The data from genome-wide association studies (GWAS) in humans are still predominantly analyzed using single marker association methods. As an alternative to Single Marker Analysis (SMA), all or subsets of markers can be tested simultaneously. This approach requires a form of Penalized Regression (PR) as the number of SNPs is much larger than the sample size. Here we review PR methods in the context of GWAS, extend them to perform penalty parameter and SNP selection by False Discovery Rate (FDR) control, and assess their performance (including penalties incorporating linkage disequilibrium) in comparison with SMA. PR methods were compared with SMA on realistically simulated GWAS data consisting of genotype data from single and multiple chromosomes and a continuous phenotype and on real data. Based on our comparisons our analytic FDR criterion may currently be the best approach to SNP selection using PR for GWAS. We found that PR with FDR control provides substantially more power than SMA with genome-wide type-I error control but somewhat less power than SMA with Benjamini-Hochberg FDR control. PR controlled the FDR conservatively while SMA-BH may not achieve FDR control in all situations. Differences among PR methods seem quite small when the focus is on variable selection with FDR control. Incorporating LD into PR by adapting penalties developed for covariates measured on graphs can improve power but also generate morel false positives or wider regions for follow-up. We recommend using the Elastic Net with a mixing weight for the Lasso penalty near 0.5 as the best method. / Ph. D.

<p>Cerebrovascular disease (stroke), especially ischemic stroke, is a major cause of death and neurological disability in adults. Because of its clinical heterogeneity, stroke is considered as a multi-factorial and polygenic disorder. Most current genetic studies of ischemic stroke focus on genetic susceptibility rather than factors determining stroke outcome. The genetic components of ischemic stroke outcome are difficult to study in humans due to environmental factors and medical intervention. Thus, we proposed to use a surgically induced, permanent, focal cerebral ischemic stroke mouse model to investigate genetic factors of ischemic stroke outcome measured by infarct volume. This model is the middle cerebral artery occlusion (MCAO) model. First, we screened infarct volumes across 32 inbred mouse strains. The infarct volume varies between strains, and this strongly suggests that infarct volume is genetically determined. To identify these genetic factors, we used genome-wide association study [Efficient Mixed-Model Association (EMMA) analysis] on infarct volume from 32 inbred mouse strains. Using the EMMA analysis, we identified 11 infarct volume-associated loci; however, most loci were mapped with missing alleles. This suggests that these loci might be false positives. Thus, we used specifically designed scripts of EMMA analysis with updated mouse SNP database to correct for potential false positives. The loci identified by the updated EMMA analyses will led us to the identification of genes involved in ischemic stroke outcome. </p><p> There are two major mechanisms were proposed to be determinants of infarct volume, the extent of native collateral circulation and neuroprotection. Using the infarct volume screening panel from 32 inbred strains, we observed that infarct volume is inversely correlated with the native collateral vessel number. However, among these inbred strains, we also observed several strains differ significantly in infarct volumes but harbor similar collateral numbers. In order to identify genetic factors determining infarct volume in a collateral-independent manner (neuroprotection), we used quantitative trait locus (QTL) mapping on mouse strains that exhibit the most difference in infarct volumes but the least difference in collateral numbers (C57BL/6J and C3H/HeJ). From the F2 B6 x C3H cross, we mapped 4 loci determining infarct volume (cerebral infarct volume QTL 4 to 7, Civq4 to Civq7). The Civq4 locus is the strongest locus (LOD 9.8) that contributes 21% of phenotypic variance in infarct volume. We also used a parallel F2 B6 x C3H cross to perform a QTL mapping on collateral vessel traits to further verify these collateral-independent loci. Among these 4 loci, the Civq4 and Civq7 loci appear to be truly collateral-independent. Based on strain-specific sequence variants and mRNA expression differences, we proposed Msr1 and Mtmr7 are the potential candidate genes of the Civq4 locus. Identification of the collateral-independent genetic factors will help to understand the genetic architecture, disease pathophysiology and potential therapeutic targets for of ischemic stroke</p> / Dissertation

Sleep disturbances and insomnia are prevalent, with around 33% of adults indicating that they experience at least one main symptom of insomnia, and bidirectional relationships exist with common psychopathology, particularly major depressive disorder (MDD). However, genetic and environmental (e.g., traumatic event exposure) contributions to the etiology of these phenotypes are not yet well understood. A genetically informative sample of approximately 12,000 Han Chinese women aged 30-60 (50% with recurrent MDD) was used to address several gaps within the sleep literature. Sleep disturbances were assessed in all individuals using a general item addressing sleeplessness (GS). A sleep within depression sum score (SDS) was also created in MDD cases, combining information from the GS and two insomnia items within MDD. A total of 11 traumatic events were assessed and additional information on childhood sexual abuse (CSA) was also obtained. First, factor analyses were conducted to determine trauma factor structure. The best-fit solution included 3 factors: interpersonal, child interpersonal, and non-assaultive, and composite variables were constructed accordingly. A series of hierarchical regressions were run to examine differential effects of trauma type and timing on sleeplessness. All traumatic events predicted sleeplessness at similar magnitudes, although population models indicated that childhood interpersonal trauma may be particularly potent. An association between CSA and sleeplessness was also replicated. A series of genetic analyses demonstrated that the single nucleotide polymorphism-based heritability of sleep phenotypes did not differ significantly from zero. Further, association analyses did not identify any genome-wide significant loci. However, using a liberal false discovery rate threshold of 0.5, two genes of interest, KCNK9 and ALDH1A2, emerged for the SDS. Polygenic risk score (PRS) analyses demonstrated genetic overlap between the SDS in MDD cases and GS in MDD controls, with PRSs explaining 0.2-0.3% of the variance. A final combined model of both genetic and environmental risk indicated that both PRS and traumatic events were significant predictors of sleeplessness. While genetic results should be interpreted with caution given the lack of heritability, additional research into the genetic and environmental contributions to insomnia, utilizing more standardized phenotypes and properly ascertained samples, is clearly warranted.

Worldwide obesity has more than doubled since 1980 and at least 2.8 million people die each year as a result of being overweight or obese. An elevated body weight is the result of the interplay between susceptibility gene variants and an obesogenic environment, and recent evidence shows that epigenetic processes are likely involved. The growing availability of high-throughput technologies has made it possible to assess quickly the entire epigenome of large samples at a relatively low cost. As a result, vast amounts of data have been generated and researchers are now confronted to both bioinformatic and biostatistic challenges to make sense of such data in the context of obesity and its complications. In this doctoral thesis, we explored associations between the human blood methylome and obesity-associated gene variants as well as dietary fat quality and quantity. We used well described preprocessing techniques and statistical methods, along with publicly available data from consortiums and other research groups, as well as tools for pathway enrichment and chromatin state inference. We found associations between obesityassociated SNPs and methylation levels at proximal promoters and enhancers, and some of these associations were replicated in multiple tissues. We also found that contrary to dietary fat quantity, dietary fat quality associates with methylation levels in the promoter of genes involved in metabolic pathways. Then, using a gene-targeted approach, we looked at the impact of an acute environmental stress (sleep loss) on the methylation and transcription levels of circadian clock genes in skeletal muscle and adipose tissue of healthy men. We found that a single night of wakefulness can alter the epigenetic and transcriptional profile of core circadian clock genes in a tissue-specific manner. Finally, we looked at the effects of chronic maternal obesity and subsequent weight loss on the transcription of epigenetic machinery genes in the fetus and placenta of mice. We found that the transcription of epigenetic machinery genes is highly sensitive to maternal weight trajectories, and particularly those of the histone acetylation pathway. Overall, this thesis demonstrated that genetics, obesogenic environment stimuli and maternal programming impact epigenetic marks at genomic locations relevant in the pathogenesis of obesity.

Lowering LDL-cholesterol with statin therapy produces substantial reductions in cardiovascular events, and larger cholesterol reductions may produce larger benefits. Rarely, myopathy occurs with statins, especially at higher doses and in combination with certain medications. Similarly strong associations might exist between myopathy with high-dose statin regimens and genetic variants, especially those affecting blood statin levels. This study aimed to find genetic variants associated with statin-induced myopathy. A feasibility study was completed to assess whether plausible effect sizes of 5 to10-fold higher risks per genetic variants could be detected among 50-100 cases with statin-induced myopathy and to consider the best study design. A genome-wide association study was then carried out using approximately 300,000 genetic markers (and additional fine-mapping) in 85 people with definite or incipient myopathy and 90 controls, who were all taking 80mg simvastatin daily in a 12,000 participant trial of 80mg vs 20mg simvastatin daily. The cases were also compared to 2,300 additional controls who had not been exposed to intensive-dose statin therapy. Replication of the myopathy result and lipid-lowering associations were tested in a 20,000 participant trial of 40mg simvastatin daily versus placebo. The genome-wide scan yielded a single strong association (p = 4x10^-9) of myopathy with the rs4363657 single nucleotide polymorphism (SNP) located within the SLCO1B1 gene on chromosome 12. This non-coding SNP was in nearly complete linkage disequilibrium (r²=0.97) with the non-synonymous rs4149056 SNP. The population prevalence of the rs4149056 C allele was 15%, and the odds ratio for myopathy was 4.5 (95% confidence interval 2.6 to 7.7) for each copy of the C allele and 16.9 (4.7 to 61.1) for CC vs TT homozygotes. Over 60% of these myopathy cases could be attributed to the C variant. The SLCO1B1 gene encodes the organic anion transport polypeptide OATP1B1, which has been shown to regulate hepatic uptake of statins. In literature reports, rs4149056 reduced statin transport and was associated with 37% (31% to 44%) higher systemic statin acid levels per C allele. The association of rs4149056 with myopathy was replicated in the trial of 40mg simvastatin daily, which also showed that it was associated with the cholesterol-lowering effects of simvastatin. No SNPs in any other region were clearly associated with myopathy (although comparison of the myopathy cases with the 2,300 controls identified a region of chromosome 1p12 that warrants further study). This study identified common variants in SLCO1B1 that influence the risks of statin-induced myopathy substantially. Genotyping these variants may be useful for tailoring both the statin dose and safety monitoring. More generally, such studies of the genetic determinants of serious adverse reactions with other drug classes may help to improve the balance between treatment efficacy and safety.

Decades of work has led researchers to believe that risk for complex behavioral phenotypes, such as alcohol use disorders, is likely influenced by multiple genes of small effect acting in conjunction with each other and the environment. Currently, the field of psychiatric genetics is developing methodologies for the identification of genetic risk variants that predispose individuals to the development of complex behavioral disorders. Several challenges related to the complex and polygenic nature of these phenotypes, must be considered. This dissertation study attempts to address these important challenges in the context of alcohol use disorders and related phenotypes. A rich twin and family study literature has indicated that 40-70% of the variance in alcohol use disorders (AUDs) is influenced by genetics. Recent attempts to identify specific x genetic risk variants associated with AUDs have been met with limited success. Meanwhile, evidence of the moderating effects of the environment on AUDs has been mounting, providing a strong rationale for examining gene-environment interaction. In the following chapters several studies will be described that integrate established twin methodologies into gene identification projects in an effort to reduce heterogeneity (both phenotypic and genotypic), elucidate environmental constructs that moderate genetic influences, and to enhance statistical power to detect the subtle genetic influences on alcohol related phenotypes.

Background: Coronary heart disease (CHD) is one of the leading causes of death worldwide, and global mortality rates are expected to continue to rise over the coming decades. In Pakistan in particular, chronic diseases are responsible for 50% of the total disease burden. Circulating lipids are strongly and linearly associated with risk of CHD; however, despite considerable efforts to demonstrate causality, available evidence is conflicting and insufficient. Study of the underlying metabolic pathways implicated in the association between lipids and CHD would help to disentangle and elucidate these complex relationships. Objectives: The primary objectives of this dissertation were to (1) identify the genetic determinants of lipid metabolites and (2) advance understanding of the effect of perturbations in lipid metabolite levels on CHD and its risk factors. Methods: Direct infusion high-resolution mass spectrometry was performed on 5662 participants from the Pakistan Risk of Myocardial Infarction Study to obtain signals for 444 known lipid metabolites. Correlations and associations of the lipids with smoking, physical activity, circulating biomarkers, and other CHD risk factors were assessed. Genome-wide analyses were conducted to analyse the association of each lipid with over 6.7 million imputed single nucleotide polymorphisms. Functional annotation and Gaussian Graphical Modelling were used to link the variants associated with each lipid to the most likely mediating gene, discern the underlying metabolic pathways, and provide a visual representation of the genetic determinants of human metabolism. Mendelian randomisation was also implemented to examine the causal effect of lipids on risk of CHD. Results: The lipids were highly correlated with each other and with levels of major circulating lipids, and they exhibited significant associations with several CHD risk factors. There were 254 lipids that had significant associations with one or more genetic variants and 355 associations between lipids and variants, with a total of 89 sentinel variants from 23 independent loci. The analyses described in this dissertation resulted in the discovery of four novel loci, identified novel relationships between genetic variants and lipids, and revealed new biological insights into lipid metabolism. Conclusion: Analyses of lipid metabolites in large epidemiological studies can contribute to enhanced understanding of mechanisms for CHD development and identification of novel causal pathways and new therapeutic targets.

Diseases that affect dairy cattle have serious economic and animal welfare implications. The inclusion of immune response (IR) traits in breeding indices has been suggested to improve inherent animal health, and decrease the use of antimicrobials. The objectives of this research were to (1) evaluate cell-mediated (CMIR) and antibody-mediated immune responses (AMIR) on 680 Holstein cows from 58 herds across Canada, (2) estimate genetic parameters of these traits (3) examine the associations with routinely evaluated traits as well as the incidence of mastitis, (4) evaluate the correlation of natural and specific antibody and (5) perform a genome-wide association study (GWAS) to determine genetic markers associated with high or low IR. In collaboration with the Canadian Bovine Mastitis Research Network cows were immunized with both a type 1 and type 2 test antigen to stimulate CMIR and AMIR, respectively. A cutaneous delayed-type hypersensitivity test to the type 1 test antigen was used as an indicator of CMIR, and serum antibody (IgG1 and IgG2) to the type 2 test antigen as an indicator of AMIR. IR phenotypes varied significantly by cow, herd and region in Canada. Heritability estimates were moderate, 0.19 for CMIR and 0.16-0.43 for AMIR depending on time in the immunization protocol and antibody isotype. Beneficial associations between AMIR and some reproductive traits were found. Using estimated breeding values, cows were classified as high, average or low responders. High AMIR cows had significantly lower incidence rates of clinical mastitis compared to average and low cows. No difference was found when cows were classified based on CMIR. Natural antibody was not genetically correlated with specific antibody nor was it associated with mastitis. The GWAS found 198 genetic markers significantly associated with AMIR, with the majority on chromosome 23 where the major histocompatability complex is located. Other significant genes involved in IR include those associated with the complement system, interleukin 17 and tumor necrosis factor. This research confirms the benefit of identifying high IR cows and gives a glimpse of current IR profiles in Canadian Holsteins. This was the first GWAS for IR traits in dairy cattle and suggests it may be possible to include IR traits in genomic selection indices. / This research was financed by grants to B.A. Mallard from National Sciences and Engineering Research Council of Canada, Alberta Milk, Dairy Farmers of New Brunswick, Nova Scotia, Ontario and Prince Edward Island, Novalait Inc., Dairy Farmers of Canada, DairyGen council of Canadian Dairy Network, Agriculture and Agri-Food Canada, Public Health Agency of Canada, Technology PEI Inc., Université de Montréal and University of Prince Edward Island through the Canadian Bovine Mastitis Research Network. Kathleen Adele Thompson Crispi was funded by the Dairy Farmers of Ontario Doctoral Research Assistantship.

Cervical stenotic myelopathy (CSM) is an important musculoskeletal and neurologic disease of the horse. Clinical disease occurs due to malformations of the vertebrae in the neck causing stenosis of the cervical vertebral canal and subsequent spinal cord compression. The disease is multifactorial in nature, therefore a clearer understanding of the etiology and pathogenesis of CSM will allow for improved management and therapeutic practices. This thesis examines issues of equine CSM diagnosis, skeletal tissue pathology, and inherited genetic determinants utilizing advances in biomedical imaging technologies and equine genomics. Magnetic resonance imaging (MRI) data provided a more complete assessment of the cervical column through image acquisition in multiple planes. First, MRI was compared to standing cervical radiographs for detection of stenosis. Using canal area or the cord canal area ratio, MRI more accurately predicted sites of compression in CSM cases. Secondly, articular process skeletal pathology localized on MRI was found to be more frequent and severe in CSM horses compared to controls. In addition, lesions were generalized throughout the cervical column and not limited to the spinal cord compression sites. A subset of lesions identified on MRI was evaluated using micro-CT and histopathology. Osteochondrosis, osseous cyst-like structures, fibrous tissue replacement of bone, and osteosclerosis were observed. These lesions support likely developmental aberrations of vertebral bone and cartilage maturation with secondary biomechanical influences. Bone cyst-like structures are a novel finding in this disease. Finally, the long-standing question of the contribution of genetic determinants to CSM was investigated using a genome wide association study (GWAS). Multiple significant loci were identified supporting the influence of a complex genetic trait in clinical disease. A simple Mendelian trait controlled by one gene is unlikely given the detection of variants across multiple chromosomes. Major contributions from this research include documentation of articular process bone and cartilage pathology in horses with CSM, support for abnormal cervical vertebrae development being an important contributing factor in the etiology and/or pathogenesis of equine CSM, and evidence that multiple genetic loci contribute to the CSM disease phenotype.

The release of the Illumina BovineSNP50 BeadChip in late 2007 has drawn attention from cattle breeders around the world to develop breeding programs that leverage association of these single nucleotide polymorphism (SNP) with economically important quantitative trait loci (QTL). In that context this project has come to study applications of the SNP panel in beef cattle. Analysis of linkage disequilibrium (LD) existing in Angus, Charolais, and crossbred animals revealed the pattern of LD within each breed group, as well as the persistence of LD phase between pairs of the breed groups. This is important for genomic selection where SNP are trained in one population and used to predict breeding value for animals in another population. Detection of chromosome regions potentially carrying QTL or causative mutations affecting the phenotypic variation in economically important traits was presented at individual SNP and haplotype levels. There were 269 SNP associated (P<0.001) with birth weight (BWT), weaning weight (WWT), average daily gain (ADG), dry matter intake (DMI), mid-test metabolic weight (MMWT), residual feed intake (RFI). They explained 1.64% - 8.06% of the phenotypic variation in these traits. There were 520 SNP associated (P<0.001) with carcass quality traits, namely hot carcass weight, back fat thickness, ribeye area, marbling scores, lean yield grade by Beef Improvement Federation, steak tenderness, and six rib dissection traits. These SNP explained 1.90 - 5.89% of the phenotypic variance of the traits. Many of the significant SNP were located on chromosome 6. Six haplotypes were found associated (P<0.05) with ADG, DMI, and RFI. In order for genomic selection to happen in beef cattle, higher density SNP panels should be made available at low genotyping cost. However, the cost of genotyping animals for high density SNP chip is still high, thus genotype imputation has come to practice. The last chapter of this thesis compared two approaches presently used in genotype imputation, investigated factors affecting imputation accuracy, as well as the impact of imputation accuracy on genomic estimated breeding value (GEBV). It proved that the highest possible accuracy of GEBV is attainable with sufficiently large groups of reference animals. / Ontario Ministry of Agriculture, Food and Rural Affairs. Ontario Cattlemen’s Association. Ontario Farm Innovation Program. Agriculture and Agri-Food Canada’s Growing Forward Program. Agriculture Adaptation Council. Ontario Research and Development Program. MITACS Accelerate. Beef Improvement Opportunities.