Marine Germain

Genetics and Beyond – The Transcriptome of Human Monocytes and Disease Susceptibility

Background Variability of gene expression in human may link gene sequence variability and phenotypes; however, non-genetic variations, alone or in combination with genetics, may also influence expression traits and have a critical role in physiological and disease processes. Methodology/Principal Findings To get better insight into the overall variability of gene expression, we assessed the transcriptome of circulating monocytes, a key cell involved in immunity-related diseases and atherosclerosis, in 1,490 unrelated individuals and investigated its association with >675,000 SNPs and 10 common cardiovascular risk factors. Out of 12,808 expressed genes, 2,745 expression quantitative trait loci were detected (P<5.78×10−12), most of them (90%) being cis-modulated. Extensive analyses showed that associations identified by genome-wide association studies of lipids, body mass index or blood pressure were rarely compatible with a mediation by monocyte expression level at the locus. At a study-wide level (P<3.9×10−7), 1,662 expression traits (13.0%) were significantly associated with at least one risk factor. Genome-wide interaction analyses suggested that genetic variability and risk factors mostly acted additively on gene expression. Because of the structure of correlation among expression traits, the variability of risk factors could be characterized by a limited set of independent gene expressions which may have biological and clinical relevance. For example expression traits associated with cigarette smoking were more strongly associated with carotid atherosclerosis than smoking itself. Conclusions/Significance This study demonstrates that the monocyte transcriptome is a potent integrator of genetic and non-genetic influences of relevance for disease pathophysiology and risk assessment.

A Novel Channelopathy in Pulmonary Arterial Hypertension

BACKGROUND Pulmonary arterial hypertension is a devastating disease with high mortality. Familial cases of pulmonary arterial hypertension are usually characterized by autosomal dominant transmission with reduced penetrance, and some familial cases have unknown genetic causes. METHODS We studied a family in which multiple members had pulmonary arterial hypertension without identifiable mutations in any of the genes known to be associated with the disease, including BMPR2, ALK1, ENG, SMAD9, and CAV1. Three family members were studied with whole-exome sequencing. Additional patients with familial or idiopathic pulmonary arterial hypertension were screened for the mutations in the gene that was identified on whole-exome sequencing. All variants were expressed in COS-7 cells, and channel function was studied by means of patch-clamp analysis. RESULTS We identified a novel heterozygous missense variant c.608 G→A (G203D) in KCNK3 (the gene encoding potassium channel subfamily K, member 3) as a disease-causing candidate gene in the family. Five additional heterozygous missense variants in KCNK3 were independently identified in 92 unrelated patients with familial pulmonary arterial hypertension and 230 patients with idiopathic pulmonary arterial hypertension. We used in silico bioinformatic tools to predict that all six novel variants would be damaging. Electrophysiological studies of the channel indicated that all these missense mutations resulted in loss of function, and the reduction in the potassium-channel current was remedied by the application of the phospholipase inhibitor ONO-RS-082. CONCLUSIONS Our study identified the association of a novel gene, KCNK3, with familial and idiopathic pulmonary arterial hypertension. Mutations in this gene produced reduced potassium-channel current, which was successfully remedied by pharmacologic manipulation. (Funded by the National Institutes of Health.)

A genome-wide association study identifies two loci associated with heart failure due to dilated cardiomyopathy

AIMS Dilated cardiomyopathy (DCM) is a major cause of heart failure with a high familial recurrence risk. So far, the genetics of DCM remains largely unresolved. We conducted the first genome-wide association study (GWAS) to identify loci contributing to sporadic DCM. METHODS AND RESULTS One thousand one hundred and seventy-nine DCM patients and 1108 controls contributed to the discovery phase. Pools of DNA stratified on disease status, population, age, and gender were constituted and used for testing association of DCM with 517 382 single nucleotide polymorphisms (SNPs). Three DCM-associated SNPs were confirmed by individual genotyping (P < 5.0 10(-7)), and two of them, rs10927875 and rs2234962, were replicated in independent samples (1165 DCM patients and 1302 controls), with P-values of 0.002 and 0.009, respectively. rs10927875 maps to a region on chromosome 1p36.13 which encompasses several genes among which HSPB7 has been formerly suggested to be implicated in DCM. The second identified locus involves rs2234962, a non-synonymous SNP (c.T757C, p. C151R) located within the sequence of BAG3 on chromosome 10q26. To assess whether coding mutations of BAG3 might cause monogenic forms of the disease, we sequenced BAG3 exons in 168 independent index cases diagnosed with familial DCM and identified four truncating and two missense mutations. Each mutation was heterozygous, present in all genotyped relatives affected by the disease and absent in a control group of 347 healthy individuals, strongly suggesting that these mutations are causing the disease. CONCLUSION This GWAS identified two loci involved in sporadic DCM, one of them probably implicates BAG3. Our results show that rare mutations in BAG3 contribute to monogenic forms of the disease, while common variant(s) in the same gene are implicated in sporadic DCM.

Genetics of Venous Thrombosis: Insights from a New Genome Wide Association Study

Background Venous Thrombosis (VT) is a common multifactorial disease associated with a major public health burden. Genetics factors are known to contribute to the susceptibility of the disease but how many genes are involved and their contribution to VT risk still remain obscure. We aimed to identify genetic variants associated with VT risk. Methodology/Principal Findings We conducted a genome-wide association study (GWAS) based on 551,141 SNPs genotyped in 1,542 cases and 1,110 controls. Twelve SNPs reached the genome-wide significance level of 2.0×10−8 and encompassed four known VT-associated loci, ABO, F5, F11 and FGG. By means of haplotype analyses, we also provided novel arguments in favor of a role of HIVEP1, PROCR and STAB2, three loci recently hypothesized to participate in the susceptibility to VT. However, no novel VT-associated loci came out of our GWAS. Using a recently proposed statistical methodology, we also showed that common variants could explain about 35% of the genetic variance underlying VT susceptibility among which 3% could be attributable to the main identified VT loci. This analysis additionally suggested that the common variants left to be identified are not uniformly distributed across the genome and that chromosome 20, itself, could contribute to ∼7% of the total genetic variance. Conclusions/Significance This study might also provide a valuable source of information to expand our understanding of biological mechanisms regulating quantitative biomarkers for VT.

Integrating genome-wide genetic variations and monocyte expression data reveals trans-regulated gene modules in humans.

One major expectation from the transcriptome in humans is to characterize the biological basis of associations identified by genome-wide association studies. So far, few cis expression quantitative trait loci (eQTLs) have been reliably related to disease susceptibility. Trans-regulating mechanisms may play a more prominent role in disease susceptibility. We analyzed 12,808 genes detected in at least 5% of circulating monocyte samples from a population-based sample of 1,490 European unrelated subjects. We applied a method of extraction of expression patterns—independent component analysis—to identify sets of co-regulated genes. These patterns were then related to 675,350 SNPs to identify major trans-acting regulators. We detected three genomic regions significantly associated with co-regulated gene modules. Association of these loci with multiple expression traits was replicated in Cardiogenics, an independent study in which expression profiles of monocytes were available in 758 subjects. The locus 12q13 (lead SNP rs11171739), previously identified as a type 1 diabetes locus, was associated with a pattern including two cis eQTLs, RPS26 and SUOX, and 5 trans eQTLs, one of which (MADCAM1) is a potential candidate for mediating T1D susceptibility. The locus 12q24 (lead SNP rs653178), which has demonstrated extensive disease pleiotropy, including type 1 diabetes, hypertension, and celiac disease, was associated to a pattern strongly correlating to blood pressure level. The strongest trans eQTL in this pattern was CRIP1, a known marker of cellular proliferation in cancer. The locus 12q15 (lead SNP rs11177644) was associated with a pattern driven by two cis eQTLs, LYZ and YEATS4, and including 34 trans eQTLs, several of them tumor-related genes. This study shows that a method exploiting the structure of co-expressions among genes can help identify genomic regions involved in trans regulation of sets of genes and can provide clues for understanding the mechanisms linking genome-wide association loci to disease.

Long-range epigenetic regulation is conferred by genetic variation located at thousands of independent loci

The interplay between genetic and epigenetic variation is only partially understood. One form of epigenetic variation is methylation at CpG sites, which can be measured as methylation quantitative trait loci (meQTL). Here we report that in a panel of lymphocytes from 1,748 individuals, methylation levels at 1,919 CpG sites are correlated with at least one distal (trans) single-nucleotide polymorphism (SNP) (P<3.2 × 10−13; FDR<5%). These trans-meQTLs include 1,657 SNP–CpG pairs from different chromosomes and 262 pairs from the same chromosome that are >1 Mb apart. Over 90% of these pairs are replicated (FDR<5%) in at least one of two independent data sets. Genomic loci harbouring trans-meQTLs are significantly enriched (P<0.001) for long non-coding transcripts (2.2-fold), known epigenetic regulators (2.3-fold), piwi-interacting RNA clusters (3.6-fold) and curated transcription factors (4.1-fold), including zinc-finger proteins (8.75-fold). Long-range epigenetic networks uncovered by this approach may be relevant to normal and disease states.

Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding.

First case of a human RASGRP2 mutation affecting Rap1 activation in platelets and causing severe bleeding.

A Follow-Up Study of a Genome-wide Association Scan Identifies a Susceptibility Locus for Venous Thrombosis on Chromosome 6p24.1

To identify genetic susceptibility factors conferring increased risk of venous thrombosis (VT), we conducted a multistage study, following results of a previously published GWAS that failed to detect loci for developing VT. Using a collection of 5862 cases with VT and 7112 healthy controls, we identified the HIVEP1 locus on chromosome 6p24.1 as a susceptibility locus for VT. Indeed, the HIVEP1 rs169713C allele was associated with an increased risk for VT, with an odds ratio of 1.20 (95% confidence interval 1.13-1.27, p = 2.86 x 10(-9)). HIVEP1 codes for a protein that participates in the transcriptional regulation of inflammatory target genes by binding specific DNA sequences in their promoter and enhancer regions. The current results provide the identification of a locus involved in VT susceptibility that lies outside the traditional coagulation/fibrinolysis pathway.

Combined analysis of three genome-wide association studies on vWF and FVIII plasma levels

BackgroundElevated levels of factor VIII (FVIII) and von Willebrand Factor (vWF) are well-established risk factors for cardiovascular diseases, in particular venous thrombosis. Although high, the heritability of these traits is poorly explained by the genetic factors known so far. The aim of this work was to identify novel single nucleotide polymorphisms (SNPs) that could influence the variability of these traits.MethodsThree independent genome-wide association studies for vWF plasma levels and FVIII activity were conducted and their results were combined into a meta-analysis totalling 1,624 subjects.ResultsNo single nucleotide polymorphism (SNP) reached the study-wide significance level of 1.12 × 10-7 that corresponds to the Bonferroni correction for the number of tested SNPs. Nevertheless, the recently discovered association of STXBP5, STX2, TC2N and CLEC4M genes with vWF levels and that of SCARA5 and STAB2 genes with FVIII levels were confirmed in this meta-analysis. Besides, among the fifteen novel SNPs showing promising association at p < 10-5 with either vWF or FVIII levels in the meta-analysis, one located in ACCN1 gene also showed weak association (P = 0.0056) with venous thrombosis in a sample of 1,946 cases and 1,228 controls.ConclusionsThis study has generated new knowledge on genomic regions deserving further investigations in the search for genetic factors influencing vWF and FVIII plasma levels, some potentially implicated in VT, as well as providing some supporting evidence of previously identified genes.

C4BPB/C4BPA is a new susceptibility locus for venous thrombosis with unknown protein S–independent mechanism: results from genome-wide association and gene expression analyses followed by case-control studies

Through its binding with protein S (PS), a key element of the coagulation/fibrinolysis cascade, the C4b-binding protein (C4BP) has been hypothesized to be involved in the susceptibility to venous thrombosis (VT). To identify genetic factors that may influence the plasma levels of the 3 C4BP existing isoforms, alpha(7)beta(1), alpha(6)beta(1), and alpha(7)beta(0), we conducted a genome-wide association study by analyzing 283 437 single nucleotide polymorphisms (SNPs) in the Genetic Analysis of Idiopathic Thrombophilia (GAIT) study composed of 352 persons. Three SNPs at the C4BPB/C4BPA locus were found genome-wide significantly associated with alpha(7)beta(0) levels. One of these SNPs was further found to explain approximately 11% of the variability of mRNA C4BPA expression in the Gutenberg Heart Study composed of 1490 persons, with no effect on C4BPB mRNA expression. The allele associated with increased alpha(7)beta(0) plasma levels and increased C4BPA expression was further found associated with increased risk of VT (odds ratio [OR] = 1.24 [1.03-1.53]) in 2 independent case-control studies (MARseille THrombosis Association study [MARTHA] and FActeurs de RIsque et de récidives de la maladie thromboembolique VEineuse [FARIVE]) gathering 1706 cases and 1379 controls. This SNP was not associated with free PS or total PS. In conclusion, we observed strong evidence that the C4BPB/C4BPA locus is a new susceptibility locus for VT through a PS-independent mechanism that remains to be elucidated.