Dissertations / Theses on the topic 'Risk variants'

This thesis includes work on the genetics of intestinal inflammatory disorders, concentrating on coeliac disease and Crohn’s disease. It explores how common genetic variants influence risk of complex phenotypes including immunological intolerance to gluten (coeliac disease) and intolerance to therapeutic agents (azathioprine and mercaptopurine) used in the treatment of intestinal inflammatory diseases. Finally it presents work aiming to move from genetic associations with complex phenotypes to understanding of how these variants modulate immunological processes. Results of a large genome wide association study that identified more than 13 new genetic risk regions influencing susceptibility to coeliac disease are presented. Results of a genome wide association study of azathioprine and 6-mercaptopurine-induced pancreatitis in inflammatory bowel disease-affected individuals are presented. Finally, a cell cytokine release assay for the prostaglandin EP4 receptor was developed, with a view to investigating how SNPs associated with Crohn’s disease in the 5p13.1 region influence EP4 receptor signalling and contribute to disease pathogenesis. This work highlights some of the challenges in moving from SNP-disease associations identified in GWASs to understanding how genetic variants change biological processes.

Corneal transplantation is the oldest, most common and usually the most successful type of solid tissue allograft. The acceptancc of corneal allografts compared to other categories of allografts is called immune privilege. However, some conditions rob the corneal allograft of its immune privilege and promote rejection, which remains the leading cause of corneal graft failure. The precise immune mechanism underlying graft failure is incompletely understood. While differences in human leukocyte antigen (HLA) molecules between donor and host contribute to the alloreactivity driving the donor-antihost response, the cytokine milieu consisting of molecules that promote and regulate the alloresponse after transplantation is also critical. Single nucleotide polymorph isms (SNPs) in the promoter and coding regions of cytokine genes are associated with differential levels of expression and therefore play an important role in transplantation immunology. Cytokines are integral components of an inflammatory response and there are several potential sources of cytokine release within the cornea and the anterior chamber. This project is a candidate gene association study, focusing on genes known to be involved in ocular immune privilege and the compromise thereof, in corneal transplant recipients at increased risk of rejection (i.e., 'high risk corneal transplantation). In view of their central roles in initiation and suppression of the inflammatory response, tumour necrosis factor alpha (Tufa), interleukin - IO (IL• I 0), interleukin- 17 ( IL-1 7), thrombospondin•1 (TSP-I), vascular endothelial growth factor-A (VEG F-A) and the glucocorticoid cortisol were investigated. All transplants had three-year follow-up and results were analysed by PHASE (maximum-likelihood) analysis is to determine haplotype frequencies. Significant association between two extended TNF-u haplotypes and corneal graft outcome was found: TCTGGA was associated with a decreased risk of cornea] graft failure (n=384, RR 0.04, Cl 002-0.671, P <0.05, Pc <0.05) and TCTAGA was associated with increased risk of failure (n=384, RH. 3.59, Cl 3.21-4.03, P <0.05, Pc <0.05). In addition, a significant association was observed between the 3-1ocus TSP-] haplotype ACA, and an increased risk of corn ca I graft failure (n==359, OR 2.27, 95% Cl 1.65-3.13, P<0.05, P, <0.05).

The main focus of my PhD has been the analysis of rare and common variants in genetic susceptibility to two complex traits: nicotine dependence (ND) and cluster headache (CH). Firstly, we conducted a genetic study to investigate the role of genetic variation at CHRNA7 and CHRFAM7A in smoking phenotypes, and to test the hypothesis that 7nAChR variation may modulate the efficacy of varenicline in smoking cessation. The study was performed on 408 regular tobacco smokers, recruited at smoking cessation centers, including 142 individuals treated with varenicline. We determined the CHRNA7 and CHRFAM7A copy number, the rs67158670 genotypes in CHRFAM7A and we resequenced the CHRNA7 proximal promoter. Our results point to a role for CHRNA7 promoter variants in tobacco addiction mechanisms; moreover, our study provides the first evidence that CHRFAM7A copy number variation could affect the response to varenicline treatment. Secondly, In order to investigate the genetic background of CH we performed a genome-wide association analysis on 99 Italian CH patients and 359 controls, using the Infinium PsychArray (Illumina). SNPs genotyping data were used to conduct genome-wide single marker case-control association analysis using common SNPs. Even if no genome-wide significant loci were reported, this analysis led to the identification of a suggestive association with a common variant of the PACAP receptor gene (ADCYAP1R1). Furthermore, we performed a gene-based association analysis considering rare protein altering variants in 745 candidate genes with a possible role in CH. This analysis provided evidence of association for a rare potentially damaging missense variant in the MME gene. ADCYAP1R1 and MME both represent very interesting candidate genes for CH, as their gene products are known to have an important function in pain mechanisms; thus, our study provides the first evidence that genetic variation in genes related to pain processing might have a role in CH susceptibility.

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.

Background: Circulating lipids levels, as well as several familial lipid metabolism disorders, are strongly associated with initiation and progression of atherosclerosis and incidence of myocardial infarction (MI). Objectives: We hypothesized that genetic variants associated with circulating lipid levels would also be associated with MI incidence, and have tested this in three independent samples. Setting and Subjects: Using age- and sex-adjusted additive genetic models, we analyzed 554 single nucleotide polymorphisms (SNPs) in 41 candidate gene regions proposed to be involved in lipid-related pathways potentially predisposing to incidence of MI in 2,602 participants of the Swedish Twin Register (STR; 57% women). All associations with nominal P&lt;0.01 were further investigated in the Uppsala Longitudinal Study of Adult Men (ULSAM; N = 1,142). Results: In the present study, we report associations of lipid-related SNPs with incident MI in two community-based longitudinal studies with in silico replication in a meta-analysis of genome-wide association studies. Overall, there were 9 SNPs in STR with nominal P-value &lt;0.01 that were successfully genotyped in ULSAM. rs4149313 located in ABCA1 was associated with MI incidence in both longitudinal study samples with nominal significance (hazard ratio, 1.36 and 1.40; P-value, 0.004 and 0.015 in STR and ULSAM, respectively). In silico replication supported the association of rs4149313 with coronary artery disease in an independent meta-analysis including 173,975 individuals of European descent from the CARDIoGRAMplusC4D consortium (odds ratio, 1.03; P-value, 0.048). Conclusions: rs4149313 is one of the few amino acid changing variants in ABCA1 known to associate with reduced cholesterol efflux. Our results are suggestive of a weak association between this variant and the development of atherosclerosis and MI.

Uterine leiomyomata (UL), commonly known as fibroids, are a major public health problem given their extreme prevalence (>70%), severity of associated symptoms, and indication for hysterectomies in women of reproductive age. Familial aggregation and twin studies have provided evidence for a genetic component to predisposition to develop UL. To date, a small number of genes involved in UL biology, including HMGA2, have been discovered through cytogenetic studies of the tumors. HMGA2 is involved in recurrent translocations in UL and a TC repeat polymorphism in the gene is associated with UL diagnosis. In this thesis, I investigate the possible role of the TC repeat in HMGA2 expression. In 293T cells, the TC repeat number did not affect promoter activation, however, in the more relevant UL and myometrial cells, HMGA2 promoter activation was severely impaired and a definitive conclusion could not be made. Genome-wide linkage and association studies provide a promising, unbiased approach for revealing additional regions of the genome associated with UL. In this thesis, I describe results from the first genome-wide linkage and association studies performed in white women affected with UL. A genome-wide linkage study of affected white sister pairs revealed two significant linkage peaks in 10p11 (LOD=4.15) and 3p21 (LOD=3.73) with five suggestive peaks (LOD>2.00) in 2q37, 5p13, 11p15, 12q14, and 17q25. A meta-analysis of genome-wide association results in two independent cohorts of white women revealed a single nucleotide polymorphism (SNP) with genome-wide significance that is associated with UL diagnosis (rs4247357, P=3.05E-08, odds ratio (OR) =1.299). The candidate SNP is located under the UL linkage peak at 17q25 and is in a large block of linkage disequilibrium (LD) which spans three genes: fatty acid synthase (FASN), coiled-coil domain containing 57 (CCDC57) and solute carrier family 16, member 3 (SLC16A3). FAS transcripts and/or protein levels are up-regulated in various neoplasms and have been implicated in tumor cell survival. By tissue microarray immunohistochemistry, we found FAS protein expression elevated three-fold in UL when compared to matched myometrial tissue implicating FASN as the first UL risk allele identified in white women by a genome-wide, unbiased approach.

The main aim of this thesis was to uncover common low penetrance variants that influence susceptibility to colorectal cancer (CRC). This was largely considered in relation to the analysis of the plethora of genetic data from our large genome‐wide association study. My work includes fine‐mapping of associated loci through additional genotyping, gene screening, and imputation for the prediction of untyped SNPs, which improved the resolution for fine-mapping and facilitated meta-analysis with datasets typed on different arrays. This led to the identification of 14 independent risk loci, while an association analysis of the X chromosome revealed evidence for two additional risk variants. I cover the detection of runs of homozygous SNPs to investigate the relationship between homozygosity and CRC and show that there is no evidence for increased homozygosity in cases in the UK population. I go on to investigate linkage based techniques to perform an analysis of chromosomal regions identical by descent (IBD), which are shared between unrelated cases more often than controls that could harbour risk variants and identified a number of good candidate genes, such as AXIN2 and E2F7, which require further analysis in additional samples. I also search for moderate penetrance susceptibility variants in several families with a dominant‐like inheritance and compare identified linkage peaks with the results of a loss of heterozygosity analysis of tumour DNA from family members to identify potential tumour suppressor genes. This analysis identified several promising regions and led to the detection of a SMAD4 mutation in one family. The associated variants identified in this study provide good evidence that the common‐disease common-variant hypothesis holds true, but that this is not the whole story as these variants account for just 8% of the familial risk. Further research and techniques will be required to uncover the remaining missing heritability.

Le cancer du sein est la néoplasie la plus fréquente chez la femme. Un ensemble de facteurs génétiques et environnementaux sont impliqués dans cette maladie complexe. Dans le cadre de mes études doctorales, je me suis intéressée à la composante génétique associée au risque de cancer du sein chez les femmes dans la population générale ainsi qu’à la modification du risque pour ce cancer chez des porteuses de mutations des gènes BRCA1 et BRCA2. Actuellement, environ la moitié de cette composante génétique est expliquée par une combinaison d'allèles à pénétrance faible, moyenne ou élevée. En outre, de récentes études ont démontré l'implication majeure de certains facteurs génétiques dans la modification du risque de cancer du sein chez des porteuses de mutations de BRCA1 et BRCA2. Dans le cadre de ce projet, nous avons étudié l’impact potentiel de certains variants génétiques dans les régions régulatrices de différents gènes et évalué leurs associations avec le risque de cancer du sein. Le projet a été divisé en deux parties : tout d'abord, nous avons évalué l'association directe entre les variants associés avec l’expression allélique différentielle et le risque de cancer du sein, afin d'identifier de nouveaux locus de susceptibilité à ce cancer. En second lieu, nous avons évalué l'impact sur l’expression génique de variants caractérisés au sein des régions promotrices de certains gènes sélectionnés, pour ensuite évaluer l’impact de ces variants sur l’expression génique. En résumé, la première partie de ce projet a conduit à l'identification d'un nouveau locus de faible pénétrance associés au risque de cancer du sein sur le locus 4q21 (rs11099601, odds ratio=1.05, p = 6.4 x 10-6), et deux nouveaux locus (11q22.3 et BRCA1- rs16942) associés avec la modification du risque de cancer du sein chez les porteuses de mutations du gène BRCA1. La seconde partie du projet a permis l'identification de nouveaux variants fonctionnels situés dans les régions promotrices des gènes ESR1, ESR2, FOXA1, RAP80, NBN et CDC7. D’autres études d’association dans de plus large cohorte ainsi que d’autres analyses fonctionnelles seront nécessaires pour confimer ces résultats, ce qui permettra de les inclure dans les nouveaux outils de prédiction de risque et ainsi assurer une estimation plus précise du risque de cancer du sein.<br>Breast cancer is the most common malignancy in women. A set of environmental and genetic factors are involved in this complex disease. This project focused on the genetic components of breast cancer susceptibility and breast cancer risk modification in BRCA1 and BRCA2 mutation carriers. Currently, about half of the inherited susceptibility to breast cancer can be imputed to a combination of high-, intermediate-, and low-risk alleles. Thus, many as yet unknown susceptibility loci remain to be identified. Moreover, recent studies have provided evidence for the involvement of genetic risk factors that might considerably modify the risk of developing breast cancer in BRCA1 and BRCA2 mutation carriers. Furthermore, genome-wide association studies have shown that several genetic variants within non-coding gene regions are associated with breast cancer risk. In this project, we focused on regulatory gene variants and their association with breast cancer risk. The project was divided in two parts. In the first section, we evaluated the direct association between single-nucleotide polymorphisms associated with differential allelic expression and breast cancer risk in order to identify new loci of breast cancer susceptibility. In the second part, we evaluated the functional impact on gene expression of variants identified within the promoter regions of selected candidate genes and then, characterize the functional impact of these variants. In summary, the first part of this project has led to the identification of a new low-penetrance locus associated with breast cancer risk on the 4q21 locus (rs11099601; odds ratio=1.05, p= 6.4 x 10-6), and two new modifiers of breast cancer risk in BRCA1 mutations carriers (11q22.3 locus and the wild type allele of BRCA1). The second part of the project allowed us to describe new functional variants within the promoters of the selected breast cancer gene candidates. Other association studies in larger cohorts and further functional analysis will be required to confirm these results, which will allow their inclusion in breast cancer risk prediction tools and thus ensure a more accurate estimation of breast cancer risk.

Congenital heart disease (CHD) is the most common congenital malformation with a birth prevalence of 7/1000. CHD may occur as Mendelian syndromic disorders or as isolated conditions. The latter represent the majority (~80%) of CHD cases. Recent technological advancements have allowed large-scale genome-wide characterization of copy number variants (CNVs), which have been proposed to contribute to the risk of sporadic CHD. This thesis presents a genome-wide CNV study involving 2256 sporadic, isolated CHD patients, 283 trio CHD families, and 1538 ancestry-matched controls that were typed on the Illumina 660W-Q SNP platform. This was followed by an extensive validation study using comparative genomic hybridization arrays, multiplex ligation-dependent probe amplification and quantitative-fluorescent PCR assays. A global enrichment of rare genic deletions was identified in CHD patients (OR = 1.8, P = 0.001), compared to controls. Rare deletions that are associated with CHD had higher gene content (P = 0.001) and higher haploinsufficiency scores (P = 0.03). Additionally, they were enriched with genes involved in the Wnt signalling pathway, known for its pivotal role in cardiac morphogenesis. Rare de novo CNVs were also identified in ~5% CHD trios; 91% of which occurred on the paternal, as opposed to the maternal chromosome (P = 0.01). They spanned previously known candidate loci as well as novel loci for CHD. Individual locus enrichments in cases vs. controls were identified for CNVs at chromosomes 1q21.1 and 15q11.2. A phenotype-specific effect was observed for the 1q21.1 CNVs, and GJA5 was identified as the causative gene for CHD in this locus. In conclusion, global rare genic deletions contribute ~4% of the population attributable risk of sporadic CHD. CNVs implicating 1q21.1, 15q11.2 and Wnt signalling genes are associated with CHD. Rare de novo CNVs identified in CHD trios exhibit a paternal origin bias possibly of relevance to the epidemiology of CHD.

The aim of this thesis is analysis and resolution of the decision-making problem "selection of merchandising and gifts supplier in an international company" using multi-criteria decision process while respecting risk. The goal of this thesis is to select a suitable supplier and offer recommendation to company's management for solving similar decision processes. The theoretical part of the thesis describes the knowledge base of methods and procedures of decision analysis that are later applied in the practical part of the thesis on the specific project with aim to optimize the decision-making process and identify the best variant. The conclusion of the thesis provides recommendations for solving similar problem in the future.

Polycyclic aromatic hydrocarbons (PAHs) are created by the incomplete combustion of fossil fuels, and are established human carcinogens. However, the effect of occupational PAH exposure on breast cancer is not well established. In addition, it is not known if genes involved in metabolizing xenobiotic compounds modify the risk of breast cancer in women exposed to PAHs. The objectives of this study were to (1) estimate the association between PAH exposure and breast cancer, (2) examine how variants of select xenobiotic metabolizing genes influence breast cancer risk, and (3) assess how these variants – several of which are involved in PAH metabolism – interact with PAH exposure to modify breast cancer risk. The relationships between PAH exposure, genetic susceptibility, and breast cancer were examined in a population-based case control study conducted in Vancouver, BC and Kingston, Ontario. A detailed questionnaire, including occupational history, and biological sample were collected from participants. Chapter 2 details how I developed a statistical model that predicts the probability of exceeding the permissible exposure limit for PAH through industry and occupation using workplace compliance testing data collected in the United States. Chapter 3 describes the use of the model to develop a job exposure matrix and estimate the association between PAH exposure and breast cancer. In Chapter 4, I assessed the associations between select gene variants and breast cancer, and evaluated whether there is evidence that those variants modify PAH exposure effect on breast cancer risk. Long term exposure to PAHs was identified as a risk factor for breast cancer, and risk was highest among premenopausal women and women with a first degree family history of breast cancer. Six variants in xenobiotic metabolizing genes were observed to be related to breast cancer risk, three of which are directly involved in PAH metabolism. In addition, there is evidence to support the notion that three of these variants modify the effect of PAH exposure, implicating the role gene-environment interactions have on modifying breast cancer risk. Evidence from this research points to the potential importance of monitoring and limiting occupational exposures to PAHs in order to reduce breast cancer risk in women.<br>Medicine, Faculty of<br>Population and Public Health (SPPH), School of<br>Graduate

Background: Genome-wide association studies have identified variants within the FTO (fat mass and obesity associated) locus as the strongest predictors of obesity amongst all obesity-associated gene loci. Recent evidence suggests that variants in FTO directly affect human adipocyte function through targeting IRX3 and IRX5 and thermogenesis regulation. Aim: We addressed the relevance of this proposed FTO-IRX pathway in adipose tissue (AT) of children. Results: Expression of IRX3 was higher in adipocytes compared to SVF. We found increased adipocyte-specific expression of IRX3 and IRX5 with the presence of the FTO risk haplotype in lean children, whereas it was unaffected by risk variants in obese peers. We further show that IRX3 expression was elevated in isolated adipocytes and AT of lean compared to obese children, particularly in UCP1-negative adipocytes, and inversely correlated with BMI SDS. Independent of BMI, IRX3 expression in adipocytes was significantly related to adipocyte hypertrophy, and subsequent associations with AT inflammation and HOMA-IR in the children. Conclusion: One interpretation of our observation of FTO risk variants linked to IRX3 expression and adipocyte size restricted to lean children, along with the decreased IRX3 expression in obese compared to lean peers, may reflect a defense mechanism for protecting body-weight, which is pertinent for lean children.

Combinations of common germline low-moderate susceptibility alleles may be responsible for some of the 90% of ovarian cancer (OC) cases not explained by known risk genes. These alleles may also affect survival of OC patients. The effects of 34 tagging single nucleotide polymorphisms (tSNPs) from candidate oncogenes (BRAF, ERBB2, KRAS, NMI and PIK3CA) and 63 tSNPs from “functionally” relevant genes (AIFM2, AKTIP, AXIN2, CASP5, FILIP1L, RBBP8, RGC32, RUVBL1 and STAG3) on the risk and survival of OC sufferers were evaluated with ~1,800 cases and 3,045 controls. Associations were found between disease risk and NMI rs11683487 (P-dominant=0.004) and RUVBL1 rs13063604 (P-trend=0.0192). These associations were not independently validated with additional samples, however, they remained significant when the results from both stages of genotyping were combined (P<0.05). Global tests of association with OC risk were significant for BRAF, ERBB2, CASP5 and RUVBL1 (P-global<0.05). However, there was no evidence of an excess of significant associations from 340 SNPs investigated with the admixture maximum likelihood test (P-trend=0.068). BRAF, FILIP1L, KRAS, RBBP8 and RUVBL1 were also associated with the survival of all OC cases (P<0.05). When analysis was restricted to the 4 main histological subtypes of OC, additional associations were identified. Although these results are of particular interest, they were based on relatively small numbers of samples and have not been corrected for multiple testing, therefore they should be treated with caution. The results from the secondary objective of the project, to evaluate whole genome amplification (WGA) of DNA and SNP multiplex platforms, are also described. To conclude, associations were identified between candidate oncogenes and functionally relevant genes on the survival and susceptibility of ovarian cancer. The performance of WGA DNA on SNP multiplex genotyping platforms highlighted the importance of comparing WGA DNA with corresponding gDNA in order to ascertain quality of genotyping on the platform.

Background Ageing is a risk factor that promotes common cardiovascular diseases such as atrial fibrillation (AF) or heart failure (HF), which in turn are associated with pathological changes in intracellular calcium homeostasis. However, the effects that ageing could have on the calcium homeostasis in human atrial cardiomyocytes are not well known. Furthermore, genetic risk variants at single nucleotide polymorphisms (SNPs) associated with a higher incidence of AF have been identified in the chromosomal region 4q25, close to the locus of the Pitx2 transcription factor that plays an important role in cardiac embryonic development. In the adult heart AF has been associated with changes in the expression of Pitx2, but findings are contradictory and the relationship between the 4q25 risk variants and Pitx2 function remain controversial. Moreover, no functional effects of the 4q25 risk variants on the calcium homeostasis have been identified so far. Therefore, in this thesis we investigated how the two risk factors, ageing and risk variants at 4q25, affect the intracellular calcium homeostasis with the intention of identifying mechanisms that underlie potentially arrhythmogenic changes in the calcium homeostasis caused by these risk factors. Hypothesis Ageing and 4q25 risk variants produce alterations in the intracellular calcium homeostasis in atrial myocytes that alone or in combination contribute to increase the propensity to atrial fibrillation. Aims of the thesis * Analyze the effects of ageing on the mechanisms that regulate the calcium homeostasis in human atrial myocytes. * Use transgenic murine models of ageing to identify molecular mechanisms underlying ageing-dependent changes in the calcium homeostasis. * Investigate how risk variants on chromosome 4q25 associated with increased AF risk, affect electrophysiological characteristics of human atrial myocytes and to identify underlying molecular mechanisms. * Investigate how ageing modulates the effects of 4q25 risk variants in human atrial myocytes. Methods Experiments were performed on isolated atrial or ventricular cells from human or murine models. Electrophysiological data were obtained using patch- clamp techniques and confocal microscopy. RT-PCR and western blot techniques were used to determine the expression levels of mRNA and the proteins studied. Results The results described in this thesis show that aging decreases the amplitude of the calcium current (ICa), an electrophysiological parameter that is also reduced by AF, the calcium content of the sarcoplasmic reticulum (SR), and the global calcium transient. These findings are corroborated by comparable changes in the expression of the proteins undertaking the corresponding calcium transport or buffering. Together, these changes likely reduce atrial contraction in the elderly. The results were reproduced in an animal model of premature aging (Zmpste24-/-) with defective lamin processing, reinforcing the notion that this mechanism may, at least partially, underlie the observed effects of aging on the calcium homeostasis in human atrial myocytes. In addition, ageing accentuated some of the effects of ageing on calcium handling in patients with a previous history of AF. Of particular interest, the ICa amplitude was further depressed by aging in patients with AF, which might contribute to maintain atrial arrhythmic episodes in these patients. The study of risk variants on chromosome 4q25 shows that the presence of the rs13143308T risk variant, alone or together with the risk variant rs2200733T, was associated with a higher frequency of spontaneous calcium release, transient inward currents (ITI) and membrane depolarizations, typical of AF. These results are the first to provide an electrophysiological mechanism that could explain a higher incidence of AF in individuals carrying risk variants at 4q25. Moreover, electrophysiological studies in right atrial myocytes from a mouse model with atrial Pitx2 insufficiency (NppaCre+Pitx2fl/-) reproduces all alterations in calcium homeostasis observed in patients with 4q25 risk variants. These results support the notion that modulation of the intracellular calcium handling by Pitx2 plays an important role in electrophysiological processes associated with AF. Analysis of potential synergies between risk variants at 4q25, ageing, and AF revealed that several effects of ageing were accentuated in patients with AF whereas aging per se does not modify the effects of the 4q25 risk variants on calcium homeostasis. However, as aging reduces the amplitude of ICa, which would reduce the atrial refractory period, this could prolong the duration of atrial arrhythmic episodes favored by the higher frequency and amplitude of spontaneous membrane depolarizations in carriers of 4q25 risk variants. Conclusions Ageing modulates calcium homeostasis in human atrial myocytes by decreasing ICa, calcium transient and SR calcium load. These changes are reproduced in a progeric mouse model, favoring a progressive decline of contractile function with age. Moreover, the observed ICa reduction is a characteristic feature of AF that may favor its maintenance in elder patients. Risk variants located on the chromosomal region 4q25, specifically the variant rs13143308T, alone or together with rs2200733T, increases spontaneous calcium release, ITIs, and membrane depolarizations. These changes are reproduced in a mouse model of Pitx2 insufficiency, and are all hallmarks of AF that could favor the initiation of arrhythmic events in carriers of the risk variants. The combined effects of 4q25 risk variants and ageing may work synergistically to promote atrial arrhythmia, with the former constituting an arrhythmogenic electrophysiological substrate that favor initiation of atrial arrhythmic episodes, and with ageing favoring their maintenance.<br>Antecedentes El envejecimiento es un factor de riesgo que favorece enfermedades cardiovasculares comunes como la fibrilación auricular (FA) o la insuficiencia cardíaca (IC), que a la vez se asocian a cambios patológicos en la homeostasis intracelular de calcio. Sin embargo, no se conocen los efectos que el envejecimiento puede tener sobre los mecanismos de la homeostasis del calcio en cardiomiocitos auriculares humanos. Por otro lado, se han descrito variantes de riesgo genéticas asociadas a la FA en la región cromosómica 4q25, próximas al factor de transcripción Pitx2 que tiene un papel importante en el desarrollo cardíaco embrionario. En el corazón adulto Pitx2 se ha asociado a predisposición a arritmias auriculares, pero la relación que existe entre los variantes de 4q25 y la función de Pitx2 es controvertida. Además, se desconocen los efectos que los variantes de riesgo 4q25 tienen sobre la homeostasis del calcio. Por ello, en esta tesis se han investigado los mecanismos que pueden subyacer a los dos factores de riesgo envejecimiento y variantes de riesgo en 4q25. Hipótesis El envejecimiento y las variantes de riesgo en 4q25 producen alteraciones en la homeostasis del calcio intracelular en miocitos auriculares, que por sí solas o en combinación contribuyen a aumentar la propensión a la fibrilación auricular. Objetivos * Analizar los efectos del envejecimiento en los mecanismos que regulan la homeostasis del calcio en miocitos auriculares humanos. * Utilizar modelos murinos transgénicos de envejecimiento para identificar los mecanismos moleculares que subyacen a los cambios en la homeostasis del calcio debido al envejecimiento. * Investigar cómo las variantes de riesgo en el cromosoma 4q25 asociadas con un mayor riesgo de FA, afectan a las características electrofisiológicas de los miocitos auriculares humanos e identificar los mecanismos moleculares subyacentes. * Investigar como la edad modula los efectos de las variantes de riesgo en 4q25 en miocitos auriculares humanos. Métodos Los experimentos se llevaron a cabo en células auriculares o ventriculares aisladas de humano o de los distintos modelos murinos. Mediante las técnicas de patch-clamp y microscopía confocal se obtuvieron los datos electrofisiológicos. Las técnicas de RT-PCR y western blot se usaron para determinar los niveles de las proteínas estudiadas. Resultados Los resultados de esta tesis muestran que el envejecimiento disminuye la corriente de calcio (ICa), parámetro electrofisiológico que también está reducido en la FA, así como del contenido de calcio del retículo sarcoplásmico (RS), del calcio transitorio global y de las principales proteínas reguladoras del calcio, lo que conjuntamente podría reducir la contracción auricular en los ancianos. Estos resultados fueron reproducidos en modelo animal de envejecimiento prematuro (Zmpste24-/-) que no procesan correctamente la proteína lamina, reforzando la noción que este mecanismo podría subyacer a los efectos del envejecimiento sobre la homeostasis del calcio en las miocitos cardíacos. Además se encontró que en pacientes con previa historia de FA, el envejecimiento tenía un efecto sumatorio sobre algunos efectos deteriorantes de la FA sobre el manejo de calcio, acentuando así a disminución de la ICa con la edad. El estudio de las variantes de riesgo en el cromosoma 4q25 muestra que la variante de riesgo rs13143308T por sí sola o junto con rs2200733T están asociadas a alteraciones electrofisiológicas típicas de la FA. Así, miocitos de pacientes portadores de estas variantes tenían una mayor frecuencia de liberación espontánea de calcio, corrientes de entrada transitorias (ITIs) y despolarizaciones de membrana espontaneas. Estos resultados son los primeros en proporcionar un mecanismo electrofisiológico que podría explicar la mayor incidencia de FA en individuos con variantes de riesgo en 4q25. Estudios electrofisiológicos en miocitos auriculares de un modelo de ratón con deficiencia auricular de Pitx2 (NppaCre+Pitx2fl/-) reproduce todas las alteraciones en la homeostasis del calcio observados en pacientes con variantes de riesgo 4q25. Estos resultados avalan la noción de que la modulación del calcio intracelular por Pitx2 juega un papel importante en procesos electrofisiológicos asociados a la FA. El análisis de potenciales efectos sinérgicos entre variantes de riesgo en 4q25, envejecimiento y la FA revelaba que el envejecimiento per se no modifica los efectos que tiene las variantes 4q25 sobre la homeostasis del calcio. Sin embargo, dado que el envejecimiento reduce la amplitud de la ICa, lo cual reduciría el periodo refractario auricular, podría favorecer la prolongación o el mantenimiento de episodios de arritmia inducidas por actividad eléctrica espontanea. Por lo tanto, es posible que las variantes de riesgo 4q25 constituyan un sustrato electrofisiológico arritmogénico que favorece el inicio de episodios arrítmicos auriculares, y que el envejecimiento actúe prolongando la duración de estos episodios al reducir el periodo refractario auricular a través de la reducción de la amplitud de la ICa. Conclusiones El envejecimiento modula la homeostasis del calcio en miocitos auriculares humanos mediante la disminución de la Ica, del calcio transitorio y del calcio acumulado en el RS. Estos cambios favorecen por un lado el deterioro progresivo de la función contráctil con la edad, y por otro el acortamiento del período refractario mediado por la reducción de la amplitud de la ICa. El modelo murino Zmpste24-/- reproduce los efectos observados reforzando el mecanismo propuesto. Las variantes de riesgo localizadas en la región cromosómica 4q25, concretamente rs13143308 por sí sola o junto con rs2200733, aumentan la liberación espontánea de calcio, ITI y despolarizaciones de la membrana, parámetros característicos de la FA, pudiendo originar eventos arrítmicos en pacientes portadores de las variantes de riesgo. El modelo murino con deficiencia parcial de Pitx2 (NppaCre+Pitx2fl/-) reproduce lo observado en humanos portadores de las variantes de riesgo, reforzando la idea de Pitx2 como nexo entre las variantes de riesgo 4q25 y las alteraciones que provocan en la homeostasis del calcio. La combinación de los efectos encontrados en este estudio propondría a las variantes de riesgo en 4q25 como un substrato arritmogénico que favorece la iniciación de episodios arrítmicos y al envejecimiento como factor que los prolongaría debido a la reducción del período refractario producido por la reducción de la amplitud de la ICa.

BACKGROUND: Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with diseases of the colon including inflammatory bowel diseases (IBD) and colorectal cancer (CRC). However, the functional role of many of these SNPs is largely unknown and tissue-specific resources are lacking. Expression quantitative trait loci (eQTL) mapping identifies target genes of disease-associated SNPs. This study provides a comprehensive eQTL map of distal colonic samples obtained from 40 healthy African Americans and demonstrates their relevance for GWAS of colonic diseases. RESULTS: 8.4 million imputed SNPs were tested for their associations with 16,252 expression probes representing 12,363 unique genes. 1,941 significant cis-eQTL, corresponding to 122 independent signals, were identified at a false discovery rate (FDR) of 0.01. Overall, among colon cis-eQTL, there was significant enrichment for GWAS variants for IBD (Crohn's disease [CD] and ulcerative colitis [UC]) and CRC as well as type 2 diabetes and body mass index. ERAP2, ADCY3, INPP5E, UBA7, SFMBT1, NXPE1 and REXO2 were identified as target genes for IBD-associated variants. The CRC-associated eQTL rs3802842 was associated with the expression of C11orf93 (COLCA2). Enrichment of colon eQTL near transcription start sites and for active histone marks was demonstrated, and eQTL with high population differentiation were identified. CONCLUSIONS: Through the comprehensive study of eQTL in the human colon, this study identified novel target genes for IBD- and CRC-associated genetic variants. Moreover, bioinformatic characterization of colon eQTL provides a tissue-specific tool to improve understanding of biological differences in diseases between different ethnic groups.

This project aimed to identify novel genetic risk variants associated with migraine in the Norfolk Island population. Statistical analysis and bioinformatics approaches such as polygenic modeling and gene clustering methods were carried out to explore genotypic and expression data from high-throughput techniques. This project had a particular focus on hormonal genes and other genetic variants and identified a modest effect size on the migraine phenotype.

The neural mechanisms underlying genetic risk for schizophrenia, a highly heritable psychiatric condition, are still under investigation. New schizophrenia risk genes discovered through genome-wide association studies (GWAS), such as neurogranin (NRGN), can be used to identify these mechanisms. In this study we examined the association of two common NRGN risk single nucleotide polymorphisms (SNPs) with functional and structural brain-based intermediate phenotypes for schizophrenia. We obtained structural, functional MRI and genotype data of 92 schizophrenia patients and 114 healthy volunteers from the multisite Mind Clinical Imaging Consortium study. Two schizophrenia-associated NRGN SNPs (rs12807809 and rs12541) were tested for association with working memory-elicited dorsolateral prefrontal cortex (DLPFC) activity and surface-wide cortical thickness. NRGN rs12541 risk allele homozygotes (TT) displayed increased working memory-related activity in several brain regions, including the left DLPFC, left insula, left somatosensory cortex and the cingulate cortex, when compared to non-risk allele carriers. NRGN rs12807809 non-risk allele (C) carriers showed reduced cortical gray matter thickness compared to risk allele homozygotes (TT) in an area comprising the right pericalcarine gyrus, the right cuneus, and the right lingual gyrus. Our study highlights the effects of schizophrenia risk variants in the NRGN gene on functional and structural brain-based intermediate phenotypes for schizophrenia. These results support recent GWAS findings and further implicate NRGN in the pathophysiology of schizophrenia by suggesting that genetic NRGN risk variants contribute to subtle changes in neural functioning and anatomy that can be quantified with neuroimaging methods.

The neural mechanisms underlying genetic risk for schizophrenia, a highly heritable psychiatric condition, are still under investigation. New schizophrenia risk genes discovered through genome-wide association studies (GWAS), such as neurogranin (NRGN), can be used to identify these mechanisms. In this study we examined the association of two common NRGN risk single nucleotide polymorphisms (SNPs) with functional and structural brain-based intermediate phenotypes for schizophrenia. We obtained structural, functional MRI and genotype data of 92 schizophrenia patients and 114 healthy volunteers from the multisite Mind Clinical Imaging Consortium study. Two schizophrenia-associated NRGN SNPs (rs12807809 and rs12541) were tested for association with working memory-elicited dorsolateral prefrontal cortex (DLPFC) activity and surface-wide cortical thickness. NRGN rs12541 risk allele homozygotes (TT) displayed increased working memory-related activity in several brain regions, including the left DLPFC, left insula, left somatosensory cortex and the cingulate cortex, when compared to non-risk allele carriers. NRGN rs12807809 non-risk allele (C) carriers showed reduced cortical gray matter thickness compared to risk allele homozygotes (TT) in an area comprising the right pericalcarine gyrus, the right cuneus, and the right lingual gyrus. Our study highlights the effects of schizophrenia risk variants in the NRGN gene on functional and structural brain-based intermediate phenotypes for schizophrenia. These results support recent GWAS findings and further implicate NRGN in the pathophysiology of schizophrenia by suggesting that genetic NRGN risk variants contribute to subtle changes in neural functioning and anatomy that can be quantified with neuroimaging methods.

Schizophrenia is a complex disorder affecting 1% of the population and is highly heritable, but the majority of contributing genetic factors has remained elusive. Current risk estimates for clinical practice are primarily determined by family history and associated empirical risk. Copy number variants (CNVs) may hold the key to explaining the missing heritability in schizophrenia research; schizophrenia risk estimates as high as 30% have been found for the most-studied CNV associated with schizophrenia, 22q11. Currently, there are methods to identify CNVs though previously collected data from SNP microarrays that would facilitate these types of studies. To determine if algorithms that call CNVs from microarray data are robust four genomic regions with putative CNVs called by the Wellcome Trust Consortium using Birdseye in Birdsuite with Affymetrix 6.0 array raw SNP intensities, primarily affecting genes CHD1L, COX5B, PAK7, ZFYVE20, were validated using Taqman real-time qPCR assays in 29 samples by research groups at VCU and Dublin. CNVs called from the algorithm were 100% validated at VCU though there were false negatives from the algorithm that were validated. Two samples at loci with putative duplications were not called by the Dublin group, which may be because of differing sensitivities of the Taqman assays to be able to detect a 50% difference in copy number between duplications and diploid controls, or because of another technical or analytical difference between the two sites. Deletion frequency of one common CNV found in the gene ERBB4, was assessed by qPCR in both Irish singleton (ICCSS) and Irish family (IHDSF) samples and compared with Irish control (Trinity Biobank) and North American control populations. The ERBB4 deletion frequency was not significantly different when comparing the Irish controls to the Irish singleton or the Irish family samples though the family samples were different when compared against the North American control population, which suggests population stratification, rather than a true association between ERBB4 and increased schizophrenia risk. Current clinical practice has been improved by knowledge and evaluation of CNV-related disorders that include risk for psychosis and additional phenotypes. Genotyping of individuals with known psychosis has led to improved patient care for non-psychosis-related phenotypes associated with CNVs. Individuals with suspected genomic disorders that are found to have CNVs can be counseled on potential psychosis risk and potential risk to their offspring. Recurrent CNVs may hold promise in future clinical practice in order to individualize risk estimates in the general patient population, and increase the number of individuals able to receive anticipatory treatment to minimize disease severity.

The human genome's strongest influences on two common diseases, systemic lupus erythematosus (SLE) and schizophrenia, arise from genetic variation in the Human Leukocyte Antigen (HLA) locus. However, the genes and functional alleles driving these genetic relationships have remained unknown. We hypothesized that a complex, multi-allelic form of structural variation in the Complement component 4 (C4) gene, within the HLA locus, underlies these relationships. Loci that exist in many structural forms and vary widely in copy number have been difficult to analyze molecularly. As a result, we know little about their population genetic properties or their influence on phenotypes. In this work, we developed molecular and statistical methods to characterize such loci and to evaluate their contribution to phenotypes. Applying these methods to the C4 locus, we found that C4 segregates in four common and at least eleven low-frequency structural forms in human populations. Although there was only partial correlation between C4 structural variation and individual single nucleotide polymorphisms (SNPs), we developed an imputation approach to enable statistical prediction of C4 structural states from flanking SNP haplotypes. C4 structural variation associated to gene expression in lymphoblastoid cell lines and human brain tissue. Applying our imputation strategy to SLE and schizophrenia case-control cohorts totaling > 75,000 individuals, we found that structural variation in C4 contributes to risk of both phenotypes in a manner predicted by its effect on gene expression in relevant tissues, and with largely opposite directions of effect - alleles that were protective for schizophrenia increased risk for SLE, and vice versa. Leveraging a natural allelic series of C4 structural forms, we developed a novel form of association testing and showed that the association to C4 is unlikely to be caused by correlation with HLA SNPs. C4 was expressed in human neurons, whereas other upstream complement pathway genes were expressed primarily by microglia. Mice lacking C4 showed a deficit in synaptic pruning that was rescued by human C4. The methods developed in this thesis enable analysis of complex structural variation, and our results identify a novel form of genome variation as making a strong contribution to phenotypes.

La maladie rénale chronique est une pathologie complexe aux nombreuses étiologies et susceptible de conduire à une insuffisance chronique terminale (IRCT), c’est-à-dire une incapacité des reins à filtrer le sang, une fonction essentielle au fonctionnement de l’organisme humain. La transplantation rénale constitue le meilleur traitement de l’IRCT. Si les taux de survie du greffon rénal à court terme sont désormais satisfaisants (plus de 90% à 1 an en France), la survie à moyen et long terme n’est pas encore optimale (autour de 60% à 5 ans en France). Bien que la compatibilité HLA ait été associée à une moins survie greffon, les mécanismes impliqués dans la perte du greffon rénal ne sont pas encore totalement connus. Ces travaux ont pour but d’identifier des variants génétiques en dehors du HLA associés au rejet et à la survie du greffon rénal. Ils s’appuient sur l’analyse de données génétiques de patients transplantés exclusivement à Nantes (N=1969 paires).La combinaison des génomes du donneur et du receveur a permis la découverte d’une nouvelle incompatibilité en amont du gène TOM1L1 associée avec la perte du greffon rénal. Ce signal a été validé dans trois cohortes indépendantes. Grâce à une granularité phénotypique inédite dans la littérature de la génétique de la transplantation rénale, un premier profil génétique de l’inflammation microvasculaire isolée (IMVi) a été établi. En plus de montrer l’influence de la survenue de l’IMVi sur la survie du greffon rénal, nous avons identifié de nombreux gènes associés à ce phénotype encore peu décrit. Nous avons montré que son fond génétique se distingue de celui du rejet humoral et d’autres sous-types de l’IMV<br>Chronic kidney disease is a complex multifactorial pathology leading to end-stage renal disease (ESRD), which is characterized by an inability of the kidneys to filter blood. Kidney transplant is currently the best treatment of ESRD both in terms of quality of life and patient survival. Despite satisfying short term survival rates (above 90% in France), mid and long-term survival are not optimal (around 60% at 5 years in France). Even if donor-recipient HLA compatibility was shown to have adverse effects on survival, kidney graft loss underlying mechanisms are not fully understood. This work aims at identifying variants outside of the HLA region associated with kidney graft loss and rejection. It relies on the analysis of genetic data of patients exclusively transplanted in Nantes (n=x). Combining donor and recipient genomes enabled the discovery a new donor-recipient genetic mismatch upstream the TOM1L1 gene associated with kidney graft loss. This signal was replicated in three independent cohorts. Thanks to an unprecedented phenotypic granularity in kidney transplant genetics literature, we described the first genetic profile of isolated microvascular inflammation (iMVI), a poorly defined phenotype, and highlighted a different genetic background from humoral rejection. We also showed that iMVI confers a higher risk of kidney graft loss and reported several genes associated with iMVI, hence contributing to better characterize this outcome

International cancer sequencing projects have generated comprehensive catalogs of alterations found in tumor genomes, as well as germline variant data for thousands of individuals. In this thesis, we describe two statistical methods exploiting these rich datasets in order to better understand tumor initiation, tumor progression and the contribution of genetic variants to the lifetime risk of developing cancer. The first method, a Bayesian inference model named cDriver, utilizes multiple signatures of positive selection acting on tumor genomes to predict cancer driver genes. Cancer cell fraction is introduced as a novel signature of positive selection on a cellular level, based on the hypothesis that cells obtaining additional advantageous driver mutations will undergo rapid proliferation and clonal expansion. We benchmarked cDriver against state of the art driver prediction methods on three cancer datasets demonstrating equal or better performance than the best competing tool. The second method, termed REWAS is a comprehensive framework for rare-variant association studies (RVAS) aiming at improving identification of cancer predisposition genes. Nonetheless, REWAS is readily applicable to any case-control study of complex diseases. Besides integrating well-established RVAS methods, we developed a novel Bayesian inference RVAS method (BATI) based on Integrated Nested Laplace Approximation (INLA). We demonstrate that BATI outperforms other methods on realistic simulated datasets, especially when meaningful biological context (e.g. functional impact of variants) is available or when risk variants in sum explain low phenotypic variance. Both methods developed during my thesis have the potential to facilitate personalized medicine and oncology through identification of novel therapeutic targets and identification of genetic predisposition facilitating prevention and early diagnosis of cancer.<br>Els distints projectes internacionals de seqüenciació de càncer duts a terme en els últims anys han generat catàlegs complets d’alteracions trobades en els genomes tumorals, així com informació de variants germinals per a milers d'individus. En aquesta tesi descrivim dos mètodes estadístics aprofitant aquestes bases de dades per tal d’entendre millor la iniciació i la progressió dels tumors, i la contribució de variants genètiques al risc de desenvolupar càncer al llarg de la vida. El primer mètode, anomenat cDriver, es basa en un model d’inferència Bayesià que utilitza múltiples senyals de la selecció positiva que ocorre en els genomes tumorals per tal de predir els gens driver del càncer. En aquest mètode, hem inclòs la fracció de cèl·lules tumorals com a nova senyal de la selecció positiva a nivell cel·lular. Aquesta es basa en la hipòtesi que les cèl·lules que adquireixen mutacions ventajoses proliferaran i s’expandiran clonalment més ràpidament. Per avaluar cDriver, aquest es va comparar amb els mètodes més utilitzats per a la predicció de gens driver actuals. L’anàlisi es va dur a terme amb conjunts de dades de tres càncer diferents i els resultats van ser iguals o millors que els obtinguts per les eines més competitives en el tema. El segon mètode, anomenat REWAS, és un marc de treball per l’estudi d’associació de variants rares (RVAS) amb l'objectiu de millorar la identificació dels gens de predisposició al càncer. Tot i això, REWAS es pot aplicar a qualsevol estudi cas-control de malalties complexes. Per una altra part, a més d'integrar mètodes RVAS ben establerts, hem desenvolupat un nou mètode d'inferència Bayesiana RVAS basat en Integrated Nested Laplace Approximation (BATI). També demostrem que BATI mostra millors resultats que altres mètodes en dades simulades amb soroll de fons real, especialment quan el context biològic (p.e. variants amb impacte funcional) està disponible or quan les variants de risc expliquen en total poca variància fenotípica.

Els distints projectes internacionals de seqüenciació de càncer duts a terme en els últims anys han generat catàlegs complets d’alteracions trobades en els genomes tumorals, així com informació de variants germinals per a milers d'individus. En aquesta tesi descrivim dos mètodes estadístics aprofitant aquestes bases de dades per tal d’entendre millor la iniciació i la progressió dels tumors, i la contribució de variants genètiques al risc de desenvolupar càncer al llarg de la vida. El primer mètode, anomenat cDriver, es basa en un model d’inferència Bayesià que utilitza múltiples senyals de la selecció positiva que ocorre en els genomes tumorals per tal de predir els gens driver del càncer. En aquest mètode, hem inclòs la fracció de cèl·lules tumorals com a nova senyal de la selecció positiva a nivell cel·lular. Aquesta es basa en la hipòtesi que les cèl·lules que adquireixen mutacions ventajoses proliferaran i s’expandiran clonalment més ràpidament. Per avaluar cDriver, aquest es va comparar amb els mètodes més utilitzats per a la predicció de gens driver actuals. L’anàlisi es va dur a terme amb conjunts de dades de tres càncer diferents i els resultats van ser iguals o millors que els obtinguts per les eines més competitives en el tema. El segon mètode, anomenat REWAS, és un marc de treball per l’estudi d’associació de variants rares (RVAS) amb l'objectiu de millorar la identificació dels gens de predisposició al càncer. Tot i això, REWAS es pot aplicar a qualsevol estudi cas-control de malalties complexes. Per una altra part, a més d'integrar mètodes RVAS ben establerts, hem desenvolupat un nou mètode d'inferència Bayesiana RVAS basat en Integrated Nested Laplace Approximation (BATI). També demostrem que BATI mostra millors resultats que altres mètodes en dades simulades amb soroll de fons real, especialment quan el context biològic (p.e. variants amb impacte funcional) està disponible or quan les variants de risc expliquen en total poca variància fenotípica.<br>International cancer sequencing projects have generated comprehensive catalogs of alterations found in tumor genomes, as well as germline variant data for thousands of individuals. In this thesis, we describe two statistical methods exploiting these rich datasets in order to better understand tumor initiation, tumor progression and the contribution of genetic variants to the lifetime risk of developing cancer. The first method, a Bayesian inference model named cDriver, utilizes multiple signatures of positive selection acting on tumor genomes to predict cancer driver genes. Cancer cell fraction is introduced as a novel signature of positive selection on a cellular level, based on the hypothesis that cells obtaining additional advantageous driver mutations will undergo rapid proliferation and clonal expansion. We benchmarked cDriver against state of the art driver prediction methods on three cancer datasets demonstrating equal or better performance than the best competing tool. The second method, termed REWAS is a comprehensive framework for rare-variant association studies (RVAS) aiming at improving identification of cancer predisposition genes. Nonetheless, REWAS is readily applicable to any case-control study of complex diseases. Besides integrating well-established RVAS methods, we developed a novel Bayesian inference RVAS method (BATI) based on Integrated Nested Laplace Approximation (INLA). We demonstrate that BATI outperforms other methods on realistic simulated datasets, especially when meaningful biological context (e.g. functional impact of variants) is available or when risk variants in sum explain low phenotypic variance. Both methods developed during my thesis have the potential to facilitate personalized medicine and oncology through identification of novel therapeutic targets and identification of genetic predisposition facilitating prevention and early diagnosis of cancer.

Background. Epithelial ovarian cancer (EOC) and age of natural menopause (ANM) are two complex traits impacting women’s health. ANM is also an important EOC risk factor. Insight into genetic factors influencing EOC and ANM could provide novel entry points for understanding EOC pathogenesis, and the normal process of ovarian aging. Methods. A two-stage genome-wide association study (GWAS) design using DNA pooling in Stage 1 was used to discover single nucleotide polymorphisms (SNPs) associated with histology-specific EOC risk, and population-specific variation in ANM. SNP-trait associations discovered in Stage 1 of these GWAS were replicated in two different consortia; EOC association in the Ovarian Cancer Association Consortium (OCAC), and ANM associations in the ReproGen Consortium. Results. Eight subtype-specific SNP-EOC associations discovery in Stage 1 of the EOC GWAS were replicated (unadjusted P <0.05) in the datasets of the OCAC: 4 in the mucinous subtype, 2 in the endometrioid and clear cell (ENCC) subtypes combined, and 2 in the low-malignancy potential (LMP) serous subtype. These associations did not achieve genome-wide significance (P < 5x10⁻⁸). However, several of the loci implicated by these SNPs harbour attractive candidate genes for ovarian cancer biology, including the mucinous locus harbouring RAD51B, the ENCC locus harbouring GRB10, and the LMP serous locus harbouring BPIL2/C22orf26. The GWAS of ANM performed in Iranian women revealed one SNP-trait association exclusive to this population (rs10140275; unadjusted P=4.0x10⁻⁴) and one shared with the European population (rs10840211). In the replication of European GWAS findings in Iranian women one SNP at the 20p12.3 locus was replicated (rs16991615, unadjusted P=0.02). SNPs tagging the 19q13.42 locus narrowly missed replication in our dataset (rs1172822, unadjusted P= 0.08). Conclusion. Eight SNP-EOC associations warrant further replication and/or fine-mapping in samples with reliable tumour information. These SNPs tag loci harbouring candidate genes involved in DNA repair, the PI3K/AKT pathway, and immune function, suggesting these pathways and processes may be important to the etiology of the rarer EOC subtypes. The generality of the European ANM GWAS findings in established in another human population (20p12.3). Further, one genetic variant influencing ANM in the Iranian population but not the European population is reported.

Epithelial ovarian cancer (EOC) is a leading cause of morbidity and mortality among women in the United States, and the etiology is incompletely understood. Common, low penetrant genetic variants such as single nucleotide polymorphisms (SNPs) likely contribute to a significant proportion of EOC. We examined whether SNPs in two understudied yet biologically important types of genes, mitochondrial-related and miRNA-related genes, may contribute to EOC susceptibility using data from a large, homogeneous study population of 1,815 EOC cases and 1,900 controls (frequency-matched on age-group and race/ethnicity) genotyped through stage 1 of an ongoing genome-wide association study. Inter-individual variation in genes involved in mitochondrial biogenesis was strongly associated with EOC risk (empirical P=0.050), especially for genes NRF1, PPARGC1A, MTERF, ESRRA, and CAMK2D. SNPs in several genes involved in the biogenesis of miRNAs (LIN28, LIN28B, AGO2, DICER, and DROSHA) also demonstrated associations with EOC risk; a joint meta-analysis and in vitro investigations reinforced evidence for a protective role of LIN28B rs12194974 (combined OR= 0.90, 95% CI: 0.82-0.98), a G>A SNP predicted to reside in a transcription factor binding site in the highly conserved LIN28B promoter. Our findings provide valuable insight into the pathogenesis of EOC, and support the consideration of variants in these genes as candidates when building risk prediction models. Most importantly, this work has provided a strong foundation for further lines of research that may aid in reducing the burden of this disease.

The gastro-intestinal (GI) tract represents the largest surface of the body and is continuously exposed to the microbial environment. In such anatomy, the survival of the host requires that the intestinal microbial flora be contained without excessive immune-reactivity to commensal bacteria while retaining the ability to respond to episodic pathogens. The discriminative recognition between beneficial commensal bacteria and potentially harmful pathogens demands an accurate interpretation by the GI mucosal immune system. Any defects in the processes of innate immune recognition and killing may lead to the development and perpetuation of chronic intestinal inflammation, namely inflammatory bowel disease (i.e. Crohn's disease (CD) and ulcerative colitis (UC)) and post-infectious irritable bowel syndrome (PI-IBS). The aim of ours studies was to evaluate the contribution of candidate genes, involved in the homeostasis and regulation of the intestinal innate immune response, to the susceptibility to CD, DC, and PI-IBS. In the first phase, we describe functional and genetic association results supporting NLRP3, encoding NALP3/cryopyrin, as a novel CD susceptibility gene. We subsequently report that the MEFV gene, encoding pyrin, known to interact with and be involved in the same pathway as NALP3/cryopyrin, does not contribute to CD and DC susceptibility. No CD or DC additional associations were observed upon NLRP3-MEFV gene-gene interaction analyses. In the third phase, we report the first association study evaluating genetic determinants for PI-IBS, using the well-characterized Walkerton population cohort. We uncovered variants in the TLR9, CDH1, and IL6 regions associated with PI-IBS susceptibility. These results are in keeping with the pathophysiologic changes observed in patients with PI-IBS, which include increased intestinal permeability and intestinal immune activation.<br>Overall, these results contribute to a better understanding of the genetic susceptibility to CD, DC and PI-IBS and shed light on new pathogenic signaling pathways in the development of these diseases.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.<br>Title from electronic title page (viewed Sep. 3, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Nutrition, School of Public Health." Discipline: Nutrition; Department/School: Public Health.

Des mutations germinales dans les gènes majeurs du cancer du sein BRCA1 et BRCA2 sont responsables de la maladie chez les patientes cumulant histoire familiale et apparition du cancer à un jeune âge. Environ 15% des femmes testées pour les mutations de BRCA1 et BRCA2 sont porteuses d’une mutation clairement pathogénique dans un des deux gènes. Cependant, des variants de signification clinique incertaine (VUS pour "variants of uncertain clinical significance") sont détectés dans 5% à 15% des cas testés. Pour évaluer la signification clinique des VUS, le Breast cancer Infomation Core (BIC) a développé un modèle Bayésien intégré, basé sur des données d'observations. Align-GVGD, un algorithme d'évaluation des substitutions faux-sens basé sur l'histoire évolutionnaire de la protéine fournit la probabilité a priori du modèle. Cependant, lorsqu'une substitution silencieuse est détectée, elle sera jugée comme neutre par l'évaluation in silico. Pourtant, une mutation au niveau de l'ARNm peut perturber la mécanique de l'épissage, par deux moyens principaux: endommagement des sites sauvages d'épissage, ou la création de sites exoniques d'épissage de novo. Notre premier objectif est de rassembler les variants déjà publiés, de les re-analyser avec le modèle d'évaluation intégrée. Nous voulons extraire le plus de variants publiés premièrement sous le statut de VUS vers un statut plus informatif, avec des recommandations cliniques associées. Par la suite, nous voulons étendre le modèle pour évaluer plus de variants, plus précisément, en intégrant l'évaluation des perturbations de l'épissage. Finalement, nous serons capable de présenter et de fournir ces informations librement sur Internet, via une interface web populaire, une Leiden Open Variation Database (LOVD)<br>Germline mutations in major breast cancer susceptibility genes BRCA1 and BRCA2 are responsible for the disease for high-risk patients (patients with early onset and familial history of breast cancer). Around 15% of screened women for BRCA1 and BRCA2 mutations carry one clearly pathogenic mutation in one of those two genes. However, variants of uncertain clinical significance (VUS) are detected in 5% to 15% of tested patients. To assess clinical significance of VUS, the Breast cancer Information Core (BIC) has developed a Bayesian integrated model, based on observational data. Align-GVGD, an algorithm evaluating damage of missense substitutions based on the evolutionary history of the protein, is providing the prior probability of the model. However, whenever a silent substitution arise, it is firstly treated as neutral by the in silico assessment. Indeed, a mutation at the mRNA level can disrupt the splicing machinery by two main means: damaging wild-type splice sites, or creating exonic de novo splice sites. Our first goal is to be a central repository of already published variants, to re-analyze them using the unified integrated evaluation model. We would like to extract the most variants from the original published status of VUS to a more informative status, with associated clinical recommendations. Then we would like to extend the model to be able to evaluate more variants more precisely by adding the splicing damages assessment in the integrated evaluation. In the end, we will be able to provide these informations freely on Internet, via a widely use web interface, a Leiden Open Variation Database (LOVD)

Small-for-gestational age (SGA) births are defined as babies who are born at less than the 10th percentile of weight for their sex and gestational age based on population growth standards1. SGA babies may be at risk for type II diabetes, cardiovascular disease, and hypertension later in life, although the exact biological mechanisms behind SGA birth are not fully understood1. Paraoxonase 1 (PON1) genetic variants are known to influence the ability of PON1 enzymes to prevent oxidative damage by hydrolyzing phospholipids, lipid peroxide by-products, and amino acid by-products such as homocysteine (Hcy)2. Infante-Rivard has previously shown that PON1 polymorphisms and Hcy concentrations (albeit in an unexpected protective direction for the latter) are associated with SGA birth3-4, however, their joint contribution to the risk of SGA birth has not been studied. The objective of this thesis is to explore the gene-environment interactions between three functional PON1 variants and plasma Hcy concentrations as well as food folate consumption (lentils) and folic acid vitamin supplementation on the risk of SGA birth. The present study uses data from births of infants at a university hospital in Montreal, Quebec. 493 case mothers and 472 control mothers provided informed consent to participate in the study. Cases were SGA babies born from May 1998 to June 2000, and controls were appropriate-for-gestational-age (AGA) babies born at the same hospital during the same time period4. Three PON1 genetic variants were analyzed in this study (C108T, L55M and Q192R). Information regarding Hcy concentrations and nutritional variables such as lentil and folate-containing vitamin consumption was collected and analyzed. Case-control, case-only and case-parent trio study designs were used and the data analyzed using unconditional logistic regression and Poisson log-linear models.Results indicate that mothers with wild type PON1 108CC genotypes and high Hcy concentrations (≥ 90th percentile) may be protected against SGA birth compared to those with lower Hcy concentrations (&lt;90th percentile) after adjustment for gestational age, sex of the fetus, and ethnicity (OR=0.52, 95% CI [0.25, 1.05]). This effect was shown to be similar for the PON1 L55M polymorphism, in both maternal and fetal models. Individuals with the PON1 55MM genotype and high Hcy levels were shown to be at an increased risk of SGA birth compared to those with low Hcy levels after adjustment for the same covariates (maternal model, OR=1.38, 95% CI [0.46, 4.17]). Results from case-only and case-parent trio designs generally concur with those of the case-control study design. PON1 polymorphisms appear to modify the effect of Hcy on the risk of SGA birth; however, the results of this study are generally non-significant and more work is needed to characterize this relationship as well as the biological mechanism behind this effect.<br>Les "naissances de faible poids par rapport à l'âge gestationnel" (Small-for-gestational age; SGA) sont définies comme l'ensemble des bébés qui sont nés avec un poids inférieur au 10ième pourcentile, selon leur sexe et leur âge gestationnel d'après des courbes établies dans la population1. Les bébés SGA peuvent être à risque pour le diabète de type II, les maladies cardiovasculaires et l'hypertension dans leur vie adulte. Les mécanismes biologiques qui déterminent le statut de SGA ne sont pas à ce jour entièrement connus1. Les variants du gène Paraoxonase 1 ( PON1) sont connus pour avoir une influence sur la capacité des enzymes PON1 de prévenir le dommage oxidatif par l'hydrolyse des phospholipides, des produits dérivés des peroxides lipides, et des produits dérivés des acides aminés, tels que l'homocystéine (Hcy)2. Infante-Rivard et coll. ont démontré que ces polymorphismes et les concentrations de Hcy (d'une manière protective inattendue) sont associés aux naissances SGA3-4, Par contre, leur contribution combinée sur le risqué de SGA n'a pas été étudiée. L'objectif de cette thèse est d'explorer les interactions gène-environnement entre trois variants fonctionnels de PON1, diverses concentrations plasmatiques de Hcy et également la consommation alimentaire de folate ainsi que sa supplementation vitaminique sur le risque de naissance SGA.La présente analyse utilise des données d'une étude faite dans un hôpital universitaire de Montréal, Québec. 493 mères de bébés de faible poids ainsi que 472 mères de bébés ayant un poids au dessus du 10ième percentile ont donné leur accord pour y participer. Les cas de naissance SGA ont été observés entre mai 1998 et juin 2000; les témoins étaient appariés pour l'âge gestationnel4, le sexe, et le group ethnique (appropriate-for-gestational-age; AGA). Trois variants génétiques PON1 ont été analysés (C108T, L55M et Q192R). Les concentrations d'homocystéine et les variables nutritionnelles (consommation de lentilles et de supplément vitaminiques) ont été également analysées. Les designs cas-contrôle (case-control), cas-seulement (case-only) et cas-parents (case-parent) ont été utilisés et les résultats analysés avec une regression logistique inconditionelle (unconditional logistic regression) et par des modèles log-linéaires Poisson (Poisson log-linear models).Les résultats indiquent que les mères avec le génotype sauvage PON1 108CC et une concentration élevée (≥90ième pourcentile) pourraient être protégées contre les naissances SGA comparées à celles ayant des concentrations Hcy plus basses (&lt;90ième pourcentile), après ajustement pour l'âge gestationnel, le sexe du foetus et l'ethnicité (OR=0.52, 95% CI [0.25, 1.05]). Cet effet a également été démontré pour le polymorphisme PON1 L55M, dans les modèles maternels et fétaux. Les porteuses de variants homozygotes de PON1, L55M (avec le génotype PON1 55MM et des concentrations élevées d'homocystéine) ont montré un risque de naissance SGA plus élevé que celles avec des concentrations Hcy plus basses (modèle maternel, OR=1.38, 95% CI [0.46, 4.17]). Les polymorphismes PON1 semblent modifier l'effet de la concentration de Hcy sur le risque de naissance SGA, par contre, plus d'investigation sera nécessaire afin de caractériser non seulement cette relation, mais également les mécanismes biologiques sous-jacents.

Genetic variants and traits in metabolic signaling pathways may interact with lifestyle factors such as obesity, physical activity, and exogenous estrogen (E), influencing postmenopausal colorectal cancer (CRC) risk, but these interrelated pathways are not fully understood. In this case-cohort study, we examined 33 single-nucleotide polymorphisms (SNPs) in genes related to insulin-like growth factor-I (IGF-I)/insulin resistance (IR) traits and signaling pathways, using data from 704 postmenopausal women in Women's Health Initiative Observation ancillary studies. Stratifying by the lifestyle modifiers, we assessed the effects of IGF-I/IR traits (fasting total and free IGF-I, IGF binding protein-3, insulin, glucose, and homeostatic model assessment-insulin resistance) on CRC risk as a mediator or influencing factor. Six SNPs in the INS, IGF-I, and IGFBP3 genes were associated with CRC risk, and those associations differed between non-obese/active and obese/inactive women and between E nonusers and users. Roughly 30% of the cancer risk due to the SNP was mediated by IGF-I/IR traits. Likewise, carriers of 11 SNPs in the IRS1 and AKT1/2 genes (signaling pathway-related genetic variants) had different associations with CRC risk between strata, and the proportion of the SNP-cancer association explained by traits varied from 30% to 50%. Our findings suggest that IGF-I/IR genetic variants interact with obesity, physical activity, and exogenous E, altering postmenopausal CRC risk, through IGF-I/IR traits, but also through different pathways. Unraveling gene-phenotype-lifestyle interactions will provide data on potential genetic targets in clinical trials for cancer prevention and intervention strategies to reduce CRC risk.

The goal of ZOFF is formulated as a change of stagnation and increase in annual profit in term of 2 years, provided adequate low risk. The paper describes the outcomes and conclusions from a marketing research that highlights the key attributes of the company and its target audience. Way out of the situation lies in identifying the problem and its subsequent formulation. To achieve the objective are used various methods of creation and comparison of the criteria which define variants created. This process comprises a decision matrix for identification and comparison of risk scenarios. The output of the work is to evaluate and compare the resulting variants and recommendation to the elimination of possible threats.

Introduction: In Africa, it is estimated that cardiovascular disease (CVD) will affect approximately 1.3 million people per annum over the following 20 years. C-reactive protein (CRP) is a predictor of CVD risk and certain CRP gene polymorphisms can result in altered CRP concentrations. The distribution of CRP gene polymorphisms is ethnic-specific and extrapolating information from other populations to the black South African population, reported to harbour considerable genetic variation, should be avoided. This highlights the fact that genetic research among black South Africans is necessary. Objectives: The main aim of this dissertation was to determine the association between various polymorphisms (reported and novel [single nucleotide polymorphisms (SNPs)] within the CRP gene with CRP concentrations [measured as high sensitivity (hs)-CRP concentrations] in a black South African population undergoing an epidemiological transition. Interactions between specific CRP polymorphisms and certain environmental factors on hs-CRP concentrations were also investigated. Methods: This cross-sectional study (n=1,588) was nested within the Prospective Urban and Rural Epidemiological (PURE) study. Genotyping was performed using Illumina VeraCode technology on the BeadXpress® platform. Hs-CRP concentrations were measured by the use of a sequential multiple analyser computer (SMAC) through a particle-enhanced immunoturbidometric assay. Results: All the SNPs adhered to the assumptions of Hardy-Weinberg equilibrium, although the distribution of several SNPs differed from that reported in other population groups. Three SNPs (rs3093058, rs3093062 and rs3093068) were associated with a significant (p ≤ 0.05) increase in CRP concentrations. Five SNPs (rs1205, rs1341665, rs2794520, rs7553007 and rs2027471) were associated with a significant (p ≤ 0.05) decrease in CRP concentrations. This difference in effect was most probably due to changes in gene function brought about by the localisation of these SNPs in the CRP gene. Men and urban individuals were more likely to present with significant associations between the SNPs investigated and CRP concentrations. The difference in the prevalence of the alleles associated with higher CRP concentrations in this population compared to non-African populations could possibly explain the increased CRP concentrations that are observed in the black South African population. Gene-gender (rs1205, rs1341665 and rs2027474) as well as gene-environmental (rs3093068) interactions were also observed. Conclusions: CRP concentrations are in themselves a complex trait and there are many factors at play that influence their expression. Numerous factors (both genetic and environmental) are involved and no single factor acting alone is likely to have enough of an influence to be used as a clinical diagnostic test of CRP concentrations. These results provide valuable information on the regulation of CRP in a black South African population as well as contribute to the literature of CRP on a global level.<br>Thesis (MSc (Nutrition))--North-West University, Potchefstroom Campus, 2013.