Arterial variants are an important consideration in the care of individuals undergoing surgery or invasive diagnostic procedures. Although there are numerous reports that describe arterial variants in localized body regions, it is unknown if some individuals have a proclivity for the development of arterial variants. The current study set out to determine if some individuals do, in fact, have a proclivity for vascular variants and further, if one arterial variant can predict the presence of another variant. To accomplish this, whole-body dissection was performed on 18 anatomical body donors. In each donor, the entire arterial tree was followed and documented. The findings from this study suggest that arterial variants are extremely common and the presence of numerous variants is frequent.

Les études d’association sur génome entier ont permis d’identifier de nombreux facteurs de risque génétiques impliqués dans des maladies complexes. Il apparaît cependant que les variants fréquents n’expliquent qu’une faible partie de l’héritabilité des maladies. Une partie non négligeable serait due à la présence de variants rares avec des effets génétiques plus forts. Tester l’association de ces variants est problématique du fait de leur faible fréquence dans la population générale. De nombreuses méthodes statistiques ont été développées avec la stratégie commune d’agréger l’information pour un groupe de variants. Cette thèse a pour objectif de comparer les principales stratégies à l’aide de simulations de différents scénarios génétiques et de l’application à de vraies données de séquençage. Nous avons aussi développé un test, appelé DoEstRare, comparant les distributions des positions des variants rares entre les cas et les témoins, afin de détecter des regroupements de variants dans des régions locales. Enfin, il a été montré qu’une structure de population est un facteur de confusion pour l’interprétation des résultats d’analyse de variants rares. Avec le recrutement de témoins pour les analyses, avec des projets tels que French Exome et VACARME, il est alors nécessaire de comprendre l’impact d’une structure à fine échelle géographique (e.g. échelle de la France) pour les différentes stratégies statistiques. La seconde partie de cette thèse consiste à évaluer cet impact au moyen de simulations de données génétiques pour des structures géographiques locales. / Genome-wide association studies have identified many common risk alleles for a wide variety of complex diseases. However these common variants explain a very small part of the heritability. A hypothesis is the presence of rare genetic variants with stronger effects. Testing the association of those rare variants is challenging due to their low frequency in populations. Many statistical methods have been developed with the strategy to aggregate the information for a group a rare variants. This thesis aims to compare the main strategies through simulating under various genetic scenarios and the application to real sequencing data. We also developed a statistical test, called DoEstRare, which can detect clustered disease-risk variants in local genetic regions, by comparing the position distributions between cases and controls. Moreover, it has been shown that population stratification represents a confounding factor in the analysis interpretations for rare variants. With the recruitment of controls, in the context of projects such as French Exome and VACARME, it is necessary to assess the impact of a very fine geographical structure (France) for different statistical strategies. The second part of this thesis consists in estimating this impact by simulating fine-scale population structures.

Le variant d'histone CENP-A est le facteur responsable de la détermination épigéné- tique du centromère. Il permet le recrutement de nombreuses protéines centromériques, et constitue ainsi la brique fondatrice du kinétochore. Il possède un domaine amino-terminal non structuré dont la fonction précise reste encore à élucider, bien qu'il soit déjà établi chez certaines espèces que ce domaine est requis pour le bon fonctionnement du cen- tromère et conséquemment le bon déroulement de la mitose. Nous avons construit des lignées cellulaires humaines exprimant stablement diverses formes mutantes de CENP-A, qui nous ont permis de réaliser des expériences de pseudogénétique en supprimant l'ex- pression de la protéine CENP-A endogène. Nous observons une augmentation drastique du taux de défauts de ségrégation des chromosomes et de cellules plurinucléées dans des cellules exprimant uniquement le domaine globulaire de CENP-A, ce qui est en accord avec les données de la littérature et confirme l'importance du domaine amino-terminal. Un phénotype similaire est observé dans des cellules exprimant une protéine CENP-A entière mais dont le domaine amino-terminal n'est pas phosphorylable. Nos résultats montrent l'implication de la phosphorylation de la sérine de CENP-A dans le bon déroulement de la mitose, et suggèrent que la fonction mitotique du domaine amino-terminal est centrée sur cette seule phosphorylation. / The histone variant CENP-A is the epigenetic factor responsible for centromere deter- mination. It allows the recruitment of a handful of centromeric proteins, and thus acts as the primary foundation for the kinetochore. It comprises an unstructured amino-terminal domain to which no precise function has yet been assigned, although it is established in some species that the mere presence of that domain is required for proper centromere func- tion and thus successful completion of mitosis. We have established several human cell lines stably expressing GFP-tagged CENP-A constructs, allowing us to perform pseudoge- netic experiments by siRNA-mediated silencing of the endogenous CENP-A. Our results show a dramatic increase of mitotic defects and plurinuclear cells when cells express only the globular domain of CENP-A; this is in accordance with the litterature and confirms the importance of the amino-terminal tail. More importantly, a similar increase of mitotic defects is observed when cells express a full-length, but non-phosphatable, CENP-A. Our results show the involvement of the phosphatable serine 7 of CENP-A in the successful completion of mitosis, and may suggest that the role of the whole amino-terminal tail of CENP-A could be reduced to this single phosphorylation event.

This dissertation aims at investigating the association between genotypes and phenotypes in human. Both common and rare regulatory variants have been studied. The phenotypes include disease risk, clinical traits and gene expression levels. This dissertation describes three different types of association study. The first study investigated the relationship between common variants and three sub-clinical traits as well as three complex diseases in the Center for Health Discovery and Well Being study (CHDWB). The second study is GWAS analysis of TNF-α and BMI/CRP conducted as a contribution to meta-GWAS analyses of these traits with investigators at the University of Groningen in the Netherlands, and the 1000 Genomes Consortium. The third study was the most original contribution of my thesis as it assessed the association between rare regulatory variants in promoter regions and gene expression levels. The results clearly show an enrichment of rare variants at both extremes of gene expression. This dissertation provides insight into how common and rare variants associate with broadly-defined quantitative phenotypes. The demonstration that rare regulatory variants make a substantial contribution to gene expression variation has important implications for personalized medicine as it implies that de novo and other rare alleles need to be considered as candidate effectors of rare disease risk.

Separate domestication events and strong selective pressures have created diverse phenotypes among existing cattle populations; however, the genetic determinants underlying most phenotypes are currently unknown. Bos taurus taurus (Bos taurus) and Bos taurus indicus (Bos indicus) cattle are subspecies of domesticated cattle that are characterized by unique morphological and metabolic traits. Because of their divergence, they are ideal model systems to understand the genetic basis of phenotypic variation. Here, we developed DNA and structural variant maps of cattle genomes representing the Bos taurus and Bos indicus breeds. Using this data, we identified genes under selection and biological processes enriched with functional coding variants between the two subspecies. Furthermore, we examined genetic variation at functional non-coding regions, which were identified through epigenetic profiling of indicative histone- and DNA-methylation modifications. Copy number variants, which were frequently not imputed by flanking or tagged SNPs, represented the largest source of genetic divergence between the subspecies, with almost half of the variants present at coding regions. We identified a number of divergent genes and biological processes between Bos taurus and Bos indicus cattle; however, the extent of functional coding variation was relatively small compared to that of functional non-coding variation. Collectively, our findings suggest that copy number and functional non-coding variants may play an important role in regulating phenotypic variation among cattle breeds and subspecies.

Copy number Variants (CNVs), which comprise deletions, insertions and inversions of genomic sequence, are a main form of genetic variation between individual genomes. CNVs are commonly present in the genomes of human and other species. However, they have not been extensively characterized as their ascertainment is challenging. I reviewed current CNV studies and CNV discovery methods, especially the algorithms which infer CNVs from whole genome Single Nucleotide Polymorphism (SNP) arrays and compared the performance of three analytical tools in order to identify the best method of CNV identification. Then I applied this method to identify CNV events in three European population isolates—the island of Vis in Croatia, the islands of Orkney in Scotland and villages in the South Tyrol in Italy - from Illumina genome-wide array data with more than 300,000 SNPs. I analyzed and compared CNV features across these three populations, including CNV frequencies, genome distribution, gene content, segmental duplication overlap and GC content. With the pedigree information for each population, I investigated the inheritance and segregation of CNVs in families. I also looked at association between CNVs and quantitative traits measured in the study samples. CNVs were widely found in study samples and reference genomes. Discrepancies were found between sets of CNVs called by different analytical tools. I detected 4016 CNVs in 1964 individuals, out of a total of 2789 participants from the three population isolates, which clustered into 743 copy number variable regions (CNVRs). Features of these CVNRs, including frequency and distribution, were compared and were shown to differ significantly between the Orcadian, South Tyrolean and Dalmatian population samples. Consistent with the inference that this indicated population-specific CNVR identity and origin, it was also demonstrated that CNV variation within each population can be used to measure genetic relatedness. Finally, I discovered that individuals who had extreme values of some metabolic traits possessed rare CNVs which overlapped with known genes more often than in individuals with moderate trait values.

For every 200 births in the UK, one will end in a stillbirth. Stillbirth is classified as a baby born dead after 24 weeks gestation. Mutations in genes that cause ion channelopathies are known to cause sudden cardiac death in adults and children. Prenatal diagnosis of LQT has been possible for decades, creating a disease spectrum where channelopathies may fatally influence pregnancy. We sequenced 35 candidate genes in 70 unexplained stillbirth cases. Thirty-nine cases harboured a predicted damaging protein missense variant. Two novel and two rare variants were observed in the transient receptor potential melastatin 7 (TRPM7) gene and five rare genetic variants were found in A-kinase anchor protein 9 (AKAP9). The aim of this PhD was to perform functional studies of these variants in TRPM7 and AKAP9. TRPM7 is an ion channel indispensable for mouse cardiogenesis. Two TRPM7 variants (p.G179V and p.T860M) showed significantly reduced current compared to wild-type channels. Conversely, cells expressing p.R494Q TRPM7, had a significant increase in current compared to WT channels, but only in CHO-K1 cells. Western blot analyses failed to detect full length TRPM7 in cells transfected with either p.G179V or p.T860M compared to wild-type expressing cells. Proteosomal inhibition using MG132 produced a small but visible band in p.T860M transfected cells. Expression of TRPM7 in iPSC-derived cardiomyocytes increases during cell maturation, and TRPM7-like current was measured in 20-23 day old cardiomyocytes. AKAP9 is required to couple adrenergic stimulation in the heart with faster cardiac repolarisation. Cells expressing WT AKAP9 alongside the KCNQ1/KCNE1 potassium channel responded to β-adrenergic stimulation, however those transfected with p.A3043T AKAP9 did not respond to treatment with forskolin. Our analyses supports two deleterious variants in TRPM7 and one in AKAP9 in unexplained stillbirth cases. These heterozygous variants could lead to haploinsufficiency and may be a cause of stillbirth.

Genome-wide association studies have been used extensively to study hundreds of phenotypes and have determined thousands of associated SNPs whose underlying biology and causation is as yet largely unknown. Many previous studies attempted to clarify the causal biology by investigating overlaps of trait-associated variants with functional annotations, but lacked statistical rigor and examined incomplete subsets of available functional annotations. Additionally, it has been difficult to disentangle the relative contributions of different annotations that may show strong correlations with one another. In this thesis, we address these shortcomings and strengthen and extend the obtained results. Two methods, permutations and logistic regression, are applied in statistically rigorous analyses of genomic annotations and their observed enrichment or depletion of trait-associated SNPs. The genomic annotations range from genic regions and regulatory features to measures of conservation and aspects of chromatin structure. Logistic regressions in a number of trait-specific subsets identify genomic annotations influencing SNPs associated with both normal variation (e.g., eye or hair colour) and diseases, suggesting some generalities in the biological underpinnings of phenotypes. SNPs associated with phenotypes of the immune system are investigated and the results highlight the distinct aetiology for this subset. Despite the heterogeneity of the studied cancers, SNPs associated to different cancers are particularly enriched for conserved regions, unlike all other trait-subsets. Nonetheless, chromatin states are, perhaps surprisingly, among the most influential genomic annotations in all trait-subsets. Evolutionary conserved regions are rarely within the top genomic annotations despite their widespread use in prioritisation methods for follow-up studies. We identify a common set of enriched or depleted genomic annotations that significantly influence all traits, but also highlight trait-­‐specific differences. These annotations may be used for the computational prioritisation of variants implicated in phenotypes of interest. The approaches developed for this thesis are further applied to studies of a specific human complex trait (height) and gene expression in atherosclerosis.

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