gene is a major cause of early-onset dystonia, but clinical disease expression occurs in only 30% of mutation carriers. To gain insight into genetic factors that may influence penetrance, we evaluated three DYT1 single-nucleotide polymorphisms, including D216H, a coding-sequence variation that moderates the effects of the DYT1 GAG deletion in cellular models. We tested DYT1 GAG-deletion carriers with (n=119) and without (n=113) clinical signs of dystonia and control individuals (n=197) and found the frequency of the 216H allele to be increased in GAG-deletion carriers without dystonia and to be decreased in carriers with dystonia, compared with the control individuals. Analysis of haplotypes demonstrated a highly protective effect of the H allele in trans with the GAG deletion; there was also suggestive evidence that the D216 allele in cis is required for the disease to be penetrant. Our findings establish, for the first time, a clinically relevant gene modifier of DYT1. [Abstract/Link to Full Text]

Golzio C, Martinovic-Bouriel J, Thomas S, Mougou-Zrelli S, Grattagliano-Bessieres B, Bonniere M, Delahaye S, Munnich A, Encha-Razavi F, Lyonnet S, Vekemans M, Attie-Bitach T, Etchevers HC
Matthew-Wood syndrome is caused by truncating mutations in the retinol-binding protein receptor gene STRA6.
Am J Hum Genet. 2007 Jun;80(6):1179-87.
Retinoic acid (RA) is a potent teratogen in all vertebrates when tight homeostatic controls on its endogenous dose, location, or timing are perturbed during early embryogenesis. STRA6 encodes an integral cell-membrane protein that favors RA uptake from soluble retinol-binding protein; its transcription is directly regulated by RA levels. Molecular analysis of STRA6 was undertaken in two human fetuses from consanguineous families we previously described with Matthew-Wood syndrome in a context of severe microphthalmia, pulmonary agenesis, bilateral diaphragmatic eventration, duodenal stenosis, pancreatic malformations, and intrauterine growth retardation. The fetuses had either a homozygous insertion/deletion in exon 2 or a homozygous insertion in exon 7 predicting a premature stop codon in STRA6 transcripts. Five other fetuses presenting at least one of the two major signs of clinical anophthalmia or pulmonary hypoplasia with at least one of the two associated signs of diaphragmatic closure defect or cardiopathy had no STRA6 mutations. These findings suggest a molecular basis for the prenatal manifestations of Matthew-Wood syndrome and suggest that phenotypic overlap with other associations may be due to genetic heterogeneity of elements common to the RA- and fibroblast growth factor-signaling cascades. [Abstract/Link to Full Text]

Mao X, Bigham AW, Mei R, Gutierrez G, Weiss KM, Brutsaert TD, Leon-Velarde F, Moore LG, Vargas E, McKeigue PM, Shriver MD, Parra EJ
A genomewide admixture mapping panel for Hispanic/Latino populations.
Am J Hum Genet. 2007 Jun;80(6):1171-8.
Admixture mapping (AM) is a promising method for the identification of genetic risk factors for complex traits and diseases showing prevalence differences among populations. Efficient application of this method requires the use of a genomewide panel of ancestry-informative markers (AIMs) to infer the population of origin of chromosomal regions in admixed individuals. Genomewide AM panels with markers showing high frequency differences between West African and European populations are already available for disease-gene discovery in African Americans. However, no such a map is yet available for Hispanic/Latino populations, which are the result of two-way admixture between Native American and European populations or of three-way admixture of Native American, European, and West African populations. Here, we report a genomewide AM panel with 2,120 AIMs showing high frequency differences between Native American and European populations. The average intermarker genetic distance is ~1.7 cM. The panel was identified by genotyping, with the Affymetrix GeneChip Human Mapping 500K array, a population sample with European ancestry, a Mesoamerican sample comprising Maya and Nahua from Mexico, and a South American sample comprising Aymara/Quechua from Bolivia and Quechua from Peru. The main criteria for marker selection were both high information content for Native American/European ancestry (measured as the standardized variance of the allele frequencies, also known as "f value") and small frequency differences between the Mesoamerican and South American samples. This genomewide AM panel will make it possible to apply AM approaches in many admixed populations throughout the Americas. [Abstract/Link to Full Text]

Jenkins D, Seelow D, Jehee FS, Perlyn CA, Alonso LG, Bueno DF, Donnai D, Josifova D, Josifiova D, Mathijssen IM, Morton JE, Orstavik KH, Sweeney E, Wall SA, Marsh JL, Nurnberg P, Passos-Bueno MR, Wilkie AO
RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity.
Am J Hum Genet. 2007 Jun;80(6):1162-70.
Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity. [Abstract/Link to Full Text]

Freathy RM, Weedon MN, Bennett A, Hypponen E, Relton CL, Knight B, Shields B, Parnell KS, Groves CJ, Ring SM, Pembrey ME, Ben-Shlomo Y, Strachan DP, Power C, Jarvelin MR, McCarthy MI, Davey Smith G, Hattersley AT, Frayling TM
Type 2 diabetes TCF7L2 risk genotypes alter birth weight: a study of 24,053 individuals.
Am J Hum Genet. 2007 Jun;80(6):1150-61.
The role of genes in normal birth-weight variation is poorly understood, and it has been suggested that the genetic component of fetal growth is small. Type 2 diabetes genes may influence birth weight through maternal genotype, by increasing maternal glycemia in pregnancy, or through fetal genotype, by altering fetal insulin secretion. We aimed to assess the role of the recently described type 2 diabetes gene TCF7L2 in birth weight. We genotyped the polymorphism rs7903146 in 15,709 individuals whose birth weight was available from six studies and in 8,344 mothers from three studies. Each fetal copy of the predisposing allele was associated with an 18-g (95% confidence interval [CI] 7-29 g) increase in birth weight (P=.001) and each maternal copy with a 30-g (95% CI 15-45 g) increase in offspring birth weight (P=2.8x10-5). Stratification by fetal genotype suggested that the association was driven by maternal genotype (31-g [95% CI 9-48 g] increase per allele; corrected P=.003). Analysis of diabetes-related traits in 10,314 nondiabetic individuals suggested the most likely mechanism is that the risk allele reduces maternal insulin secretion (disposition index reduced by ~0.15 standard deviation; P=1x10-4), which results in increased maternal glycemia in pregnancy and hence increased offspring birth weight. We combined information with the other common variant known to alter fetal growth, the -30G-->A polymorphism of glucokinase (rs1799884). The 4% of offspring born to mothers carrying three or four risk alleles were 119 g (95% CI 62-172 g) heavier than were the 32% born to mothers with none (for overall trend, P=2x10-7), comparable to the impact of maternal smoking during pregnancy. In conclusion, we have identified the first type 2 diabetes-susceptibility allele to be reproducibly associated with birth weight. Common gene variants can substantially influence normal birth-weight variation. [Abstract/Link to Full Text]

Muers MR, Sharpe JA, Garrick D, Sloane-Stanley J, Nolan PM, Hacker T, Wood WG, Higgs DR, Gibbons RJ
Defining the cause of skewed X-chromosome inactivation in X-linked mental retardation by use of a mouse model.
Am J Hum Genet. 2007 Jun;80(6):1138-49.
Extreme skewing of X-chromosome inactivation (XCI) is rare in the normal female population but is observed frequently in carriers of some X-linked mutations. Recently, it has been shown that various forms of X-linked mental retardation (XLMR) have a strong association with skewed XCI in female carriers, but the mechanisms underlying this skewing are unknown. ATR-X syndrome, caused by mutations in a ubiquitously expressed, chromatin-associated protein, provides a clear example of XLMR in which phenotypically normal female carriers virtually all have highly skewed XCI biased against the X chromosome that harbors the mutant allele. Here, we have used a mouse model to understand the processes causing skewed XCI. In female mice heterozygous for a null Atrx allele, we found that XCI is balanced early in embryogenesis but becomes skewed over the course of development, because of selection favoring cells expressing the wild-type Atrx allele. Unexpectedly, selection does not appear to be the result of general cellular-viability defects in Atrx-deficient cells, since it is restricted to specific stages of development and is not ongoing throughout the life of the animal. Instead, there is evidence that selection results from independent tissue-specific effects. This illustrates an important mechanism by which skewed XCI may occur in carriers of XLMR and provides insight into the normal role of ATRX in regulating cell fate. [Abstract/Link to Full Text]

Lou XY, Chen GB, Yan L, Ma JZ, Zhu J, Elston RC, Li MD
A generalized combinatorial approach for detecting gene-by-gene and gene-by-environment interactions with application to nicotine dependence.
Am J Hum Genet. 2007 Jun;80(6):1125-37.
The determination of gene-by-gene and gene-by-environment interactions has long been one of the greatest challenges in genetics. The traditional methods are typically inadequate because of the problem referred to as the "curse of dimensionality." Recent combinatorial approaches, such as the multifactor dimensionality reduction (MDR) method, the combinatorial partitioning method, and the restricted partition method, have a straightforward correspondence to the concept of the phenotypic landscape that unifies biological, statistical genetics, and evolutionary theories. However, the existing approaches have several limitations, such as not allowing for covariates, that restrict their practical use. In this study, we report a generalized MDR (GMDR) method that permits adjustment for discrete and quantitative covariates and is applicable to both dichotomous and continuous phenotypes in various population-based study designs. Computer simulations indicated that the GMDR method has superior performance in its ability to identify epistatic loci, compared with current methods in the literature. We applied our proposed method to a genetics study of four genes that were reported to be associated with nicotine dependence and found significant joint action between CHRNB4 and NTRK2. Moreover, our example illustrates that the newly proposed GMDR approach can increase prediction ability, suggesting that its use is justified in practice. In summary, GMDR serves the purpose of identifying contributors to population variation better than do the other existing methods. [Abstract/Link to Full Text]

Dahlman I, Dicker A, Jiao H, Kere J, Blomqvist L, van Harmelen V, Hoffstedt J, Borch-Johnsen K, Jorgensen T, Hansen T, Pedersen O, Laakso M, Arner P
A common haplotype in the G-protein-coupled receptor gene GPR74 is associated with leanness and increased lipolysis.
Am J Hum Genet. 2007 Jun;80(6):1115-24.
The G-protein-coupled receptor GPR74 is a novel candidate gene for body weight regulation. In humans, it is predominantly expressed in brain, heart, and adipose tissue. We report a haplotype in the GPR74 gene, ATAG, with allele frequency ~4% in Scandinavian cohorts, which was associated with protection against obesity in two samples selected for obese and lean phenotypes (odds ratio for obesity 0.48 and 0.62; nominal P=.0014 and .014; n=1,013 and 1,423, respectively). In a population-based sample, it was associated with lower waist (P=.02) among 3,937 men and with obesity protection (odds ratio 0.36; P=.036) among those selected for obese or lean phenotypes. The ATAG haplotype was associated with increased adipocyte lipid mobilization (lipolysis) in vivo and in vitro. In human fat cells, GPR74 receptor stimulation and inhibition caused a significant and marked decrease and increase, respectively, of lipolysis, which could be linked to catecholamine stimulation of adipocytes through beta -adrenergic receptors. These findings suggest that a common haplotype in the GPR74 gene protects against obesity, which, at least in part, is caused by a relief of inhibition of lipid mobilization from adipose tissue. The latter involves a cross-talk between GPR74 and beta -adrenoceptor signaling to lipolysis in fat cells. [Abstract/Link to Full Text]

Balaci L, Spada MC, Olla N, Sole G, Loddo L, Anedda F, Naitza S, Zuncheddu MA, Maschio A, Altea D, Uda M, Pilia S, Sanna S, Masala M, Crisponi L, Fattori M, Devoto M, Doratiotto S, Rassu S, Mereu S, Giua E, Cadeddu NG, Atzeni R, Pelosi U, Corrias A, Perra R, Torrazza PL, Pirina P, Ginesu F, Marcias S, Schintu MG, Del Giacco GS, Manconi PE, Malerba G, Bisognin A, Trabetti E, Boner A, Pescollderungg L, Pignatti PF, Schlessinger D, Cao A, Pilia G
IRAK-M is involved in the pathogenesis of early-onset persistent asthma.
Am J Hum Genet. 2007 Jun;80(6):1103-14.
Asthma is a multifactorial disease influenced by genetic and environmental factors. In the past decade, several loci and >100 genes have been found to be associated with the disease in at least one population. Among these loci, region 12q13-24 has been implicated in asthma etiology in multiple populations, suggesting that it harbors one or more asthma susceptibility genes. We performed linkage and association analyses by transmission/disequilibrium test and case-control analysis in the candidate region 12q13-24, using the Sardinian founder population, in which limited heterogeneity of pathogenetic alleles for monogenic and complex disorders as well as of environmental conditions should facilitate the study of multifactorial traits. We analyzed our cohort, using a cutoff age of 13 years at asthma onset, and detected significant linkage to a portion of 12q13-24. We identified IRAK-M as the gene contributing to the linkage and showed that it is associated with early-onset persistent asthma. We defined protective and predisposing SNP haplotypes and replicated associations in an outbred Italian population. Sequence analysis in patients found mutations, including inactivating lesions, in the IRAK-M coding region. Immunohistochemistry of lung biopsies showed that IRAK-M is highly expressed in epithelial cells. We report that IRAK-M is involved in the pathogenesis of early-onset persistent asthma. IRAK-M, a negative regulator of the Toll-like receptor/IL-1R pathways, is a master regulator of NF- kappa B and inflammation. Our data suggest a mechanistic link between hyperactivation of the innate immune system and chronic airway inflammation and indicate IRAK-M as a potential target for therapeutic intervention against asthma. [Abstract/Link to Full Text]

Miyazawa H, Kato M, Awata T, Kohda M, Iwasa H, Koyama N, Tanaka T, Huqun S, Okazaki Y, Hagiwara K
Homozygosity haplotype allows a genomewide search for the autosomal segments shared among patients.
Am J Hum Genet. 2007 Jun;80(6):1090-102.
A promising strategy for identifying disease susceptibility genes for both single- and multiple-gene diseases is to search patients' autosomes for shared chromosomal segments derived from a common ancestor. Such segments are characterized by the distinct identity of their haplotype. The methods and algorithms currently available have only a limited capability for determining a high-resolution haplotype genomewide. We herein introduce the homozygosity haplotype (HH), a haplotype described by the homozygous SNPs that are easily obtained from high-density SNP genotyping data. The HH represents haplotypes of both copies of homologous autosomes, allowing for direct comparisons of the autosomes among multiple patients and enabling the identification of the shared segments. The HH successfully detected the shared segments from members of a large family with Marfan syndrome, which is an autosomal dominant, single-gene disease. It also detected the shared segments from patients with model multigene diseases originating with common ancestors who lived 10-25 generations ago. The HH is therefore considered to be useful for the identification of disease susceptibility genes in both single- and multiple-gene diseases. [Abstract/Link to Full Text]

Modamio-Hoybjor S, Mencia A, Goodyear R, del Castillo I, Richardson G, Moreno F, Moreno-Pelayo MA
A mutation in CCDC50, a gene encoding an effector of epidermal growth factor-mediated cell signaling, causes progressive hearing loss.
Am J Hum Genet. 2007 Jun;80(6):1076-89.
We previously mapped a novel autosomal dominant deafness locus, DFNA44, by studying a family with postlingual, progressive, nonsyndromic hearing loss. We report here on the identification of a mutation in CCDC50 as the cause of hearing loss in the family. CCDC50 encodes Ymer, an effector of epidermal growth factor (EGF)-mediated cell signaling that is ubiquitously expressed in different organs and has been suggested to inhibit down-regulation of the EGF receptor. We have examined its expression pattern in mouse inner ear. Western blotting and cell transfection results indicate that Ymer is a soluble, cytoplasmic protein, and immunostaining shows that Ymer is expressed in a complex spatiotemporal pattern during inner ear development. In adult inner ear, the expression of Ymer is restricted to the pillar cells of the cochlea, the stria vascularis, and the vestibular sensory epithelia, where it shows spatial overlap with the microtubule-based cytoskeleton. In dividing cells, Ymer colocalizes with microtubules of the mitotic apparatus. We suggest that DFNA44 hearing loss may result from a time-dependent disorganization of the microtubule-based cytoskeleton in the pillar cells and stria vascularis of the adult auditory system. [Abstract/Link to Full Text]

Costa MF, Oliveira AG, Feitosa-Santana C, Zatz M, Ventura DF
Red-green color vision impairment in Duchenne muscular dystrophy.
Am J Hum Genet. 2007 Jun;80(6):1064-75.
The present study evaluated the color vision of 44 patients with Duchenne muscular dystrophy (DMD) (mean age 14.8 years; SD 4.9) who were submitted to a battery of four different color tests: Cambridge Colour Test (CCT), Neitz Anomaloscope, Ishihara, and American Optical Hardy-Rand-Rittler (AO H-R-R). Patients were divided into two groups according to the region of deletion in the dystrophin gene: upstream of exon 30 (n=12) and downstream of exon 30 (n=32). The control group was composed of 70 age-matched healthy male subjects with no ophthalmological complaints. Of the patients with DMD, 47% (21/44) had a red-green color vision defect in the CCT, confirmed by the Neitz Anomaloscope with statistical agreement (P<.001). The Ishihara and the AO H-R-R had a lower capacity to detect color defects--5% and 7%, respectively, with no statistical similarity between the results of these two tests nor between CCT and Anomaloscope results (P>.05). Of the patients with deletion downstream of exon 30, 66% had a red-green color defect. No color defect was found in the patients with deletion upstream of exon 30. A negative correlation between the color thresholds and age was found for the controls and patients with DMD, suggesting a nonprogressive color defect. The percentage (66%) of patients with a red-green defect was significantly higher than the expected <10% for the normal male population (P<.001). In contrast, patients with DMD with deletion upstream of exon 30 had normal color vision. This color defect might be partially explained by a retina impairment related to dystrophin isoform Dp260. [Abstract/Link to Full Text]

Yang T, Vidarsson H, Rodrigo-Blomqvist S, Rosengren SS, Enerback S, Smith RJ
Transcriptional control of SLC26A4 is involved in Pendred syndrome and nonsyndromic enlargement of vestibular aqueduct (DFNB4).
Am J Hum Genet. 2007 Jun;80(6):1055-63.
Although recessive mutations in the anion transporter gene SLC26A4 are known to be responsible for Pendred syndrome (PS) and nonsyndromic hearing loss associated with enlarged vestibular aqueduct (EVA), also known as "DFNB4," a large percentage of patients with this phenotype lack mutations in the SLC26A4 coding region in one or both alleles. We have identified and characterized a key transcriptional regulatory element in the SLC26A4 promoter that binds FOXI1, a transcriptional activator of SLC26A4. In nine patients with PS or nonsyndromic EVA, a novel c.-103T-->C mutation in this regulatory element interferes with FOXI1 binding and completely abolishes FOXI1-mediated transcriptional activation. We have also identified six patients with mutations in FOXI1 that compromise its ability to activate SLC26A4 transcription. In one family, the EVA phenotype segregates in a double-heterozygous mode in the affected individual who carries single mutations in both SLC26A4 and FOXI1. This finding is consistent with our observation that EVA occurs in the Slc26a4(+/-); Foxi1(+/-) double-heterozygous mouse mutant. These results support a novel dosage-dependent model for the molecular pathogenesis of PS and nonsyndromic EVA that involves SLC26A4 and its transcriptional regulatory machinery. [Abstract/Link to Full Text]

Yang Y, Chung EK, Wu YL, Savelli SL, Nagaraja HN, Zhou B, Hebert M, Jones KN, Shu Y, Kitzmiller K, Blanchong CA, McBride KL, Higgins GC, Rennebohm RM, Rice RR, Hackshaw KV, Roubey RA, Grossman JM, Tsao BP, Birmingham DJ, Rovin BH, Hebert LA, Yu CY
Gene copy-number variation and associated polymorphisms of complement component C4 in human systemic lupus erythematosus (SLE): low copy number is a risk factor for and high copy number is a protective factor against SLE susceptibility in European Americans.
Am J Hum Genet. 2007 Jun;80(6):1037-54.
Interindividual gene copy-number variation (CNV) of complement component C4 and its associated polymorphisms in gene size (long and short) and protein isotypes (C4A and C4B) probably lead to different susceptibilities to autoimmune disease. We investigated the C4 gene CNV in 1,241 European Americans, including patients with systemic lupus erythematosus (SLE), their first-degree relatives, and unrelated healthy subjects, by definitive genotyping and phenotyping techniques. The gene copy number (GCN) varied from 2 to 6 for total C4, from 0 to 5 for C4A, and from 0 to 4 for C4B. Four copies of total C4, two copies of C4A, and two copies of C4B were the most common GCN counts, but each constituted only between one-half and three-quarters of the study populations. Long C4 genes were strongly correlated with C4A (R=0.695; P<.0001). Short C4 genes were correlated with C4B (R=0.437; P<.0001). In comparison with healthy subjects, patients with SLE clearly had the GCN of total C4 and C4A shifting to the lower side. The risk of SLE disease susceptibility significantly increased among subjects with only two copies of total C4 (patients 9.3%; unrelated controls 1.5%; odds ratio [OR] = 6.514; P=.00002) but decreased in those with > or =5 copies of C4 (patients 5.79%; controls 12%; OR=0.466; P=.016). Both zero copies (OR=5.267; P=.001) and one copy (OR=1.613; P=.022) of C4A were risk factors for SLE, whereas > or =3 copies of C4A appeared to be protective (OR=0.574; P=.012). Family-based association tests suggested that a specific haplotype with a single short C4B in tight linkage disequilibrium with the -308A allele of TNFA was more likely to be transmitted to patients with SLE. This work demonstrates how gene CNV and its related polymorphisms are associated with the susceptibility to a human complex disease. [Abstract/Link to Full Text]

Price AL, Patterson N, Yu F, Cox DR, Waliszewska A, McDonald GJ, Tandon A, Schirmer C, Neubauer J, Bedoya G, Duque C, Villegas A, Bortolini MC, Salzano FM, Gallo C, Mazzotti G, Tello-Ruiz M, Riba L, Aguilar-Salinas CA, Canizales-Quinteros S, Menjivar M, Klitz W, Henderson B, Haiman CA, Winkler C, Tusie-Luna T, Ruiz-Linares A, Reich D
A genomewide admixture map for Latino populations.
Am J Hum Genet. 2007 Jun;80(6):1024-36.
Admixture mapping is an economical and powerful approach for localizing disease genes in populations of recently mixed ancestry and has proven successful in African Americans. The method holds equal promise for Latinos, who typically inherit a mix of European, Native American, and African ancestry. However, admixture mapping in Latinos has not been practical because of the lack of a map of ancestry-informative markers validated in Native American and other populations. To address this, we screened multiple databases, containing millions of markers, to identify 4,186 markers that were putatively informative for determining the ancestry of chromosomal segments in Latino populations. We experimentally validated each of these markers in at least 232 new Latino, European, Native American, and African samples, and we selected a subset of 1,649 markers to form an admixture map. An advantage of our strategy is that we focused our map on markers distinguishing Native American from other ancestries and restricted it to markers with very similar frequencies in Europeans and Africans, which decreased the number of markers needed and minimized the possibility of false disease associations. We evaluated the effectiveness of our map for localizing disease genes in four Latino populations from both North and South America. [Abstract/Link to Full Text]

Zweier C, Peippo MM, Hoyer J, Sousa S, Bottani A, Clayton-Smith J, Reardon W, Saraiva J, Cabral A, Gohring I, Devriendt K, de Ravel T, Bijlsma EK, Hennekam RC, Orrico A, Cohen M, Dreweke A, Reis A, Nurnberg P, Rauch A
Haploinsufficiency of TCF4 causes syndromal mental retardation with intermittent hyperventilation (Pitt-Hopkins syndrome).
Am J Hum Genet. 2007 May;80(5):994-1001.
Pitt-Hopkins syndrome is a rarely reported syndrome of so-far-unknown etiology characterized by mental retardation, wide mouth, and intermittent hyperventilation. By molecular karyotyping with GeneChip Human Mapping 100K SNP arrays, we detected a 1.2-Mb deletion on 18q21.2 in one patient. Sequencing of the TCF4 transcription factor gene, which is contained in the deletion region, in 30 patients with significant phenotypic overlap revealed heterozygous stop, splice, and missense mutations in five further patients with severe mental retardation and remarkable facial resemblance. Thus, we establish the Pitt-Hopkins syndrome as a distinct but probably heterogeneous entity caused by autosomal dominant de novo mutations in TCF4. Because of its phenotypic overlap, Pitt-Hopkins syndrome evolves as an important differential diagnosis to Angelman and Rett syndromes. Both null and missense mutations impaired the interaction of TCF4 with ASCL1 from the PHOX-RET pathway in transactivating an E box-containing reporter construct; therefore, hyperventilation and Hirschsprung disease in patients with Pitt-Hopkins syndrome might be explained by altered development of noradrenergic derivatives. [Abstract/Link to Full Text]

Amiel J, Rio M, de Pontual L, Redon R, Malan V, Boddaert N, Plouin P, Carter NP, Lyonnet S, Munnich A, Colleaux L
Mutations in TCF4, encoding a class I basic helix-loop-helix transcription factor, are responsible for Pitt-Hopkins syndrome, a severe epileptic encephalopathy associated with autonomic dysfunction.
Am J Hum Genet. 2007 May;80(5):988-93.
Pitt-Hopkins syndrome (PHS) is a rare syndromic encephalopathy characterized by daily bouts of hyperventilation and a facial gestalt. We report a 1.8-Mb de novo microdeletion on chromosome 18q21.1, identified by array-comparative genomic hybridization in one patient with PHS. We subsequently identified two de novo heterozygous missense mutations of a conserved amino acid in the basic region of the TCF4 gene in three additional subjects with PHS. These findings demonstrate that TCF4 anomalies are responsible for PHS and provide the first evidence of a human disorder related to class I basic helix-loop-helix transcription-factor defects (also known as "E proteins"). Moreover, our data may shed new light on the normal processes underlying autonomic nervous system development and maintenance of an appropriate ventilatory neuronal circuitry. [Abstract/Link to Full Text]

Raymond FL, Tarpey PS, Edkins S, Tofts C, O'Meara S, Teague J, Butler A, Stevens C, Barthorpe S, Buck G, Cole J, Dicks E, Gray K, Halliday K, Hills K, Hinton J, Jones D, Menzies A, Perry J, Raine K, Shepherd R, Small A, Varian J, Widaa S, Mallya U, Moon J, Luo Y, Shaw M, Boyle J, Kerr B, Turner G, Quarrell O, Cole T, Easton DF, Wooster R, Bobrow M, Schwartz CE, Gecz J, Stratton MR, Futreal PA
Mutations in ZDHHC9, which encodes a palmitoyltransferase of NRAS and HRAS, cause X-linked mental retardation associated with a Marfanoid habitus.
Am J Hum Genet. 2007 May;80(5):982-7.
We have identified one frameshift mutation, one splice-site mutation, and two missense mutations in highly conserved residues in ZDHHC9 at Xq26.1 in 4 of 250 families with X-linked mental retardation (XLMR). In three of the families, the mental retardation phenotype is associated with a Marfanoid habitus, although none of the affected individuals meets the Ghent criteria for Marfan syndrome. ZDHHC9 is a palmitoyltransferase that catalyzes the posttranslational modification of NRAS and HRAS. The degree of palmitoylation determines the temporal and spatial location of these proteins in the plasma membrane and Golgi complex. The finding of mutations in ZDHHC9 suggests that alterations in the concentrations and cellular distribution of target proteins are sufficient to cause disease. This is the first XLMR gene to be reported that encodes a posttranslational modification enzyme, palmitoyltransferase. Furthermore, now that the first palmitoyltransferase that causes mental retardation has been identified, defects in other palmitoylation transferases become good candidates for causing other mental retardation syndromes. [Abstract/Link to Full Text]

Crisponi L, Crisponi G, Meloni A, Toliat MR, Nurnberg G, Usala G, Uda M, Masala M, Hohne W, Becker C, Marongiu M, Chiappe F, Kleta R, Rauch A, Wollnik B, Strasser F, Reese T, Jakobs C, Kurlemann G, Cao A, Nurnberg P, Rutsch F
Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1.
Am J Hum Genet. 2007 May;80(5):971-81.
Crisponi syndrome is a severe autosomal recessive condition that is phenotypically characterized by abnormal, paroxysmal muscular contractions resembling neonatal tetanus, large face, broad nose, anteverted nares, camptodactyly, hyperthermia, and sudden death in most cases. We performed homozygosity mapping in five Sardinian and three Turkish families with Crisponi syndrome, using high-density single-nucleotide polymorphism arrays, and identified a critical region on chromosome 19p12-13.1. The most prominent candidate gene was CRLF1, recently found to be involved in the pathogenesis of cold-induced sweating syndrome type 1 (CISS1). CISS1 belongs to a group of conditions with overlapping phenotypes, also including cold-induced sweating syndrome type 2 and Stuve-Wiedemann syndrome. All these syndromes are caused by mutations of genes of the ciliary neurotrophic factor (CNTF)-receptor pathway. Here, we describe the identification of four different CRLF1 mutations in eight different Crisponi-affected families, including a missense mutation, a single-nucleotide insertion, and a nonsense and an insertion/deletion (indel) mutation, all segregating with the disease trait in the families. Comparison of the mutation spectra of Crisponi syndrome and CISS1 suggests that neither the type nor the location of the CRLF1 mutations points to a phenotype/genotype correlation that would account for the most severe phenotype in Crisponi syndrome. Other, still-unknown molecular factors may be responsible for the variable phenotypic expression of the CRLF1 mutations. We suggest that the syndromes can comprise a family of "CNTF-receptor-related disorders," of which Crisponi syndrome would be the newest member and allelic to CISS1. [Abstract/Link to Full Text]

Dagoneau N, Bellais S, Blanchet P, Sarda P, Al-Gazali LI, Di Rocco M, Huber C, Djouadi F, Le Goff C, Munnich A, Cormier-Daire V
Mutations in cytokine receptor-like factor 1 (CRLF1) account for both Crisponi and cold-induced sweating syndromes.
Am J Hum Genet. 2007 May;80(5):966-70.
Crisponi syndrome is a rare autosomal recessive disorder characterized by congenital muscular contractions of facial muscles, with trismus in response to stimuli, dysmorphic features, bilateral camptodactyly, major feeding and respiratory difficulties, and access of hyperthermia leading to death in the first months of life. The overlap with Stuve-Wiedemann syndrome (SWS) is striking, but the two conditions differ in that congenital lower limb bowing is absent in Crisponi syndrome, whereas it is a cardinal feature of SWS. We report here the exclusion of the leukemia inhibitory factor receptor gene in Crisponi syndrome and the identification of homozygote or compound heterozygote cytokine receptor-like factor 1 (CRLF1) mutations in four children from three unrelated families. The four mutations were located in the immunoglobulin-like and type III fibronectin domains, and three of them predicted premature termination of translation. Using real-time quantitative polymerase chain reaction, we found a significant decrease in CRLF1 mRNA expression in patient fibroblasts, which is suggestive of a mutation-mediated decay of the abnormal transcript. CRLF1 forms a heterodimer complex with cardiotrophin-like cytokine factor 1, and this heterodimer competes with ciliary neurotrophic factor for binding to the ciliary neurotrophic factor receptor (CNTFR) complex. The identification of CRLF1 mutations in Crisponi syndrome supports the key role of the CNTFR pathway in the function of the autonomic nervous system. [Abstract/Link to Full Text]

Gao X, Gordon D, Zhang D, Browne R, Helms C, Gillum J, Weber S, Devroy S, Swaney S, Dobbs M, Morcuende J, Sheffield V, Lovett M, Bowcock A, Herring J, Wise C
CHD7 gene polymorphisms are associated with susceptibility to idiopathic scoliosis.
Am J Hum Genet. 2007 May;80(5):957-65.
Idiopathic scoliosis (IS) is the most common spinal deformity in children, and its etiology is unknown. To refine the search for genes underlying IS susceptibility, we ascertained a new cohort of 52 families and conducted a follow-up study of genomewide scans that produced evidence of linkage and association with 8q12 loci (multipoint LOD 2.77; P=.0028). Further fine mapping in the region revealed significant evidence of disease-associated haplotypes (P<1.0 x 10-4) centering over exons 2-4 of the CHD7 gene associated with the CHARGE (coloboma of the eye, heart defects, atresia of the choanae, retardation of growth and/or development, genital and/or urinary abnormalities, and ear abnormalities and deafness) syndrome of multiple developmental anomalies. Resequencing CHD7 exons and conserved intronic sequence blocks excluded coding changes but revealed at least one potentially functional polymorphism that is overtransmitted (P=.005) to affected offspring and predicts disruption of a caudal-type (cdx) transcription-factor binding site. Our results identify the first gene associated with IS susceptibility and suggest etiological overlap between the rare, early-onset CHARGE syndrome and common, later-onset IS. [Abstract/Link to Full Text]

Bauchet M, McEvoy B, Pearson LN, Quillen EE, Sarkisian T, Hovhannesyan K, Deka R, Bradley DG, Shriver MD
Measuring European population stratification with microarray genotype data.
Am J Hum Genet. 2007 May;80(5):948-56.
A proper understanding of population genetic stratification--differences in individual ancestry within a population--is crucial in attempts to find genes for complex traits through association mapping. We report on genomewide typing of approximately 10,000 single-nucleotide polymorphisms in 297 individuals, to explore population structure in Europeans of known and unknown ancestry. The results reveal the presence of several significant axes of stratification, most prominently in a northern-southeastern trend, but also along an east-west axis. We also demonstrate the selection and application of EuroAIMs (European ancestry informative markers) for ancestry estimation and correction. The Coriell Caucasian and CEPH (Centre d'Etude du Polymorphisme Humain) Utah sample panels, often used as proxies for European populations, are found to reflect different subsets of the continent's ancestry. [Abstract/Link to Full Text]

Balciuniene J, Feng N, Iyadurai K, Hirsch B, Charnas L, Bill BR, Easterday MC, Staaf J, Oseth L, Czapansky-Beilman D, Avramopoulos D, Thomas GH, Borg A, Valle D, Schimmenti LA, Selleck SB
Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities.
Am J Hum Genet. 2007 May;80(5):938-47.
Low-copy repeats (LCRs) are genomic features that affect chromosome stability and can produce disease-associated rearrangements. We describe members of three families with deletions in 10q22.3-q23.31, a region harboring a complex set of LCRs, and demonstrate that rearrangements in this region are associated with behavioral and neurodevelopmental abnormalities, including cognitive impairment, autism, hyperactivity, and possibly psychiatric disease. Fine mapping of the deletions in members of all three families by use of a custom 10q oligonucleotide array-based comparative genomic hybridization (NimbleGen) and polymerase chain reaction-based methods demonstrated a different deletion in each family. In one proband, the deletion breakpoints are associated with DNA fragments containing noncontiguous sequences of chromosome 10, whereas, in the other two families, the breakpoints are within paralogous LCRs, removing approximately 7.2 Mb and 32 genes. Our data provide evidence that the 10q22-q23 genomic region harbors one or more genes important for cognitive and behavioral development and that recurrent deletions affecting this interval define a novel genomic disorder. [Abstract/Link to Full Text]

Hart CE, Race V, Achouri Y, Wiame E, Sharrard M, Olpin SE, Watkinson J, Bonham JR, Jaeken J, Matthijs G, Van Schaftingen E
Phosphoserine aminotransferase deficiency: a novel disorder of the serine biosynthesis pathway.
Am J Hum Genet. 2007 May;80(5):931-7.
We present the first two identified cases of phosphoserine aminotransferase deficiency. This disorder of serine biosynthesis has been identified in two siblings who showed low concentrations of serine and glycine in plasma and cerebrospinal fluid. Clinically, the index patient presented with intractable seizures, acquired microcephaly, hypertonia, and psychomotor retardation and died at age 7 mo despite supplementation with serine (500 mg/kg/d) and glycine (200 mg/kg/d) from age 11 wk. The younger sibling received treatment from birth, which led to a normal outcome at age 3 years. Measurement of phosphoserine aminotransferase activity in cultured fibroblasts in the index patient was inconclusive, but mutational analysis revealed compound heterozygosity for two mutations in the PSAT1 gene--one frameshift mutation (c.delG107) and one missense mutation (c.299A-->C [p.Asp100Ala])--in both siblings. Expression studies of the p.Asp100Ala mutant protein revealed a V(max) of only 15% of that of the wild-type protein. [Abstract/Link to Full Text]

Epstein MP, Allen AS, Satten GA
A simple and improved correction for population stratification in case-control studies.
Am J Hum Genet. 2007 May;80(5):921-30.
Population stratification remains an important issue in case-control studies of disease-marker association, even within populations considered to be genetically homogeneous. Campbell et al. (Nature Genetics 2005;37:868-872) illustrated this by showing that stratification induced a spurious association between the lactase gene (LCT) and tall/short status in a European American sample. Furthermore, existing approaches for controlling stratification by use of substructure-informative loci (e.g., genomic control, structured association, and principal components) could not resolve this confounding. To address this problem, we propose a simple two-step procedure. In the first step, we model the odds of disease, given data on substructure-informative loci (excluding the test locus). For each participant, we use this model to calculate a stratification score, which is that participant's estimated odds of disease calculated using his or her substructure-informative-loci data in the disease-odds model. In the second step, we assign subjects to strata defined by stratification score and then test for association between the disease and the test locus within these strata. The resulting association test is valid even in the presence of population stratification. Our approach is computationally simple and less model dependent than are existing approaches for controlling stratification. To illustrate these properties, we apply our approach to the data from Campbell et al. and find no association between the LCT locus and tall/short status. Using simulated data, we show that our approach yields a more appropriate correction for stratification than does principal components or genomic control. [Abstract/Link to Full Text]

Wang T, Zhu X, Elston RC
Improving power in contrasting linkage-disequilibrium patterns between cases and controls.
Am J Hum Genet. 2007 May;80(5):911-20.
Genetic association studies offer an opportunity to find genetic variants underlying complex human diseases. The success of this approach depends on the linkage disequilibrium (LD) between markers and the disease variant(s) in a local region of the genome. Because, in the region with a disease mutation, the LD pattern among markers may differ between cases and controls, in some scenarios, it is useful to compare a measure of this LD, to map disease mutations. For example, using the composite correlation to characterize the LD among markers, Zaykin et al. recently suggested an "LD contrast" test and showed that it has high power under certain haplotype-driven disease models. Furthermore, it is likely that individual variants observed at different positions in a gene act jointly with each other to influence the phenotype, and the LD contrast test is also a useful method to detect such joint action. However, the LD among markers introduced by mutations and their joint action is usually confounded by background LD, which is measured at the population level, especially in a local region with disease mutations. Because the measures of LD that are usually used, such as the composite correlation, represent both effects, they may not be optimal for the purpose of detecting association when high background LD exists. Here, we describe a test that improves the LD contrast test by taking into account the background LD. Because the proposed test is developed in a regression framework, it is very flexible and can be extended to continuous traits and to incorporate covariates. Our simulation results demonstrate the validity and substantially higher power of the proposed method over current methods. Finally, we illustrate our new method by applying it to real data from the International Collaborative Study on Hypertension in Blacks. [Abstract/Link to Full Text]

Kalb R, Neveling K, Hoehn H, Schneider H, Linka Y, Batish SD, Hunt C, Berwick M, Callen E, Surralles J, Casado JA, Bueren J, Dasi A, Soulier J, Gluckman E, Zwaan CM, van Spaendonk R, Pals G, de Winter JP, Joenje H, Grompe M, Auerbach AD, Hanenberg H, Schindler D
Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype.
Am J Hum Genet. 2007 May;80(5):895-910.
FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA. [Abstract/Link to Full Text]

Gayden T, Cadenas AM, Regueiro M, Singh NB, Zhivotovsky LA, Underhill PA, Cavalli-Sforza LL, Herrera RJ
The Himalayas as a directional barrier to gene flow.
Am J Hum Genet. 2007 May;80(5):884-94.
High-resolution Y-chromosome haplogroup analyses coupled with Y-short tandem repeat (STR) haplotypes were used to (1) investigate the genetic affinities of three populations from Nepal--including Newar, Tamang, and people from cosmopolitan Kathmandu (referred to as "Kathmandu" subsequently)--as well as a collection from Tibet and (2) evaluate whether the Himalayan mountain range represents a geographic barrier for gene flow between the Tibetan plateau and the South Asian subcontinent. The results suggest that the Tibetans and Nepalese are in part descendants of Tibeto-Burman-speaking groups originating from Northeast Asia. All four populations are represented predominantly by haplogroup O3a5-M134-derived chromosomes, whose Y-STR-based age (+/-SE) was estimated at 8.1+/-2.9 thousand years ago (KYA), more recent than its Southeast Asian counterpart. The most pronounced difference between the two regions is reflected in the opposing high-frequency distributions of haplogroups D in Tibet and R in Nepal. With the exception of Tamang, both Newar and Kathmandu exhibit considerable similarities to the Indian Y-haplogroup distribution, particularly in their haplogroup R and H composition. These results indicate gene flow from the Indian subcontinent and, in the case of haplogroup R, from Eurasia as well, a conclusion that is also supported by the admixture analysis. In contrast, whereas haplogroup D is completely absent in Nepal, it accounts for 50.6% of the Tibetan Y-chromosome gene pool. Coalescent analyses suggest that the expansion of haplogroup D derivatives--namely, D1-M15 and D3-P47 in Tibet--involved two different demographic events (5.1+/-1.8 and 11.3+/-3.7 KYA, respectively) that are more recent than those of D2-M55 representatives common in Japan. Low frequencies, relative to Nepal, of haplogroup J and R lineages in Tibet are also consistent with restricted gene flow from the subcontinent. Yet the presence of haplogroup O3a5-M134 representatives in Nepal indicates that the Himalayas have been permeable to dispersals from the east. These genetic patterns suggest that this cordillera has been a biased bidirectional barrier. [Abstract/Link to Full Text]

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Abrusan G, Krambeck HJ, Junier T, Giordano J, Warburton PE
Biased distributions and decay of LINEs in the chicken genome.
Genetics. 2007 Oct 18;
The genomes of birds are much smaller than mammalian genomes, and transposable elements (TEs) make up only 10% of the chicken genome, compared with the 45% of the human genome. To study the mechanisms that constrain the copy numbers of TEs, and in consequence the genome size of birds, we analysed the distributions of LINEs (CR1s) and SINEs (MIRs) on the chicken autosomes and Z chromosome. We show, that: 1) CR1 repeats are longest on the Z chromosome, and their length is negatively correlated with the local GC content, 2) The decay of CR1 elements is highly biased, the 5' ends of the insertions are lost much faster than their 3' ends, 3) The GC distribution of CR1 repeats shows a bimodal pattern, repeats are enriched in both AT rich and GC rich regions of the genome, but the CR1 families show large differences in their GC distribution and their change over time. 4) The few MIRs in the chicken are most abundant in regions with intermediate GC content. Our results indicate that the primary mechanism that removes repeats from the chicken genome is ectopic exchange, and the low abundance of repeats in avian genomes is likely to be the consequence of their high recombination rates. [Abstract/Link to Full Text]

Zhang K, Rosenberg NA
On the genealogy of a duplicated microsatellite.
Genetics. 2007 Dec;177(4):2109-22.
When a microsatellite locus is duplicated in a diploid organism, a single pair of PCR primers may amplify as many as four distinct alleles. To study the evolution of a duplicated microsatellite, we consider a coalescent model with symmetric stepwise mutation. Conditional on the time of duplication and a mutation rate, both in a model of completely unlinked loci and in a model of completely linked loci, we compute the probabilities for a sampled diploid individual to amplify one, two, three, or four distinct alleles with one pair of microsatellite PCR primers. These probabilities are then studied to examine the nature of their dependence on the duplication time and the mutation rate. The mutation rate is observed to have a stronger effect than the duplication time on the four probabilities, and the unlinked and linked cases are seen to behave similarly. Our results can be useful for helping to interpret genetic variation at microsatellite loci in species with a very recent history of gene and genome duplication. [Abstract/Link to Full Text]

Davies JL, Simancík F, Lyngsř R, Mailund T, Hein J
On recombination-induced multiple and simultaneous coalescent events.
Genetics. 2007 Dec;177(4):2151-60.
Coalescent theory deals with the dynamics of how sampled genetic material has spread through a population from a single ancestor over many generations and is ubiquitous in contemporary molecular population genetics. Inherent in most applications is a continuous-time approximation that is derived under the assumption that sample size is small relative to the actual population size. In effect, this precludes multiple and simultaneous coalescent events that take place in the history of large samples. If sequences do not recombine, the number of sequences ancestral to a large sample is reduced sufficiently after relatively few generations such that use of the continuous-time approximation is justified. However, in tracing the history of large chromosomal segments, a large recombination rate per generation will consistently maintain a large number of ancestors. This can create a major disparity between discrete-time and continuous-time models and we analyze its importance, illustrated with model parameters typical of the human genome. The presence of gene conversion exacerbates the disparity and could seriously undermine applications of coalescent theory to complete genomes. However, we show that multiple and simultaneous coalescent events influence global quantities, such as total number of ancestors, but have negligible effect on local quantities, such as linkage disequilibrium. Reassuringly, most applications of the coalescent model with recombination (including association mapping) focus on local quantities. [Abstract/Link to Full Text]

Huerta-Sanchez E, Durrett R, Bustamante CD
Population genetics of polymorphism and divergence under fluctuating selection.
Genetics. 2007 Oct 18;
Current methods for detecting fluctuating selection require time series data on genotype frequencies. Here, we propose an alternative approach which makes use of DNA polymorphism data from a sample of individuals collected at a single point in time. Our method uses classical diffusion approximations to model temporal fluctuations in the selection coefficients in order to find the expected distribution of mutation frequencies in the population (TAKAHATA et al. (1975); GILLESPIE (1973)). Using the Poisson Random Field setting (SAWYER and HARTL (1992), HARTL et al. (1994)), we derive the site frequency spectrum (SFS) for three different models of fluctuating selection. We find that the general effect of fluctuating selection is to produce a more "U" shaped site-frequency spectrum with an excess of high frequency derived mutations at the expense of middle frequency variants. We present likelihood ratio tests comparing the fluctuating selection models to the neutral model using SFS data, and use Monte Carlo simulations to assess their power. We find that we have sufficient power to reject a neutral hypothesis using samples on the order of a few hundred SNPs and a sample size of about 20, and also power to distinguish between selection that varies in time and constant selection. We also find that fluctuating selection increases the probability of fixation of selected sites even if, on average, there is no difference in selection among a pair of alleles segregating at the locus. Fluctuating selection will, therefore, lead to an increase in the ratio of divergence to polymorphism similar to that observed under positive directional selection. [Abstract/Link to Full Text]

Tanaka K, Takehana Y, Naruse K, Hamaguchi S, Sakaizumi M
Evidence for different origins of sex chromosomes in closely related oryzias fishes: substitution of the master sex-determining gene.
Genetics. 2007 Dec;177(4):2075-81.
The medaka Oryzias latipes and its two sister species, O. curvinotus and O. luzonensis, possess an XX-XY sex-determination system. The medaka sex-determining gene DMY has been identified on the orthologous Y chromosome [O. latipes linkage group 1 (LG1)] of O. curvinotus. However, DMY has not been discovered in other Oryzias species. These results and molecular phylogeny suggest that DMY was generated recently [ approximately 10 million years ago (MYA)] by gene duplication of DMRT1 in a common ancestor of O. latipes and O. curvinotus. We identified seven sex-linked markers from O. luzonensis (sister species of O. curvinotus) and constructed a sex-linkage map. Surprisingly, all seven sex-linked markers were located on an autosomal linkage group (LG12) of O. latipes. As suggested by the phylogenetic tree, the sex chromosomes of O. luzonensis should be "younger" than those of O. latipes. In the lineage leading to O. luzonensis after separation from O. curvinotus approximately 5 MYA, a novel sex-determining gene may have arisen and substituted for DMY. Oryzias species should provide a useful model for evolution of the master sex-determining gene and differentiation of sex chromosomes from autosomes. [Abstract/Link to Full Text]

Powell AE, Nicotra ML, Moreno MA, Lakkis FG, Dellaporta SL, Buss LW
Differential Effect of Allorecognition Loci on Phenotype in Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa).
Genetics. 2007 Dec;177(4):2101-7.
The allorecognition complex of Hydractinia symbiolongicarpus is a chromosomal interval containing two loci, alr1 and alr2, that controls fusion between genetically distinct colonies. Recombination between these two loci has been associated with a heterogeneous class of phenotypes called transitory fusion. A large-scale backcross was performed to generate a population of colonies (N = 106) with recombination breakpoints within the allorecognition complex. Two distinct forms of transitory fusion were correlated with reciprocal recombination products, suggesting that alr1 and alr2 contributed differentially to the allorecognition response. Specifically, type I transitory fusion is associated with rapid and persistent separation of allogeneic tissues, whereas type II transitory fusion generates a patchwork of continuously fusing and separating tissues. [Abstract/Link to Full Text]

Pinkel D
Analytical description of mutational effects in competing asexual populations.
Genetics. 2007 Dec;177(4):2135-49.
The adaptation of a population to a new environment is a result of selection operating on a suite of stochastically occurring mutations. This article presents an analytical approach to understanding the population dynamics during adaptation, specifically addressing a system in which periods of growth are separated by selection in bottlenecks. The analysis derives simple expressions for the average properties of the evolving population, including a quantitative description of progressive narrowing of the range of selection coefficients of the predominant mutant cells and of the proportion of mutant cells as a function of time. A complete statistical description of the bottlenecks is also presented, leading to a description of the stochastic behavior of the population in terms of effective mutation times. The effective mutation times are related to the actual mutation times by calculable probability distributions, similar to the selection coefficients being highly restricted in their probable values. This analytical approach is used to model recently published experimental data from a bacterial coculture experiment, and the results are compared to those of a numerical model published in conjunction with the data. Finally, experimental designs that may improve measurements of fitness distributions are suggested. [Abstract/Link to Full Text]

Dole J, Weber DF
Detection of quantitative trait Loci influencing recombination using recombinant inbred lines.
Genetics. 2007 Dec;177(4):2309-19.
The genetic basis of variation in recombination in higher plants is polygenic and poorly understood, despite its theoretical and practical importance. Here a method of detecting quantitative trait loci (QTL) influencing recombination in recombinant inbred lines (RILs) is proposed that relies upon the fact that genotype data within RILs carry the signature of past recombination. Behavior of the segregational genetic variance in numbers of chromosomal crossovers (recombination) over generations is described for self-, full-sib-, and half-sib-generated RILs with no dominance in true crossovers. This genetic variance, which as a fraction of the total phenotypic variance contributes to the statistical power of the method, was asymptotically greatest with half sibbing, less with sibbing, and least with selfing. The statistical power to detect a recombination QTL declined with diminishing QTL effect, genome target size, and marker density. For reasonably tight marker linkage power was greater with less intense inbreeding for later generations and vice versa for early generations. Generational optima for segregation variance and statistical power were found, whose onset and narrowness varied with marker density and mating design, being more pronounced for looser marker linkage. Application of this method to a maize RIL population derived from inbred lines Mo17 and B73 and developed by selfing suggested two putative QTL (LOD > 2.4) affecting certain chromosomes, and using a canonical transformation another putative QTL was detected. However, permutation tests failed to support their presence (experimentwise alpha = 0.05). Other populations with more statistical power and chosen specifically for recombination QTL segregation would be more effective. [Abstract/Link to Full Text]

Wheeler D, Newbigin E
Expression of 10 S-Class SLF-like Genes in Nicotiana alata Pollen and Its Implications for Understanding the Pollen Factor of the S Locus.
Genetics. 2007 Dec;177(4):2171-80.
The S locus of Nicotiana alata encodes a polymorphic series of ribonucleases (S-RNases) that determine the self-incompatibility (SI) phenotype of the style. The pollen product of the S locus (pollen S) in N. alata is unknown, but in species from the related genus Petunia and in self-incompatible members of the Plantaginaceae and Rosaceae, this function has been assigned to an F-box protein known as SLF or SFB. Here we describe the identification of 10 genes (designated DD1-10) encoding SLF-related proteins that are expressed in N. alata pollen. Because our approach to cloning the DD genes was based on sequences of SLFs from other species, we presume that one of the DD genes encodes the N. alata SLF ortholog. Seven of the DD genes were exclusively expressed in pollen and a low level of sequence variation was found in alleles of each DD gene. Mapping studies confirmed that all 10 DD genes were linked to the S locus and that at least three were located in the same chromosomal segment as pollen S. Finally, the different topologies of the phylogenetic trees produced using available SLF-related sequences and those produced using S-RNase sequences suggests that pollen S and the S-RNase have different evolutionary histories. [Abstract/Link to Full Text]

Kim TS, Logsdon BA, Park S, Mezey JG, Lee K
Quantitative Trait Loci for the Circadian Clock in Neurospora crassa.
Genetics. 2007 Dec;177(4):2335-47.
Neurospora crassa has been a model organism for the study of circadian clocks for the past four decades. Among natural accessions of Neurospora crassa, there is significant variation in clock phenotypes. In an attempt to investigate natural allelic variants contributing to quantitative variation, we used a quantitative trait loci mapping approach to analyze three independent mapping populations whose progenitors were collected from geographically isolated locations. Two circadian clock phenotypes, free-running period and entrained phase, were evaluated in the 188 F(1) progeny of each mapping population. To identify the clock QTL, we applied two QTL mapping analyses: composite interval mapping (CIM) and Bayesian multiple QTL analysis (BMQ). When controlling false positive rates </=0.05, BMQ appears to be the more sensitive of the two approaches. BMQ confirmed most of the QTL from CIM (18 QTL) and identified 23 additional QTL. While 13 QTL colocalize with previously identified clock genes, we identified 30 QTL that were not linked with any previously characterized clock genes. These are candidate regions where clock genes may be located and are expected to lead to new insights in clock regulation. [Abstract/Link to Full Text]

Burgio G, Szatanik M, Guénet JL, Arnau MR, Panthier JJ, Montagutelli X
Interspecific Recombinant Congenic Strains Between C57BL/6 and Mice of the Mus spretus Species: A Powerful Tool to Dissect Genetic Control of Complex Traits.
Genetics. 2007 Dec;177(4):2321-33.
Complex traits are under the genetic control of multiple genes, often with weak effects and strong epistatic interactions. We developed two new collections of mouse strains to improve genetic dissection of complex traits. They are derived from several backcrosses of the Mus spretus SEG/Pas or STF/Pas strains on the C57BL/6J background. Each of the 55 interspecific recombinant congenic strains (IRCSs) carries up to eight SEG/Pas chromosomal segments with an average size of 11.7 Mb, totalizing 1.37% of the genome. The complete series covers 39.7% of the SEG/Pas genome. As a complementary resource, six partial or complete interspecific consomic strains were developed and increased genome coverage to 45.6%. To evaluate the usefulness of these strains for QTL mapping, 16 IRCSs were compared with C57BL/6J for seven hematological parameters. Strain 66H, which carries three SEG/Pas chromosomal segments, had lower red blood cell volume and higher platelet count than C57BL/6J. Each chromosomal segment was isolated in a congenic strain to evaluate individual effects. Congenic strains were combined to assess epistasis. Our data show that both traits were controlled by several genes with complex epistatic interactions. IRCSs are therefore useful to unravel QTL with small effects and gene-by-gene interactions. [Abstract/Link to Full Text]

Jeffress JK, Page SL, Royer SK, Belden ED, Blumenstiel JP, Anderson LK, Hawley RS
The formation of the central element of the synaptonemal complex may occur by multiple mechanisms: the roles of the N- and C-terminal domains of the Drosophila c(3)g protein in mediating synapsis and recombination.
Genetics. 2007 Dec;177(4):2445-56.
In Drosophila melanogaster oocytes, the C(3)G protein comprises the transverse filaments (TFs) of the synaptonemal complex (SC). Like other TF proteins, such as Zip1p in yeast and SCP1 in mammals, C(3)G is composed of a central coiled-coil-rich domain flanked by N- and C-terminal globular domains. Here, we analyze in-frame deletions within the N- and C-terminal regions of C(3)G in Drosophila oocytes. As is the case for Zip1p, a C-terminal deletion of C(3)G fails to attach to the lateral elements of the SC. Instead, this C-terminal deletion protein forms a large cylindrical polycomplex structure. EM analysis of this structure reveals a polycomplex of concentric rings alternating dark and light bands. However, unlike both yeast and mammals, all three proteins deleted for N-terminal regions completely abolished both SC and polycomplex formation. Both the N- and C-terminal deletions significantly reduce or abolish meiotic recombination similarly to c(3)G null homozygotes. To explain these data, we propose that in Drosophila the N terminus, but not the C-terminal globular domain, of C(3)G is critical for the formation of antiparallel pairs of C(3)G homodimers that span the central region and thus for assembly of complete TFs, while the C terminus is required to affix these homodimers to the lateral elements. [Abstract/Link to Full Text]

Mather KA, Caicedo AL, Polato NR, Olsen KM, McCouch S, Purugganan MD
The Extent of Linkage Disequilibrium in Rice (Oryza sativa L.).
Genetics. 2007 Dec;177(4):2223-32.
Despite its status as one of the world's major crops, linkage disequilibrium (LD) patterns have not been systematically characterized across the genome of Asian rice (Oryza sativa). Such information is critical to fully exploit the genome sequence for mapping complex traits using association techniques. Here we characterize LD in five 500-kb regions of the rice genome in three major cultivated rice varieties (indica, tropical japonica, and temperate japonica) and in the wild ancestor of Asian rice, Oryza rufipogon. Using unlinked SNPs to determine the amount of background linkage disequilibrium in each population, we find that the extent of LD is greatest in temperate japonica (probably >500 kb), followed by tropical japonica ( approximately 150 kb) and indica ( approximately 75 kb). LD extends over a shorter distance in O. rufipogon (<40 kb) than in any of the O. sativa groups assayed here. The differences in the extent of LD among these groups are consistent with differences in outcrossing and recombination rate estimates. As well as heterogeneity between groups, our results suggest variation in LD patterns among genomic regions. We demonstrate the feasibility of genomewide association mapping in cultivated Asian rice using a modest number of SNPs. [Abstract/Link to Full Text]

Bolkan BJ, Booker R, Goldberg ML, Barbash DA
Developmental and cell cycle progression defects in Drosophila hybrid males.
Genetics. 2007 Dec;177(4):2233-41.
Matings between D. melanogaster females and males of sibling species in the D. melanogaster complex yield hybrid males that die prior to pupal differentiation. We have reexamined a previous report suggesting that the developmental defects in these lethal hybrid males reflect a failure in cell proliferation that may be the consequence of problems in mitotic chromosome condensation. We also observed a failure in cell proliferation, but find in contrast that the frequencies of mitotic figures and of nuclei staining for the mitotic marker phosphohistone H3 in the brains of hybrid male larvae are extremely low. We also found that very few of these brain cells in male hybrids are in S phase, as determined by BrdU incorporation. These data suggest that cells in hybrid males are arrested in either the G(1) or G(2) phases of the cell cycle. The cells in hybrid male brains appear to be particularly sensitive to environmental stress; our results indicate that certain in vitro incubation conditions induce widespread cellular necrosis in these brains, causing an abnormal nuclear morphology noted by previous investigators. We also document that hybrid larvae develop very slowly, particularly during the second larval instar. Finally, we found that the frequency of mitotic figures in hybrid male larvae mutant for Hybrid male rescue (Hmr) is increased relative to lethal hybrid males, although not to wild-type levels, and that chromosome morphology in Hmr(-) hybrid males is also not completely normal. [Abstract/Link to Full Text]

Weber A, Clark RM, Vaughn L, de Jesús Sánchez-Gonzalez J, Yu J, Yandell BS, Bradbury P, Doebley J
Major Regulatory Genes in Maize Contribute to Standing Variation in Teosinte (Zea mays ssp. parviglumis).
Genetics. 2007 Dec;177(4):2349-59.
In plants, many major regulatory genes that control plant growth and development have been identified and characterized. Despite a detailed knowledge of the function of these genes little is known about how they contribute to the natural variation for complex traits. To determine whether major regulatory genes of maize contribute to standing variation in Balsas teosinte we conducted association mapping in 584 Balsas teosinte individuals. We tested 48 markers from nine candidate regulatory genes against 13 traits for plant and inflorescence architecture. We identified significant associations using a mixed linear model that controls for multiple levels of relatedness. Ten associations involving five candidate genes were significant after correction for multiple testing, and two survive the conservative Bonferroni correction. zfl2, the maize homolog of FLORICAULA of Antirrhinum, was associated with plant height. zap1, the maize homolog of APETALA1 of Arabidopsis, was associated with inflorescence branching. Five SNPs in the maize domestication gene, teosinte branched1, were significantly associated with either plant or inflorescence architecture. Our data suggest that major regulatory genes in maize do play a role in the natural variation for complex traits in teosinte and that some of the minor variants we identified may have been targets of selection during domestication. [Abstract/Link to Full Text]

Shpak M
Selection against demographic stochasticity in age-structured populations.
Genetics. 2007 Dec;177(4):2181-94.
It has been shown that differences in fecundity variance can influence the probability of invasion of a genotype in a population; i.e., a genotype with lower variance in offspring number can be favored in finite populations even if it has a somewhat lower mean fitness than a competitor. In this article, Gillespie's results are extended to population genetic systems with explicit age structure, where the demographic variance (variance in growth rate) calculated in the work of Engen and colleagues is used as a generalization of "variance in offspring number" to predict the interaction between deterministic and random forces driving change in allele frequency. By calculating the variance from the life-history parameters, it is shown that selection against variance in the growth rate will favor a genotypes with lower stochasticity in age-specific survival and fertility rates. A diffusion approximation for selection and drift in a population with two genotypes with different life-history matrices (and therefore different mean growth rates and demographic variances) is derived and shown to be consistent with individual-based simulations. It is also argued that for finite populations, perturbation analyses of both the mean and the variance in growth rate may be necessary to determine the sensitivity of fitness to changes in the life-history parameters. [Abstract/Link to Full Text]

Evertts AG, Plymire C, Craig NL, Levin HL
The Hermes Transposon of Musca domestica Is an Efficient Tool for the Mutagenesis of Schizosaccharomyces pombe.
Genetics. 2007 Dec;177(4):2519-23.
Currently, no transposon-based method for the mutagenesis of Schizosaccharomyces pombe exists. We have developed such a system based on the introduction of the hermes transposon from the housefly into S. pombe. This system efficiently disrupts open reading frames and allows the insertion sites to be readily identified. [Abstract/Link to Full Text]

Biedler JK, Shao H, Tu Z
Evolution and Horizontal Transfer of a DD37E DNA Transposon in Mosquitoes.
Genetics. 2007 Dec;177(4):2553-8.
ITmD37E, a unique class II transposable element (TE) with an ancient origin, appears to have been involved in multiple horizontal transfers in mosquitoes as ITmD37E sequences from 10 mosquito species of five genera share high nucleotide (nt) identities. For example, ITmD37E sequences from Aedes aegypti and Anopheles gambiae, which have an estimated common ancestor of 145-200 million years ago, display 92% nt identity. The comparison of ITmD37E and host mosquito phylogenies shows a lack of congruence. The wide distribution of conserved ITmD37Es in mosquitoes and the presence of intact copies suggest that this element may have been recently active. [Abstract/Link to Full Text]

Chen C, Yu Q, Hou S, Li Y, Eustice M, Skelton RL, Veatch O, Herdes RE, Diebold L, Saw J, Feng Y, Qian W, Bynum L, Wang L, Moore PH, Paull RE, Alam M, Ming R
Construction of a sequence-tagged high-density genetic map of papaya for comparative structural and evolutionary genomics in brassicales.
Genetics. 2007 Dec;177(4):2481-91.
A high-density genetic map of papaya (Carica papaya L.) was constructed using microsatellite markers derived from BAC end sequences and whole-genome shot gun sequences. Fifty-four F(2) plants derived from varieties AU9 and SunUp were used for linkage mapping. A total of 707 markers, including 706 microsatellite loci and the morphological marker fruit flesh color, were mapped into nine major and three minor linkage groups. The resulting map spanned 1069.9 cM with an average distance of 1.5 cM between adjacent markers. This sequence-based microsatellite map resolved the very large linkage group 2 (LG 2) of the previous high-density map using amplified fragment length polymorphism markers. The nine major LGs of our map represent papaya's haploid nine chromosomes with LG 1 of the sex chromosome being the largest. This map validates the suppression of recombination at the male-specific region of the Y chromosome (MSY) mapped on LG 1 and at potential centromeric regions of other LGs. Segregation distortion was detected in a large region on LG 1 surrounding the MSY region due to the abortion of the YY genotype and in a region of LG6 due to an unknown cause. This high-density sequence-tagged genetic map is being used to integrate genetic and physical maps and to assign genome sequence scaffolds to papaya chromosomes. It provides a framework for comparative structural and evolutional genomic research in the order Brassicales. [Abstract/Link to Full Text]

Andreescu C, Avendano S, Brown SR, Hassen A, Lamont SJ, Dekkers JC
Linkage disequilibrium in related breeding lines of chickens.
Genetics. 2007 Dec;177(4):2161-9.
High-density genotyping of single-nucleotide polymorphisms (SNPs) enables detection of quantitative trait loci (QTL) by linkage disequilibrium (LD) mapping using LD between markers and QTL and the subsequent use of this information for marker-assisted selection (MAS). The success of LD mapping and MAS depends on the extent of LD in the populations of interest and the use of associations across populations requires LD between loci to be consistent across populations. To assess the extent and consistency of LD in commercial broiler breeding populations, we used genotype data for 959 and 398 SNPs on chromosomes 1 and 4 on 179-244 individuals from each of nine commercial broiler chicken breeding lines. Results show that LD measured by r(2) extends over shorter distances than reported previously in other livestock breeding populations. The LD at short distance (within 1 cM) tended to be consistent across related populations; correlations of LD measured by r for pairs of lines ranged from 0.17 to 0.94 and closely matched the line relationships based on marker allele frequencies. In conclusion, LD-based correlations are good estimates of line relationships and the relationship between a pair of lines a good predictor of LD consistency between the lines. [Abstract/Link to Full Text]

Aladzsity I, Tóth ML, Sigmond T, Szabó E, Bicsák B, Barna J, Regos A, Orosz L, Kovács AL, Vellai T
Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans.
Genetics. 2007 Sep;177(1):655-60.
Here we show that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes unc-51/atg1 and bec-1/atg6/beclin1 results in small body size without affecting cell number. Furthermore, loss-of-function mutations in unc-51 and bec-1 suppress the giant phenotype of mutant animals with aberrant insulin-like growth factor-1 (insulin/IGF-1) or transforming growth factor-beta (TGF-beta) signaling. This function for unc-51 and bec-1 in cell size control and their interaction with these two growth modulatory pathways may represent a link between the hormonal and nutritional regulation of cell growth. [Abstract/Link to Full Text]

Papaceit M, Avila V, Aguadé M, García-Dorado A
The dynamics of the roo transposable element in mutation-accumulation lines and segregating populations of Drosophila melanogaster.
Genetics. 2007 Sep;177(1):511-22.
We estimated the number of copies for the long terminal repeat (LTR) retrotransposable element roo in a set of long-standing Drosophila melanogaster mutation-accumulation full-sib lines and in two large laboratory populations maintained with effective population size approximately 500, all of them derived from the same isogenic origin. Estimates were based on real-time quantitative PCR and in situ hybridization. Considering previous estimates of roo copy numbers obtained at earlier stages of the experiment, the results imply a strong acceleration of the insertion rate in the accumulation lines. The detected acceleration is consistent with a model where only one (maybe a few) of the approximately 70 roo copies in the ancestral isogenic genome was active and each active copy caused new insertions with a relatively high rate ( approximately 10(-2)), with new inserts being active copies themselves. In the two laboratory populations, however, a stabilized copy number or no accelerated insertion was found. Our estimate of the average deleterious viability effects per accumulated insert [E(s) < 0.003] is too small to account for the latter finding, and we discuss the mechanisms that could contain copy number. [Abstract/Link to Full Text]

Lili LN, Britton NF, Feil EJ
The persistence of parasitic plasmids.
Genetics. 2007 Sep;177(1):399-405.
The conditions under which plasmids are predicted to persist remain controversial. Here, we reevaluate the ordinary differential equations used previously to model plasmid persistence and conclude that the parameter space required for maintenance is far less stringent than has been supposed. Strikingly, our model demonstrates that purely parasitic plasmids may persist, even in the absence of heterogeneity in the host population, and that this persistence is expressed by oscillations or damped oscillations between the plasmid-bearing and the plasmid-free class. [Abstract/Link to Full Text]

Díaz-Castillo C, Golic KG
Evolution of gene sequence in response to chromosomal location.
Genetics. 2007 Sep;177(1):359-74.
Evolutionary forces acting on the repetitive DNA of heterochromatin are not constrained by the same considerations that apply to protein-coding genes. Consequently, such sequences are subject to rapid evolutionary change. By examining the Troponin C gene family of Drosophila melanogaster, which has euchromatic and heterochromatic members, we find that protein-coding genes also evolve in response to their chromosomal location. The heterochromatic members of the family show a reduced CG content and increased variation in DNA sequence. We show that the CG reduction applies broadly to the protein-coding sequences of genes located at the heterochromatin:euchromatin interface, with a very strong correlation between CG content and the distance from centric heterochromatin. We also observe a similar trend in the transition from telomeric heterochromatin to euchromatin. We propose that the methylation of DNA is one of the forces driving this sequence evolution. [Abstract/Link to Full Text]

Rawls AS, Schultz SA, Mitra RD, Wolff T
Bedraggled, a putative transporter, influences the tissue polarity complex during the R3/R4 fate decision in the Drosophila eye.
Genetics. 2007 Sep;177(1):313-28.
The tissue polarity pathway is required for the establishment of epithelial polarity in a variety of vertebrate and invertebrate organs. Core tissue polarity proteins act in a dynamically regulated complex to direct the polarization of the Drosophila eye. We report the identification and characterization of bedraggled (bdg), a novel gene that regulates one output of the tissue polarity pathway--the establishment of the R3/R4 photoreceptor fates. bdg encodes a novel, putative transporter protein and interacts genetically with all of the core polarity genes to influence the specification of the R3 and R4 cell fates. Finally, bdg is required for both viability and the initial stages of imaginal disc development. [Abstract/Link to Full Text]

Kiernan AE, Li R, Hawes NL, Churchill GA, Gridley T
Genetic background modifies inner ear and eye phenotypes of jag1 heterozygous mice.
Genetics. 2007 Sep;177(1):307-11.
Mice heterozygous for missense mutations of the Notch ligand Jagged1 (Jag1) exhibit head-shaking behavior indicative of an inner ear vestibular defect. In contrast, mice heterozygous for a targeted deletion of the Jag1 gene (Jag1del1) do not demonstrate obvious head-shaking behavior. To determine whether the differences in inner ear phenotypes were due to the types of Jag1 mutations or to differences in genetic background, we crossed Jag1del1 heterozygous mice onto the same genetic background as the missense mutants. This analysis revealed that variation of the Jag1 mutant inner ear phenotype is caused by genetic background differences and is not due to the type of Jag1 mutation. Genome scans of N2 backcross mice identified a significant modifier locus on chromosome 7, as well as a suggestive locus on chromosome 14. We also analyzed modifiers of an eye defect in Jag1del1 heterozygous mice from this same cross. [Abstract/Link to Full Text]

Ma J, Jin R, Jia X, Dobry CJ, Wang L, Reggiori F, Zhu J, Kumar A
An interrelationship between autophagy and filamentous growth in budding yeast.
Genetics. 2007 Sep;177(1):205-14.
Over the last 15 years, yeast pseudohyphal growth (PHG) has been the focus of intense research interest as a model of fungal pathogenicity. Specifically, PHG is a stress response wherein yeast cells deprived of nitrogen form filaments of elongated cells. Nitrogen limitation also induces autophagy, a ubiquitous eukaryotic stress response in which proteins are trafficked to the vacuole/lysosome for degradation and recycling. Although autophagy and filamentous growth are both responsive to nitrogen stress, a link between these processes has not been investigated to date. Here, we present several studies describing an interrelationship between autophagy and filamentous growth. By microarray-based expression profiling, we detect extensive upregulation of the pathway governing autophagy during early PHG and find both processes active under conditions of nitrogen stress in a filamentous strain of budding yeast. Inhibition of autophagy results in increased PHG, and autophagy-deficient yeast induce PHG at higher concentrations of available nitrogen. Our results suggest a model in which autophagy mitigates nutrient stress, delaying the onset of PHG; conversely, inhibition of autophagy exacerbates nitrogen stress, resulting in precocious and overactive PHG. This physiological connection highlights the central role of autophagy in regulating the cell's nutritional state and the responsiveness of PHG to that state. [Abstract/Link to Full Text]

Rodin S, Kyrchanova O, Pomerantseva E, Parshikov A, Georgiev P
New properties of Drosophila fab-7 insulator.
Genetics. 2007 Sep;177(1):113-21.
In the Abd-B 3' cis-regulatory region, which is subdivided into a series of iab domains, boundary elements have previously been detected, including the Fab-7 element providing for the autonomous functioning of the iab-6 and iab-7 cis-regulatory domains. Here, it has been shown that a single copy of the 860-bp Fab-7 insulator effectively blocks the yellow and white enhancers. The eye and testis enhancers can stimulate the white promoter across the pair of Fab-7, which is indicative of a functional interaction between the insulators. Unexpectedly, Fab-7 has proved to lose the enhancer-blocking activity when placed near the white promoter. It seems likely that Fab-7 strengthens the relatively weak white promoter, which leads to the efficient enhancer-promoter interaction and insulator bypass. [Abstract/Link to Full Text]

Williams BR, Bateman JR, Novikov ND, Wu CT
Disruption of topoisomerase II perturbs pairing in drosophila cell culture.
Genetics. 2007 Sep;177(1):31-46.
Homolog pairing refers to the alignment and physical apposition of homologous chromosomal segments. Although commonly observed during meiosis, homolog pairing also occurs in nonmeiotic cells of several organisms, including humans and Drosophila. The mechanism underlying nonmeiotic pairing, however, remains largely unknown. Here, we explore the use of established Drosophila cell lines for the analysis of pairing in somatic cells. Using fluorescent in situ hybridization (FISH), we assayed pairing at nine regions scattered throughout the genome of Kc167 cells, observing high levels of homolog pairing at all six euchromatic regions assayed and variably lower levels in regions in or near centromeric heterochromatin. We have also observed extensive pairing in six additional cell lines representing different tissues of origin, different ploidies, and two different species, demonstrating homolog pairing in cell culture to be impervious to cell type or culture history. Furthermore, by sorting Kc167 cells into G1, S, and G2 subpopulations, we show that even progression through these stages of the cell cycle does not significantly change pairing levels. Finally, our data indicate that disrupting Drosophila topoisomerase II (Top2) gene function with RNAi and chemical inhibitors perturbs homolog pairing, suggesting Top2 to be a gene important for pairing. [Abstract/Link to Full Text]

Culbertson MR
Navigating without a road map.
Genetics. 2007 Sep;177(1):1-7. [Abstract/Link to Full Text]

Recent Articles in Cell & Chromosome

Fabian L, Troscianczuk J, Forer A
Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement.
Cell Chromosome. 2007;61.
Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise. [Abstract/Link to Full Text]

Vadakkan KI, Li B, De Boni U
Trend towards varying combinatorial centromere association in morphologically identical clusters in Purkinje neurons.
Cell Chromosome. 2006;5(1):1.
Neurons with similar morphology and neurotransmitter content located at a specific brain region may be part of the same or functionally separate networks. To address the question whether morphologically similar neurons have similar structural architecture at the chromosomal level, we studied Purkinje neurons in the cerebellum. Previous studies have shown that in Purkinje neurons centromeres of several chromosomes form clusters and that the number and size of these clusters remain stable in the adult brain. We examined whether the same set of centromeres form clusters in all the Purkinje neurons. Fluorescent in situ hybridization (FISH) with chromosome-specific para-centromeric probes provided an indirect evidence for a trend towards varying contributions from different chromosomes forming the centromeric clusters in adjacent Purkinje neurons. The results of the study indicate that the individual Purkinje neurons are likely unique in inter-chromosomal spatial associations. [Abstract/Link to Full Text]

Sills ES, Kim JJ, Witt MA, Palermo GD
Non-obstructive azoospermia and maturation arrest with complex translocation 46,XY t(9;13;14)(p22;q21.2;p13) is consistent with the Luciani-Guo hypothesis of latent aberrant autosomal regions and infertility.
Cell Chromosome. 2005 Sep 14;42.
OBJECTIVE: To describe clinical and histological features observed in the setting of an unusual complex translocation involving three autosomes (9, 13, and 14) identified in an otherwise healthy male referred for infertility consultation. MATERIALS AND METHODS: The patient was age 30 and no family history was available (adopted). Total azoospermia was confirmed on multiple semen analyses. Peripheral karyotype showed a 46,XY t(9;13;14)(p22:q21.2;p13) genotype; no Y-chromosome microdeletions were identified. Cystic fibrosis screening was negative. Bilateral testis biopsy revealed uniform maturation arrest and peritubular fibrosis. RESULTS: Formal genetic counseling was obtained and the extant literature reviewed with the couple. Given the low probability of obtaining sperm on testicular biopsy, as well as the high risk of any retrieved sperm having an unbalanced genetic rearrangement, the couple elected to proceed with fertility treatment using anonymous donor sperm for insemination. CONCLUSION: Although genes mapped to the Y-chromosome have been established as critical to normal testicular development and spermatogenesis, certain autosomal genes are now also recognized as important in these processes. Here we present clinical evidence to support the Luciani-Guo hypothesis (first advanced in 1984 and refined in 2002), which predicts severe spermatogenic impairment with aberrations involving chromosomes 9, 13, and/or 14, independent of Y-chromosome status. Additional study including fluorescent in situ hybridization and molecular analysis of specific chromosomal regions is needed to characterize more fully the contribution(s) of these autosomes to male testicular development and spermatogenesis. [Abstract/Link to Full Text]

Fadl-Elmula I
Chromosomal changes in uroepithelial carcinomas.
Cell Chromosome. 2005 Aug 7;41.
This article reviews and summarizes chromosomal changes responsible for the initiation and progression of uroepithelial carcinomas. Characterization of these alterations may lead to a better understanding of the genetic mechanisms and open the door for molecular markers that can be used for better diagnosis and prognosis of the disease. Such information might even help in designing new therapeutic strategies geared towards prevention of tumor recurrences and more aggressive approach in progression-prone cases. The revision of 205 cases of uroepithelial carcinomas reported with abnormal karyotypes showed karyotypic profile characterized by nonrandom chromosomal aberrations varying from one or few changes in low-grade and early stage tumors to massively rearranged karyotypes in muscle invasive ones. In general, the karyotypic profile was dominated by losses of chromosomal material seen as loss of entire chromosome and/or deletions of genetic materials. Rearrangements of chromosome 9 resulting in loss of material from 9p, 9q, or of the entire chromosome were the most frequent cytogenetic alterations, seen in 45% of the cases. Whereas loss of material from chromosome arms 1p, 8p, and 11p, and gains of chromosome 7, and chromosome arm 1q, and 8q seem to be an early, but secondary, changes appearing in superficial and well differentiated tumors, the formation of an isochromosome for 5p and loss of material from 17p are associated with more aggressive tumor phenotypes. Upper urinary tract TCCs have identical karyotypic profile to that of bladder TCCs, indicating the same pathogenetic mechanisms are at work in both locales. Intratumor cytogenetic heterogeneity was not seen except in a few post-radiation uroepithelial carcinomas in which distinct karyotypic and clonal pattern were characterized by massive intratumor heterogeneity (cytogenetic polyclonality) with near-diploid clones and simple balanced and/or unbalanced translocations. In the vast majority of cases strong correlation between the tumors grade/stage and karyotypic complexity was seen, indicating that progressive accumulation of acquired genetic alterations is the driving force behind multistep bladder TCC carcinogenesis. Although most of these cytogenetic alterations have been identified for many years, the molecular consequences and relevant cancer genes of these alterations have not yet been identified. However, loss of TSG(s) from chromosome 9 seems to be the primary and important event(s) in uroepithelial carcinogenesis. [Abstract/Link to Full Text]

Daniel A, St Heaps L
Chromosome loops arising from intrachromosomal tethering of telomeres occur at high frequency in G1 (non-cycling) mitotic cells: Implications for telomere capture.
Cell Chromosome. 2004 9 29;3(1):3.
BACKGROUND: To investigate potential mechanisms for telomere capture the spatial arrangement of telomeres and chromosomes was examined in G1 (non-cycling) mitotic cells with diploid or triploid genomes. This was examined firstly by directly labelling the respective short arm (p) and long arm subtelomeres (q) with different fluorophores and probing cell preparations using a number of subtelomere probe pairs, those for chromosomes 1, 3, 4, 5, 6, 7, 9, 10, 12, 17, 18, and 20. In addition some interstitial probes (CEN15, PML and SNRPN on chromosome 15) and whole chromosome paint probes (e.g. WCP12) were jointly hybridised to investigate the co-localization of interphase chromosome domains and tethered subtelomeres. Cells were prepared by omitting exposure to colcemid and hypotonic treatments. RESULTS: In these cells a specific interphase chromosome topology was detected. It was shown that the p and q telomeres of the each chromosome associate frequently (80% pairing) in an intrachromosomal manner, i.e. looped chromosomes with homologues usually widely spaced within the nucleus. This p-q tethering of the telomeres from the one chromosome was observed with large (chromosomes 3, 4, 5), medium sized (6, 7, 9, 10, 12), or small chromosomes (17, 18, 20). When triploid nuclei were probed there were three tetherings of p-q subtelomere signals representing the three widely separated looped chromosome homologues. The separate subtelomere pairings were shown to coincide with separate chromosome domains as defined by the WCP and interstitial probes. The 20% of apparently unpaired subtelomeric signals in diploid nuclei were partially documented to be pairings with the telomeres of other chromosomes. CONCLUSIONS: A topology for telomeres was detected where looped chromosome homologues were present at G1 interphase. These homologues were spatially arranged with respect to one-another independently of other chromosomes, i.e. there was no chromosome order on different sides of the cell nuclei and no segregation into haploid sets was detected. The normal function of this high frequency of intrachromosomal loops is unknown but a potential role is likely in the genesis of telomere captures whether of the intrachromosomal type or between non-homologues. This intrachromosomal tethering of telomeres cannot be related to telomeric or subtelomeric sequences since these are shared in varying degree with other chromosomes. In our view, these intrachromosomal telomeric tetherings with the resulting looped chromosomes arranged in a regular topology must be important to normal cell function since non-cycling cells in G1 are far from quiescent, are in fact metabolically active, and these cells represent the majority status since only a small proportion of cells are normally dividing. [Abstract/Link to Full Text]

Nelson SM, Ferguson LR, Denny WA
DNA and the chromosome - varied targets for chemotherapy.
Cell Chromosome. 2004 May 24;3(1):2.
The nucleus of the cell serves to maintain, regulate, and replicate the critical genetic information encoded by the genome. Genomic DNA is highly associated with proteins that enable simple nuclear structures such as nucleosomes to form higher-order organisation such as chromatin fibres. The temporal association of regulatory proteins with DNA creates a dynamic environment capable of quickly responding to cellular requirements and distress. The response is often mediated through alterations in the chromatin structure, resulting in changed accessibility of specific DNA sequences that are then recognized by specific proteins. Anti-cancer drugs that target cellular DNA have been used clinically for over four decades, but it is only recently that nuclease specific drugs have been developed to not only target the DNA but also other components of the nuclear structure and its regulation. In this review, we discuss some of the new drugs aimed at primary DNA sequences, DNA secondary structures, and associated proteins, keeping in mind that these agents are not only important from a clinical perspective but also as tools for understanding the nuclear environment in normal and cancer cells. [Abstract/Link to Full Text]

Heng HH, Stevens JB, Liu G, Bremer SW, Ye CJ
Imaging genome abnormalities in cancer research.
Cell Chromosome. 2004 Jan 13;3(1):1.
Increasing attention is focusing on chromosomal and genome structure in cancer research due to the fact that genomic instability plays a principal role in cancer initiation, progression and response to chemotherapeutic agents. The integrity of the genome (including structural, behavioral and functional aspects) of normal and cancer cells can be monitored with direct visualization by using a variety of cutting edge molecular cytogenetic technologies that are now available in the field of cancer research. Examples are presented in this review by grouping these methodologies into four categories visualizing different yet closely related major levels of genome structures. An integrated discussion is also presented on several ongoing projects involving the illustration of mitotic and meiotic chromatin loops; the identification of defective mitotic figures (DMF), a new type of chromosomal aberration capable of monitoring condensation defects in cancer; the establishment of a method that uses Non-Clonal Chromosomal Aberrations (NCCAs) as an index to monitor genomic instability; and the characterization of apoptosis related chromosomal fragmentations caused by drug treatments. [Abstract/Link to Full Text]

Cooper S, Shedden K
Microarray analysis of gene expression during the cell cycle.
Cell Chromosome. 2003 Sep 19;2(1):1.
Microarrays have been applied to the determination of genome-wide expression patterns during the cell cycle of a number of different cells. Both eukaryotic and prokaryotic cells have been studied using whole-culture and selective synchronization methods. The published microarray data on yeast, mammalian, and bacterial cells have been uniformly interpreted as indicating that a large number of genes are expressed in a cell-cycle-dependent manner. These conclusions are reconsidered using explicit criteria for synchronization and precise criteria for identifying gene expression patterns during the cell cycle. The conclusions regarding cell-cycle-dependent gene expression based on microarray analysis are weakened by arguably problematic choices for synchronization methodology (e.g., whole-culture methods that do not synchronize cells) and questionable statistical rigor for identifying cell-cycle-dependent gene expression. Because of the uncertainties in synchrony methodology, as well as uncertainties in microarray analysis, one should be somewhat skeptical of claims that there are a large number of genes expressed in a cell-cycle-dependent manner. [Abstract/Link to Full Text]

Sharif WD, Glick GG, Davidson MK, Wahls WP
Distinct functions of S. pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II.
Cell Chromosome. 2002 Sep 19;1(1):1.
BACKGROUND: In most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction. We report that these functions are separable in the fission yeast Schizosaccharomyces pombe. RESULTS: Intron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination. rec12-117, rec12-D15 (null), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products. Since S. pombe contains only three chromosome pairs, many of those aneuploid products were viable. The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products. The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II. Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors. CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I. Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I. In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II. [Abstract/Link to Full Text]

Mailhes JB, Hilliard C, Lowery M, London SN
MG-132, an inhibitor of proteasomes and calpains, induced inhibition of oocyte maturation and aneuploidy in mouse oocytes.
Cell Chromosome. 2002 Oct 8;1(1):2.
BACKGROUND: Although chromosome missegregation during oocyte maturation (OM) is a significant contributor to human morbidity and mortality, very little is known about the causes and mechanisms of aneuploidy. Several investigators have proposed that temporal perturbations during OM predispose oocytes to aberrant chromosome segregation. One approach for testing this proposal is to temporarily inhibit the activity of protein proteolysis during OM. We used the reversible proteasome inhibitor MG-132 to transiently perturb the temporal sequence of events during OM and subsequently analyzed mouse metaphase II (MII) for cytogenetic abnormalities. The transient inhibition of proteasome activity by MG-132 resulted in elevated levels of oocytes containing extra chromatids and chromosomes. RESULTS: The transient inhibition of proteasome-mediated proteolysis during OM by MG-132 resulted in dose-response delays during OM and elevated levels of aneuploid MII oocytes. Oocytes exposed in vitro to MG-132 exhibited greater delays during metaphase I (MI) as demonstrated by significantly (p < 0.01) higher levels of MI arrested oocytes and lower frequencies of premature sister chromatid separation in MII oocytes. Furthermore, the proportions of MII oocytes containing single chromatids and extra chromosomes significantly (p < 0.01) increased with MG-132 dosage. CONCLUSIONS: These data suggest that the MG-132-induced transient delay of proteasomal activity during mouse OM in vitro predisposed oocytes to abnormal chromosome segregation. Although these findings support a relationship between disturbed proteasomal activity and chromosome segregation, considerable additional data are needed to further investigate the roles of proteasome-mediated proteolysis and other potential molecular mechanisms on chromosome segregation during OM. [Abstract/Link to Full Text]

Recent Articles in Genome Biology

Sivasubbu S, Balciunas D, Amsterdam A, Ekker SC
Insertional mutagenesis strategies in zebrafish.
Genome Biol. 2007;8 Suppl 1S9.
We review here some recent developments in the field of insertional mutagenesis in zebrafish. We highlight the advantages and limitations of the rich body of retroviral methodologies, and we focus on the mechanisms and concepts of new transposon-based mutagenesis approaches under development, including prospects for conditional 'gene trapping' and 'gene breaking' approaches. [Abstract/Link to Full Text]

Korzh V
Transposons as tools for enhancer trap screens in vertebrates.
Genome Biol. 2007;8 Suppl 1S8.
DNA transposons are efficient tools in transgenesis and have therefore become popular in the analysis of the regulatory genome in vertebrates via enhancer trap screens. Here, I discuss recent progress in this field of research, with a focus on the application of one of these transposons, namely the medaka fish derived Tol2, to enhancer trapping in zebrafish, and how this approach compares with others that have a similar objective. [Abstract/Link to Full Text]

Kawakami K
Tol2: a versatile gene transfer vector in vertebrates.
Genome Biol. 2007;8 Suppl 1S7.
The medaka fish Tol2 element is an autonomous transposon that encodes a fully functional transposase. The transposase protein can catalyze transposition of a transposon construct that has 200 and 150 base pairs of DNA from the left and right ends of the Tol2 sequence, respectively. These sequences contain essential terminal inverted repeats and subterminal sequences. DNA inserts of fairly large sizes (as large as 11 kilobases) can be cloned between these sequences without reducing transpositional activity. The Tol2 transposon system has been shown to be active in all vertebrate cells tested thus far, including zebrafish, Xenopus, chicken, mouse, and human. In this review I describe and discuss how the Tol2 transposon is being applied to transgenic studies in these vertebrates, and possible future applications. [Abstract/Link to Full Text]

Parinov S, Emelyanov A
Transposable elements in fish functional genomics: technical challenges and perspectives.
Genome Biol. 2007;8 Suppl 1S6.
The recent introduction of several transposable elements in zebrafish opens new frontiers for genetic manipulation in this important vertebrate model. This review discusses transposable elements as mutagenesis tools for fish functional genomics. We review various mutagenesis strategies that were previously applied in other genetic models, such as Drosophila, Arabidopsis, and mouse, that may be beneficial if applied in fish. We also discuss the forthcoming challenges of high-throughput functional genomics in fish. [Abstract/Link to Full Text]

Collier LS, Largaespada DA
Transposable elements and the dynamic somatic genome.
Genome Biol. 2007;8 Suppl 1S5.
Although alterations in the genomes of somatic cells cannot be passed on to future generations, they can have beneficial or detrimental effects on the host organism, depending on the context in which they occur. This review outlines the ways in which transposable elements have important consequences for somatic cell genomes. [Abstract/Link to Full Text]

Kikuta H, Fredman D, Rinkwitz S, Lenhard B, Becker TS
Retroviral enhancer detection insertions in zebrafish combined with comparative genomics reveal genomic regulatory blocks - a fundamental feature of vertebrate genomes.
Genome Biol. 2007;8 Suppl 1S4.
A large-scale enhancer detection screen was performed in the zebrafish using a retroviral vector carrying a basal promoter and a fluorescent protein reporter cassette. Analysis of insertional hotspots uncovered areas around developmental regulatory genes in which an insertion results in the same global expression pattern, irrespective of exact position. These areas coincide with vertebrate chromosomal segments containing identical gene order; a phenomenon known as conserved synteny and thought to be a vestige of evolution. Genomic comparative studies have found large numbers of highly conserved noncoding elements (HCNEs) spanning these and other loci. HCNEs are thought to act as transcriptional enhancers based on the finding that many of those that have been tested direct tissue specific expression in transient or transgenic assays. Although gene order in hox and other gene clusters has long been known to be conserved because of shared regulatory sequences or overlapping transcriptional units, the chromosomal areas found through insertional hotspots contain only one or a few developmental regulatory genes as well as phylogenetically unrelated genes. We have termed these regions genomic regulatory blocks (GRBs), and show that they underlie the phenomenon of conserved synteny through all sequenced vertebrate genomes. After teleost whole genome duplication, a subset of GRBs were retained in two copies, underwent degenerative changes compared with tetrapod loci that exist as single copy, and that therefore can be viewed as representing the ancestral form. We discuss these findings in light of evolution of vertebrate chromosomal architecture and the identification of human disease mutations. [Abstract/Link to Full Text]

Sasakura Y, Oogai Y, Matsuoka T, Satoh N, Awazu S
Transposon mediated transgenesis in a marine invertebrate chordate: Ciona intestinalis.
Genome Biol. 2007;8 Suppl 1S3.
Achievement of transposon mediated germline transgenesis in a basal chordate, Ciona intestinalis, is discussed. A Tc1/mariner superfamily transposon, Minos, has excision and transposition activities in Ciona. Minos enables the creation of stable transgenic lines, enhancer detection, and insertional mutagenesis. [Abstract/Link to Full Text]

Pavlopoulos A, Oehler S, Kapetanaki MG, Savakis C
The DNA transposon Minos as a tool for transgenesis and functional genomic analysis in vertebrates and invertebrates.
Genome Biol. 2007;8 Suppl 1S2.
Transposons are powerful tools for conducting genetic manipulation and functional studies in organisms that are of scientific, economic, or medical interest. Minos, a member of the Tc1/mariner family of DNA transposons, exhibits a low insertional bias and transposes with high frequency in vertebrates and invertebrates. Its use as a tool for transgenesis and genome analysis of rather different animal species is described. [Abstract/Link to Full Text]

Ostertag EM, Madison BB, Kano H
Mutagenesis in rodents using the L1 retrotransposon.
Genome Biol. 2007;8 Suppl 1S16.
LINE1 (L1) retrotransposons are genetic elements that are present in all mammalian genomes. L1s are active in both humans and mice, and are capable of copying themselves and inserting the copy into a new genomic location. These de novo insertions occasionally result in disease. Endogenous L1 retrotransposons can be modified to increase their activity and mutagenic power in a variety of ways. Here we outline the advantages of using modified L1 retrotransposons for performing random mutagenesis in rodents and discuss several potential applications. [Abstract/Link to Full Text]

Collier LS, Largaespada DA
Transposons for cancer gene discovery: Sleeping Beauty and beyond.
Genome Biol. 2007;8 Suppl 1S15.
The use of Sleeping Beauty transposons as somatic mutagens to discover cancer genes in hematopoietic tumors and sarcomas has been documented. Here, we discuss the future of Sleeping Beauty for cancer genetic studies and the potential use of additional transposable elements for somatic mutagenesis. [Abstract/Link to Full Text]

Takeda J, Keng VW, Horie K
Germline mutagenesis mediated by Sleeping Beauty transposon system in mice.
Genome Biol. 2007;8 Suppl 1S14.
Following the descovery of its transposition activity in mammalian culture systems, the Sleeping Beauty (SB) transposon has since been applied to achieve germline mutagenesis in mice. Initially, the transposition efficiency was found to be low in cultured systems, but its activity in germ cells was unexpectedly high. This difference in transposition efficiency was found to be largely dependent on chromosomal status of the host genomic DNA and transposon vector design. The SB transposon system has been found to be suitable for comprehensive mutagenesis in mice. Therefore, it is an effective tool as a forward genetics screen for tagged insertional mutagenesis in mice. [Abstract/Link to Full Text]

Clark KJ, Carlson DF, Fahrenkrug SC
Pigs taking wing with transposons and recombinases.
Genome Biol. 2007;8 Suppl 1S13.
Swine production has been an important part of our lives since the late Mesolithic or early Neolithic periods, and ranks number one in world meat production. Pig production also contributes to high-value-added medical markets in the form of pharmaceuticals, heart valves, and surgical materials. Genetic engineering, including the addition of exogenous genetic material or manipulation of the endogenous genome, holds great promise for changing pig phenotypes for agricultural and medical applications. Although the first transgenic pigs were described in 1985, poor survival of manipulated embryos; inefficiencies in the integration, transmission, and expression of transgenes; and expensive husbandry costs have impeded the widespread application of pig genetic engineering. Sequencing of the pig genome and advances in reproductive technologies have rejuvenated efforts to apply transgenesis to swine. Pigs provide a compelling new resource for the directed production of pharmaceutical proteins and the provision of cells, vascular grafts, and organs for xenotransplantation. Additionally, given remarkable similarities in the physiology and size of people and pigs, swine will increasingly provide large animal models of human disease where rodent models are insufficient. We review the challenges facing pig transgenesis and discuss the utility of transposases and recombinases for enhancing the success and sophistication of pig genetic engineering. 'The paradise of my fancy is one where pigs have wings.' (GK Chesterton). [Abstract/Link to Full Text]

Hackett CS, Geurts AM, Hackett PB
Predicting preferential DNA vector insertion sites: implications for functional genomics and gene therapy.
Genome Biol. 2007;8 Suppl 1S12.
Viral and transposon vectors have been employed in gene therapy as well as functional genomics studies. However, the goals of gene therapy and functional genomics are entirely different; gene therapists hope to avoid altering endogenous gene expression (especially the activation of oncogenes), whereas geneticists do want to alter expression of chromosomal genes. The odds of either outcome depend on a vector's preference to integrate into genes or control regions, and these preferences vary between vectors. Here we discuss the relative strengths of DNA vectors over viral vectors, and review methods to overcome barriers to delivery inherent to DNA vectors. We also review the tendencies of several classes of retroviral and transposon vectors to target DNA sequences, genes, and genetic elements with respect to the balance between insertion preferences and oncogenic selection. Theoretically, knowing the variables that affect integration for various vectors will allow researchers to choose the vector with the most utility for their specific purposes. The three principle benefits from elucidating factors that affect preferences in integration are as follows: in gene therapy, it allows assessment of the overall risks for activating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment; in genomic studies, it allows one to discern random from selected integration events; and in gene therapy as well as functional genomics, it facilitates design of vectors that are better targeted to specific sequences, which would be a significant advance in the art of transgenesis. [Abstract/Link to Full Text]

Yergeau DA, Mead PE
Manipulating the Xenopus genome with transposable elements.
Genome Biol. 2007;8 Suppl 1S11.
The study of amphibian embryogenesis has provided important insight into the mechanisms of vertebrate development. The frog Xenopus laevis has been an important model of vertebrate cell biology and development for many decades. Genetic studies in this organism are not practical because of the tetraploid nature of the genome and the long generation time of this species. Recently, a closely related frog, namely Xenopus tropicalis, has been proposed as an alternative system; it shares all of the physical characteristics that make X. laevis a useful model but has the advantage of a diploid genome and short generation time. The rapid accumulation of genetic resources for this animal and the success of pilot mutagenesis screens have helped propel this model system forward. Transposable elements will provide invaluable tools for manipulating the frog genome. These integration systems are ideally suited to transgenesis and insertional mutagenesis strategies in the frog. The high fecundity of the frog combined with the ability to remobilize transposon transgenes integrated into frog genome will allow large-scale insertional mutagenesis screens to be performed in laboratories with modest husbandry capacities. [Abstract/Link to Full Text]

Grabher C, Wittbrodt J
Meganuclease and transposon mediated transgenesis in medaka.
Genome Biol. 2007;8 Suppl 1S10.
From among a plethora of various gene delivery methods, the researcher must choose the right one according to availability for a given species and the precise application the transgenic animal is intended for. Here we review the progress in meganuclease and Sleeping Beauty transposon mediated transgenesis over recent years with a focus on medaka and zebrafish. We present a side-by-side comparison of these two approaches based on their biologic properties and provide interesting perspectives for future experiments and applications, which are different for the two techniques because of their distinct modes of action. [Abstract/Link to Full Text]

Mátés L, Izsvák Z, Ivics Z
Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives.
Genome Biol. 2007;8 Suppl 1S1.
To meet the increasing demand of linking sequence information to gene function in vertebrate models, genetic modifications must be introduced and their effects analyzed in an easy, controlled, and scalable manner. In the mouse, only about 10% (estimate) of all genes have been knocked out, despite continuous methodologic improvement and extensive effort. Moreover, a large proportion of inactivated genes exhibit no obvious phenotypic alterations. Thus, in order to facilitate analysis of gene function, new genetic tools and strategies are currently under development in these model organisms. Loss of function and gain of function mutagenesis screens based on transposable elements have numerous advantages because they can be applied in vivo and are therefore phenotype driven, and molecular analysis of the mutations is straightforward. At present, laboratory harnessing of transposable elements is more extensive in invertebrate models, mostly because of their earlier discovery in these organisms. Transposons have already been found to facilitate functional genetics research greatly in lower metazoan models, and have been applied most comprehensively in Drosophila. However, transposon based genetic strategies were recently established in vertebrates, and current progress in this field indicates that transposable elements will indeed serve as indispensable tools in the genetic toolkit for vertebrate models. In this review we provide an overview of transposon based genetic modification techniques used in higher and lower metazoan model organisms, and we highlight some of the important general considerations concerning genetic applications of transposon systems. [Abstract/Link to Full Text]

Brown KR, Jurisica I
Unequal evolutionary conservation of human protein interactions in interologous networks.
Genome Biol. 2007;8(5):R95.
BACKGROUND: Protein-protein interaction (PPI) networks have been transferred between organisms using interologs, allowing model organisms to supplement the interactomes of higher eukaryotes. However, the conservation of various network components has not been fully explored. Unequal conservation of certain network components may limit the ability to fully expand the target interactomes using interologs. RESULTS: In this study, we transfer high quality human interactions to lower eukaryotes, and examine the evolutionary conservation of individual network components. When human proteins are mapped to yeast, we find a strong positive correlation (r = 0.50, P = 3.9 x 10(-4)) between evolutionary conservation and the number of interacting proteins, which is also found when mapped to other model organisms. Examining overlapping PPI networks, Gene Ontology (GO) terms, and gene expression data, we are able to demonstrate that protein complexes are conserved preferentially, compared to transient interactions in the network. Despite the preferential conservation of complexes, and the fact that the human interactome comprises an abundance of transient interactions, we demonstrate how transferring human PPIs to yeast augments this well-studied protein interaction network, using the coatomer complex and replisome as examples. CONCLUSION: Human proteins, like yeast proteins, show a correlation between the number of interacting partners and evolutionary conservation. The preferential conservation of proteins with higher degree leads to enrichment in protein complexes when interactions are transferred between organisms using interologs. [Abstract/Link to Full Text]

Tamames J, Moya A, Valencia A
Modular organization in the reductive evolution of protein-protein interaction networks.
Genome Biol. 2007;8(5):R94.
BACKGROUND: The variation in the sizes of the genomes of distinct life forms remains somewhat puzzling. The organization of proteins into domains and the different mechanisms that regulate gene expression are two factors that potentially increase the capacity of genomes to create more complex systems. High-throughput protein interaction data now make it possible to examine the additional complexity generated by the way that protein interactions are organized. RESULTS: We have studied the reduction in genome size of Buchnera compared to its close relative Escherichia coli. In this well defined evolutionary scenario, we found that among all the properties of the protein interaction networks, it is the organization of networks into modules that seems to be directly related to the evolutionary process of genome reduction. CONCLUSION: In Buchnera, the apparently non-random reduction of the modular structure of the networks and the retention of essential characteristics of the interaction network indicate that the roles of proteins within the interaction network are important in the reductive process. [Abstract/Link to Full Text]

Wong CW, Heng CL, Wan Yee L, Soh SW, Kartasasmita CB, Simoes EA, Hibberd ML, Sung WK, Miller LD
Optimization and clinical validation of a pathogen detection microarray.
Genome Biol. 2007;8(5):R93.
DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics. [Abstract/Link to Full Text]

Del Sol A, Araúzo-Bravo MJ, Amoros D, Nussinov R
Modular architecture of protein structures and allosteric communications: potential implications for signaling proteins and regulatory linkages.
Genome Biol. 2007;8(5):R92.
BACKGROUND: Allosteric communications are vital for cellular signaling. Here we explore a relationship between protein architectural organization and shortcuts in signaling pathways. RESULTS: We show that protein domains consist of modules interconnected by residues that mediate signaling through the shortest pathways. These mediating residues tend to be located at the inter-modular boundaries, which are more rigid and display a larger number of long-range interactions than intra-modular regions. The inter-modular boundaries contain most of the residues centrally conserved in the protein fold, which may be crucial for information transfer between amino acids. Our approach to modular decomposition relies on a representation of protein structures as residue-interacting networks, and removal of the most central residue contacts, which are assumed to be crucial for allosteric communications. The modular decomposition of 100 multi-domain protein structures indicates that modules constitute the building blocks of domains. The analysis of 13 allosteric proteins revealed that modules characterize experimentally identified functional regions. Based on the study of an additional functionally annotated dataset of 115 proteins, we propose that high-modularity modules include functional sites and are the basic functional units. We provide examples (the Galphas subunit and P450 cytochromes) to illustrate that the modular architecture of active sites is linked to their functional specialization. CONCLUSION: Our method decomposes protein structures into modules, allowing the study of signal transmission between functional sites. A modular configuration might be advantageous: it allows signaling proteins to expand their regulatory linkages and may elicit a broader range of control mechanisms either via modular combinations or through modulation of inter-modular linkages. [Abstract/Link to Full Text]

Sultan M, Piccini I, Balzereit D, Herwig R, Saran NG, Lehrach H, Reeves RH, Yaspo ML
Gene expression variation in Down's syndrome mice allows prioritization of candidate genes.
Genome Biol. 2007;8(5):R91.
BACKGROUND: Down's syndrome (DS), or trisomy 21, is a complex developmental disorder that exhibits many clinical signs that vary in occurrence and severity among patients. The molecular mechanisms responsible for DS have thus far remained elusive. We argue here that normal variation in gene expression in the population contributes to the heterogeneous clinical picture of DS, and we estimated the amplitude of this variation in 50 mouse orthologs of chromosome 21 genes in brain regions of Ts65Dn (a mouse model of DS). We analyzed the RNAs of eight Ts65Dn and eight euploid mice by real-time polymerase chain reaction. RESULTS: In pooled RNAs, we confirmed that trisomic/euploid gene expression ratios were close to 1.5. However, we observed that inter-individual gene expression levels spanned a broad range of values. We identified three categories of genes: genes with expression levels consistently higher in Ts65Dn than in euploids (9, 17, and 7 genes in cerebellum, cortex, and midbrain, respectively); genes whose expression levels partially overlap between the two groups (10, 9, and 14 genes); and genes with intermingled expression, which cannot be used to differentiate trisomics from euploids (12, 5 and 9 genes). Of the genes in the first category, App, Cbr1, and Mrps6 exhibited tight regulation in the three tissues and are therefore attractive candidates for further research. CONCLUSION: This is the first analysis addressing inter-individual gene expression levels as a function of trisomy. We propose a strategy allowing discrimination between candidates for the constant features of DS and those genes that may contribute to the partially penetrant signs of DS. [Abstract/Link to Full Text]

Jiménez JL, Hegemann B, Hutchins JR, Peters JM, Durbin R
A systematic comparative and structural analysis of protein phosphorylation sites based on the mtcPTM database.
Genome Biol. 2007;8(5):R90.
mtcPTM is an online repository of human and mouse phosphosites in which data are hierarchically organized to preserve biologically relevant experimental information, thus allowing straightforward comparisons of phosphorylation patterns found under different conditions. The database also contains the largest available collection of atomic models of phosphorylatable proteins. Detailed analysis of this structural dataset reveals that phosphorylation sites are found in a heterogeneous range of structural and sequence contexts. mtcPTM is available on the web [Abstract/Link to Full Text]

Beste DJ, Hooper T, Stewart G, Bonde B, Avignone-Rossa C, Bushell ME, Wheeler P, Klamt S, Kierzek AM, McFadden J
GSMN-TB: a web-based genome-scale network model of Mycobacterium tuberculosis metabolism.
Genome Biol. 2007;8(5):R89.
BACKGROUND: An impediment to the rational development of novel drugs against tuberculosis (TB) is a general paucity of knowledge concerning the metabolism of Mycobacterium tuberculosis, particularly during infection. Constraint-based modeling provides a novel approach to investigating microbial metabolism but has not yet been applied to genome-scale modeling of M. tuberculosis. RESULTS: GSMN-TB, a genome-scale metabolic model of M. tuberculosis, was constructed, consisting of 849 unique reactions and 739 metabolites, and involving 726 genes. The model was calibrated by growing Mycobacterium bovis bacille Calmette Guérin in continuous culture and steady-state growth parameters were measured. Flux balance analysis was used to calculate substrate consumption rates, which were shown to correspond closely to experimentally determined values. Predictions of gene essentiality were also made by flux balance analysis simulation and were compared with global mutagenesis data for M. tuberculosis grown in vitro. A prediction accuracy of 78% was achieved. Known drug targets were predicted to be essential by the model. The model demonstrated a potential role for the enzyme isocitrate lyase during the slow growth of mycobacteria, and this hypothesis was experimentally verified. An interactive web-based version of the model is available. CONCLUSION: The GSMN-TB model successfully simulated many of the growth properties of M. tuberculosis. The model provides a means to examine the metabolic flexibility of bacteria and predict the phenotype of mutants, and it highlights previously unexplored features of M. tuberculosis metabolism. [Abstract/Link to Full Text]

Lobo NF, Campbell KS, Thaner D, Debruyn B, Koo H, Gelbart WM, Loftus BJ, Severson DW, Collins FH
Analysis of 14 BAC sequences from the Aedes aegypti genome: a benchmark for genome annotation and assembly.
Genome Biol. 2007;8(5):R88.
BACKGROUND: Aedes aegypti is the principal vector of yellow fever and dengue viruses throughout the tropical world. To provide a set of manually curated and annotated sequences from the Ae. aegypti genome, 14 mapped bacterial artificial chromosome (BAC) clones encompassing 1.57 Mb were sequenced, assembled and manually annotated using a combination of computational gene-finding, expressed sequence tag (EST) matches and comparative protein homology. PCR and sequencing were used to experimentally confirm expression and sequence of a subset of these transcripts. RESULTS: Of the 51 manual annotations, 50 and 43 demonstrated a high level of similarity to Anopheles gambiae and Drosophila melanogaster genes, respectively. Ten of the 12 BAC sequences with more than one annotated gene exhibited synteny with the A. gambiae genome. Putative transcripts from eight BAC clones were found in multiple copies (two copies in most cases) in the Aedes genome assembly, which point to the probable presence of haplotype polymorphisms and/or misassemblies. CONCLUSION: This study not only provides a benchmark set of manually annotated transcripts for this genome that can be used to assess the quality of the auto-annotation pipeline and the assembly, but it also looks at the effect of a high repeat content on the genome assembly and annotation pipeline. [Abstract/Link to Full Text]

Smith LK, Gomez MJ, Shatalin KY, Lee H, Neyfakh AA
Monitoring of gene knockouts: genome-wide profiling of conditionally essential genes.
Genome Biol. 2007;8(5):R87.
We have developed a new microarray-based genetic technique, named MGK (Monitoring of Gene Knockouts), for genome-wide identification of conditionally essential genes. MGK identified bacterial genes that are critical for fitness in the absence of aromatic amino acids, and was further applied to identify genes whose inactivation causes bacterial cell death upon exposure to the bacteriostatic antibiotic chloramphenicol. Our findings suggest that MGK can serve as a robust tool in functional genomics studies. [Abstract/Link to Full Text]

Zhang X, De la Cruz O, Pinto JM, Nicolae D, Firestein S, Gilad Y
Characterizing the expression of the human olfactory receptor gene family using a novel DNA microarray.
Genome Biol. 2007;8(5):R86.
BACKGROUND: Olfactory receptor (OR) genes were discovered more than a decade ago, when Buck and Axel observed that, in rats, certain G-protein coupled receptors are expressed exclusively in the olfactory epithelium. Subsequently, protein sequence similarity was used to identify entire OR gene repertoires of a number of mammalian species, but only in mouse were these predictions followed up by expression studies in olfactory epithelium. To rectify this, we have developed a DNA microarray that contains probes for most predicted human OR loci and used that array to examine OR gene expression profiles in olfactory epithelium tissues from three individuals. RESULTS: We detected expression of 437 (76%) human OR genes in these olfactory epithelia. Interestingly, we detected widespread expression of OR pseudogenes, an observation that may shed light on the mechanism of OR gene choice in the olfactory sensory neurons. To address the hypothesis that OR genes may carry out additional functions, we also characterized the expression of OR genes in a number of non-olfactory tissues. CONCLUSION: While our results corroborate the functional annotation of the majority of predicted human odorant receptors, we find that a large number of putative human OR genes are expressed in non-olfactory tissues, sometimes exclusively so. Our evolutionary analysis of ectopically expressed human OR genes does not lend support to the hypothesis that these genes have alternative functions. [Abstract/Link to Full Text]

Poustka AJ, Kühn A, Groth D, Weise V, Yaguchi S, Burke RD, Herwig R, Lehrach H, Panopoulou G
A global view of gene expression in lithium and zinc treated sea urchin embryos: new components of gene regulatory networks.
Genome Biol. 2007;8(5):R85.
BACKGROUND: The genome of the sea urchin Strongylocentrotus purpuratus has recently been sequenced because it is a major model system for the study of gene regulatory networks. Embryonic expression patterns for most genes are unknown, however. RESULTS: Using large-scale screens on arrays carrying 50% to 70% of all genes, we identified novel territory-specific markers. Our strategy was based on computational selection of genes that are differentially expressed in lithium-treated embryos, which form excess endomesoderm, and in zinc-treated embryos, in which endomesoderm specification is blocked. Whole-mount in situ hybridization (WISH) analysis of 700 genes indicates that the apical organ region is eliminated in lithium-treated embryos. Conversely, apical and specifically neural markers are expressed more broadly in zinc-treated embryos, whereas endomesoderm signaling is severely reduced. Strikingly, the number of serotonergic neurons is amplified by at least tenfold in zinc-treated embryos. WISH analysis further indicates that there is crosstalk between the Wnt (wingless int), Notch, and fibroblast growth factor signaling pathways in secondary mesoderm cell specification and differentiation, similar to signaling cascades that function during development of presomitic mesoderm in mouse embryogenesis. We provide differential expression data for more than 4,000 genes and WISH patterns of more than 250 genes, and more than 2,400 annotated WISH images. CONCLUSION: Our work provides tissue-specific expression patterns for a large fraction of the sea urchin genes that have not yet been included in existing regulatory networks and await functional integration. Furthermore, we noted neuron-inducing activity of zinc on embryonic development; this is the first observation of such activity in any organism. [Abstract/Link to Full Text]

Mahony S, Corcoran DL, Feingold E, Benos PV
Regulatory conservation of protein coding and microRNA genes in vertebrates: lessons from the opossum genome.
Genome Biol. 2007;8(5):R84.
BACKGROUND: Being the first noneutherian mammal sequenced, Monodelphis domestica (opossum) offers great potential for enhancing our understanding of the evolutionary processes that take place in mammals. This study focuses on the evolutionary relationships between conservation of noncoding sequences, cis-regulatory elements, and biologic functions of regulated genes in opossum and eight vertebrate species. RESULTS: Analysis of 145 intergenic microRNA and all protein coding genes revealed that the upstream sequences of the former are up to twice as conserved as the latter among mammals, except in the first 500 base pairs, where the conservation is similar. Comparison of promoter conservation in 513 protein coding genes and related transcription factor binding sites (TFBSs) showed that 41% of the known human TFBSs are located in the 6.7% of promoter regions that are conserved between human and opossum. Some core biologic processes exhibited significantly fewer conserved TFBSs in human-opossum comparisons, suggesting greater functional divergence. A new measure of efficiency in multigenome phylogenetic footprinting (base regulatory potential rate [BRPR]) shows that including human-opossum conservation increases specificity in finding human TFBSs. CONCLUSION: Opossum facilitates better estimation of promoter conservation and TFBS turnover among mammals. The fact that substantial TFBS numbers are located in a small proportion of the human-opossum conserved sequences emphasizes the importance of marsupial genomes for phylogenetic footprinting-based motif discovery strategies. The BRPR measure is expected to help select genome combinations for optimal performance of these algorithms. Finally, although the etiology of the microRNA upstream increased conservation remains unknown, it is expected to have strong implications for our understanding of regulation of their expression. [Abstract/Link to Full Text]

De Bleser P, Hooghe B, Vlieghe D, van Roy F
A distance difference matrix approach to identifying transcription factors that regulate differential gene expression.
Genome Biol. 2007;8(5):R83.
We introduce a method that considers target genes of a transcription factor, and searches for transcription factor binding sites (TFBSs) of secondary factors responsible for differential responses among these targets. Based on the distance difference matrix concept, the method simultaneously integrates statistical overrepresentation and co-occurrence of TFBSs. Our approach is validated on datasets of differentially regulated human genes and is shown to be highly effective in detecting TFBSs responsible for the observed differential gene expression. [Abstract/Link to Full Text]

Kapur K, Xing Y, Ouyang Z, Wong WH
Exon arrays provide accurate assessments of gene expression.
Genome Biol. 2007;8(5):R82.
We have developed a strategy for estimating gene expression on Affymetrix Exon arrays. The method includes a probe-specific background correction and a probe selection strategy in which a subset of probes with highly correlated intensities across multiple samples are chosen to summarize gene expression. Our results demonstrate that the proposed background model offers improvements over the default Affymetrix background correction and that Exon arrays may provide more accurate measurements of gene expression than traditional 3' arrays. [Abstract/Link to Full Text]

Recent Articles in Nuclear Receptor

Nunn AV, Bell J, Barter P
The integration of lipid-sensing and anti-inflammatory effects: how the PPARs play a role in metabolic balance.
Nucl Recept. 2007;5(1):1.
The peroxisomal proliferating-activated receptors (PPARs) are lipid-sensing transcription factors that have a role in embryonic development, but are primarily known for modulating energy metabolism, lipid storage, and transport, as well as inflammation and wound healing. Currently, there is no consensus as to the overall combined function of PPARs and why they evolved. We hypothesize that the PPARs had to evolve to integrate lipid storage and burning with the ability to reduce oxidative stress, as energy storage is essential for survival and resistance to injury/infection, but the latter increases oxidative stress and may reduce median survival (functional longevity). In a sense, PPARs may be an evolutionary solution to something we call the 'hypoxia-lipid' conundrum, where the ability to store and burn fat is essential for survival, but is a 'double-edged sword', as fats are potentially highly toxic. Ways in which PPARs may reduce oxidative stress involve modulation of mitochondrial uncoupling protein (UCP) expression (thus reducing reactive oxygen species, ROS), optimising forkhead box class O factor (FOXO) activity (by improving whole body insulin sensitivity) and suppressing NFkB (at the transcriptional level). In light of this, we therefore postulate that inflammation-induced PPAR downregulation engenders many of the signs and symptoms of the metabolic syndrome, which shares many features with the acute phase response (APR) and is the opposite of the phenotype associated with calorie restriction and high FOXO activity. In genetically susceptible individuals (displaying the naturally mildly insulin resistant 'thrifty genotype'), suboptimal PPAR activity may follow an exaggerated but natural adipose tissue-related inflammatory signal induced by excessive calories and reduced physical activity, which normally couples energy storage with the ability to mount an immune response. This is further worsened when pancreatic decompensation occurs, resulting in gluco-oxidative stress and lipotoxicity, increased inflammatory insulin resistance and oxidative stress. Reactivating PPARs may restore a metabolic balance and help to adapt the phenotype to a modern lifestyle. [Abstract/Link to Full Text]

Doney AS, Fischer B, Lee SP, Morris AD, Leese G, Palmer CN
Association of common variation in the PPARA gene with incident myocardial infarction in individuals with type 2 diabetes: a Go-DARTS study.
Nucl Recept. 2005 Nov 25;34.
BACKGROUND: Common variants of the PPARA gene have been found to associate with ischaemic heart disease in non diabetic men. The L162V variant was found to be protective while the C2528G variant increased risk. L162V has also been associated with altered lipid measures. We therefore sought to determine the effect of PPARA gene variation on susceptibility to myocardial infarction in patients with type 2 diabetes. 1810 subjects with type 2 diabetes from the prospective Go-DARTS study were genotyped for the L162V and C2528G variants in the PPARA gene and the association of the variants with incident non-fatal myocardial infarction was examined. Cox's proportional hazards was used to interrogate time to event from recruitment, and linear regression for analysing association of genotype with quantitative clinical traits. RESULTS: The V162 allele was associated with decreased risk of non-fatal myocardial infarction (HR = 0.31, 95%CI 0.10-0.93 p = 0.037) whereas the C2528 allele was associated with increased risk (HR = 2.77 95%CI 1.34-5.75 p = 0.006). Similarly V162 was associated with a later mean age of diagnosis with type 2 diabetes and C2582 an earlier age of diagnosis. C2528 was also associated with increased total cholesterol and LDL cholesterol, which did not account for the observed increased risk. Haplotype analysis demonstrated that when both rare variants occurred on the same haplotype the effect of each was abrogated. CONCLUSION: Genetic variation at the PPARA locus is important in determining cardiovascular risk in both male and female patients with diabetes. This genotype associated risk appears to be independent of the effect of these genotypes on lipid profiles and age of diagnosis with diabetes. [Abstract/Link to Full Text]

Tachibana K, Kobayashi Y, Tanaka T, Tagami M, Sugiyama A, Katayama T, Ueda C, Yamasaki D, Ishimoto K, Sumitomo M, Uchiyama Y, Kohro T, Sakai J, Hamakubo T, Kodama T, Doi T
Gene expression profiling of potential peroxisome proliferator-activated receptor (PPAR) target genes in human hepatoblastoma cell lines inducibly expressing different PPAR isoforms.
Nucl Recept. 2005 Oct 3;33.
BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and commonly play an important role in the regulation of lipid homeostasis. To identify human PPARs-responsive genes, we established tetracycline-regulated human hepatoblastoma cell lines that can be induced to express each human PPAR and investigated the gene expression profiles of these cells. RESULTS: The expression of each introduced PPAR gene was investigated using the various concentrations of doxycycline in the culture media. We found that the expression of each PPAR subtype was tightly controlled by the concentration of doxycycline in these established cell lines. DNA microarray analyses using these cell lines were performed with or without adding each subtype ligand and provided much important information on the PPAR target genes involved in lipid metabolism, transport, storage and other activities. Interestingly, it was noted that while ligand-activated PPARdelta induced target gene expression, unliganded PPARdelta repressed these genes. The real-time RT-PCR was used to verify the altered expression of selected genes by PPARs and we found that these genes were induced to express in the same pattern as detected in the microarray analyses. Furthermore, we analysed the 5'-flanking region of the human adipose differentiation-related protein (adrp) gene that responded to all subtypes of PPARs. From the detailed analyses by reporter assays, the EMSAs, and ChIP assays, we determined the functional PPRE of the human adrp gene. CONCLUSION: The results suggest that these cell lines are important tools used to identify the human PPARs-responsive genes. [Abstract/Link to Full Text]

Krasowski MD, Yasuda K, Hagey LR, Schuetz EG
Evolutionary selection across the nuclear hormone receptor superfamily with a focus on the NR1I subfamily (vitamin D, pregnane X, and constitutive androstane receptors).
Nucl Recept. 2005 Sep 30;32.
BACKGROUND: The nuclear hormone receptor (NR) superfamily complement in humans is composed of 48 genes with diverse roles in metabolic homeostasis, development, and detoxification. In general, NRs are strongly conserved between vertebrate species, and few examples of molecular adaptation (positive selection) within this superfamily have been demonstrated. Previous studies utilizing two-species comparisons reveal strong purifying (negative) selection of most NR genes, with two possible exceptions being the ligand-binding domains (LBDs) of the pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3), two proteins involved in the regulation of toxic compound metabolism and elimination. The aim of this study was to apply detailed phylogenetic analysis using maximum likelihood methods to the entire complement of genes in the vertebrate NR superfamily. Analyses were carried out both across all vertebrates and limited to mammals and also separately for the two major domains of NRs, the DNA-binding domain (DBD) and LBD, in addition to the full-length sequences. Additional functional data is also reported for activation of PXR and the vitamin D receptor (VDR; NR1I1) to gain further insight into the evolution of the NR1I subfamily. RESULTS: The NR genes appear to be subject to strong purifying selection, particularly in the DBDs. Estimates of the ratio of the non-synonymous to synonymous nucleotide substitution rates (the omega ratio) revealed that only the PXR LBD had a sub-population of codons with an estimated omega ratio greater than 1. CAR was also unusual in showing high relative omega ratios in both the DBD and LBD, a finding that may relate to the recent appearance of the CAR gene (presumably by duplication of a pre-mammalian PXR gene) just prior to the evolution of mammals. Functional analyses of the NR1I subfamily show that human and zebrafish PXRs show similar activation by steroid hormones and early bile salts, properties not shared by sea lamprey, mouse, or human VDRs, or by Xenopus laevis PXRs. CONCLUSION: NR genes generally show strong sequence conservation and little evidence for positive selection. The main exceptions are PXR and CAR, genes that may have adapted to cross-species differences in toxic compound exposure. [Abstract/Link to Full Text]

Santos GM, Pantoja CJ, Costa E Silva A, Rodrigues MC, Ribeiro RC, Simeoni LA, Lomri N, Neves FA
Thyroid hormone receptor binding to DNA and T3-dependent transcriptional activation are inhibited by uremic toxins.
Nucl Recept. 2005 Apr 4;3(1):1.
BACKGROUND: There is a substantial clinical overlap between chronic renal failure (CRF) and hypothyroidism, suggesting the presence of hypothyroidism in uremic patients. Although CRF patients have low T3 and T4 levels with normal thyroid-stimulating hormone (TSH), they show a higher prevalence of goiter and evidence for blunted tissue responsiveness to T3 action. However, there are no studies examining whether thyroid hormone receptors (TRs) play a role in thyroid hormone dysfunction in CRF patients. To evaluate the effects of an uremic environment on TR function, we investigated the effect of uremic plasma on TRbeta1 binding to DNA as heterodimers with the retinoid X receptor alpha (RXRalpha) and on T3-dependent transcriptional activity. RESULTS: We demonstrated that uremic plasma collected prior to hemodialysis (Pre-HD) significantly reduced TRbeta1-RXRalpha binding to DNA. Such inhibition was also observed with a vitamin D receptor (VDR) but not with a peroxisome proliferator-activated receptor gamma (PPARgamma). A cell-based assay confirmed this effect where uremic pre-HD ultrafiltrate inhibited the transcriptional activation induced by T3 in U937 cells. In both cases, the inhibitory effects were reversed when the uremic plasma and the uremic ultrafiltrate were collected and used after hemodialysis (Post-HD). CONCLUSION: These results suggest that dialyzable toxins in uremic plasma selectively block the binding of TRbeta1-RXRalpha to DNA and impair T3 transcriptional activity. These findings may explain some features of hypothyroidism and thyroid hormone resistance observed in CRF patients. [Abstract/Link to Full Text]

Handschin C, Blättler S, Roth A, Looser R, Oscarson M, Kaufmann MR, Podvinec M, Gnerre C, Meyer UA
The evolution of drug-activated nuclear receptors: one ancestral gene diverged into two xenosensor genes in mammals.
Nucl Recept. 2004 10 12;2(1):7.
BACKGROUND: Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals. RESULTS: To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR. CONCLUSION: Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species. [Abstract/Link to Full Text]

Kanno Y, Otsuka S, Hiromasa T, Nakahama T, Inouye Y
Diurnal difference in CAR mRNA expression.
Nucl Recept. 2004 Aug 28;2(1):6.
BACKGROUND: The constitutive androstane receptor (CAR, NR1I3) plays a key role in the transcriptional activation of genes that encode xenobiotic/steroid and drug metabolizing enzymes. RESULTS: The expression of CAR mRNA throughout the circadian rhythm is reported for the first time in phase with the clock gene Bmal1 and in antiphase with the clock-controlled gene Rev-erbalpha mRNAs, with a peak at Zeitgeber time (ZT) 20 and a trough at ZT8, and a peak/trough ratio of 2.0. The diurnal difference in CAR mRNA expression might underlie the 1.7-fold difference in the magnitude of the PB-dependent induction of CYP2B1/2 mRNA. CONCLUSION: The circadian oscillation of xenosensor gene CAR mRNA expression is partially responsible for chronopharmacokinetics and chronopharmacology in disease. [Abstract/Link to Full Text]

Taguchi Y, Koslowski M, Bodenner DL
Binding of estrogen receptor with estrogen conjugated to bovine serum albumin (BSA).
Nucl Recept. 2004 Aug 19;2(1):5.
BACKGROUND: The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E2-BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E2-BSA does not compete for E2 binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E2 and E2-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E2-BSA preparations with unconjugated E2 (usually between 3-5%), the latter was carefully removed by ultrafiltration. RESULTS: As previously reported, a 10-to 1000-fold molar excess of E2-BSA was unable to compete with 3H-E2 binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E2-BSA, the binding of 3H-E2 was significantly reduced. E2-BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E2-BSA (E2-BSA-FITC). Staining was restricted to the cell membrane when E2-BSA-FITC was incubated with stable transfectants of the murine ERalpha within ER-negative HeLa cells and with MC7 cells that endogenously produce ERalpha. This staining appeared highly specific since it was competed by pre-incubation with E2 in a dose dependent manner and with the competitor ICI-182,780. CONCLUSIONS: These results demonstrate that E2-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E2-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E2. More importantly, these findings demonstrate that in intact cells that express ER, E2-BSA binding is localized to the cell membrane, strongly suggesting a membrane bound form of the ER. [Abstract/Link to Full Text]

Ghose R, Zimmerman TL, Thevananther S, Karpen SJ
Endotoxin leads to rapid subcellular re-localization of hepatic RXRalpha: A novel mechanism for reduced hepatic gene expression in inflammation.
Nucl Recept. 2004 Aug 16;2(1):4.
BACKGROUND: Lipopolysaccharide (LPS) treatment of animals down-regulates the expression of hepatic genes involved in a broad variety of physiological processes, collectively known as the negative hepatic acute phase response (APR). Retinoid X receptor alpha (RXRalpha), the most highly expressed RXR isoform in liver, plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. Many of the genes regulated by RXRalpha are repressed during the negative hepatic APR, although the underlying mechanism is not known. We hypothesized that inflammation-induced alteration of the subcellular location of RXRalpha was a common mechanism underlying the negative hepatic APR. RESULTS: Nuclear RXRalpha protein levels were significantly reduced (~50%) within 1-2 hours after low-dose LPS treatment and remained so for at least 16 hours. RXRalpha was never detected in cytosolic extracts from saline-treated mice, yet was rapidly and profoundly detectable in the cytosol from 1 hour, to at least 4 hours, after LPS administration. These effects were specific, since the subcellular localization of the RXRalpha partner, the retinoic acid receptor (RARalpha), was unaffected by LPS. A potential cell-signaling modulator of RXRalpha activity, c-Jun-N-terminal kinase (JNK) was maximally activated at 1-2 hours, coincident with maximal levels of cytoplasmic RXRalpha. RNA levels of RXRalpha were unchanged, while expression of 6 sentinel hepatic genes regulated by RXRalpha were all markedly repressed after LPS treatment. This is likely due to reduced nuclear binding activities of regulatory RXRalpha-containing heterodimer pairs. CONCLUSION: The subcellular localization of native RXRalpha rapidly changes in response to LPS administration, correlating with induction of cell signaling pathways. This provides a novel and broad-ranging molecular mechanism for the suppression of RXRalpha-regulated genes in inflammation. [Abstract/Link to Full Text]

Björnström L, Sjöberg M
Estrogen receptor-dependent activation of AP-1 via non-genomic signalling.
Nucl Recept. 2004 Jun 14;2(1):3.
BACKGROUND: Ligand-bound estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) modulate AP-1-dependent transcription via protein-protein interactions on DNA, in a manner that depends on the type of cells and the subtype of ER. We present here evidence for an additional mechanism by which ERs modulate the transcriptional activity of AP-1. RESULTS: We show that ERs located in the cytoplasm efficiently activate transcription at AP-1 sites in response to 17beta-estradiol, while ERs present in the nucleus repress transcription under the same conditions. 17beta-estradiol-induced activation of the coll-73-luc reporter correlated with cytoplasmic localization of various ERalpha and ERbeta mutant receptors, and was inhibited in the presence of the full estrogen antagonist ICI 182,780 and the MAP-kinase inhibitor UO126. We also show that the selective estrogen receptor modulator (SERM) tamoxifen is as potent as 17beta-estradiol in inducing activation of AP-1 when ERalpha is present in the cytoplasm. CONCLUSIONS: These results suggest that non-genomic signalling is involved in the mechanism by which ERalpha and ERbeta influence AP-1-dependent transcription. We have previously shown that Stat3 and Stat5 are targeted by non-genomic actions of ERs, and the results presented here allow us to conclude that ERs bound to 17beta-estradiol mediate the transcriptional activation of promoters regulated by AP-1 and by Stat proteins via different combinations of signal transduction pathways. Our observations thereby provide new insights into the mechanisms by which ERs act at alternate response elements, and suggest a mechanism by which tamoxifen exerts its action as a tissue-selective agonist. [Abstract/Link to Full Text]

Uht RM, Webb P, Nguyen P, Price Jr RH, Valentine C, Favre H, Kushner PJ
A conserved lysine in the estrogen receptor DNA binding domain regulates ligand activation profiles at AP-1 sites, possibly by controlling interactions with a modulating repressor.
Nucl Recept. 2004 May 7;2(1):2.
BACKGROUND: Estrogen receptors alpha and beta (ERalpha and ERbeta) differentially activate genes with AP-1 elements. ERalpha activates AP-1 targets via activation functions with estrogens (the AF-dependent pathway), whereas ERbeta, and a short version of ERalpha (ERalpha DBD-LBD) activate only with anti-estrogens (AF-independent pathway). The DNA binding domain (DBD) plays an important role in both pathways, even though neither pathway requires ERE recognition. RESULTS: Mutations of a highly conserved DBD lysine (ERalpha.K206A/G), lead to super-activation of AP-1 through activation function dependent pathways, up to 200 fold. This super-activity can be elicited either through ER AFs 1 or 2, or that of a heterologous activation function (VP16). The homologous substitution in ERbeta, K170A, or in ERalpha DBD-LBD leads to estrogen-dependent AP-1 activation and loss of the usually potent anti-estrogen effects. Each of numerous K206 substitutions in ERalpha, except K206R, eliminates anti-estrogen activation and this loss correlates perfectly with a loss of ability to titrate a repressive function from the RU486 bound progesterone receptor. CONCLUSION: We conclude that ER DBDs contain a complex regulatory function that influences ligand activation profiles at AP-1. This function, which requires the integrity of the conserved lysine, both allows for activation at AP-1 with anti-estrogens (with ERbeta and ERalpha DBD-LBD), and prevents ERalpha from becoming superactive at AP-1 with estrogens. We discuss the possibility that a repressor interaction with the DBD both mediates the AF-independent pathway and dampens the AF dependent pathway. Mutations in the conserved lysine might, by this model, disrupt the binding or function of the repressor. [Abstract/Link to Full Text]

Arnold KA, Eichelbaum M, Burk O
Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor.
Nucl Recept. 2004 Mar 25;2(1):1.
BACKGROUND: The constitutive androstane receptor (CAR) plays a key role in the control of drug metabolism and transport by mediating the phenobarbital-type induction of many phase I and II drug metabolizing enzymes and drug transporters. RESULTS: We identified transcripts generated by four different alternative splicing events in the human CAR gene. Two of the corresponding ligand binding domain isoforms demonstrated novel functional properties: First, CAR(SV3), which is encoded by a transcript containing an lengthened exon 7, differentially transactivated target gene promoters. Second, CAR(SV2), which results from the use of an alternative 3' splice site lengthening exon 8, showed ligand-dependent instead of constitutive interaction with coactivators. Furthermore, alternatively spliced transcripts demonstrated a tissue-specific expression pattern. In most tissues, only transcripts generated by alternative splicing within exon 9 were expressed. The encoded variant demonstrated a loss-of-function phenotype. Correct splicing of exon 8 to exon 9 is restricted to only a few tissues, among them liver and small intestine for which CAR function has been demonstrated, and is associated with the induction of CAR expression during differentiation of intestinal cells. CONCLUSION: Due to their specific activities, CAR variant proteins SV2 and SV3 may modulate the activity of reference CAR(SV1). Furthermore, we propose that transcriptional activation and regulation of splicing of exon 9 may be coupled to ensure appropriate tissue- and differentiation state-specific expression of transcripts encoding functional CAR protein. Altogether, alternative splicing seems to be of utmost importance for the regulation of CAR expression and function. [Abstract/Link to Full Text]

Vosper H, Khoudoli GA, Palmer CN
The peroxisome proliferator activated receptor delta is required for the differentiation of THP-1 monocytic cells by phorbol ester.
Nucl Recept. 2003 Dec 11;1(1):9.
BACKGROUND: PPARdelta (NR1C2) promotes lipid accumulation in human macrophages in vitro and has been implicated in the response of macrophages to vLDL. We have investigated the role of PPARdelta in PMA-stimulated macrophage differentiation.The THP-1 monocytic cell line which displays macrophage like differentiation in response to phorbol esters was used as a model system. We manipulated the response to PMA using a potent synthetic agonist of PPARdelta, compound F. THP-1 sub-lines that either over-expressed PPARdelta protein, or expressed PPARdelta anti-sense RNA were generated. We then explored the effects of these genetic modulations on the differentiation process. RESULTS: The PPARdelta agonist, compound F, stimulated differentiation in the presence of sub-nanomolar concentrations of phorbol ester. Several markers of differentiation were induced by compound F in a synergistic fashion with phorbol ester, including CD68 and IL8. Over-expression of PPARdelta also sensitised THP-1 cells to phorbol ester and correspondingly, inhibition of PPARdelta by anti-sense RNA completely abolished this response. CONCLUSIONS: These data collectively demonstrate that PPARdelta plays a fundamental role in mediating a subset of cellular effects of phorbol ester and supports observations from mouse knockout models that PPARdelta is involved in macrophage-mediated inflammatory responses. [Abstract/Link to Full Text]

Val P, Lefrançois-Martinez AM, Veyssičre G, Martinez A
SF-1 a key player in the development and differentiation of steroidogenic tissues.
Nucl Recept. 2003 Sep 18;1(1):8.
Since its discovery in the early 1990s, the orphan nuclear receptor SF-1 has been attributed a central role in the development and differentiation of steroidogenic tissues. SF-1 controls the expression of all the steroidogenic enzymes and cholesterol transporters required for steroidogenesis as well as the expression of steroidogenesis-stimulating hormones and their cognate receptors. SF-1 is also an essential regulator of genes involved in the sex determination cascade. The study of SF-1 null mice and of human mutants has been of great value to demonstrate the essential role of this factor in vivo, although the complete adrenal and gonadal agenesis in knock-out animals has impeded studies of its function as a transcriptional regulator. In particular, the role of SF-1 in the hormonal responsiveness of steroidogenic genes promoters is still a subject of debate. This extensive review takes into account recent data obtained from SF-1 haploinsufficient mice, pituitary-specific knock-outs and from transgenic mice experiments carried out with SF-1 target gene promoters. It also summarizes the pros and cons regarding the presumed role of SF-1 in cAMP signalling. [Abstract/Link to Full Text]

Henry II KW, Spencer ML, Theodosiou M, Lou D, Noonan DJ
A neuronal-specific differentiation protein that directly modulates retinoid receptor transcriptional activation.
Nucl Recept. 2003 Sep 10;1(1):7.
BACKGROUND: The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes. This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex. Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription. RESULTS: A novel yeast screen sensitive to retinoid-X receptor (RXR) transcriptional activation resulted in the isolation of the rat homologue of the mouse NPDC-1 gene. NPDC-1 has been shown to be involved in the control of neural cell proliferation and differentiation, possibly through interactions with the cell cycle promoting transcription factor E2F-1. Although the amino acid sequence of NPDC-1 is highly conserved between mouse, rat and human homologues, their tissue specific expression was seen to vary. A potential for direct protein:protein interaction between NPDC-1, RXR and retinoic acid receptor beta (RARbeta) was observed in vitro and NPDC-1 facilitated RXR homodimer and RAR-RXR heterodimer DNA binding in vitro. Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner. CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression. As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation. [Abstract/Link to Full Text]

Martin PJ, Delmotte MH, Formstecher P, Lefebvre P
PLZF is a negative regulator of retinoic acid receptor transcriptional activity.
Nucl Recept. 2003 Sep 6;1(1):6.
BACKGROUND: Retinoic acid receptors (RARs) are ligand-regulated transcription factors controlling cellular proliferation and differentiation. Receptor-interacting proteins such as corepressors and coactivators play a crucial role in specifying the overall transcriptional activity of the receptor in response to ligand treatment. Little is known however on how receptor activity is controlled by intermediary factors which interact with RARs in a ligand-independent manner. RESULTS: We have identified the promyelocytic leukemia zinc finger protein (PLZF), a transcriptional corepressor, to be a RAR-interacting protein using the yeast two-hybrid assay. We confirmed this interaction by GST-pull down assays and show that the PLZF N-terminal zinc finger domain is necessary and sufficient for PLZF to bind RAR. The RAR ligand binding domain displayed the highest affinity for PLZF, but corepressor and coactivator binding interfaces did not contribute to PLZF recruitment. The interaction was ligand-independent and correlated to a decreased transcriptional activity of the RXR-RAR heterodimer upon overexpression of PLZF. A similar transcriptional interference could be observed with the estrogen receptor alpha and the glucocorticoid receptor. We further show that PLZF is likely to act by preventing RXR-RAR heterodimerization, both in-vitro and in intact cells. CONCLUSION: Thus RAR and PLZF interact physically and functionally. Intriguingly, these two transcription factors play a determining role in hematopoiesis and regionalization of the hindbrain and may, upon chromosomal translocation, form fusion proteins. Our observations therefore define a novel mechanism by which RARs activity may be controlled. [Abstract/Link to Full Text]

Jiang S, Tanaka T, Iwanari H, Hotta H, Yamashita H, Kumakura J, Watanabe Y, Uchiyama Y, Aburatani H, Hamakubo T, Kodama T, Naito M
Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4alpha (HNF4alpha) isoforms in human and rats.
Nucl Recept. 2003 Aug 8;1(1):5.
BACKGROUND: Hepatocyte nuclear factor-4alpha (HNF4alpha; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4alpha causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4alpha in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4alpha protein, due, in part, to the limited availability of human HNF4alpha-specific antibodies. RESULTS: Monoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4alpha fusion proteins as the immunizing agent. The mouse anti-human HNF4alpha monoclonal antibody (K9218) generated against human HNF4alpha1/alpha2/alpha3 amino acids 3-49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4alpha proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4alpha protein, but HEK293 showed no expression of HNF4alpha protein. Nuclear-specific localization of the HNF4alpha protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4alpha protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4alpha protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4alpha in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4alpha mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis. CONCLUSION: These results suggest that this method has the potential to produce valuable antibodies without the need for a protein purification step. Immunohistochemical studies indicate the tissue and subcellular specific localization of HNF4alpha and demonstrate the utility of K9218 for the detection of P1 promoter-driven HNF4alpha isoforms in humans and in several other mammalian species. [Abstract/Link to Full Text]

Watanabe Y, Tanaka T, Uchiyama Y, Takeno T, Izumi A, Yamashita H, Kumakura J, Iwanari H, Shu-Ying J, Naito M, Mangelsdorf DJ, Hamakubo T, Kodama T
Establishment of a monoclonal antibody for human LXRalpha: Detection of LXRalpha protein expression in human macrophages.
Nucl Recept. 2003 May 9;1(1):1.
Liver X activated receptor alpha (LXRalpha) forms a functional dimeric nuclear receptor with RXR that regulates the metabolism of several important lipids, including cholesterol and bile acids. As compared with RXR, the LXRalpha protein level in the cell is low and the LXRalpha protein itself is very hard to detect. We have previously reported that the mRNA for LXRalpha is highly expressed in human cultured macrophages. In order to confirm the presence of the LXRalpha protein in the human macrophage, we have established a monoclonal antibody against LXRalpha, K-8607. The binding of mAb K-8607 to the human LXRalpha protein was confirmed by a wide variety of different techniques, including immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). By immunoblotting with this antibody, the presence of native LXR protein in primary cultured human macrophage was demonstrated, as was its absence in human monocytes. This monoclonal anti-LXRalpha antibody should prove to be a useful tool in the analysis of the human LXRalpha protein. [Abstract/Link to Full Text]

Tzameli I, Chua SS, Cheskis B, Moore DD
Complex effects of rexinoids on ligand dependent activation or inhibition of the xenobiotic receptor, CAR.
Nucl Recept. 2003 Jun 6;1(1):2.
BACKGROUND: CAR/RXR heterodimers bind a variety of hormone response elements and activate transcription in the absence of added ligands. This constitutive activity of murine CAR can be inhibited by the inverse agonist ligand androstanol or increased by the agonist TCPOBOP. RXR agonists activate some RXR heterodimer complexes, which are termed permissive, while other non-permissive complexes are not responsive to such ligands. RESULTS: Direct protein-protein interaction studies demonstrate that the RXR agonist 9-cis-RA increases interaction of CAR/RXR heterodimers with the coactivator SRC-3, but also inhibits the ability of TCPOBOP to increase and androstanol to decrease coactivator binding. CAR transactivation of a response element with a five nucleotide spacer (DR-5) is unaffected by 9-cis-RA or the synthetic RXR agonist LG1069. In agreement with the inhibitory effect observed in vitro, these rexinoids block both the TCPOBOP mediated transactivation of this element and the androstanol dependent inhibition. In contrast, CAR transactivation of other response elements is increased by rexinoids. Stable expression of CAR in a HepG2 derived cell line increases expression of the endogenous CAR target CYP2B6. This expression is further increased by TCPOBOP but decreased by either androstanol or LG1069, and LG1069 blocks the stimulatory effect of TCPOBOP but not the inhibitory effect of androstanol. CONCLUSION: We conclude that CAR/RXR heterodimers are neither strictly permissive nor non-permissive for RXR signaling. Instead, rexinoids have distinct effects in different contexts. These results expand the potential regulatory mechanisms of rexinoids and suggest that such compounds may have complex and variable effects on xenobiotic responses. [Abstract/Link to Full Text]

De Miguel F, Lee SO, Onate SA, Gao AC
Stat3 enhances transactivation of steroid hormone receptors.
Nucl Recept. 2003 Jun 13;1(1):3.
BACKGROUND: Steroid hormone receptors (SHRs) are members of the superfamily of ligand-activated transcription factors that regulate many biological processes. Co-regulators act as bridging molecules between the SHR and general transcription factors to enhance transactivation of target genes. Previous studies demonstrated that Stat3 is constitutively activated in prostate cancer and can enhance prostate specific antigen (PSA) expression and promote androgen independent growth. In this study, we investigate whether Stat3 can enhance steroid hormone receptors activation. METHODS: CV-1 cells in which plasmids expressing androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR) or estrogen receptor (ER) were cotransfected with a constitutively active STAT3 mutant. RESULTS: Stat3 stimulates the transcriptional activity of all four SHR tested, AR, GR, PR and ER, in a hormone-dependent manner. Stat3 acts in a synergistic fashion with other coactivators such as SRC-1, pCAF, CBP, and TIF-2 on the transcriptional activity of these SHR. In addition, Stat3 significantly enhanced the sensitivity of androgen receptor in response to androgen. STAT3 did not affect the specificity of AR for other steroid hormones other than androgen or binding of AR to other hormone responsive elements. CONCLUSIONS: These findings suggest that Stat3 can enhance the transactivation of AR, GR, PR and ER, and activated Stat3 could have a role in the development or progression of a hypersensitive AR. [Abstract/Link to Full Text]

Webb P, Valentine C, Nguyen P, Price RH, Marimuthu A, West BL, Baxter JD, Kushner PJ
ERbeta Binds N-CoR in the Presence of Estrogens via an LXXLL-like Motif in the N-CoR C-terminus.
Nucl Recept. 2003 Jun 28;1(1):4.
Nuclear receptors (NRs) usually bind the corepressors N-CoR and SMRT in the absence of ligand or in the presence of antagonists. Agonist binding leads to corepressor release and recruitment of coactivators. Here, we report that estrogen receptor beta (ERbeta) binds N-CoR and SMRT in the presence of agonists, but not antagonists, in vitro and in vivo. This ligand preference differs from that of ERalpha interactions with corepressors, which are inhibited by estradiol, and resembles that of ERbeta interactions with coactivators. ERbeta /N-CoR interactions involve ERbeta AF-2, which also mediates coactivator recognition. Moreover, ERbeta recognizes a sequence (PLTIRML) in the N-CoR C-terminus that resembles coactivator LXXLL motifs. Inhibition of histone deacetylase activity specifically potentiates ERbeta LBD activity, suggesting that corepressors restrict the activity of AF-2. We conclude that the ER isoforms show completely distinct modes of interaction with a physiologically important corepressor and discuss our results in terms of ER isoform specificity in vivo. [Abstract/Link to Full Text]

Recent Articles in Molecular & Cellular Proteomics

Abu-Farha M, Lambert JP, Al-Madhoun AS, Elisma F, Skerjanc IS, Figeys D
The tale of two domains: Proteomic and genomic analysis of SMYD2, a new histone methyltransferase.
Mol Cell Proteomics. 2007 Dec 7; .
Very little is known about SMYD2 (SET and MYND containing protein 2), a member of the SMYD protein family. However, the interest in better understanding the roles of SMYD2 has grown due to recent reports indicating that SMYD2 methylates p53 and histone H3. In this paper, we present a combined proteomic and genomic study of SMYD2 designed to elucidate its molecular roles. We report the cytosolic and nuclear interactome of SMYD2 using a combination of IP-HTMS, ChIP-HTMS, and co-immunoprecipitation methods. In particular, we report that SMYD2 interacts with HSP90alpha independently of the SET and MYND domain; with EBP41L3 through the MYND domain; and with p53 through the SET domain. We demonstrated that the interaction of SMYD2 with HSP90alpha enhances SMYD2 histone methyltransferase activity and specificity for histone H3 at lysine 4 (H3K4) in vitro. Interestingly, histone H3K36 methyltransferase activity is independent of its interaction with HSP90alpha similarly to LSD1 dependency on the androgen receptor. We also show that the SET domain is required for the methylation at H3K4. We demonstrated using a modified chromatin IP protocol that the SMYD2 gain of function leads to an increase in H3K4 methylation in vivo, while no observable levels of H3K36 were observed. We also report that the SMYD2 gain of function is correlated with the up-regulation of 37 and down-regulation of 4 genes, the majority of which are involved in the cell cycle, chromatin remodeling, and transcriptional regulation. We validate that the up-regulation of TACC2 by SMYD2 occurs as a result of SMYD2 binding to the TACC2 promoter where it methylates H3K4. Furthermore, the combination of the SMYD2 interactome with the gene expressions data suggests that some of the genes regulated by SMYD2 are closely associated with SMYD2 interacting proteins. [Abstract/Link to Full Text]

Karamessinis PM, Malamitsi-Puchner A, Boutsikou T, Makridakis M, Vougas K, Fountoulakis M, Vlahou A, Chrousos G
Marked defects in the expression and glycosylation of alpha -2-HS glycoprotein/fetuin-A in plasma from neonates with intrauterine growth restriction: Proteomic screening and potential clinical implications.
Mol Cell Proteomics. 2007 Dec 7;
Intrauterine growth restriction (IUGR) has been associated with increased perinatal morbidity and mortality and increased morbidity and metabolic abnormalities later in life. IUGR is characterized as the failure of a fetus to achieve his or her genetic growth potential in utero. Altered protein expression profiles associated with IUGR may be informative on the pathologic mechanisms of this condition and might reveal potential markers for postnatal complications. The aim of this study was to compare protein profiles of umbilical cord (UC) plasma from IUGR and appropriate for gestational age (AGA) full-term neonates. Blood samples from doubly clamped UC at delivery from ten IUGR and ten AGA full-term neonates were analyzed by two dimensional electrophoresis (2-DE) and mass spectrometry (MS). Prominent changes of the alpha-2-HS glycoprotein/fetuin-A, were observed in IUGR cases. Specifically, we show that these changes occur primarily at the level of post-translational modifications of the protein. Using a combination of mass spectrometry and classical biochemical assays, single and heavy chain forms of fetuin-A were found to lack the normally present O-linked sialic acids in IUGR neonates. Fetuin A is a glycoprotein that has been associated with promotion of in vitro cell replication, fetal growth and osteogenesis, and protection from Gram- bacterial endotoxins. Prominent defects in glycosylation/sialylation of fetuin-A revealed by our study, might be responsible for impaired function of fetuin-A, leading to deficient fetal growth, especially osteogenesis, and/or to the development of complications frequently seen later in the lives of IUGR neonates. [Abstract/Link to Full Text]

Couté Y, Kindbeiter K, Belin S, Dieckmann R, Duret L, Bezin L, Sanchez JC, Diaz JJ
ISG20L2, a novel vertebrate nucleolar exoribonuclease involved in Ribosome biogenesis.
Mol Cell Proteomics. 2007 Dec 6;
Proteomic analyses of human nucleoli provided molecular bases for an understanding of the multiple functions fulfilled by these nuclear domains. However, the biological roles of about 100 of the identified proteins are unpredictable. The present article describes the functional characterization of one of these proteins, ISG20L2. We demonstrate that ISG20L2 is a 3' to 5' exoribonuclease involved in ribosome biogenesis at the level of 5.8S rRNA maturation, more specifically in the processing of the 12S pre-rRNA. The use of truncated forms of ISG20L2 demonstrated that its N-terminal half promotes the nucleolar localization and suggested that its C-terminal half bears the exoribonuclease activity. Identification of the binding partners of ISG20L2 confirms its involvement in the biogenesis of the large ribosomal subunit. These results strongly support the notion that, in human, as it was demonstrated in yeast, 5.8S rRNA maturation requires several proteins in addition to the exosome complex. Furthermore, this observation sustains greatly the idea that the extremely conserved need for correctly processed rRNAs in vertebrates and yeast are achieved by close but different mechanisms. [Abstract/Link to Full Text]

Zheng X, Hong L, Shi L, Guo J, Sun Z, Zhou J
Proteomic analysis of host cells infected with infectious bursal disease virus.
Mol Cell Proteomics. 2007 Dec 4;
The effect of infectious bursal disease virus (IBDV) infection on cellular protein expression is essential for viral pathogenesis. To characterize the cellular response to IBDV infection, the differential proteomes of chicken embryo fibroblasts (CEFs), with and without IBDV infection, were analyzed at different time points with two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF identification. Comparative analysis of multiple 2-DE gels revealed that the majority of protein expression changes appeared at 48 h and 96 h after IBDV infection. Mass spectrometry identified 51 altered cellular proteins, including 13 up-regulated proteins and 38 down-regulated proteins 12 to 96 h after infection. Notably, 2-DE analysis revealed that IBDV infection induced the overexpression of polyubiquitin, apolipoprotein A-1, heat shock 27 kDa protein 1, actins, tubulins, eukaryotic translation initiation factor 4A isoform 2, acidic ribosomal phosphoprotein, and ribosomal protein SA isoform 2. In addition, IBDV infection considerably suppressed those cellular proteins involved in ubiquitin-mediated protein degradation, energy metabolism, intermediate filaments, host translational apparatus, and signal transduction. Moreover, 38 corresponding genes of the differentially expressed proteins were quantitated by real-time RT-PCR to examine the transcriptional profiles between infected and uninfected CEFs. Western blot further confirmed the inhibition of Rho protein GDP dissociation inhibitor expression and the induction of polyubiquitin during IBDV infection. Subcellular distribution analysis of the cytoskeletal proteins vimentin and ss-tubulin clearly demonstrated that IBDV infection induced the disruption of the vimentin network and microtubules late in IBDV infection. Thus, this work effectively provides useful dynamic protein-related information to facilitate further investigation of the underlying mechanism of IBDV infection and pathogenesis. [Abstract/Link to Full Text]

Trinidad JC, Thalhammer A, Specht CG, Lynn AJ, Baker PR, Schoepfer R, Burlingame AL
Quantitative analysis of synaptic phosphorylation and protein expression.
Mol Cell Proteomics. 2007 Dec 3;
The postsynaptic density (PSD) signaling machinery contains proteins with diverse functions. Brain region-specific variations in PSD components mediate distinct physiological responses to synaptic activation. We have developed mass spectrometry based methods to comprehensively compare both relative protein expression and phosphorylation status from proteins present in biochemical preparations of postsynaptic density. Using these methods, we determined the relative expression of 2159 proteins and 1564 phosphorylation sites in PSD preparations from murine cortex, midbrain, cerebellum, and hippocampus. These experiments were conducted twice, using independent biological replicates, which allowed us to assess the experimental and biological variability in this system. Concerning protein expression, cluster analysis revealed that known functionally associated proteins display coordinated synaptic expression. Therefore, proteins identified as co-clustering with known protein complexes are prime candidates for assignment as previously unrecognized components. Concerning degree of phosphorylation, we observed more extensive phosphorylation sites on NMDA receptors than AMPA receptors, consistent with the central role of NMDA receptors in processing synaptic transmission patterns. Average kinase and phosphatase levels were highest in the hippocampus, correlating with a higher overall phosphopeptide abundance present in this brain region. These findings suggest the hippocampus utilizes reversible protein phosphorylation to a greater extent than other brain regions, when modifying synaptic strength. [Abstract/Link to Full Text]

Gramolini AO, Kislinger T, Alikhani-Koopaei R, Fong V, Thompson NJ, Isserlin R, Sharma P, Oudit GY, Trivieri MG, Fagan A, Kannan A, Higgins D, Huedig H, Hess G, Arab S, Seidman JG, Seidman CE, Frey B, Perry M, Backx PH, Liu PP, Maclennan DH, Emili A
Comparative proteomic profiling of a phospholamban mutant mouse model of dilated cardiomyopathy reveals progressive intracellular stress responses.
Mol Cell Proteomics. 2007 Nov 30;
Defective mobilization of Ca2+ by cardiomyocytes predisposes to cardiac insufficiency, but the causative mechanisms leading to congestive heart failure (HF) remain unclear. Here, we perform exhaustive global proteomic surveys of cardiac ventricle isolated from a mouse model of cardiomyopathy overexpressing a phospholamban mutant, R9C (PLN R9C) and exhibiting impaired Ca2+ handling and death at 24 weeks and compare them with normal control littermates. The relative expression patterns of 6190 high-confidence proteins were monitored by shotgun tandem mass spectrometry at 8, 16 and 24 weeks of disease progression. Significant differential abundance of 593 proteins was detected. These proteins mapped to select biological pathways such as ER-stress response, cytoskeletal remodeling and apoptosis, and included known biomarkers of HF (e.g. BNP/ANF, ACE) and other indicators of pre-symptomatic functional impairment. These altered proteomic profiles were concordant with cognate mRNA patterns recorded in parallel using high-density mRNA microarrays, and top candidates were validated by RT-PCR and Western-blotting. Mapping of our highest ranked proteins against a human diseased explant and to available datasets indicate that many of these proteins could serve as markers of disease. Indeed, we show that several of these proteins are detectable in mouse and human plasma, and display differential abundance in the plasma of diseased mice and affected patients. These results offer a systems-wide perspective of the dynamic mal-adaptions associated with impaired Ca2+ homeostasis that perturb myocyte function and ultimately converge to cause HF. [Abstract/Link to Full Text]

Xie H, Onsongo G, Popko J, de Jong EP, Cao J, Carlis JV, Griffin RJ, Rhodus NL, Griffin TJ
Proteomic analysis of cells in whole saliva from oral cancer patients via value-added three-dimensional peptide fractionation and tandem mass spectrometry.
Mol Cell Proteomics. 2007 Nov 28;
Whole human saliva possesses tremendous potential in clinical diagnostics, particularly for conditions within the oral cavity, such as oral cancer. Although many have studied the soluble fraction of whole saliva, few have taken advantage of the diagnostic potential of the cells present in saliva, and none have taken advantage of proteomic capabilities for their study. We report on a novel proteomic method with which we characterized for the first time cells contained in whole saliva from patients diagnosed with oral squamous cell carcinoma. Our method employs three dimensions of peptide fractionation, combining these steps: preparative IEF using Free Flow Electrophoresis; strong cation exchange step-gradient chromatography; and microcapillary reverse-phase liquid chromatography. We determined that the whole saliva samples contained enough cells, mostly exfoliated epithelial cells, providing adequate amounts of total protein for proteomic analysis. From a mixture of four oral cancer patient samples, the analysis resulted in a catalogue of over one thousand human proteins, each identified from at least two peptides, including numerous proteins with a role in oral squamous cell carcinoma signaling and tumorigenesis pathways. Additionally, proteins from over 30 different bacteria were identified, some of which putatively contribute to cancer development. The combination of preparative IEF followed by strong cation exchange chromatography effectively fractionated the complex peptide mixtures, despite the closely related physiochemical peptide properties of these separations (pI and solution phase charge, respectively). Furthermore, compared to our two-step method combining preparative IEF and reverse phase liquid chromatography, our three-step method identified significantly more cellular proteins while retaining higher confidence protein identification enabled by peptide pI information gained through IEF. Thus, for detecting salivary markers of oral cancer, and possibly other conditions of the oral cavity, the results confirm both the potential of analyzing the cells in whole saliva and doing so with our proteomic method. [Abstract/Link to Full Text]

Graumann J, Hubner NC, Kim JB, Ko K, Moser M, Kumar C, Cox J, Schoeler H, Mann M
SILAC-labeling and proteome quantitation of mouse embryonic stem cells to a depth of 5111 proteins.
Mol Cell Proteomics. 2007 Nov 28;
Embryonic stem (ES) cells are pluripotent cells isolated from mammalian preimplantation embryos. They are capable of differentiating into all cell types and therefore hold great promise in regenerative medicine. Here we show that murine ES cells can be fully SILAC-labeled when grown feeder-free during the last phase of cell culture. We fractionated the SILAC-labeled ES cell proteome by one dimensional gel electrophoresis and by isoelectric focusing of peptides. High resolution analysis on a linear ion trap-orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 5,000 distinct proteins. This is the largest quantified proteome reported to date and contains prominent stem cell markers such as Oct4, Nanog, Sox2 and Utf1 along with the embryonic form of Ras (ERas). We also quantify the proportion of the ES cell proteome present in cytosolic, nucleoplasmic and membrane/chromatin fractions. We compared two different preparation approaches - cell fractionation followed by 1D gel separation and in-solution digestion of total cell lysate combined by isoelectric focusing, and found comparable proteome coverage with no apparent bias for any functional protein classes for either approach. Bioinformatic analysis of the ES cell proteome reveals a broad distribution of cellular functions with overrepresentation of proteins involved in proliferation. We compare the proteome with a recently published map of chromatin states of promoters in ES cells and find excellent correlation between protein expression and the presence of active and repressive chromatin marks. [Abstract/Link to Full Text]

Hsieh SY, Tseng CL, Lee YS, Kuo AJ, Sun CF, Lin YH, Chen JK
Highly efficient classification and identification of human pathogenic bacteria by MALDI-TOF MS.
Mol Cell Proteomics. 2007 Nov 27;
Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional workflows are time-consuming and procedures are multi-faceted. Mass spectrometry (MS) can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising 6 human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to 6. In parallel, classification models were constructed via the Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover, bacteria fewer than 104 cells, and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance, even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future. [Abstract/Link to Full Text]

Williamson AJ, Smith DL, Blinco D, Unwin RD, Pearson S, Wilson C, Miller C, Lancashire L, Lacaud G, Kouskoff V, Whetton AD
Quantitative proteomic analysis demonstrates post-transcriptional regulation of embryonic stem cell differentiation to hematopoiesis.
Mol Cell Proteomics. 2007 Nov 27;
Embryonic stem (ES) cells can differentiate in vitro to produce the endothelial and hematopoietic precursor, the hemangioblasts, which are derived from the mesoderm germ layer. Differentiation of BryGFP/+ ES cell to hemangioblasts can be followed by the expression of the BryGFP/+ and Flk1 genes. Proteomic and transcriptomic changes during this differentiation process were analyzed to identify mechanisms for phenotypic change during early differentiation. Three populations of differentiating BryGFP ES cells were obtained by flow cytometric sorting, GFP-Flk1- (epiblast), GFP+Flk1- (mesoderm) and GFP+Flk1+ (hemangioblast). Microarray analyses and relative quantification 2 dimensional liquid chromatography/tandem mass spectrometry (LCLCMSMS) on nuclear extracts were performed. We identified and quantified 2389 proteins, 1057 of which were associated to their microarray probeset. These included a variety of low abundance transcription factors, e.g. UTF1, Sox2, Oct4 and E2F4, demonstrating a high level of proteomic penetrance. When paired comparisons of changes in the mRNA and protein expression levels were performed low levels of correlation were found. A strong correlation between isobaric tag-derived relative quantification and western blot analysis was found for a number of nuclear proteins. Pathway and ontology analysis identified proteins known to be involved in the regulation of stem cell differentiation and proteins with no described function in early ES cell development were also shown to change markedly at the proteome level only. ES cell development is regulated at the mRNA and protein level. [Abstract/Link to Full Text]

Thingholm TE, Jensen ON, Robinson PJ, Larsen MR
SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides.
Mol Cell Proteomics. 2007 Nov 26;
The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphoryated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 microg of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides. [Abstract/Link to Full Text]

Pavelka N, Fournier ML, Swanson SK, Pelizzola M, Ricciardi-Castagnoli P, Florens L, Washburn MP
Statistical similarities between transcriptomics and quantitative shotgun proteomics data.
Mol Cell Proteomics. 2007 Nov 19;
If the large collection of microarray-specific statistical tools was applicable to the analysis of quantitative shotgun proteomics datasets, it would certainly foster an important advancement of proteomics research. Here, we analyze two large multi-dimensional protein identification technology (MudPIT) datasets - one containing 8 replicates of the soluble fraction of a yeast whole-cell lysate, one containing 9 replicates of a human immuno-precipitate - to test whether normalized spectral abundance factor (NSAF) values share substantially similar statistical properties with transcript abundance values from Affymetrix GeneChip data. First, we show similar dynamic range and distribution properties of these two types of numeric values. Next, we observe that the standard deviation (SD) of a protein's NSAF values is dependent on the average NSAF value of the protein itself, following a power law. This relationship can be modeled by a power law global error model (PLGEM), initially developed to describe the variance-versus-mean dependence that exists in GeneChip data. PLGEM parameters obtained from NSAF datasets prove to be surprisingly similar to the typical parameters observed in GeneChip datasets. The most important common feature identified by this approach is that, although in absolute terms the SD of replicated abundance values increases as a function of increasing average abundance, the coefficient of variation - a relative measure of variability - becomes progressively smaller under the same conditions. We next show that PLGEM parameters are reasonably stable to decreasing numbers of replicates. We finally illustrate one possible application of PLGEM in the identification of differentially abundant proteins, which might potentially outperform standard statistical tests. In summary, we believe that this body of work lays the foundation for the application of microarray-specific tools in the analysis of NSAF datasets. [Abstract/Link to Full Text]

Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Ponten F, Brismar H, Uhlen M, Andersson-Svahn H
Towards a confocal subcellular atlas of the human proteome.
Mol Cell Proteomics. 2007 Nov 19;
Information of protein localization on subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here, we report on a pilot study of 466 proteins in three human cell lines aimed to allow large-scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large-scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization. [Abstract/Link to Full Text]

Nanavati D, Gucek M, Milne JL, Subramaniam S, Markey SP
Stoichiometry and absolute quantification of proteins with mass spectrometry using fluorescent and isotope labeled concatenated peptide standards.
Mol Cell Proteomics. 2007 Nov 19;
We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in E. coli to function as a spectroscopic standard for the quantification of an analogous stable isotope labeled, non-fluorescent fusion protein (GAB*), and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that derive from the a, ss and protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB*, and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits a, ss and was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with an RSD of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic or antibody-based technologies. [Abstract/Link to Full Text]

Marcantonio M, Trost M, Courcelles M, Desjardins M, Thibault P
Combined enzymatic and data mining approaches for comprehensive phosphoproteome analyses; application to cell signaling events of interferon- stimulated macrophages.
Mol Cell Proteomics. 2007 Nov 14;
Protein phosphorylation is a central cell signaling event that underlies a broad spectrum of key physiological processes. Advances in affinity chromatography and mass spectrometry are now providing the ability to identify and quantitate thousands of phosphorylation sites simultaneously. In spite of these remarkable advances, comprehensive phosphoproteome analyses still present sizable analytical challenges in view of suppression effects affecting positive ion detection of phosphopeptides and the variable quality of MS/MS spectra limiting sequence assignment and identification of modification sites. This work presents an integrated enzymatic and data mining approach enabling the comprehensive detection of native and putative phosphopeptides following alkaline phosphatase digestion of TiO2-enriched cell extracts. The correlation of retention times of more than 750 phospho- and dephosphopeptide pairs from J774 macrophage cell extracts indicated that removal of the phosphate groups can impart a gain or a loss in hydrophobicity that is partly explained by the formation of salt bridge with proximal amino groups. Dephosphorylation also led to an average 2-fold increase in MS sensitivity which facilitated peptide sequencing. More importantly, alkaline phosphatase digestion enhanced the overall population of putative phosphopeptides from TiO2-enriched cell extracts providing a unique approach to profile multi-phosphorylated cognates that would have remained otherwise undetected. The application of this approach is demonstrated for differential phosphoproteome analyses of mouse macrophages exposed to interferon-gammafor 5 min. TiO2 enrichment enabled the identification of 1144 phosphopeptides from 433 different proteins of which 125 phosphopeptides showed a 2-fold change upon interferon-gamma. The use of alkaline phosphatase nearly doubled the number of putative phosphopeptides assignments leading to the observation of key interferon-gamma signaling events involved in vesicle trafficking, production of reactive oxygen species and mRNA translation. [Abstract/Link to Full Text]

Kim JY, Song HJ, Lim HJ, Shin MG, Kim JS, Kim HJ, Kim BY, Lee SW
Platelet factor-4 is an indicator of blood count recovery in acute myeloid leukemia patients in complete remission.
Mol Cell Proteomics. 2007 Nov 12;
To investigate whether serum biomarkers can be used to indicate the responsiveness of acute myeloid leukemia (AML) to remission induction chemotherapy, we performed MALDI-TOF protein profile analysis of patient sera. The resulting spectra revealed a protein (or peptide) peak at m/z 7764 that varied in intensity; its intensity was much higher in samples from patients in complete remission than in those from patients with resistant disease, or in samples taken prior to treatment (at the time of diagnosis). Using fractionation, trypsin digestion, and MS/MS and protein molecular weight analyses, we identified the m/z 7764 protein as platelet factor-4 (PF4). This identification was confirmed by a magnetic bead-based MALDI immunoassay. Statistical comparison of PF4 levels and platelet counts in patient sera revealed a significant positive correlation between the two variables. This study demonstrates that PF4 protein levels are a good indicator for the recovery of blood count in the complete remission of AML. The linear positive correlation curve indicates that blood count recovery of platelets to >100,000/mm3 is equivalent to a serum PF4 recovery level of >2.492 microg/mL. [Abstract/Link to Full Text]

Würtz SO, Mřller S, Mouridsen H, Hertel PB, Friis E, Brünner N
Plasma and serum levels of tissue inhibitor of metalloproteinases-1 are associated with prognosis in node-negative breast cancer - a prospective study.
Mol Cell Proteomics. 2007 Nov 12;
The tumor level of TIMP-1 has been suggested as a new prognostic marker in breast cancer. The purpose of this study was to investigate whether TIMP-1 also carries prognostic information when measured in blood as this is a much more preferable material compared with tumor extracts. Using ELISA, TIMP-1 was measured in prospectively collected pre-operative plasma and serum samples from 519 patients with primary breast cancer and the measurements were related to patient outcome. The median age of the patients was 58 years (range 38-80 years) and the median follow up time was 1043 days (range 300-1630 days). Plasma and serum TIMP-1 measurements correlated significantly with each other with a Pearson correlation coefficient of 0.75 (p <0.0001). For univariate survival analysis, patients were divided into quartiles according to increasing TIMP-1 levels (Q1-Q4). Analysis of all patients showed that high TIMP-1 plasma levels were significantly associated with a shorter disease free survival. Sub-group analysis showed that plasma TIMP-1 significantly predicted the prognosis of node-negative patients but not of node-positive patients. Importantly, plasma TIMP-1 was able to further stratify low-risk node negative patients. High serum TIMP-1 levels were associated with a shorter disease free survival however the association was not statistically significant. In contrast, serum TIMP-1 significantly predicted the prognosis of node-negative and low-risk patients. In multivariate survival analysis of node negative patients including all the classical prognostic parameters, plasma TIMP-1 remained significantly associated with prognosis when comparing Q1 with Q2 and Q4. Serum TIMP-1 remained significant when comparing Q1 with Q4. Taken together, this study is to our knowledge the first large prospective study suggesting that TIMP-1 carries independent prognostic information when measured in blood, especially plasma. This was especially true in the node-negative group of patients and in patients already defined as low-risk patients using the currently available prognostic parameters. [Abstract/Link to Full Text]

Sarioglu H, Brandner S, Haberger M, Jacobsen C, Lichtmannegger J, Wormke M, Andrae U
Analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome changes in 5L rat hepatoma cells reveals novel targets of dioxin action including the mitochondrial apoptosis regulator VDAC2.
Mol Cell Proteomics. 2007 Nov 12;
As part of a comprehensive survey of the impact of the environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the proteome of hepatic cells, we have performed a high resolution 2-DE study on the rat hepatoma cell line 5L. 78 protein species corresponding to 73 different proteins were identified as up- or down-regulated following exposure of the cells to 1 nM TCDD for 8 hours. There was an overlap of only nine proteins with those detected as altered by TCDD in our recent study employing the non-gel based isotope-coded protein label (ICPL) method (Sarioglu et al., Proteomics 2006, 6, 2407-2421) indicating a strong complementarity of the two approaches. For the majority of the altered proteins, an effect of TCDD on their abundance or posttranslational modifications had not been known before. Several observations suggest that a sizeable fraction of the proteins with altered abundance was induced as an adaptive response to TCDD-induced oxidative stress that was demonstrated using the fluorescent probe dihydrorhodamine 123. A prominent group of these proteins comprised various enzymes for which there is evidence that their expression is regulated via the Keap1/Nrf2/ARE pathway. Other proteins included several involved in the maintenance of mitochondrial energy production and the regulation of the mitochondrial apoptotic pathway. A particularly intriguing finding was the up-regulation of the mitochondrial outer membrane pore protein, voltage-dependent anion channel-selective protein 2 (VDAC2), which was dependent on the presence of a functional Ah receptor. The regulatability of VDAC2 protein abundance has not been described previously. In view of the recently discovered central role of VDAC2 as an inhibitor of the activation of the proapoptotic protein BAK and the mitochondrial apoptotic pathway, the present data point to a hitherto unrecognised mechanism by which TCDD may affect cellular homeostasis and survival. [Abstract/Link to Full Text]

Chaurand P, Rahman MA, Hunt T, Mobley JA, Gu G, Latham JC, Caprioli RM, Kasper S
Monitoring mouse prostate development by profiling and imaging mass spectrometry.
Mol Cell Proteomics. 2007 Nov 8;
Mass spectrometry-based tissue profiling and imaging are technologies which allow identification and visualization of protein signals directly on thin sections cut from fresh frozen tissue specimens. These technologies were utilized to evaluate protein expression profiles in the normal mouse prostate during development (1 to 5 weeks of age), sexual maturation (6 weeks of age) and adult prostate (at 10, 15 or 40 weeks of age). The evolution of protein expression during normal prostate development and maturation were subsequently compared to 15 week prostate tumors derived from genetically engineered mice carrying the Large T antigen gene under regulation of the prostate-specific probasin promoter (LPB-Tag mouse model for prostate cancer). This approach identified proteins differentially expressed at specific time points during prostate development. Furthermore, expression of some of these proteins, for example probasin and spermine binding protein, were associated with prostate maturation, and prostate tumor formation resulted in their loss of expression. Cyclophilin A, a protein found in other cancers, was differentially a-acetylated on the N-terminal and both isoforms appeared during normal prostate and prostate tumor development. Imaging mass spectrometry localized the protein signals to specific prostatic lobes or regions. Thus, tissue profiling and imaging can be utilized to analyze the ontogeny of protein expression during prostate morphogenesis and tumorigenesis and identify proteins which could potentially serve as biomarkers for prostate cancer. [Abstract/Link to Full Text]

Barceló-Batllori S, Kalko SG, Esteban Y, Moreno S, Carmona MC, Gomis R
Integration of DIGE and bioinformatics analyses reveals a role of the anti-obesity agent tungstate in redox and energy homeostasis pathways in brown adipose tissue.
Mol Cell Proteomics. 2007 Nov 13;
Our previous results demonstrated that tungstate decreased weight gain and adiposity in obese rats through increased thermogenesis and lipid oxidation, suggesting that brown adipose tissue was one of the targets of its anti-obesity effect. To identify potential targets of tungstate, we used DIGE to compare brown adipose tissue protein extracts from the following experimental groups: untreated lean, tungstate treated lean, untreated obese and tungstate treated obese rats. In order to distinguish between direct targets of tungstate action from those that are secondary to body weight loss, we also included in the analysis an additional group consisting of obese rats that lose weight by caloric restriction. Hierarchical clustering of Anova and T-test contrasts clearly separated the different experimental groups. DIGE analysis identified 20 proteins as tungstate obesity-direct targets, involved in: Krebs cycle, glycolysis, lipolysis and fatty acid oxidation, electron transport and redox. Protein oxidation was decreased by tungstate treatment which confirmed a role in redox processes; however palmitate oxidation, as a measure of fatty acid beta oxidation, was not altered by tungstate, thus questioning its putative function on fatty acid oxidation. Protein network analyses using Ingenuity pathways highlighted peroxisome proliferator activated receptor coactivator 1 alpha (PGC-1 alpha) as a potential target. We confirmed by real-time PCR that indeed tungstate up-regulates PGC-1 alpha and its major target, uncoupling protein 1, was also increased as shown by western blot. These results illustrate the utility of proteomics and bioinformatics approaches to identify targets of obesity therapies, and suggest that in brown adipose tissue, tungstate modulates redox processes and increases energy dissipation through uncoupling and PGC-1 alpha up-regulation, thus contributing to its overall anti-obesity effect. [Abstract/Link to Full Text]

Miura Y, Hato M, Shinohara Y, Kuramoto H, Furukawa JI, Kurogochi M, Shimaoka H, Tada M, Nakanishi K, Ozaki M, Todo S, Nishimura SI
BlotGlycoABCTM: An integrated glycoblotting technique for rapid and large-scale clinical glycomics.
Mol Cell Proteomics. 2007 Nov 5;
Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important, since the proteomics research targets glycosylation, a posttranslational modification. Here we report an "all-in-one" protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABCTM bead, on-bead stabilization of sialic acids, and fluorescent-labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOFMS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery. [Abstract/Link to Full Text]

Villanueva J, Nazarian A, Lawlor K, Yi SS, Robbins RJ, Tempst P
A sequence-specific exopeptidase activity test (SSEAT) for 'functional' biomarker discovery.
Mol Cell Proteomics. 2007 Nov 6;
One form of functional proteomics entails profiling of genuine activities, as opposed to surrogates of activity or active 'states', in a complex biological matrix. For example, tracking enzyme-catalyzed changes, in real time, ranging from simple modifications to complex anabolic or catabolic reactions. Here, we present a test to compare defined exoprotease activities within individual proteomes of two or more groups of biological samples. It tracks degradation of artificial substrates, under strictly controlled conditions, using semi-automated MALDI-TOF mass spectrometric analysis of the resulting patterns. Each fragment is quantitated by comparison with double-labeled, non-degradable internal standards (all-D-amino acid peptides), spiked into the samples at the same time as the substrates to reflect adsorptive and processing-related losses. The full array of metabolites is then quantitated (CVs of 6.3 to 14.3% over 5 replicates) and subjected to multivariate statistical analysis. Using this approach, we tested serum samples of 48 metastatic thyroid cancer patients and 48 healthy controls, with selected peptide substrates taken from earlier standard peptidomic screens (i.e., the 'discovery' phase), and obtained class predictions with 94% sensitivity and 90% specificity, without prior feature selection (24 features). The test all but eliminates reproducibility problems related to sample collection, storage and handling, as well as to possible variability in endogenous peptide precursor levels owing to hemostatic alterations in cancer patients. [Abstract/Link to Full Text]

Berlanda Scorza F, Doro F, Rodríguez-Ortega MJ, Stella M, Liberatori S, Taddei AR, Serino L, Gomes Moriel D, Nesta B, Fontana MR, Spagnuolo A, Pizza M, Norais N, Grandi G
Proteomic characterization of outer membrane vesicles from the extraintestinal pathogenic Escherichia coli tolR IHE3034 mutant.
Mol Cell Proteomics. 2007 Nov 2;
Extra Intestinal Pathogenic Escherichia coli (ExPEC) are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis or septicemia. In this paper, we focused our attention on the identification of the outer-membrane proteins of the pathogen in consideration of their important biological role and of their being potential targets for prophylactic and therapeutic intervention. To this aim, we generated a tolR mutant of the pathogenic IHE3034 strain, which spontaneously released a large quantity of OMVs in the culture supernatant. The vesicles were analysed by 2D-electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which belonging to the outer membrane and periplasmic compartments. Interestingly, based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally, we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that OMVs represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis. [Abstract/Link to Full Text]

Winkler W, Zellner M, Diestinger M, Babeluk R, Marchetti M, Goll A, Zehetmayer S, Bauer P, Rappold E, Miller I, Roth E, Allmaier G, Oehler R
Biological variation of the platelet proteome in the elderly population and its implication for biomarker research.
Mol Cell Proteomics. 2007 Oct 25;
Knowledge about the extent of total variation experienced between samples from different individuals is of great importance for the design of not only proteomic, but every clinical study. This variation defines the smallest statistically significant detectable signal difference when comparing two groups of individuals. We isolated platelets from 20 healthy human volunteers aged 56 100 years, because this age group is most commonly encountered in the clinics. We determined the technical and total variation experienced in a proteome analysis using 2D-DIGE with IPGs in the pI ranges 4-7 and 6-9. Only spots which were reproducibly detectable in at least 90% of all gels (n=908) were included in the study. All spots have a similar technical variation with a median coefficient of variation (cv) of 7%. In contrast, spots show a more diverse total variation between individuals with a surprisingly low median cv of only 18%. Because most known biomarkers show an effect size in a 1 to 2-fold range of their cv, any future clinical proteomics study with platelets will require an analytical method which is able to detect such small quantitative differences. In addition, we calculated the minimal number of samples (sample size) needed to detect given protein expression differences with statistical significance. [Abstract/Link to Full Text]

Pflieger D, Jünger M, Müller M, Rinner O, Lee H, Gehrig P, Gstaiger M, Aebersold R
Quantitative proteomic analysis of protein complexes: Concurrent identification of interactors and their state of phosphorylation.
Mol Cell Proteomics. 2007 Oct 23;
Protein complexes have largely been studied by immuno-affinity purification and (mass spectrometric) analysis. While this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications and detecting quantitative changes in complex composition or state of modification of complex components. We have developed a protocol which enables us to determine, in a single LC-MS/MS analysis, the true protein constituents of a complex, to detect changes in the complex composition and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of four-plex iTRAQ isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate (IRS) homologue Chico. These experiments allowed us to identify 14-3-3e, 14-3-3 and the insulin receptor as specific Chico interactors. In a further experiment, we compared the immunopurified materials obtained from tagged-Chico-expressing cells that were either treated by insulin or left unstimulated. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins. [Abstract/Link to Full Text]

Huang H, Li L, Wu C, Schibli D, Colwill K, Ma S, Li C, Roy P, Ho K, Songyang Z, Pawson T, Gao Y, Li SS
Defining the specificity space of the human src-homology 2 domain.
Mol Cell Proteomics. 2007 Oct 22;
Src-homology 2 (SH2) domains are the largest family of interaction modules encoded by the human genome to recognize tyrosine-phosphorylated sequences and thereby play pivotal roles in transducing and controlling cellular signals emanated from protein tyrosine kinases. Different SH2 domains select for distinct phosphopeptides, and the function of a given SH2 domain is often dictated by the specific motifs that it recognizes. Therefore, deciphering the phosphotyrosyl peptide motif recognized by an SH2 domain is the key to understanding its cellular function. Here we cloned all 120 SH2 domains identified in the human genome and determined the phosphotyrosyl peptide-binding properties of 76 SH2 domains by screening an oriented peptide array library (OPAL). Of these 76, we defined the selectivity for 43 SH2 domains and refined the binding motifs for another 33 SH2 domains. A number of novel binding motifs have been identified, which is exemplified by the BRDG1 SH2 domain that selects specifically for a bulky, hydrophobic residue at P+4 relative to the pTyr residue. Based on the OPAL data, we developed SMALI (or scoring matrix-assisted ligand identification), a web-based program for predicting binding partners for SH2-containing proteins. When applied to SH2D1A/SAP, a protein whose mutation or deletion underlies the X-linked lymphoproliferative syndrome, SMALI not only recapitulated known interactions but also identified a number of novel interacting proteins for this disease-associated protein. SMALI also identified a number of potential interactors for BRDG1, a protein whose function is largely unknown. Peptide in-solution binding analysis demonstrated that a SMALI score correlates well with the binding energy of a peptide to a given SH2 domain. The definition of the specificity space of the human SH2 domain provides both the necessary molecular basis and a platform for future exploration of the functions for SH2-containing proteins in cells. [Abstract/Link to Full Text]

Pierce A, Unwin RD, Evans CA, Griffiths S, Carney L, Zhang L, Jaworska E, Lee CF, Blinco D, Okoniewski MJ, Miller CJ, Bitton DA, Spooncer E, Whetton AD
Eight-channel iTRAQ enables comparison of the activity of 6 leukaemogenic tyrosine kinases.
Mol Cell Proteomics. 2007 Oct 21;
There are a number of leukemogenic protein tyrosine kinases (PTKs) associated with leukaemic transformation. Whilst each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of 6 leukemogenic PTKs (BCR/ABL, TEL/PDGFRbeta, FIP1/PDGFRalpha, D816V KIT, NPM/ALK and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we have assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyse the effects of these 6 leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRalpha. The proteomic effects of the 4 type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five out of 6 PTKs affected the motility-related proteins CapG and vimentin, though this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels, but also revealed alternative patterns of regulation of the CapG protein by different oncogenes, illustrating the utility of such a combined approach. [Abstract/Link to Full Text]

Rothbauer U, Zolghadr K, Muyldermans S, Schepers A, Cardoso MC, Leonhardt H
A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins.
Mol Cell Proteomics. 2007 Oct 21;
Green fluorescent proteins (GFP) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13 kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover, GBP is also suitable for chromatin immunoprecipitations (ChIPs) from cells expressing fluorescent DNA binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. According to the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical and functional analyses with one and the same protein. [Abstract/Link to Full Text]

Lu JY, Lin YY, Qian J, Tao SC, Zhu J, Pickart C, Zhu H
Functional dissection of an HECT ubiquitin E3 ligase.
Mol Cell Proteomics. 2007 Oct 19;
SUMMARY Ubiquitination is one of the most prevalent protein posttranslational modifications in eukaryotes, and its malfunction is associated with a variety of human diseases. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitination remain largely unknown. Here, we have used a combination of yeast proteome chip assays, genetic screening, and in vitro/in vivo biochemical analyses to identify and characterize eight novel in vivo substrates of the ubiquitinating enzyme Rsp5, a homolog of the human ubiquitin-ligating enzyme Nedd4 in yeast. Our analysis of the effects of a deubiquitinating enzyme, Ubp2, has demonstrated that an accumulation of K63-linked poly-ubiquitin chains results in processed forms of two substrates, Sla1 and Ygr068c. Finally, we have shown that the localization of another newly identified substrate, Rnr2, is Rsp5-dependent. We believe that our approach constitutes a paradigm for the functional dissection of an enzyme with pleiotropic effects. [Abstract/Link to Full Text]

Zimmerli LU, Schiffer E, Zürbig P, Good DM, Kellmann M, Mouls L, Pitt AR, Coon JJ, Schmieder RE, Peter K, Mischak H, Kolch W, Delles C, Dominiczak AF
Urinary proteomic biomarkers in coronary artery disease.
Mol Cell Proteomics. 2007 Oct 19;
Urinary proteomics is emerging as a powerful non-invasive tool for diagnosis and monitoring of variety of human diseases. We tested whether signatures of urinary polypeptides can contribute to the existing biomarkers for coronary artery disease (CAD). We examined a total of 359 urine samples from 88 patients with severe CAD and 282 controls. Spot urine was analyzed using capillary electrophoresis online coupled to electrospray ionization- time of flight mass spectrometry (CE-ESI-TOF-MS) enabling characterization of more than 1000 polypeptides per sample. In a first step a "training set" for biomarker definition was created. Multiple biomarker patterns clearly distinguished healthy controls from CAD patients and we extracted 15 peptides that define a characteristic CAD signature panel. In a second step, the ability of the CAD specific panel to predict presence of CAD was evaluated in a blinded study using a "test set". The signature panel showed sensitivity of 98% [95% CI 88.7 to 99.6] and 83% specificity [95% CI 51.6 to 97.4]. Further, the peptide pattern significantly changed towards the healthy signature correlating with the level of physical activity after therapeutic intervention. Our results show that urinary proteomics can identify CAD patients with high confidence, and might also play a role in monitoring the effects of therapeutic interventions. The workflow is amenable to clinical routine testing suggesting that non-invasive proteomic analysis can become a valuable addition to other biomarkers used in cardiovascular risk assessment. [Abstract/Link to Full Text]

Recent Articles in Proteome Science

Mi J, Garcia-Arcos I, Alvarez R, Cristobal S
Age-related subproteomic analysis of mouse liver and kidney peroxisomes.
Proteome Sci. 2007 Nov 27;5(1):19.
ABSTRACT: BACKGROUND: Despite major recent advances in the understanding of peroxisomal functions and how peroxisomes arise, only scant information is available regarding this organelle in cellular aging. The aim of this study was to characterize the changes in the protein expression profile of aged versus young liver and kidney peroxisome-enriched fractions from mouse and to suggest possible mechanisms underlying peroxisomal aging. Peroxisome-enriched fractions from 10 weeks, 18 months and 24 months C57bl/6J mice were analyzed by quantitative proteomics. RESULTS: Peroxisomal proteins were enriched by differential and density gradient centrifugation and proteins were separated by two-dimensional electrophoresis (2-DE), quantified and identified by mass spectrometry (MS). In total, sixty-five proteins were identified in both tissues. Among them, 14 proteins were differentially expressed in liver and 21 proteins in kidney. The eight proteins differentially expressed in both tissues were involved in b-oxidation, a-oxidation, isoprenoid biosynthesis, amino acid metabolism, and stress response. Quantitative proteomics, clustering methods, and prediction of transcription factors, all indicated that there is a decline in protein expression at 18 months and a recovery at 24 months. CONCLUSIONS: These results indicate that some peroxisomal proteins show a tissue-specific functional response to aging. This response is probably dependent on their differential regeneration capacity. The differentially expressed proteins could lead several cellular effects: such as alteration of fatty acid metabolism that could alert membrane protein functions, increase of the oxidative stress and contribute to decline in bile salt synthesis. The ability to detect age-related variations in the peroxisomal proteome can help in the search for reliable and valid aging biomarkers. [Abstract/Link to Full Text]

Tsai H, Low TY, Freeby S, Paulus A, Ramnarayanan K, Cheng CP, Leung HC
Increase in local protein concentration by field-inversion gel electrophoresis.
Proteome Sci. 2007 Sep 26;5(1):18.
ABSTRACT: BACKGROUND: Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE). RESULTS: Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. CONCLUSION: The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool. [Abstract/Link to Full Text]

Yu H, Wakim B, Li M, Halligan B, Tint GS, Patel SB
Quantifying raft proteins in neonatal mouse brain by 'tube-gel' protein digestion label-free shotgun proteomics.
Proteome Sci. 2007;517.
ABSTRACT: BACKGROUND: The low concentration and highly hydrophobic nature of proteins in lipid raft samples present significant challenges for the sensitive and accurate proteomic analyses of lipid raft proteins. Elimination of highly enriched lipids and interfering substances from raft samples is generally required before mass spectrometric analyses can be performed, but these procedures often lead to excessive protein loss and increased sample variability. For accurate analyses of the raft proteome, simplified protocols are needed to avoid excessive sample handling and purification steps. RESULTS: We have devised a simple protocol using a 'tube-gel' protein digestion that, when combined with mass spectrometry, can be used to obtain comprehensive and reproducible identification and quantitation of the lipid raft proteome prepared from neonatal mouse brain. Lipid rafts (detergent-resistant membranes using Triton X-100 extraction) prepared from neonatal mouse brain were directly incorporated into a polyacrylamide tube-gel matrix without prior protein separation. After in-gel digestion of proteins, nanospray LC-MS/MS was used to analyze the extracted peptides, and the resulting spectra were searched to identify the proteins present in the sample. Using the standard 'label-free' proteomics approach, the total number of MS/MS spectra for the identified proteins was used to provide a measure of relative protein abundances. This approach was successfully applied to lipid rafts prepared from neonatal mouse brain. A total of 216 proteins were identified: 127 proteins (58.8%) were predicted to be membrane proteins, or membrane-associated proteins and 175 proteins (~80%) showed less than a 2-fold variation in the relative abundance in replicate samples. CONCLUSION: The tube-gel protein digestion protocol coupled with nanospray LC-MS/MS (TubeGeLC-MS/MS) offers a simple and reproducible method for identifying and quantifying the changes of relative abundances in lipid raft proteins from neonatal mouse brain and could become a useful approach for studying lipid raft proteins from various tissues. [Abstract/Link to Full Text]

Gagné JP, Ethier C, Gagné P, Mercier G, Bonicalzi ME, Mes-Masson AM, Droit A, Winstall E, Isabelle M, Poirier GG
Comparative proteome analysis of human epithelial ovarian cancer.
Proteome Sci. 2007;516.
ABSTRACT: BACKGROUND: Epithelial ovarian cancer is a devastating disease associated with low survival prognosis mainly because of the lack of early detection markers and the asymptomatic nature of the cancer until late stage. Using two complementary proteomics approaches, a differential protein expression profile was carried out between low and highly transformed epithelial ovarian cancer cell lines which realistically mimic the phenotypic changes observed during evolution of a tumour metastasis. This investigation was aimed at a better understanding of the molecular mechanisms underlying differentiation, proliferation and neoplastic progression of ovarian cancer. RESULTS: The quantitative profiling of epithelial ovarian cancer model cell lines TOV-81D and TOV-112D generated using iTRAQ analysis and two-dimensional electrophoresis coupled to liquid chromatography tandem mass spectrometry revealed some proteins with altered expression levels. Several of these proteins have been the object of interest in cancer research but others were unrecognized as differentially expressed in a context of ovarian cancer. Among these, series of proteins involved in transcriptional activity, cellular metabolism, cell adhesion or motility and cytoskeleton organization were identified, suggesting their possible role in the emergence of oncogenic pathways leading to aggressive cellular behavior. CONCLUSION: The differential protein expression profile generated by the two proteomics approaches combined to complementary characterizations studies will open the way to more exhaustive and systematic representation of the disease and will provide valuable information that may be helpful to uncover the molecular mechanisms related to epithelial ovarian cancer. [Abstract/Link to Full Text]

Levels JH, Bleijlevens B, Rezaee F, Aerts JM, Meijers JC
SELDI-TOF mass spectrometry of High-Density Lipoprotein.
Proteome Sci. 2007;515.
ABSTRACT: BACKGROUND: High-Density Lipoprotein (HDL), one of the main plasma lipoproteins, serves as a docking station for proteins involved in inflammation, coagulation, and lipid metabolism. METHODS: To elucidate the protein composition of HDL, we employed SELDI-TOF mass spectrometry as a potential high-throughput proteomic candidate for protein profiling of HDL. HDL derived from normolipemic individuals was captured on PS20 protein-chips using covalently bound antibodies against apo A-I or A-II. RESULTS: After optimisation, on-chip capture of HDL particles directly from plasma or from pre-purified HDL resulted in comparable fingerprints confirming specific capture of HDL. Depending on the capture antibody some differences in the fingerprint were observed. The most detailed fingerprint was observed up to 50 kDa; approximately 95 peaks were detected in the 3-50 kDa molecular mass range. Between 50 and 160 kDa, 27 more peaks were detected. CONCLUSION: Based on these results, SELDI-TOF MS may be a suitable high-throughput candidate for HDL protein profiling and marker search. This approach may be used to i) investigate the underlying mechanisms that lead to increased atherothrombotic risk and ii) to investigate the atherothrombotic state of an individual. [Abstract/Link to Full Text]

Whistler T, Rollin D, Vernon SD
A method for improving SELDI-TOF mass spectrometry data quality.
Proteome Sci. 2007;514.
ABSTRACT: BACKGROUND: Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a powerful tool for rapidly generating high-throughput protein profiles from a large number of samples. However, the events that occur between the first and last sample run are likely to introduce technical variation in the results. METHODS: We fractionated and analyzed quality control and investigational serum samples on 3 Protein Chips and used statistical methods to identify poor-quality spectra and to identify and reduce technical variation. RESULTS: Using diagnostic plots, we were able to visually depict all spectra and to identify and remove those that were of poor quality. We detected a technical variation associated with when the samples were run (referred to as batch effect) and corrected for this variation using analysis of variance. These corrections increased the number of peaks that were reproducibly detected. CONCLUSION: By removing poor-quality, outlier spectra, we were able to increase peak detection, and by reducing the variance introduced when samples are processed and analyzed in batches, we were able to increase the reproducibility of peak detection. [Abstract/Link to Full Text]

Okumichi H, Kanamoto T, Souchelnytskyi N, Tanimoto S, Tanaka K, Kiuchi Y
Proteomic analyses of retina of excitatory amino acid carrier 1 deficient mice.
Proteome Sci. 2007;513.
ABSTRACT: BACKGROUND: Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter found in neuronal tissues and is extensively expressed in the retina. EAAC1 plays a role in a variety of neural functions, but its biological functions in the retina has not been fully determined. The purpose of this study was to identify proteins regulated by EAAC1 in the retina of mice. To accomplish this, we used a proteomics-based approach to identify proteins that are up- or down-regulated in EAAC1-deficient (EAAC1-/-) mice. RESULTS: Proteomic analyses and two-dimensional gel electorphoresis were performed on the retina of EAAC1-/- mice, and the results were compared to that of wild type mice. The protein spots showing significant differences were selected for identification by mass spectrometric analyses. Thirteen proteins were differentially expressed; nine proteins were up-regulated and five proteins were down-regulated in EAAC1-/- retina. Functional clustering showed that identified proteins are involved in various cellular process, e.g. cell cycle, cell death, transport and metabolism. CONCLUSION: We identified thirteen proteins whose expression is changed in EAAC-/- mice retinas. These proteins are known to regulate cell proliferation, death, transport, metabolism, cell organization and extracellular matrix. [Abstract/Link to Full Text]

Jethwaney D, Islam MR, Leidal KG, de Bernabe DB, Campbell KP, Nauseef WM, Gibson BW
Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils.
Proteome Sci. 2007;512.
ABSTRACT: BACKGROUND: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. RESULTS: To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis. CONCLUSION: Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli. [Abstract/Link to Full Text]

Walz A, Mujer CV, Connolly JP, Alefantis T, Chafin R, Dake C, Whittington J, Kumar SP, Khan AS, DelVecchio VG
Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins.
Proteome Sci. 2007;511.
BACKGROUND: The secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2-) during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax. RESULTS: A total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites), lipoprotein signal peptides (13 have SpII sites), and N-terminal membrane helices (9 have transmembrane helices). The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices.Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa) of protective antigen (PA) were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase. CONCLUSION: This is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and accumulation may be relevant in elucidation of the progression of pathogenicity, identification of therapeutics and diagnostic markers, and vaccine development. This study also adds to the continuously growing list of identified Bacillus anthracis secretome proteins. [Abstract/Link to Full Text]

Wolschin F, Amdam GV
Comparative proteomics reveal characteristics of life-history transitions in a social insect.
Proteome Sci. 2007;510.
BACKGROUND: Honey bee (Apis mellifera) workers are characterized by complex social behavior. Their life-history is dominated by a period of within-nest activity followed by a phase of long-distance flights and foraging. General insights into insect metabolism imply that foraging onset is associated with fundamental metabolic changes, and theory on social evolution suggests metabolic adaptations that are advantageous for the colony as a whole. RESULTS: Here we address the life-history characteristics of workers with LC-MS/MS based relative quantification of major proteins. Our approach includes: i. Calculation of a false positive rate for the identifications, ii. Support of relative protein quantification results obtained from spectral count by non-parametric statistics, and iii. Correction for Type 1 error inflation using a bootstrap iteration analysis. Our data are consistent with the use of glucose as the main fuel for honey bee flight. Moreover, the data delivers information on the expression of ATPsynthases/ATPases, and provide new insights into nurse- and forager-specific patterns of protection against oxidative stress. CONCLUSION: The results show the suitability of this approach to investigate fundamental biochemical changes in an insect, and provide new evidence for metabolic specializations that occur during the social ontogeny of worker honey bees. [Abstract/Link to Full Text]

Rollin D, Whistler T, Vernon SD
Laboratory methods to improve SELDI peak detection and quantitation.
Proteome Sci. 2007;59.
BACKGROUND: Protein profiling with surface-enhanced laser desorption-ionisation time-of-flight mass spectrometry (SELDI-TOF MS) is a promising approach for biomarker discovery. Some candidate biomarkers have been identified using SELDI-TOF, but validation of these can be challenging because of technical parameters that effect reproducibility. Here we describe steps to improve the reproducibility of peak detection. METHODS: SELDI-TOF mass spectrometry was performed using a system manufactured by Ciphergen Biosystems along with their ProteinChip System. Serum from 10 donors was pooled and used for all experiments. Serum was fractionated with Expression Difference Mapping kit-Serum Fractionation from the same company and applied to three different ProteinChips. The fractionations were run over a one month period to examine the contribution of sample batch and time to peak detection variability. Spectra were processed and peaks detected using the Ciphergen Express software and variance measured. RESULTS: Experimental parameters specific to the serum fraction and ProteinChip, including spot protocols (laser intensity and detector sensitivity) were optimized to decrease peak detection variance. Optimal instrument settings, regular calibration along with controlled sample handling and processing nearly doubled the number of peaks detected and decreased intensity variance. CONCLUSION: This report assesses the variation across fractionated sera processed over a one-month period. The optimizations reported decreased the variance and increased the number of peaks detected. [Abstract/Link to Full Text]

Howes CG, Foster LJ
PrestOMIC, an open source application for dissemination of proteomic datasets by individual laboratories.
Proteome Sci. 2007;58.
BACKGROUND: Technological advances in mass spectrometry and other detection methods are leading to larger and larger proteomics datasets. However, when papers describing such information are published the enormous volume of data can typically only be provided as supplementary data in a tabular form through the journal website. Several journals in the proteomics field, together with the Human Proteome Organization's (HUPO) Proteomics Standards Initiative and institutions such as the Institute for Systems Biology are working towards standardizing the reporting of proteomics data, but just defining standards is only a means towards an end for sharing data. Data repositories such as and the Open Proteomics Database allow for public access to proteomics data but provide little, if any, interpretation. RESULTS & CONCLUSION: Here we describe PrestOMIC, an open source application for storing mass spectrometry-based proteomic data in a relational database and for providing a user-friendly, searchable and customizable browser interface to share one's data with the scientific community. The underlying database and all associated applications are built on other existing open source tools, allowing PrestOMIC to be modified as the data standards evolve. We then use PrestOMIC to present a recently published dataset from our group through our website. [Abstract/Link to Full Text]

Mazzatti DJ, Pawelec G, Longdin R, Powell JR, Forsey RJ
SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence.
Proteome Sci. 2007;57.
BACKGROUND: The adaptive immune response requires waves of T-cell clonal expansion on contact with pathogen and elimination after clearance of the source of antigen. However, lifelong persistent infections with common viruses cause chronic antigenic stimulation which takes its toll on adaptive immunity in late life. Chronic antigenic stress results in deregulation of the T-cell response and accumulation of anergic cells. Longitudinal studies of the elderly show that this impacts on survival. Identifying the nature of the defects in chronically-stimulated T-cells and protein bio-markers of these dysfunctional cells would help to understand age-associated compromised T-cell function (immunosenescence) and facilitate the development of targeted intervention strategies.The purpose of this work was to use surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to analyse proteins associated with T-cell senescence in order to identify potential bio-markers. Clonal populations of T-cells isolated from elderly octogenarian and centenarian donors were grown in vitro until senescence, and early passage and late passage (pre-senescent) cells were analysed using SELDI-TOF-MS ProteinChip arrays. RESULTS: Discriminant analysis identified several protein or peptide peaks in the region of 14.5-16.5 kDa that were associated with T-cell clone senescence. Human profilin-1, a ubiquitous protein associated with actin remodelling and cellular motility was unambiguously identified. Altered expression of profilin-1 in senescent T-cell clones was confirmed by Western blot analysis. CONCLUSION: Due to the proposed roles of profilin-1 in cellular survival, cytoskeleton remodelling, motility, and proliferation, it is hypothesised that differential expression of profilin-1 in ageing may contribute directly to immunosenescence. [Abstract/Link to Full Text]

Pastorelli R, Saletta F, Carpi D, Campagna R, dell'Osta C, Schiarea S, Vineis P, Airoldi L, Matullo G
Proteome characterization of a human urothelial cell line resistant to the bladder carcinogen 4-aminobiphenyl.
Proteome Sci. 2007;56.
BACKGROUND: The aromatic amine 4-aminobiphenyl (4-ABP) is an environmental and occupational contaminant known to be a major etiological agent of human bladder cancer. 4-ABP metabolites are able to form DNA adducts that may induce mutations and initiate bladder carcinogenesis. Cells exposed to 4-ABP may develop resistance to the carcinogen. The aim of the present study was to detect and identify proteins whose expression is altered in the bladder carcinoma RT112 sub-lines selected for acquired resistance to 4-ABP, in order to disentangle the mechanisms. RESULTS: Differential proteome analysis of cell lysates showed an overall perturbation in cell metabolism and energy pathways in the 4-ABP-resistant human urothelial clones, with over-expression of membrane trafficking proteins such as annexin 2. The resistant clones had altered expression of many proteins linked directly (i.e. lamin A/C, programmed cell death 6 interacting protein) or indirectly (i.e. 94 kDa glucose-regulated protein, fatty acid-binding protein) to decreased apoptosis, suggesting that resistance to 4-ABP might be associated with low apoptotic activity. CONCLUSION: Our data provide evidence that deregulation of apoptosis and membrane trafficking proteins might be strongly implicated in the selection of carcinogen resistant cells. Some of these proteins might have potential as biomarkers of resistance and cancer risk. [Abstract/Link to Full Text]

Vorum H, Řstergaard M, Rice GE, Honoré B, Bek T
Identification of differentially regulated proteins in a patient with Leber's Congenital Amaurosis--a proteomic study.
Proteome Sci. 2007;55.
BACKGROUND: To identify the pattern of protein expression in the retina from a patient with Leber's Congenital Amaurosis (LCA) secondary to a mutation in the AIPL1 gene. The retina from one eye of a patient with LCA and 7 control eyes were studied. The tissue was subjected to high resolution two-dimensional gel electrophoresis, image analysis and mass spectrometry, in an effort to identify differentially regulated proteins. RESULTS: In the LCA retina seven protein spots were differentially expressed. Six proteins were significantly up-regulated of which three could be identified as: alphaA-crystallin, triosephophate isomerase, and an N-terminal fragment of the beta-chain of ATP synthase. One protein spot that was down-regulated in the LCA retina was identified as a C-terminal fragment of beta-tubulin. CONCLUSION: Retinal tissue in LCA is characterised by an up-regulation of alphaA-crystallin, triosephosphate isomerase, and ATP synthase (beta-chain fragment) and down-regulation of a fragment of beta-tubulin. These proteins/protein fragments may play a crucial role for the retinal degeneration processes in LCA and other retinal dystrophies. [Abstract/Link to Full Text]

Siepen JA, Swainston N, Jones AR, Hart SR, Hermjakob H, Jones P, Hubbard SJ
An informatic pipeline for the data capture and submission of quantitative proteomic data using iTRAQ.
Proteome Sci. 2007;54.
BACKGROUND: Proteomics continues to play a critical role in post-genomic science as continued advances in mass spectrometry and analytical chemistry support the separation and identification of increasing numbers of peptides and proteins from their characteristic mass spectra. In order to facilitate the sharing of this data, various standard formats have been, and continue to be, developed. Still not fully mature however, these are not yet able to cope with the increasing number of quantitative proteomic technologies that are being developed. RESULTS: We propose an extension to the PRIDE and mzData XML schema to accommodate the concept of multiple samples per experiment, and in addition, capture the intensities of the iTRAQ reporter ions in the entry. A simple Java-client has been developed to capture and convert the raw data from common spectral file formats, which also uses a third-party open source tool for the generation of iTRAQ reported intensities from Mascot output, into a valid PRIDE XML entry. CONCLUSION: We describe an extension to the PRIDE and mzData schemas to enable the capture of quantitative data. Currently this is limited to iTRAQ data but is readily extensible for other quantitative proteomic technologies. Furthermore, a software tool has been developed which enables conversion from various mass spectrum file formats and corresponding Mascot peptide identifications to PRIDE formatted XML. The tool represents a simple approach to preparing quantitative and qualitative data for submission to repositories such as PRIDE, which is necessary to facilitate data deposition and sharing in public domain database. The software is freely available from [Abstract/Link to Full Text]

Liu J, Bell AW, Bergeron JJ, Yanofsky CM, Carrillo B, Beaudrie CE, Kearney RE
Methods for peptide identification by spectral comparison.
Proteome Sci. 2007;53.
BACKGROUND: Tandem mass spectrometry followed by database search is currently the predominant technology for peptide sequencing in shotgun proteomics experiments. Most methods compare experimentally observed spectra to the theoretical spectra predicted from the sequences in protein databases. There is a growing interest, however, in comparing unknown experimental spectra to a library of previously identified spectra. This approach has the advantage of taking into account instrument-dependent factors and peptide-specific differences in fragmentation probabilities. It is also computationally more efficient for high-throughput proteomics studies. RESULTS: This paper investigates computational issues related to this spectral comparison approach. Different methods have been empirically evaluated over several large sets of spectra. First, we illustrate that the peak intensities follow a Poisson distribution. This implies that applying a square root transform will optimally stabilize the peak intensity variance. Our results show that the square root did indeed outperform other transforms, resulting in improved accuracy of spectral matching. Second, different measures of spectral similarity were compared, and the results illustrated that the correlation coefficient was most robust. Finally, we examine how to assemble multiple spectra associated with the same peptide to generate a synthetic reference spectrum. Ensemble averaging is shown to provide the best combination of accuracy and efficiency. CONCLUSION: Our results demonstrate that when combined, these methods can boost the sensitivity and specificity of spectral comparison. Therefore they are capable of enhancing and complementing existing tools for consistent and accurate peptide identification. [Abstract/Link to Full Text]

Roelofsen H, Alvarez-Llamas G, Schepers M, Landman K, Vonk RJ
Proteomics profiling of urine with surface enhanced laser desorption/ionization time of flight mass spectrometry.
Proteome Sci. 2007;52.
BACKGROUND: Urine consists of a complex mixture of peptides and proteins and therefore is an interesting source of biomarkers. Because of its high throughput capacity SELDI-TOF-MS is a proteomics technology frequently used in biomarker studies. We compared the performance of seven SELDI protein chip types for profiling of urine using standard chip protocols. RESULTS: Performance was assessed by determining the number of detectable peaks and spot to spot variation for the seven array types and two different matrices: SPA and CHCA. A urine sample taken from one healthy volunteer was applied in eight-fold for each chip type/matrix combination. Data were analyzed for total number of detected peaks (S/N > 5). Spot to spot variation was determined by calculating the average CV of peak intensities. In addition, an inventory was made of detectable peaks with each chip and matrix type. Also the redundancy in peaks detected with the different chip/matrix combinations was determined. A total of 425 peaks (136 non-redundant peaks) could be detected when combining the data from the seven chip types and the two matrices. Most peaks were detected with the CM10 chip with CHCA (57 peaks). The Q10 with CHCA (51 peaks), SEND (48 peaks) and CM10 with SPA (48 peaks) also performed well. The CM10 chip with CHCA also has the best reproducibility with an average CV for peak intensity of 13%. CONCLUSION: The combination of SEND, CM10 with CHCA, CM10 with SPA, IMAC-Cu with SPA and H50 with CHCA provides the optimal information from the urine sample with good reproducibility. With this combination a total of 217 peaks (71 non-redundant peaks) can be detected with CV's ranging from 13 to 26%, depending on the chip and matrix type. Overall, CM10 with CHCA is the best performing chip type. [Abstract/Link to Full Text]

Emadali A, Metrakos PP, Kalantari F, Boutros T, Boismenu D, Chevet E
Proteomic analysis of tyrosine phosphorylation during human liver transplantation.
Proteome Sci. 2007;51.
BACKGROUND: Ischemia-reperfusion (I/R) causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. RESULTS: Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. CONCLUSION: Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes. [Abstract/Link to Full Text]

Neasta J, Uttenweiler-Joseph S, Chaoui K, Monsarrat B, Meunier JC, Moulédous L
Effect of long-term exposure of SH-SY5Y cells to morphine: a whole cell proteomic analysis.
Proteome Sci. 2006;423.
BACKGROUND: Opiate addiction reflects plastic changes that endurably alter synaptic transmission within relevant neuronal circuits. The biochemical mechanisms of these adaptations remain largely unknown and proteomics-based approaches could lead to a broad characterization of the molecular events underlying adaptations to chronic drug exposure. RESULTS: Thus, we have started proteomic analyses of the effects of chronic morphine exposure in a recombinant human neuroblastoma SH-SY5Y clone that stably overexpresses the mu-opioid receptor. Cells were treated with morphine for 6, 24 and 72 hours, the proteins were separated by 2-D gel electrophoresis and stained with Coomassie blue, and the protein map was compared with that obtained from untreated cells. Spots showing a statistically significant variation were selected for identification using mass spectrometric analyses. CONCLUSION: A total of 45 proteins were identified, including proteins involved in cellular metabolism, cytoskeleton organization, vesicular trafficking, transcriptional and translational regulation, and cell signaling. [Abstract/Link to Full Text]

Sundsten T, Eberhardson M, Göransson M, Bergsten P
The use of proteomics in identifying differentially expressed serum proteins in humans with type 2 diabetes.
Proteome Sci. 2006;422.
BACKGROUND: The aim of the study was to optimize protocols for finding and identifying serum proteins that are differentially expressed in persons with normal glucose tolerance (NGT) compared to individuals with type 2 diabetes mellitus (T2DM). Serum from persons with NGT and persons with T2DM was profiled using ProteinChip arrays and time-of-flight mass spectra were generated by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). RESULTS: Mass spectra from NGT- and T2DM-groups were compared. Fifteen proteins ranging from 5 to 79 kDa were differentially expressed (p < 0.05). Five of these proteins showed decreased and ten showed increased serum levels in individuals with T2DM. To be able to identify the proteins, the complexity of the sample was reduced by fractionation approaches. Subsequently, the purified fractions containing biomarkers were separated by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in two identical lanes. Protein bands of the first lane were excised and subjected to passive elution to recapture the biomarkers on ProteinChip arrays. The corresponding bands of the second lane were subjected to peptide-mass fingerprinting (PMF). Using this approach four of the differentially expressed proteins were identified as apolipoprotein C3 (9.4 kDa), transthyretin (13.9 kDa), albumin (66 kDa) and transferrin (79 kDa). Whereas apolipoprotein C3 and transthyretin were up-regulated, albumin and transferrin were down-regulated in T2DM. CONCLUSION: Protocols for protein profiling by SELDI-TOF MS and protein identification by fractionation, SDS-PAGE and PMF were optimized for serum from humans with T2DM. With these protocols differentially expressed proteins were discovered and identified when serum from NGT- and T2DM-individuals was analyzed. [Abstract/Link to Full Text]

Gu Q, Sivanandam TM, Kim CA
Signal stability of Cy3 and Cy5 on antibody microarrays.
Proteome Sci. 2006;421.
BACKGROUND: The antibody microarray technique is a newly emerging proteomics tool for differential protein expression analyses that uses fluorescent dyes Cy 3 and Cy 5. Environmental factors, such as light exposure, can affect the signal intensity of fluorescent dyes on microarray slides thus, it is logical to scan microarray slides immediately after the final wash and drying processes. However, no research data are available concerning time-dependent changes of fluorescent signals on antibody microarray slides to this date. In the present study, microarray slides were preserved at -20 degrees C after regular microarray experiments and were rescanned at day 10, 20 and 30 to evaluate change in signal intensity. RESULTS: Fluorescent intensities of microarray spots were detected using a confocal laser scanner after the experiment at day 0, and re-examined at day 10, 20 and 30, respectively. Fluorescent intensities of rescanned microarray spots did not show significant changes when compared with those scanned immediately after standard microarray experiments. CONCLUSION: Microarray slides can be preserved and rescanned multiple times using a confocal laser scanner over a period of days or weeks. [Abstract/Link to Full Text]

Roche S, Tiers L, Provansal M, Piva MT, Lehmann S
Interest of major serum protein removal for Surface-Enhanced Laser Desorption/Ionization - Time Of Flight (SELDI-TOF) proteomic blood profiling.
Proteome Sci. 2006;420.
BACKGROUND: Surface-Enhanced Laser Desorption/Ionization - Time Of Flight (SELDI-TOF) has been proposed as new approach for blood biomarker discovery. However, results obtained so far have been often disappointing as this technique still has difficulties to detect low-abundant plasma and serum proteins. RESULTS: We used a serum depletion scheme using chicken antibodies against various abundant proteins to realized a pre-fractionation of serum prior to SELDI-TOF profiling. Depletion of major serum proteins by immunocapture was confirmed by 1D and 2D gel electrophoresis. SELDI-TOF analysis of bound and unbound (depleted) serum fractions revealed that this approach allows the detection of new low abundant protein peaks with satisfactory reproducibility. CONCLUSION: The combination of immunocapture and SELDI-TOF analysis opens new avenues into proteomic profiling for the discovery of blood biomarkers. [Abstract/Link to Full Text]

Wang W, Hollmann R, Deckwer WD
Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase.
Proteome Sci. 2006;419.
High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320), both carrying a heterologous dextransucrase (dsrS) gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction) the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available.High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses.The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the mutated genes in WH320 in addition to the known lacZ.In view of the results of this proteomic study it seems that at high cell density conditions and hence low growth rates MS941, in contrast to WH320, does not maintain a vegetative growth which is essential for the expression of the foreign dsrS gene by using the xylA promoter. It is conceivable that applications of a promoter which is highly active under nutrient-limited cultivation conditions is necessary, at least for MS941, for the overexpression of recombinant genes in such B. megaterium fed-batch cultivation process. However to obtain a heterologous protein in secreted and properly folded form stills remains a big challenge. [Abstract/Link to Full Text]

Wolski WE, Farrow M, Emde AK, Lehrach H, Lalowski M, Reinert K
Analytical model of peptide mass cluster centres with applications.
Proteome Sci. 2006;418.
BACKGROUND: The elemental composition of peptides results in formation of distinct, equidistantly spaced clusters across the mass range. The property of peptide mass clustering is used to calibrate peptide mass lists, to identify and remove non-peptide peaks and for data reduction. RESULTS: We developed an analytical model of the peptide mass cluster centres. Inputs to the model included, the amino acid frequencies in the sequence database, the average length of the proteins in the database, the cleavage specificity of the proteolytic enzyme used and the cleavage probability. We examined the accuracy of our model by comparing it with the model based on an in silico sequence database digest. To identify the crucial parameters we analysed how the cluster centre location depends on the inputs. The distance to the nearest cluster was used to calibrate mass spectrometric peptide peak-lists and to identify non-peptide peaks. CONCLUSION: The model introduced here enables us to predict the location of the peptide mass cluster centres. It explains how the location of the cluster centres depends on the input parameters. Fast and efficient calibration and filtering of non-peptide peaks is achieved by a distance measure suggested by Wool and Smilansky. [Abstract/Link to Full Text]

Monsinjon T, Andersen OK, Leboulenger F, Knigge T
Data processing and classification analysis of proteomic changes: a case study of oil pollution in the mussel, Mytilus edulis.
Proteome Sci. 2006;4(1):17.
BACKGROUND: Proteomics may help to detect subtle pollution-related changes, such as responses to mixture pollution at low concentrations, where clear signs of toxicity are absent. The challenges associated with the analysis of large-scale multivariate proteomic datasets have been widely discussed in medical research and biomarker discovery. This concept has been introduced to ecotoxicology only recently, so data processing and classification analysis need to be refined before they can be readily applied in biomarker discovery and monitoring studies. RESULTS: Data sets obtained from a case study of oil pollution in the Blue mussel were investigated for differential protein expression by retentate chromatography-mass spectrometry and decision tree classification. Different tissues and different settings were used to evaluate classifiers towards their discriminatory power. It was found that, due the intrinsic variability of the data sets, reliable classification of unknown samples could only be achieved on a broad statistical basis (n > 60) with the observed expression changes comprising high statistical significance and sufficient amplitude. The application of stringent criteria to guard against overfitting of the models eventually allowed satisfactory classification for only one of the investigated data sets and settings. CONCLUSION: Machine learning techniques provide a promising approach to process and extract informative expression signatures from high-dimensional mass-spectrometry data. Even though characterisation of the proteins forming the expression signatures would be ideal, knowledge of the specific proteins is not mandatory for effective class discrimination. This may constitute a new biomarker approach in ecotoxicology, where working with organisms, which do not have sequenced genomes render protein identification by database searching problematic. However, data processing has to be critically evaluated and statistical constraints have to be considered before supervised classification algorithms are employed. [Abstract/Link to Full Text]

Jin M, Diaz PT, Bourgeois T, Eng C, Marsh CB, Wu HM
Two-dimensional gel proteome reference map of blood monocytes.
Proteome Sci. 2006;416.
BACKGROUND: Blood monocytes play a central role in regulating host inflammatory processes through chemotaxis, phagocytosis, and cytokine production. However, the molecular details underlying these diverse functions are not completely understood. Understanding the proteomes of blood monocytes will provide new insights into their biological role in health and diseases. RESULTS: In this study, monocytes were isolated from five healthy donors. Whole monocyte lysates from each donor were then analyzed by 2D gel electrophoresis, and proteins were detected using Sypro Ruby fluorescence and then examined for phosphoproteomes using ProQ phospho-protein fluorescence dye. Between 1525 and 1769 protein spots on each 2D gel were matched, analyzed, and quantified. Abundant protein spots were then subjected to analysis by mass spectrometry. This report describes the protein identities of 231 monocyte protein spots, which represent 164 distinct proteins and their respective isoforms or subunits. Some of these proteins had not been previously characterized at the protein level in monocytes. Among the 231 protein spots, 19 proteins revealed distinct modification by protein phosphorylation. CONCLUSION: The results of this study offer the most detailed monocyte proteomic database to date and provide new perspectives into the study of monocyte biology. [Abstract/Link to Full Text]

Delom F, Chevet E
Phosphoprotein analysis: from proteins to proteomes.
Proteome Sci. 2006;415.
Characterization of protein modification by phosphorylation is one of the major tasks that have to be accomplished in the post-genomic era. Phosphorylation is a key reversible modification occurring mainly on serine, threonine and tyrosine residues that can regulate enzymatic activity, subcellular localization, complex formation and degradation of proteins. The understanding of the regulatory role played by phosphorylation begins with the discovery and identification of phosphoproteins and then by determining how, where and when these phosphorylation events take place. Because phosphorylation is a dynamic process difficult to quantify, we must at first acquire an inventory of phosphoproteins and characterize their phosphorylation sites. Several experimental strategies can be used to explore the phosphorylation status of proteins from individual moieties to phosphoproteomes. In this review, we will examine and catalogue how proteomics techniques can be used to answer specific questions related to protein phosphorylation. Hence, we will discuss the different methods for enrichment of phospho-proteins and -peptides, and then the various technologies for their identification, quantitation and validation. [Abstract/Link to Full Text]

Braitbard O, Bishara-Shieban J, Glickstein H, Kott-Gutkowski M, Pace U, Rund DG, Stein WD
An ELISA-based procedure for assaying proteins in digests of human leukocytes and cell lines, using specifically selected peptides and appropriate antibodies.
Proteome Sci. 2006;414.
BACKGROUND: We describe the application of an ELISA-based assay (the Peptidomatrix) that can be used to simultaneously identify and quantitate a number of proteins in biological samples. The biological sample (blood component, biopsy, culture or other) is first lysed to release all the proteins, without any additional separation. The denatured proteins in the sample are then digested in bulk with the desired proteolytic enzyme(s). The peptides in the digest are then assayed by appropriate antibodies, using a competition ELISA protocol. RESULTS: As an example of its use, the present paper applies the Peptidomatrix to the assay of four membrane proteins MDR1 (P-glycoprotein or ABCB1), MRP1 (ABCC1), BCRP/MXR (ABCG2) and the alpha subunit of the Na, K_ATPase (ATP1A1), present in a number of cell lines and in human lymphocytes. We show that we can detect and quantitate these proteins, using a series of peptide-antibody pairs, and that we can differentiate between cell lines or cell preparations that express the target proteins and those that do not. CONCLUSION: We have devised a simple, ELISA-based proteomics assay that enables the quantitation of designated proteins in a cell or tissue sample, and that can be used in any laboratory, with minimal specialized equipment. [Abstract/Link to Full Text]

Hurst RE, Kyker KD, Dozmorov MG, Takemori N, Singh A, Matsumoto H, Saban R, Betgovargez E, Simonian MH
Proteome-level display by 2-dimensional chromatography of extracellular matrix-dependent modulation of the phenotype of bladder cancer cells.
Proteome Sci. 2006;413.
BACKGROUND: The extracellular matrix can have a profound effect upon the phenotype of cancer cells. Previous work has shown that growth of bladder cancer cells on a matrix derived from normal basement membrane suppresses many malignant features that are displayed when the cells are grown on a matrix that has been modified by malignant tumors. This work was undertaken to investigate proteome-level changes as determined by a new commercially available proteome display involving 2-dimensional chromatography for bladder cancer cells grown on different extracellular matrix preparations that modulate the expression of the malignant phenotype. RESULTS: Depending on the matrix, between 1300 and 2000 distinct peaks were detected by two-dimensional chromatographic fractionation of 2.1-4.4 mg of total cellular protein. The fractions eluting from the reversed-phase fractionation were suitable for mass spectrometric identification following only lyophilization and trypsin digestion and achieved approximately 10-fold higher sensitivity than was obtained with gel-based separations. Abundant proteins that were unique to cells grown on one of the matrices were identified by mass spectrometry. Following concentration, peaks of 0.03 AU provided unambiguous identification of protein components when 10% of the sample was analyzed, whereas peaks of 0.05 AU was approximately the lower limit of detection when the entire sample was separated on a gel and in-gel digestion was used. Although some fractions were homogeneous, others were not, and up to 3 proteins per fraction were identified. Strong evidence for post-translational modification of the unique proteins was noted. All 13 of the unique proteins from cells grown on Matrigel were related to MYC pathway. CONCLUSION: The system provides a viable alternative to 2-dimensional gel electrophoresis for proteomic display of biological systems. The findings suggest the importance of MYC to the malignant phenotype of bladder cancer cells. [Abstract/Link to Full Text]