Genetic and Epigenetic Determinants of Thrombin Generation Potential : an epidemiological approach

Thrombin Generation Potential (TGP) is a promising in vitro measurement that allows quantifying thrombin activity, in a close way to what happens in vivo. It is sensitive to coagulation factors deficiencies, anticoagulant proteins and is associated to thrombotic disorders. There exists two polymorphisms located in the F2 (prothrombin) gene known to influence TGP levels, and altogether they explain 11.3% of the TGP inter-individual variability. With the aims of identifying novel genetic and epigenetic factors that influence TGP variability, I have performed two different studies in the present work. First, I conducted the first genome-wide association study for the three TGP biomarkers (ETP, Peak and Lagtime) using imputation data from two French studies. The most significant single nucleotide polymorphisms (SNPs) were then replicated in two independent French studies. This analysis lead to the discovery of ORM1 as a new gene participating to the control of TGP. Second, I followed a similar strategy using this time whole blood DNA methylation levels at CpG sites to identify DNA methylation marks involved in TGP variability. I analyzed the association between methylation-wide patterns from a French study and a French-Canadian families measured for TGP. Unfortunately, I did not identify robust associations between whole DNA methylation levels and thrombin generation.Le potentiel de génération thrombine (TGP en anglais) est une nouvelle mesure qui permet de quantifier in vitro l'activité globale de la thrombine reflétant bien les mécanismes in vivo de la coagulation. Ce méthode de dosage est sensible aux déficits de facteurs de coagulation, à la prise d'anti-coagulants et à de nombreux troubles de la coagulation. Au moment où j'ai débuté ma thèse, seuls deux polymorphismes génétiques, tous les deux situés dans le gène F2 codant pour la prothrombine, étaient connus pour influencer la variabilité plasmatique du TGP. Mon projet de thèse avait pour objectifs d'identifier de nouveaux facteurs génétiques, mais également épigénétiques, pouvant influencer les taux plasmatiques de TGP. Dans une première partie, j'ai mené la toute première étude d'association génome-entier (GWAS pour Genome Wide Association Study en anglais) sur 3 biomarqueurs (temps de latence, quantité totale de thrombine produite et niveau maximal de thrombine produite) du TGP dans deux études françaises rassemblant 1267 sujets et j'ai répliqué les résultats les plus significatifs dans deux autres études françaises indépendantes de 1344 sujets. Cette stratégie a permis de mettre en évidence qu'un polymorphisme génétique du gène ORM1 était associé de manière robuste au temps de latence, biomarqueur caractérisant le temps nécessaire pour initier la coagulation après induction. Dans la seconde partie de ma thèse, en suivant une stratégie similaire mais cette fois-ci en étudiant non plus des polymorphismes génétiques mais des marques de méthylation d'ADN, j'ai recherché si des niveaux de méthylation de site CpG, mesurés à partir d'ADN sanguin et couvrant l'ensemble du génome, pouvaient être associés à la variabilité des 3 mêmes biomarqueurs de TGP. Malheureusement, à partir de deux échantillons mis à ma disposition et rassemblant 425 sujets, je n'ai pas pu mettre en évidence d'association robuste entre des marques de méthylation sanguine et la génération trombine

Déterminants génétiques et épigénétiques du potentiel de génération de thrombine par une approche épidémiologique

Le potentiel de génération thrombine (TGP en anglais) est une nouvelle mesure qui permet de quantifier in vitro l'activité globale de la thrombine reflétant bien les mécanismes in vivo de la coagulation. Ce méthode de dosage est sensible aux déficits de facteurs de coagulation, à la prise d'anti-coagulants et à de nombreux troubles de la coagulation. Au moment où j'ai débuté ma thèse, seuls deux polymorphismes génétiques, tous les deux situés dans le gène F2 codant pour la prothrombine, étaient connus pour influencer la variabilité plasmatique du TGP. Mon projet de thèse avait pour objectifs d'identifier de nouveaux facteurs génétiques, mais également épigénétiques, pouvant influencer les taux plasmatiques de TGP. Dans une première partie, j'ai mené la toute première étude d'association génome-entier (GWAS pour Genome Wide Association Study en anglais) sur 3 biomarqueurs (temps de latence, quantité totale de thrombine produite et niveau maximal de thrombine produite) du TGP dans deux études françaises rassemblant 1267 sujets et j'ai répliqué les résultats les plus significatifs dans deux autres études françaises indépendantes de 1344 sujets. Cette stratégie a permis de mettre en évidence qu'un polymorphisme génétique du gène ORM1 était associé de manière robuste au temps de latence, biomarqueur caractérisant le temps nécessaire pour initier la coagulation après induction. Dans la seconde partie de ma thèse, en suivant une stratégie similaire mais cette fois-ci en étudiant non plus des polymorphismes génétiques mais des marques de méthylation d'ADN, j'ai recherché si des niveaux de méthylation de site CpG, mesurés à partir d'ADN sanguin et couvrant l'ensemble du génome, pouvaient être associés à la variabilité des 3 mêmes biomarqueurs de TGP. Malheureusement, à partir de deux échantillons mis à ma disposition et rassemblant 425 sujets, je n'ai pas pu mettre en évidence d'association robuste entre des marques de méthylation sanguine et la génération trombine.Thrombin Generation Potential (TGP) is a promising in vitro measurement that allows quantifying thrombin activity, in a close way to what happens in vivo. It is sensitive to coagulation factors deficiencies, anticoagulant proteins and is associated to thrombotic disorders. There exists two polymorphisms located in the F2 (prothrombin) gene known to influence TGP levels, and altogether they explain 11.3% of the TGP inter-individual variability. With the aims of identifying novel genetic and epigenetic factors that influence TGP variability, I have performed two different studies in the present work. First, I conducted the first genome-wide association study for the three TGP biomarkers (ETP, Peak and Lagtime) using imputation data from two French studies. The most significant single nucleotide polymorphisms (SNPs) were then replicated in two independent French studies. This analysis lead to the discovery of ORM1 as a new gene participating to the control of TGP. Second, I followed a similar strategy using this time whole blood DNA methylation levels at CpG sites to identify DNA methylation marks involved in TGP variability. I analyzed the association between methylation-wide patterns from a French study and a French-Canadian families measured for TGP. Unfortunately, I did not identify robust associations between whole DNA methylation levels and thrombin generation

Close genetic relationships in vast territories: autosomal and X chromosome Alu diversity in Yakuts from Siberia.

International audienc

Genome-wide investigation of DNA methylation marks associated with FV Leiden mutation.

In order to investigate whether DNA methylation marks could contribute to the incomplete penetrance of the FV Leiden mutation, a major genetic risk factor for venous thrombosis (VT), we measured genome-wide DNA methylation levels in peripheral blood samples of 98 VT patients carrying the mutation and 251 VT patients without the mutation using the dedicated Illumina HumanMethylation450 array. The genome-wide analysis of 388,120 CpG probes identified three sites mapping to the SLC19A2 locus whose DNA methylation levels differed significantly (p<3 10-8) between carriers and non-carriers. The three sites replicated (p<2 10-7) in an independent sample of 214 individuals from five large families ascertained on VT and FV Leiden mutation among which 53 were carriers and 161 were non-carriers of the mutation. In both studies, these three CpG sites were also associated (2.33 10-11<p<3.02 10-4) with biomarkers of the Protein C pathway known to be influenced by the FV Leiden mutation. A comprehensive linkage disequilibrium (LD) analysis of the whole locus revealed that the original associations were due to LD between the FV Leiden mutation and a block of single nucleotide polymorphisms (SNP) located in SLC19A2. After adjusting for this block of SNPs, the FV Leiden mutation was no longer associated with any CpG site (p>0.05). In conclusion, our work clearly illustrates some promises and pitfalls of DNA methylation investigations on peripheral blood DNA in large epidemiological cohorts. DNA methylation levels at SLC19A2 are influenced by SNPs in LD with FV Leiden, but these DNA methylation marks do not explain the incomplete penetrance of the FV Leiden mutation

Manhattan plot of the MWAS results at 388,120 CpG sites.

<p>Manhattan plot of the MWAS results at 388,120 CpG sites.</p

Association of rs970740 with <i>SLC19A2</i> cg1658605, cg0483076 and cg09671955 levels.

<p>p-values were adjusted for age, sex, batch, chip and cell type composition.</p><p>In the F5L-families study, the rs970740 was not genotyped but substituted by the proxy rs2420371 that is in complete association (r² = 1) with it.</p><p>Association of rs970740 with <i>SLC19A2</i> cg1658605, cg0483076 and cg09671955 levels.</p

Association⁽¹⁾ of <i>SLC19A2</i> CpG sites with ACVn (MARTHA) and APCR (F5L-families) phenotypes.

(1)<p> Association is expressed as % change in phenotype (95% Confidence Interval) for every 0.1 unit increase in methylation β-value.</p>(2)<p> Analysis were adjusted for age, sex, batch, chip and cell type composition.</p><p>Association^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108087#nt105" target="_blank">(1)</a>} of <i>SLC19A2</i> CpG sites with ACVn (MARTHA) and APCR (F5L-families) phenotypes.</p

Characteristics of the studied populations.

(1)<p> In MARTHA, ACVn ratio was significantly (p = 1.63 10⁻³⁸) decreased in <i>F5</i> rs6025 carriers compared to non-carriers.</p>(2)<p> In families, APCR ratio was significantly (p = 9.98 10⁻⁴⁷) decreased in <i>F5</i> rs6205 carriers compared to non-carriers.</p><p>Characteristics of the studied populations.</p

Runs of homozygosity – association with coronary artery disease and gene expression in monocytes and macrophages

Runs of homozygosity (ROHs) are recognised signature of recessive inheritance. Contributions of ROHs to the genetic architecture of coronary artery disease and regulation of gene expression in cells relevant to atherosclerosis are not known. Our combined analysis of 24,320 individuals from 11 populations of white European ethnicity showed an association between coronary artery disease and both the count and the size of ROHs. Individuals with coronary artery disease had approximately 0.63 (95% CI: 0.4-0.8) excess of ROHs when compared to coronary artery disease-free controls (P=1.49x10-9 ). The average total length of ROHs was approximately 1046.92 (95% CI: 634.4-1459.5) kb greater in individuals with coronary artery disease than controls (P=6.61x10-7 ). None of the identified individual ROHs was associated with coronary artery disease after correction for multiple testing. However, in aggregate burden analysis, ROHs favouring increased risk of coronary artery disease were much more common than those showing the opposite direction of association with coronary artery disease (P=2.69x10-33). Individual ROHs showed significant associations with monocyte and macrophage expression of genes in their close proximity – subjects with several individual ROHs showed significant differences in the expression of 44 mRNAs in monocytes and 17 mRNAs in macrophages when compared to subjects without those ROHs. This study provides evidence for an excess of homozygosity in coronary artery disease in outbred populations and suggest the potential biological relevance of ROHs in cells of importance to the pathogenesis of atherosclerosis