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Phocine distemper virus (PDV) has caused two mass mortalities of European harbour seals (Phoca vitulina) in recent decades. Levels of mortality varied considerably among European populations in both the 1988 and 2002 epidemics, with higher mortality in continental European populations in comparison to UK populations. High levels of genetic differentiation at neutral makers among seal populations allow for the possibility that there could be potential genetic differences at functional loci that may account for some of the variation in mortality. Recent genome sequencing of carnivore species and development of genomic tools have now made it possible to explore the possible contribution of variation in candidate genes from harbour seals in relation to the differential mortality patterns. We assessed variation in eight genes (CD46, IFNG, IL4, IL8, IL10, RARa, SLAM and TLR2) encoding key proteins involved in host cellular interactions with Morbilliviruses and the relationship of variants to disease status. This work constitutes the first genetic association study for Morbillivirus disease susceptibility in a non-model organism, and for a natural mortality event. We found no variation in harbour seals from across Europe in the protein coding domains of the viral receptors SLAM and CD46, but SNPs were present in SLAM intron 2. SNPs were also present in IL8 p2 and RARa exon 1. There was no significant association of SLAM or RARa polymorphisms with disease status implying no role of these genes in determining resistance to PDV induced mortality, that could be detected with the available samples and the small number of polymorphisms indentified. However there was significant differentiation of allele frequencies among populations. PDV and other morbilliviruses are important models for wildlife epidemiology, host switches and viral evolution. Despite a negative result in this case, full sequencing of pinniped and other 'non-model' carnivore genomes will help in refining understanding the role of host genetics in disease susceptibility for these viruses.

Alu elements are sequences of approximately 300 basepairs that together comprise more than 10% of the human genome. Due to their recent origin in primate evolution some Alu elements are polymorphic in humans, present in some individuals while absent in others. We present PopAlu, a tool to detect polymorphic Alu elements on a population scale from paired-end sequencing data. PopAlu uses read pair distance and orientation as well as split reads to identify the location and precise breakpoints of polymorphic Alus. Genotype calling enables us to differentiate between homozygous and heterozygous carriers, making the output of PopAlu suitable for use in downstream analyses such as genome-wide association studies (GWAS). We show on a simulated dataset that PopAlu calls Alu elements inserted and deleted with respect to a reference genome with high accuracy and high precision. Our analysis of real data of a human trio from the 1000 Genomes Project confirms that PopAlu is able to produce highly accurate genotype calls. To our knowledge, PopAlu is the first tool that identifies polymorphic Alu elements from multiple individuals simultaneously, pinpoints the precise breakpoints and calls genotypes with high accuracy.

Advances in next-generation DNA sequencing (NGS) now make it possible, and affordable, to sequence the entire genome of an individual. Routine clinical application is on the horizon. There is a consensus that some subsets of genetic information should be disclosed to patients, but disclosure to their relatives is less consensual. This issue becomes especially salient after a patient's death, when permission can no longer be sought. There has however been little debate on postmortem disclosure. We identify and explain the arguments in favor of and against disclosure of genetic information to the relatives of a deceased patient. We conclude that there are valid reasons to communicate some subsets of genetic information to family members after death, and we propose a passive postmortem disclosure policy.

An important component of plant evolution is the plethora of pathways producing more than 200,000 biochemically diverse specialized metabolites with pharmacological, nutritional and ecological significance. To unravel dynamics underlying metabolic diversification, it is critical to determine lineage-specific gene family expansion in a phylogenomics framework. However, robust functional annotation is often only available for core enzymes catalyzing committed reaction steps within few model systems. In a genome informatics approach, we extracted information from early-draft gene-space assemblies and non-redundant transcriptomes to identify protein families involved in isoprenoid biosynthesis. Isoprenoids comprise terpenoids with various roles in plant-environment interaction, such as pollinator attraction or pathogen defense. Combining lines of evidence provided by synteny, sequence homology and Hidden-Markov-Modelling, we screened 17 genomes including 12 major crops and found evidence for 1,904 proteins associated with terpenoid biosynthesis. Our terpenoid genes set contains evidence for 840 core terpene-synthases and 338 triterpene-specific synthases. We further identified 190 prenyltransferases, 39 isopentenyl-diphosphate isomerases as well as 278 and 219 proteins involved in mevalonate and methylerithrol pathways, respectively. Assessing the impact of gene and genome duplication to lineage-specific terpenoid pathway expansion, we illustrated key events underlying terpenoid metabolic diversification within 250 million years of flowering plant radiation. By quantifying Angiosperm-wide versatility and phylogenetic relationships of pleiotropic gene families in terpenoid modular pathways, our analysis offers significant insight into evolutionary dynamics underlying diversification of plant secondary metabolism. Furthermore, our data provide a blueprint for future efforts to identify and more rapidly clone terpenoid biosynthetic genes from any plant species.

The recent sequencing of several gymnosperm genomes has greatly facilitated studying the evolution of their genes and gene families. In this study, we examine the evidence for expression-mediated selection in the first two fully sequenced representatives of the gymnosperm plant clade (Picea abies and Picea glauca). We use genome-wide estimates of gene expression (> 50,000 expressed genes) to study the relationship between gene expression, codon bias, rates of sequence divergence, protein length, and gene duplication. We found that gene expression is correlated with rates of sequence divergence and codon bias, suggesting that natural selection is acting on Picea protein-coding genes for translational efficiency. Gene expression, rates of sequence divergence, and codon bias are correlated with the size of gene families, with large multicopy gene families having, on average, a lower expression level and breadth, lower codon bias, and higher rates of sequence divergence than single-copy gene families. Tissue-specific patterns of gene expression were more common in large gene families with large gene expression divergence than in single-copy families. Recent family expansions combined with large gene expression variation in paralogs and increased rates of sequence evolution suggest that some Picea gene families are rapidly evolving to cope with biotic and abiotic stress. Our study highlights the importance of gene expression and natural selection in shaping the evolution of protein-coding genes in Picea species, and sets the ground for further studies investigating the evolution of individual gene families in gymnosperms.

International audience; Eucalypts are the world's most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

Meningiomas are frequent central nervous system neoplasms, which despite their predominant benignity, show sporadically malignant behavior. Type 2 neurofibromatosis and polymorphisms in several genes have been associated with meningioma risk and are probably involved in its pathogenesis. Although GWAS studies have found loci related to meningioma risk, little is known about the factors determining malignant transformation. Thus, this study is aimed to identify the genomic and transcriptomic factors influencing evolution from benignity toward aggressive phenotypes. By applying an integrative bioinformatics pipeline combining public information on a wealth of biological layers of complexity (from genetic polymorphisms to protein interactions), this study identified a module of co-expressed genes highly correlated with tumor stage and statistically linked to several genomic regions (module Quantitative Trait Loci, mQTLs). Ontology analysis of the transcription hub genes identified microtubule-associated cell-cycle processes as key drivers of such network. mQTLs and single nucleotide polymorphisms associated with meningioma stage were replicated in an alternative meningioma cohort, and integration of these results with up-to-date scientific literature and several databases retrieved a list of genes and pathways with a potentially important role in meningioma malignancy. As a result, cytoskeleton and cell–cell adhesion pathways, calcium-channels and glutamate receptors, as well as oxidoreductase and endoplasmic reticulum-associated degradation pathways were found to be the most important and redundant findings associated to meningioma progression. This study presents an integrated view of the pathways involved in meningioma malignant conversion and paves the way for the development of new research lines that will improve our understanding of meningioma biology.

Background Recent advances in DNA sequencing techniques resulted in more than forty sequenced plant genomes representing a diverse set of taxa of agricultural, energy, medicinal and ecological importance. However, gene family curation is often only inferred from DNA sequence homology and lacks insights into evolutionary processes contributing to gene family dynamics. In a comparative genomics framework, we integrated multiple lines of evidence provided by gene synteny, sequence homology and protein-based Hidden Markov Modelling to extract homologous super-clusters composed of multi-domain resistance (R)-proteins of the NB-LRR type (for NUCLEOTIDE BINDING/LEUCINE-RICH REPEATS), that are involved in plant innate immunity. Results To assess the diversity of R-proteins within and between species, we screened twelve eudicot plant genomes including six major crops and found a total of 2,363 NB-LRR genes. Our curated R-proteins set shows a 50% average for tandem duplicates and a 22% fraction of gene copies retained from ancient polyploidy events (ohnologs). We provide evidence for strong positive selection and show significant differences in molecular evolution rates (Ka/Ks-ratio) among tandem- (mean = 1.59), ohnolog (mean = 1.36) and singleton (mean = 1.22) R-gene duplicates. To foster the process of gene-edited plant breeding, we report species-specific presence/absence of all 140 NB-LRR genes present in the model plant Arabidopsis and describe four distinct clusters of NB-LRR “gatekeeper” loci sharing syntenic orthologs across all analyzed genomes. Conclusion By curating a near-complete set of multi-domain R-protein clusters in an eudicot-wide scale, our analysis offers significant insight into evolutionary dynamics underlying diversification of the plant innate immune system. Furthermore, our methods provide a blueprint for future efforts to identify and more rapidly clone functional NB-LRR genes from any plant species. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-966) contains supplementary material, which is available to authorized users.

Genome sequencing has demonstrated that besides frequent small-scale duplications, large-scale duplication events such as whole genome duplications (WGDs) are found on many branches of the evolutionary tree of life. Especially in the plant lineage, there is evidence for recurrent WGDs, and the ancestor of all angiosperms was in fact most likely a polyploid species. The number of WGDs found in sequenced plant genomes allows us to investigate questions about the roles of WGDs that were hitherto impossible to address. An intriguing observation is that many plant WGDs seem associated with periods of increased environmental stress and/or fluctuations, a trend that is evident for both present-day polyploids and palaeopolyploids formed around the Cretaceous–Palaeogene (K–Pg) extinction at 66 Ma. Here, we revisit the WGDs in plants that mark the K–Pg boundary, and discuss some specific examples of biological innovations and/or diversifications that may be linked to these WGDs. We review evidence for the processes that could have contributed to increased polyploid establishment at the K–Pg boundary, and discuss the implications on subsequent plant evolution in the Cenozoic.

The physiopathology of drug hypersensitivity (HS) are only partially known. A genetic background for such drug allergy is still demonstrated but only few genes are identified. Using a candidate gene approach, we tested the association of NOD1 and 2 genes with betalactam HS and the association of several cytokines genes with some drug HS. Using a whole genome approach, we tried to discover new candidate gene for DRESS. Among 368 italian cases and controls and 387 spanish cases and 326 controls, we identified one polymorphism of NOD2 gene associated with a protective effect for italians and another polymorphism associated with higher risk of druh HS for spanians. No association with NOD1 polymorphims was identified. Among 118 cases and 236 controls, we noticed that IL1 polymorphisms (IL1-RN-A2 and IL1-? -511) and IL10 polymorphism (-592A) were associated with DRESS.Ending, among 18 DRESS, a whole-genome array let us identify variations containing KLRC2 and CESP1 genes. These studies demonstrate the implication of several genes involved in inflammation, antivirus response or drug metabolism in different drug HS.Fonctionnal studies are needed to confirm these results, Les mécanismes physiopathologiques des hypersensibilités (HS) médicamenteuses ne sont que partiellement connus. Un terrain génétique favorisant est connu de longue date, mais peu de facteurs de risques sont formellement identifiés. A l’aide d’une approche par gènes candidats, nous avons évalué l’association entre des polymorphismes des gènes NOD1 et 2 et l’HS aux bétalactamines ; l’association entre plusieurs polymorphismes cytokiniques et différentes HS médicamenteuses. Enfin, nous avons utilisé une approche pangénomique à la recherche de gènes candidats au cours d’une HS spécifique, le DRESS. Parmi 368 cas et autant de contrôles italiens ainsi que 387 cas et 326 contrôles espagnols, nous avons mis en évidence une association entre l’un des polymorphismes de NOD2 et un faible risque d’HS immédiate aux bétalactamines chez les patients italiens, tandis qu’un autre polymorphisme de NOD2 était associé à un risque augmenté d’HS immédiate aux bétalactamines chez les patients espagnols. Aucune association avec le polymorphisme de NOD1 n’était identifiée. Parmi 118 patients et 236 contrôles, nous avons identifiés l’association entre les polymorphismes de l’IL1 (IL1-RN-A2 et IL1-[bêta] -511) et de l’IL10 (-592A) avec le risque de DRESS. Enfin, parmi 18 DRESS, l’analyse de puces CNV pangénomique nous a permis d’identifier des variations comportant les gènes KLRC2 et CESP1.Au total, nous avons pu démontrer l’implication de gènes modulant l’inflammation, la réponse antivirale ou le métabolisme des médicaments dans différentes HS médicamenteuses. Une confirmation à l’étage fonctionnelle de ces résultats est nécessaire

Background Chinese bayberry (Myrica rubra Sieb. and Zucc.) is a subtropical evergreen tree originating in China. It has been cultivated in southern China for several thousand years, and annual production has reached 1.1 million tons. The taste and high level of health promoting characters identified in the fruit in recent years has stimulated its extension in China and introduction to Australia. A limited number of co-dominant markers have been developed and applied in genetic diversity and identity studies. Here we report, for the first time, a survey of whole genome shotgun data to develop a large number of simple sequence repeat (SSR) markers to analyse the genetic diversity of the common cultivated Chinese bayberry and the relationship with three other Myrica species. Results The whole genome shotgun survey of Chinese bayberry produced 9.01Gb of sequence data, about 26x coverage of the estimated genome size of 323 Mb. The genome sequences were highly heterozygous, but with little duplication. From the initial assembled scaffold covering 255 Mb sequence data, 28,602 SSRs (≥5 repeats) were identified. Dinucleotide was the most common repeat motif with a frequency of 84.73%, followed by 13.78% trinucleotide, 1.34% tetranucleotide, 0.12% pentanucleotide and 0.04% hexanucleotide. From 600 primer pairs, 186 polymorphic SSRs were developed. Of these, 158 were used to screen 29 Chinese bayberry accessions and three other Myrica species: 91.14%, 89.87% and 46.84% SSRs could be used in Myrica adenophora, Myrica nana and Myrica cerifera, respectively. The UPGMA dendrogram tree showed that cultivated Myrica rubra is closely related to Myrica adenophora and Myrica nana, originating in southwest China, and very distantly related to Myrica cerifera, originating in America. These markers can be used in the construction of a linkage map and for genetic diversity studies in Myrica species. Conclusion Myrica rubra has a small genome of about 323 Mb with a high level of heterozygosity. A large number of SSRs were identified, and 158 polymorphic SSR markers developed, 91% of which can be transferred to other Myrica species.

Background The CHRNA5-CHRNA3-CHRNB4 gene cluster on 15q25 has consistently been associated with smoking quantity, nicotine dependence and lung cancer. Recent research also points towards its involvement in cardiovascular homeostasis, but studies in large human samples are lacking, especially on the role of the gene cluster in blood pressure regulation. Methodology/Principal Findings We studied the associations between 18 single nucleotide polymorphisms (SNPs) in CHRNA5-CHRNA3-CHRNB4 and systolic blood pressure (SBP), diastolic blood pressure (DBP), and body mass index (BMI) in 5402 young adults from the Northern Finland Birth Cohort 1966. We observed some evidence for associations between two SNPs and SBP and between six SNPs and BMI; the evidence for associations with DBP was weaker. The associations with the three phenotypes were driven by different loci with low linkage disequilibrium with each other. The associations appeared more pronounced in smokers, such that the smoking-increasing alleles would predict lower SBP and BMI. Each additional copy of the rs1948 G-allele and the rs950776 A-allele reduced SBP on average by −1.21 (95% CI −2.01, −0.40) mmHg in smokers. The variants associated with BMI included rs2036534, rs6495309, rs1996371, rs6495314, rs4887077 and rs11638372 and had an average effect size of −0.38 (−0.68, −0.08) kg/m2 per an additional copy of the risk allele in smokers. Formal assessments of interactions provided weaker support for these findings, especially after adjustment for multiple testing. Conclusions Variation at 15q25 appears to interact with smoking status in influencing SBP and BMI. The genetic loci associated with SBP were in low linkage disequilibrium with those associated with BMI suggesting that the gene cluster might regulate SBP through biological mechanisms that partly differ from those regulating BMI. Further studies in larger samples are needed for more precise evaluation of the possible interactions, and to understand the mechanisms behind.

Background Oomycetes of the genus Phytophthora are pathogens that infect a wide range of plant species. For dicot hosts such as tomato, potato and soybean, Phytophthora is even the most important pathogen. Previous analyses of Phytophthora genomes uncovered many genes, large gene families and large genome sizes that can partially be explained by significant repeat expansion patterns. Results Analysis of the complete genomes of three different Phytophthora species, using a newly developed approach, unveiled a large number of small duplicated blocks, mainly consisting of two or three consecutive genes. Further analysis of these duplicated genes and comparison with the known gene and genome duplication history of ten other eukaryotes including parasites, algae, plants, fungi, vertebrates and invertebrates, suggests that the ancestor of P. infestans, P. sojae and P. ramorum most likely underwent a whole genome duplication (WGD). Genes that have survived in duplicate are mainly genes that are known to be preferentially retained following WGDs, but also genes important for pathogenicity and infection of the different hosts seem to have been retained in excess. As a result, the WGD might have contributed to the evolutionary and pathogenic success of Phytophthora. Conclusions The fact that we find many small blocks of duplicated genes indicates that the genomes of Phytophthora species have been heavily rearranged following the WGD. Most likely, the high repeat content in these genomes have played an important role in this rearrangement process. As a consequence, the paucity of retained larger duplicated blocks has greatly complicated previous attempts to detect remnants of a large-scale duplication event in Phytophthora. However, as we show here, our newly developed strategy to identify very small duplicated blocks might be a useful approach to uncover ancient polyploidy events, in particular for heavily rearranged genomes.

The number of sequenced genomes of representatives within the green lineage is rapidly increasing. Consequently, comparative sequence analysis has significantly altered our view on the complexity of genome organization, gene function, and regulatory pathways. To explore all this genome information, a centralized infrastructure is required where all data generated by different sequencing initiatives is integrated and combined with advanced methods for data mining. Here, we describe PLAZA, an online platform for plant comparative genomics (http://bioinformatics.psb.ugent.be/plaza/). This resource integrates structural and functional annotation of published plant genomes together with a large set of interactive tools to study gene function and gene and genome evolution. Precomputed data sets cover homologous gene families, multiple sequence alignments, phylogenetic trees, intraspecies whole-genome dot plots, and genomic colinearity between species. Through the integration of high confidence Gene Ontology annotations and tree-based orthology between related species, thousands of genes lacking any functional description are functionally annotated. Advanced query systems, as well as multiple interactive visualization tools, are available through a user-friendly and intuitive Web interface. In addition, detailed documentation and tutorials introduce the different tools, while the workbench provides an efficient means to analyze user-defined gene sets through PLAZA's interface. In conclusion, PLAZA provides a comprehensible and up-to-date research environment to aid researchers in the exploration of genome information within the green plant lineage.

Genome-wide scanning for signals of recent positive selection is essential for a comprehensive and systematic understanding of human adaptation. Here, we present a genomic survey of recent local selective sweeps, especially aimed at those nearly or recently completed. A novel approach was developed for such signals, based on contrasting the extended haplotype homozygosity (EHH) profiles between populations. We applied this method to the genome single nucleotide polymorphism (SNP) data of both the International HapMap Project and Perlegen Sciences, and detected widespread signals of recent local selection across the genome, consisting of both complete and partial sweeps. A challenging problem of genomic scans of recent positive selection is to clearly distinguish selection from neutral effects, given the high sensitivity of the test statistics to departures from neutral demographic assumptions and the lack of a single, accurate neutral model of human history. We therefore developed a new procedure that is robust across a wide range of demographic and ascertainment models, one that indicates that certain portions of the genome clearly depart from neutrality. Simulations of positive selection showed that our tests have high power towards strong selection sweeps that have undergone fixation. Gene ontology analysis of the candidate regions revealed several new functional groups that might help explain some important interpopulation differences in phenotypic traits., Author Summary The evolution of new functions and adaptation to new environments occurs by positive selection, whereby beneficial mutations increase in frequency and eventually become fixed in a population. Detecting such selection in humans is crucial for understanding the importance of past genetic adaptations and their role in contemporary common diseases. Methods have already been developed for detecting the signature of positive selection in large, genome-scale datasets (such as the “HapMap”). Positive selection is expected to more rapidly increase the frequency of an allele, and hence, the length of the haplotype (extent of DNA segment) associated with the selected allele, relative to those that are not under selection. Such methods compare haplotype lengths within a single population. Here, we introduce a new method that compares the lengths of haplotypes associated with the same allele in different populations. We demonstrate that our method has greater power to detect selective sweeps that are fixed or nearly so, and we construct a statistical framework that shows that our method reliably detects positive selection. We applied our method to the HapMap data and identified approximately 500 candidate regions in the human genome that show a signature of recent positive selection. Further targeted studies of these regions should reveal important genetic adaptations in our past., The authors present a novel genome-scan approach for detecting local positive selection. The approach is designed to detect selective events that have resulted in complete or near-complete fixation of a beneficial allele.

elPrep is a high-performance tool for preparing sequence alignment/map files for variant calling in sequencing pipelines. It can be used as a replacement for SAMtools and Picard for preparation steps such as filtering, sorting, marking duplicates, reordering contigs, and so on, while producing identical results. What sets elPrep apart is its software architecture that allows executing preparation pipelines by making only a single pass through the data, no matter how many preparation steps are used in the pipeline. elPrep is designed as a multithreaded application that runs entirely in memory, avoids repeated file I/O, and merges the computation of several preparation steps to significantly speed up the execution time. For example, for a preparation pipeline of five steps on a whole-exome BAM file (NA12878), we reduce the execution time from about 1: 40 hours, when using a combination of SAMtools and Picard, to about 15 minutes when using elPrep, while utilising the same server resources, here 48 threads and 23GB of RAM. For the same pipeline on whole-genome data (NA12878), elPrep reduces the runtime from 24 hours to less than 5 hours. As a typical clinical study may contain sequencing data for hundreds of patients, elPrep can remove several hundreds of hours of computing time, and thus substantially reduce analysis time and cost.

AB - Microarray experiments can reveal important information about transcriptional regulation. In our case, we look for potential promoter regulatory elements in the upstream region of coexpressed genes. Here we present two modifications of the original Gibbs sampling algorithm for motif finding (Lawrence et al., 1993). First, we introduce the use of a probability distribution to estimate the number of copies of the motif in a sequence. Second, we describe the technical aspects of the incorporation of a higher-order background model whose application we discussed in Thijs et al. (2001). Our implementation is referred to as the Motif Sampler. We successfully validate our algorithm on several data sets. First, we show results for three sets of upstream sequences containing known motifs: 1) the G-box light-response element in plants, 2) elements involved in methionine response in Saccharomyces cerevisiae, and 3) the FNR O(2)-responsive element in bacteria. We use these data sets to explain the influence of the parameters on the performance of our algorithm. Second, we show results for upstream sequences from four clusters of coexpressed genes identified in a microarray experiment on wounding in Arabidopsis thaliana. Several motifs could be matched to regulatory elements from plant defence pathways in our database of plant cis-acting regulatory elements (PlantCARE). Some other strong motifs do not have corresponding motifs in PlantCARE but are promising candidates for further analysis ispartof: Journal of computational biology vol:9 issue:3 pages:447-464 ispartof: location:CANADA, MONTREAL status: published

Summary Objective To elucidate disease-specific molecular changes in osteoarthritis (OA) by analyzing the differential gene expression profile of damaged vs intact cartilage areas within the same joint of patients with OA of the knee using a combination of a novel RNA extraction technique and whole-genome oligonucleotide arrays. Methods The transcriptome of macroscopically affected vs intact articular cartilage as determined by visual assessment was analyzed using an optimized mill-based total RNA isolation directly from the tissue and high density synthetic oligonucleotide arrays. Articular cartilage samples were obtained from patients with OA of the knee. Expression of differentially regulated genes was validated by real-time quantitative polymerase chain reaction and immunohistochemistry. Results The amount of RNA obtained by the optimized extraction procedure was at least 1μg per 500mg of cartilage and fulfilled the common quality requirements. After hybridization onto HG-U133 Plus 2.0 GeneChips (Affymetrix), 28.6–51.7% of the probe sets on the microarray showed a detectable signal above the signal threshold in the individual samples. A subset of 411 transcripts, which appeared to be differentially expressed, was obtained when applying predefined filtering criteria. Of these, six genes were found to be up-regulated in the affected cartilage of all patients, including insulin-like growth factor binding protein 3 (IGFBP-3), wnt-1-inducible signaling protein 1 (WISP-1), aquaporin 1 (AQP-1), delta/notch-like EGF-repeat containing transmembrane (DNER), decay accelerating factor (DAF), complement factor I (IF). Conclusion The optimized methodical approach reported here not only allows to determine area-specific gene expression profiles of intraindividually different low-RNA containing OA cartilage specimens. In addition, this study also revealed novel genes not yet reported to play a role in the pathophysiology of joint destruction in OA.

The mapping resolution of the physical map for chicken Chromosome 4 (GGA4) was improved by a combination of radiation hybrid (RH) mapping and bacterial artificial chromosome (BAC) mapping. The ChickRH6 hybrid panel was used to construct an RH map of GGA4. Eleven microsatellites known to be located on GGA4 were included as anchors to the genetic linkage map for this chromosome. Based on the known conserved synteny between GGA4 and human Chromosomes 4 and X, sequences were identified for the orthologous chicken genes from these human chromosomes by BLAST analysis. These sequences were subsequently used for the development of STS markers to be typed on the RH panel. Using a logarithm of the odds (LOD) threshold of 5.0, nine linkage groups could be constructed which were aligned with the genetic linkage map of this chromosome. The resulting RH map consisted of the 11 microsatellite markers and 50 genes. To further increase the number of genes on the map and to provide additional anchor points for the physical BAC map of this chromosome, BAC clones were identified for 22 microsatellites and 99 genes. The combined RH and BAC mapping approach resulted in the mapping of 61 genes on GGA4 increasing the resolution of the chicken-human comparative map for this chromosome. This enhanced comparative mapping resolution enabled the identification of multiple rearrangements between GGA4 and human Chromosomes 4q and Xp.