Genome-wide DNA polymorphism analyses using VariScan

BACKGROUND: DNA sequence polymorphisms analysis can provide valuable information on the evolutionary forces shaping nucleotide variation, and provides an insight into the functional significance of genomic regions. The recent ongoing genome projects will radically improve our capabilities to detect specific genomic regions shaped by natural selection. Current available methods and software, however, are unsatisfactory for such genome-wide analysis. RESULTS: We have developed methods for the analysis of DNA sequence polymorphisms at the genome-wide scale. These methods, which have been tested on a coalescent-simulated and actual data files from mouse and human, have been implemented in the VariScan software package version 2.0. Additionally, we have also incorporated a graphical-user interface. The main features of this software are: i) exhaustive population-genetic analyses including those based on the coalescent theory; ii) analysis adapted to the shallow data generated by the high-throughput genome projects; iii) use of genome annotations to conduct a comprehensive analyses separately for different functional regions; iv) identification of relevant genomic regions by the sliding-window and wavelet-multiresolution approaches; v) visualization of the results integrated with current genome annotations in commonly available genome browsers. CONCLUSION: VariScan is a powerful and flexible suite of software for the analysis of DNA polymorphisms. The current version implements new algorithms, methods, and capabilities, providing an important tool for an exhaustive exploratory analysis of genome-wide DNA polymorphism data.

A genome-wide association study of anorexia nervosa.

Anorexia nervosa (AN) is a complex and heritable eating disorder characterized by dangerously low body weight. Neither candidate gene studies nor an initial genome-wide association study (GWAS) have yielded significant and replicated results. We performed a GWAS in 2907 cases with AN from 14 countries (15 sites) and 14 860 ancestrally matched controls as part of the Genetic Consortium for AN (GCAN) and the Wellcome Trust Case Control Consortium 3 (WTCCC3). Individual association analyses were conducted in each stratum and meta-analyzed across all 15 discovery data sets. Seventy-six (72 independent) single nucleotide polymorphisms were taken forward for in silico (two data sets) or de novo (13 data sets) replication genotyping in 2677 independent AN cases and 8629 European ancestry controls along with 458 AN cases and 421 controls from Japan. The final global meta-analysis across discovery and replication data sets comprised 5551 AN cases and 21 080 controls. AN subtype analyses (1606 AN restricting; 1445 AN binge-purge) were performed. No findings reached genome-wide significance. Two intronic variants were suggestively associated: rs9839776 (P=3.01 × 10(-7)) in SOX2OT and rs17030795 (P=5.84 × 10(-6)) in PPP3CA. Two additional signals were specific to Europeans: rs1523921 (P=5.76 × 10(-)(6)) between CUL3 and FAM124B and rs1886797 (P=8.05 × 10(-)(6)) near SPATA13. Comparing discovery with replication results, 76% of the effects were in the same direction, an observation highly unlikely to be due to chance (P=4 × 10(-6)), strongly suggesting that true findings exist but our sample, the largest yet reported, was underpowered for their detection. The accrual of large genotyped AN case-control samples should be an immediate priority for the field.

Genome-wide RNA polymerase II profiles and RNA accumulation reveal kinetics of transcription and associated epigenetic changes during diurnal cycles.

Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II) as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver.

Sequencing of Lp-PLA2-encoding PLA2G7 gene in 2000 Europeans reveals several rare loss-of-function mutations.

Elevated plasma levels of lipoprotein-associated phospholipase A(2) (Lp-PLA2) activity have been shown to be associated with increased risk of coronary heart disease and an inhibitor of this enzyme is under development for the treatment of that condition. A Val279Phe null allele in this gene, that may influence patient eligibility for treatment, is relatively common in East Asians but has not been observed in Europeans. We investigated the existence and functional effects of low frequency alleles in a Western European population by re-sequencing the exons of PLA2G7 in 2000 samples. In all, 19 non-synonymous single-nucleotide polymorphisms (nsSNPs) were found, 14 in fewer than four subjects (minor allele frequency <0.1%). Lp-PLA2 activity was significantly lower in rare nsSNP carriers compared with non-carriers (167.8±63.2 vs 204.6±41.8, P=0.01) and seven variants had enzyme activities consistent with a null allele. The cumulative frequency of these null alleles was 0.25%, so <1 in 10,000 Europeans would be expected to be homozygous, and thus not potentially benefit from treatment with an Lp-PLA2 inhibitor.

Novel method to estimate the phenotypic variation explained by genome-wide association studies reveals large fraction of the missing heritability.

Genome-wide association studies (GWAS) are conducted with the promise to discover novel genetic variants associated with diverse traits. For most traits, associated markers individually explain just a modest fraction of the phenotypic variation, but their number can well be in the hundreds. We developed a maximum likelihood method that allows us to infer the distribution of associated variants even when many of them were missed by chance. Compared to previous approaches, the novelty of our method is that it (a) does not require having an independent (unbiased) estimate of the effect sizes; (b) makes use of the complete distribution of P-values while allowing for the false discovery rate; (c) takes into account allelic heterogeneity and the SNP pruning strategy. We applied our method to the latest GWAS meta-analysis results of the GIANT consortium. It revealed that while the explained variance of genome-wide (GW) significant SNPs is around 1% for waist-hip ratio (WHR), the observed P-values provide evidence for the existence of variants explaining 10% (CI=[8.5-11.5%]) of the phenotypic variance in total. Similarly, the total explained variance likely to exist for height is estimated to be 29% (CI=[28-30%]), three times higher than what the observed GW significant SNPs give rise to. This methodology also enables us to predict the benefit of future GWA studies that aim to reveal more associated genetic markers via increased sample size.

Multiplex targeted high-throughput sequencing for Mendelian cardiac disorders.

Mendelian cardiomyopathies and arrhythmias are characterized by an important genetic heterogeneity, rendering Sanger sequencing very laborious and expensive. As a proof of concept, we explored multiplex targeted high-throughput sequencing (HTS) as a fast and cost-efficient diagnostic method for individuals suffering from Mendelian cardiac disorders. We designed a DNA capture assay including all exons from 130 genes involved in cardiovascular Mendelian disorders and analysed simultaneously four samples by multiplexing. Two patients had familial hypertrophic cardiomyopathy (HCM) and two patients suffered from long QT syndrome (LQTS). In patient 1 with HCM, we identified two known pathogenic missense variants in the two most frequently mutated sarcomeric genes MYH7 and MYBPC. In patient 2 with HCM, a known acceptor splice site variant in MYBPC3 was found. In patient 3 with LQTS, two missense variants in the genes SCN5A and KCNQ were identified. Finally, in patient 4 with LQTS a known missense variant was found in MYBPC3, which is usually mutated in patients with cardiomyopathy. Our results showed that multiplex targeted HTS works as an efficient and cost-effective tool for molecular diagnosis of heterogeneous disorders in clinical practice and offers new insights in the pathogenesis of these complex diseases.

Genome-wide association study of recurrent major depressive disorder in two European case-control cohorts.

Major depressive disorder (MDD) is a highly prevalent disorder with substantial heritability. Heritability has been shown to be substantial and higher in the variant of MDD characterized by recurrent episodes of depression. Genetic studies have thus far failed to identify clear and consistent evidence of genetic risk factors for MDD. We conducted a genome-wide association study (GWAS) in two independent datasets. The first GWAS was performed on 1022 recurrent MDD patients and 1000 controls genotyped on the Illumina 550 platform. The second was conducted on 492 recurrent MDD patients and 1052 controls selected from a population-based collection, genotyped on the Affymetrix 5.0 platform. Neither GWAS identified any SNP that achieved GWAS significance. We obtained imputed genotypes at the Illumina loci for the individuals genotyped on the Affymetrix platform, and performed a meta-analysis of the two GWASs for this common set of approximately half a million SNPs. The meta-analysis did not yield genome-wide significant results either. The results from our study suggest that SNPs with substantial odds ratio are unlikely to exist for MDD, at least in our datasets and among the relatively common SNPs genotyped or tagged by the half-million-loci arrays. Meta-analysis of larger datasets is warranted to identify SNPs with smaller effects or with rarer allele frequencies that contribute to the risk of MDD.

The genome and development-dependent transcriptomes of Pyronema confluens: a window into fungal evolution

Fungi are a large group of eukaryotes found in nearly all ecosystems. More than 250 fungal genomes have already been sequenced, greatly improving our understanding of fungal evolution, physiology, and development. However, for the Pezizomycetes, an early-diverging lineage of filamentous ascomycetes, there is so far only one genome available, namely that of the black truffle, Tuber melanosporum, a mycorrhizal species with unusual subterranean fruiting bodies. To help close the sequence gap among basal filamentous ascomycetes, and to allow conclusions about the evolution of fungal development, we sequenced the genome and assayed transcriptomes during development of Pyronema confluens, a saprobic Pezizomycete with a typical apothecium as fruiting body. With a size of 50 Mb and ~13,400 protein-coding genes, the genome is more characteristic of higher filamentous ascomycetes than the large, repeat-rich truffle genome; however, some typical features are different in the P. confluens lineage, e.g. the genomic environment of the mating type genes that is conserved in higher filamentous ascomycetes, but only partly conserved in P. confluens. On the other hand, P. confluens has a full complement of fungal photoreceptors, and expression studies indicate that light perception might be similar to distantly related ascomycetes and, thus, represent a basic feature of filamentous ascomycetes. Analysis of spliced RNA-seq sequence reads allowed the detection of natural antisense transcripts for 281 genes. The P. confluens genome contains an unusually high number of predicted orphan genes, many of which are upregulated during sexual development, consistent with the idea of rapid evolution of sex-associated genes. Comparative transcriptomics identified the transcription factor gene pro44 that is upregulated during development in P. confluens and the Sordariomycete Sordaria macrospora. The P. confluens pro44 gene (PCON_06721) was used to complement the S. macrospora pro44 deletion mutant, showing functional conservation of this developmental regulator.

Developmental constraints on vertebrate genome evolution.

Constraints in embryonic development are thought to bias the direction of evolution by making some changes less likely, and others more likely, depending on their consequences on ontogeny. Here, we characterize the constraints acting on genome evolution in vertebrates. We used gene expression data from two vertebrates: zebrafish, using a microarray experiment spanning 14 stages of development, and mouse, using EST counts for 26 stages of development. We show that, in both species, genes expressed early in development (1) have a more dramatic effect of knock-out or mutation and (2) are more likely to revert to single copy after whole genome duplication, relative to genes expressed late. This supports high constraints on early stages of vertebrate development, making them less open to innovations (gene gain or gene loss). Results are robust to different sources of data -- gene expression from microarrays, ESTs, or in situ hybridizations; and mutants from directed KO, transgenic insertions, point mutations, or morpholinos. We determine the pattern of these constraints, which differs from the model used to describe vertebrate morphological conservation ("hourglass" model). While morphological constraints reach a maximum at mid-development (the "phylotypic" stage), genomic constraints appear to decrease in a monotonous manner over developmental time.

Impact of phenotype definition on genome-wide association signals: empirical evaluation in human immunodeficiency virus type 1 infection.

Discussion on improving the power of genome-wide association studies to identify candidate variants and genes is generally centered on issues of maximizing sample size; less attention is given to the role of phenotype definition and ascertainment. The authors used genome-wide data from patients infected with human immunodeficiency virus type 1 (HIV-1) to assess whether differences in type of population (622 seroconverters vs. 636 seroprevalent subjects) or the number of measurements available for defining the phenotype resulted in differences in the effect sizes of associations between single nucleotide polymorphisms and the phenotype, HIV-1 viral load at set point. The effect estimate for the top 100 single nucleotide polymorphisms was 0.092 (95% confidence interval: 0.074, 0.110) log(10) viral load (log(10) copies of HIV-1 per mL of blood) greater in seroconverters than in seroprevalent subjects. The difference was even larger when the authors focused on chromosome 6 variants (0.153 log(10) viral load) or on variants that achieved genome-wide significance (0.232 log(10) viral load). The estimates of the genetic effects tended to be slightly larger when more viral load measurements were available, particularly among seroconverters and for variants that achieved genome-wide significance. Differences in phenotype definition and ascertainment may affect the estimated magnitude of genetic effects and should be considered in optimizing power for discovering new associations.

Identification of cancer/testis-antigen genes by massively parallel signature sequencing.

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.

Length variation in HIV-1 gp120 as the product of DNA misalignment mechanism

Background: To determine whether misalignment structures such as duplications, repeats, and palindromes are associated to insertions/deletions (indels) in gp120, indicating that indels are indeed frameshift mutations generated by DNA misalignment mechanism. Methods: Cloning and sequencing of a fragment of HIV-1 gp120 spanning C2-C4 derived from plasma RNA in 12 patients with early chronic disease and naïve to antiretroviral therapy. Results: Indels in V4 involved always insertion and deletion of duplicated nucleotide segments, and AAT repeats, and were associated to the presence of palindromic sequences. No duplications were detected in V3 and C3. Palindromic sequences occurred with similar frequencies in V3, C3 and V4; the frequency of palindromes in individual genes was found to be significantly higher in structural (gp120, p ≤ 3.00E-7) and significantly lower in regulatory (Tat, p ≤ 9.00E-7) genes, as compared to the average frequency calculated over the full genome. Discussion: Indels in V4 are associated to misalignment structures (i.e. duplications repeat and palindromes) indicating DNA misalignment as the mechanism underlying length variation in V4. The finding that indels in V4 are caused by DNA misalignment has some very important implications: 1) indels in V4 are likely to occur in proviral DNA (and not in RNA), after integration of HIV into the host genome; 2) they are likely to occur as progressive modifications of the early founder virus during chronic infection, as more and more cells get infected; 3) frameshift mutations involving any number of base pairs are likely to occur evenly across gp120; however, only those mutants carrying a functional gp120 (indels as multiples of three base pairs) will be able to perpetuate the virus cycle and to keep spreading through the population.

Computational analysis of the genetic and environmental contributions to disease-related human phenotypes: From predicting adverse lipid response of HIV patients receiving antiretroviral therapy to genome-wide association studies of cardiovascular and lipid related disorders

Genetic variants influence the risk to develop certain diseases or give rise to differences in drug response. Recent progresses in cost-effective, high-throughput genome-wide techniques, such as microarrays measuring Single Nucleotide Polymorphisms (SNPs), have facilitated genotyping of large clinical and population cohorts. Combining the massive genotypic data with measurements of phenotypic traits allows for the determination of genetic differences that explain, at least in part, the phenotypic variations within a population. So far, models combining the most significant variants can only explain a small fraction of the variance, indicating the limitations of current models. In particular, researchers have only begun to address the possibility of interactions between genotypes and the environment. Elucidating the contributions of such interactions is a difficult task because of the large number of genetic as well as possible environmental factors.In this thesis, I worked on several projects within this context. My first and main project was the identification of possible SNP-environment interactions, where the phenotypes were serum lipid levels of patients from the Swiss HIV Cohort Study (SHCS) treated with antiretroviral therapy. Here the genotypes consisted of a limited set of SNPs in candidate genes relevant for lipid transport and metabolism. The environmental variables were the specific combinations of drugs given to each patient over the treatment period. My work explored bioinformatic and statistical approaches to relate patients' lipid responses to these SNPs, drugs and, importantly, their interactions. The goal of this project was to improve our understanding and to explore the possibility of predicting dyslipidemia, a well-known adverse drug reaction of antiretroviral therapy. Specifically, I quantified how much of the variance in lipid profiles could be explained by the host genetic variants, the administered drugs and SNP-drug interactions and assessed the predictive power of these features on lipid responses. Using cross-validation stratified by patients, we could not validate our hypothesis that models that select a subset of SNP-drug interactions in a principled way have better predictive power than the control models using "random" subsets. Nevertheless, all models tested containing SNP and/or drug terms, exhibited significant predictive power (as compared to a random predictor) and explained a sizable proportion of variance, in the patient stratified cross-validation context. Importantly, the model containing stepwise selected SNP terms showed higher capacity to predict triglyceride levels than a model containing randomly selected SNPs. Dyslipidemia is a complex trait for which many factors remain to be discovered, thus missing from the data, and possibly explaining the limitations of our analysis. In particular, the interactions of drugs with SNPs selected from the set of candidate genes likely have small effect sizes which we were unable to detect in a sample of the present size (<800 patients).In the second part of my thesis, I performed genome-wide association studies within the Cohorte Lausannoise (CoLaus). I have been involved in several international projects to identify SNPs that are associated with various traits, such as serum calcium, body mass index, two-hour glucose levels, as well as metabolic syndrome and its components. These phenotypes are all related to major human health issues, such as cardiovascular disease. I applied statistical methods to detect new variants associated with these phenotypes, contributing to the identification of new genetic loci that may lead to new insights into the genetic basis of these traits. This kind of research will lead to a better understanding of the mechanisms underlying these pathologies, a better evaluation of disease risk, the identification of new therapeutic leads and may ultimately lead to the realization of "personalized" medicine.

Ambiguous nucleotide calls from population-based sequencing of HIV-1 are a marker for viral diversity and the age of infection.

Background. The time passed since the infection of a human immunodeficiency virus (HIV)-infected individual (the age of infection) is an important but often only poorly known quantity. We assessed whether the fraction of ambiguous nucleotides obtained from bulk sequencing as done for genotypic resistance testing can serve as a proxy of this parameter. Methods. We correlated the age of infection and the fraction of ambiguous nucleotides in partial pol sequences of HIV-1 sampled before initiation of antiretroviral therapy (ART). Three groups of Swiss HIV Cohort Study participants were analyzed, for whom the age of infection was estimated on the basis of Bayesian back calculation (n = 3,307), seroconversion (n = 366), or diagnoses of primary HIV infection (n = 130). In addition, we studied 124 patients for whom longitudinal genotypic resistance testing was performed while they were still ART-naive. Results. We found that the fraction of ambiguous nucleotides increased with the age of infection with a rate of .2% per year within the first 8 years but thereafter with a decreasing rate. We show that this pattern is consistent with population-genetic models for realistic parameters. Finally, we show that, in this highly representative population, a fraction of ambiguous nucleotides of >.5% provides strong evidence against a recent infection event < 1 year prior to sampling (negative predictive value, 98.7%). Conclusions. These findings show that the fraction of ambiguous nucleotides is a useful marker for the age of infection.