Yes / Drug resistance diagnostics that rely on the detection of resistance-related mutations could expedite patient care and TB eradication. We perform minimum inhibitory concentration testing for 12 anti-TB drugs together with Illumina whole-genome sequencing on 1452 clinical Mycobacterium tuberculosis (MTB) isolates. We evaluate genome-wide associations between mutations in MTB genes or non-coding regions and resistance, followed by validation in an independent data set of 792 patient isolates. We confirm associations at 13 non-canonical loci, with two involving non-coding regions. Promoter mutations are measured to have smaller average effects on resistance than gene body mutations. We estimate the heritability of the resistance phenotype to 11 anti-TB drugs and identify a lower than expected contribution from known resistance genes. This study highlights the complexity of the genomic mechanisms associated with the MTB resistance phenotype, including the relatively large number of potentially causal loci, and emphasizes the contribution of the non-coding portion of the genome. / Biomedical research grant from the American Lung Association (PI MF, RG-270912-N), a K01 award from the BD2K initiative (PI MF, ES026835), and an NIAID U19 CETR grant (P.I. M.M., AI109755), the Belgian Science Policy (Belspo) (L.R., C.J.M.).

No description available.

Poliomyelitis is a foremost cause of paralysis among preventable diseases among children and adolescents globally. It is caused by persistent infection with poliovirus (PV). The PV infection does not always cause paralysis. A lack of immunization always increases the risk of paralytic polio. Genetic factors also been shown to affect the risk of developing the disease. The aim of this thesis is to investigate whether there are any genetic associations to paralytic poliomyelitis. This is based on a model for understanding its nature as a complex disease, where many genes are involved in contributing to the disease state. This is a population-based case-control study to identify genetic loci that influence disease risk. The study examined the association of genetic variation in single nucleotide polymorphisms (SNPs) across the genome with paralytic poliomyelitis susceptibility in the United States and Canadian survivors of poliomyelitis population, using a genome-wide association study (GWAS) approach. No association was observed. Loci that have been previously implicated were not found to affect the susceptibility to poliomyelitis in this study. The thesis consists of four chapters. Chapter 1 describes the epidemiology, pathogenesis and management of poliomyelitis. Chapter 2 gives an overview of the genomics of infectious diseases in general. Chapter 3 introduces the study population and presents the genome-wide analysis and associations with logistic regression to identify loci explore genes that might be associated with paralytic poliomyelitis and presents results. Chapter 4 discusses the implications of the results and explains future directions. / Thesis / Master of Science (MSc)

Hedgehog (Hh) est un morphogène conservé au cours de l’évolution, et qui est impliqué dans un grand nombre de processus développementaux. Ma thèse vise à comprendre comment la sécrétion, et la mise en place du gradient d’Hh sont régulées à partir de son site de production, en utilisant la drosophile comme modèle animal. Pour identifier les régulateurs positifs impliqués dans la maturation du signal Hh, nous avons conçu et réalisé un crible génétique couvrant l’ensemble du génome, par ARNs interférents (ARNi). Grâce à ce crible, nous avons identifié la petite protéine GTPase Rab8 qui serait impliquée spécifiquement dans le routage intracellulaire de Hh. Selon notre modèle proposé, la protéine Hh serait secrétée de 2 façons. Sa sécrétion du coté apical est nécessaire à l'activation de gènes cibles à longue distance, alors que sa sécrétion du coté baso-latéral permettrait l'activation de gènes cibles à courte distance. La façon par laquelle Hh est transportée de la membrane apicale à la membrane basale à l’intérieur des cellules productrices n’est pas connue. La perte de fonction de Rab8 dans les cellules productrices de Hh induit une augmentation de l’activation des gènes cibles à courtes distances, alors l’expression des gènes cibles activés à longues distances est réduite. De plus, en utilisant des expériences sur tissus vivant pour suivre la dynamique de l'internalisation de la protéine endogène d’Hh, nous avons constaté que la perte de Rab8 n'a pas d’effet sur sa sécrétion primaire, mais entraine des défauts dans l’endocytose de Hh, affectant, par la suite, la mise en place du gradient morphogénétique. / Hedgehog (Hh) is a conserved secreted morphogen involved in an array of developmental processes. Using Drosophila as a model, during my thesis we aimed to ask how the secretion, extraction and transport of Hh protein are regulated at the site of its production. To understand the positive regulators of Hh secretion and transport we designed and performed a genome-wide RNAi screen in Drosophila to identify new regulators of Hh transport and identified the small GTPase Rab8 as a novel component required for Hh trafficking. According to our proposed model, there are two pools of secreted Hh. The apical pool is needed for long range target gene activation, and basolateral pool for short range target gene activation. It is not clear how Hh is sorted apico-basally in the producing cells. Interfering with Rab8 function in the Hh producing cells extends Hh short range targets. Conversely, it reduces the long range Hh targets, suggesting that interfering with Rab8 function in the Hh producing cells impairs Hh trafficking, thus hampering the fine tuning between the two secreted pools of Hh. Moreover, using live assays to track the dynamics of endogenous Hh internalization, we observed that loss of Rab8 in Hh producing cells does not affect its primary secretion, but causes defects in Hh endocytosis, subsequently affecting its gradient activity. We hypothesize a model where Hh is targeted for primary secretion to the apical side of the wing disc, which then is internalized, and this internalized Hh is then directed for recycling which is essential for its long range activity.

Copy number variation (CNV) is an important polymorphism that is associated with a wide range of traits in human, wild and livestock species. In chicken, an important source of animal protein and a developmental model organism, CNV is associated with several phenotypes and evolutionary footprints. However, identification and characterization of CNV inheritance on chicken genome lacks further investigation. We screened CNVs in chicken using two distinct populations with known pedigree. In 826 broilers we identified 25,819 CNVs (4,299 deletions and 21,520 duplications) of which 21,077 were inherited, 201 showed no inheritance and 4,541 were classified as de novo CNVs. In 514 F2 animals (layer and broiler cross) we identified 21,796 CNVs (2,254 deletions and 19,543 duplications) of which 18,230 were inherited, 587 not inherited and 2,979 were classified as de novo CNVs. After a strict filtering step to remove potential false positives and negative CNVs, only 220 (4.84%) and 430 (14.43%) de novo CNVs remained in the broiler and F2 populations, respectively. A total of 33.11% (50 out of 151) of the inherited CNVs identified in ten animals were validated by sequencing data. From the validated CNVs, 64% had more than 80% of their size (bp) validated. A total of 59% and 48.8% were classified as novel CNVs regions (CNVRs) in the broiler and F2, respectively. Considering the Bonferroni-corrected p-values for multiple testing and statistically significant p-values ≤ 0.01, we found two CNV segments significantly associated with breast weight, one with breast weight yield, six with breast meat weight, 18 CNV segments with breast meat yield, four with breast filet weight and two with breast yield. These CNV segments that were significantly associated overlapped with 181 protein-coding genes. The CNVseg 300, that was associated with all traits and encompass six CNVRs, overlapped a total of 26 protein-coding genes. Among these genes, the gene MYL1 (Myosin Light Chain 1) is expressed in the fast skeletal muscle fibers, and the genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) and RAB17 (Member RAS Oncogene Family), that were associated with the lavender phenotype (feather blue-grey color) and regulation of homeothermy and the metabolism. The present study improves our knowledge about CNV in the chicken genome and provides insight in the distribution and of different classes of CNVs, i.e. inherited and de novo CNVs, in two experimental chicken populations. In addition, the genome-wide association analyses were the first performed on broiler population with breast muscle traits, that are important characteristics for poultry production. The GWAS results allow us to understand the probably relationship between some genes and CNVRs that are significantly associated with breast muscle traits. / A variação de número de cópias (CNV) é um polimorfismo importante que está associado a uma ampla gama de características em seres humanos, espécies selvagens e domésticas. Em frango, que é uma importante fonte de proteína e considerado um modelo biológico, CNVs foram associados a vários fenótipos e passos evolutivos. No entanto, nenhum estudo foi realizado para a identificação e caracterização da herança da CNV no genoma da galinha. Identificamos as CNVs no genoma da galinha usando duas populações experimentais e com pedigree conhecido: uma população de frangos de corte e uma F2. Em 826 frangos de corte, identificamos 25.819 CNVs (4.299 deleções e 21.520 duplicações), dos quais 21.077 foram herdados, 201 não foram herdados e 4.541 foram CNVs denominados de novo. Em 514 animais F2, identificamos 21.796 CNVs (2.254 deleções e 19.543 duplicações) das quais 18.230 foram herdadas, 587 não foram herdadas e 2.979 foram de novo CNVs. Após a etapa de filtragem nos de novo CNVs, apenas 220 (4,84%) e 430 (14,43%) permaneceram nas populações de frango de corte e F2, respectivamente. Um total de 33,11% (50 de 151) das CNV identificadas por dados de genotipagem em dez animais foram validados por dados de sequenciamento. Dos validados, 64% tinham mais de 80% do tamanho (pb) validados. Um total de 59% e 48,8% foram classificados como novas regiões de CNVs (CNVRs) nas populações de frango de corte e F2, respectivamente. Considerando os p-values corrigidos por Bonferroni para testes múltiplos e estatisticamente significativos (≤ 0,01), encontramos dois segmentos de CNV significativamente associados ao peso do peito, um ao rendimento de peso de peito, seis ao peso de carne de peito, 18 ao rendimento de carne de peito, quatro ao peso de filé de peito e dois ao rendimento do filé de peito. Esses segmentos de CNV significativamente associados estão sobrepostos com 181 genes codificadores de proteínas. O CNVseg 300, que foi associado a todas as características e abrange seis CNVRs, foram sobrepostos a um total de 26 genes codificadores de proteínas. Entre estes genes, o gene MYL1 (Myosin Light Chain 1) é expresso nas fibras rápidas do músculo esquelético, e os genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) e RAB17 (Member RAS Oncogene Family), que foram anteiromente associados ao fenótipo de cor azul acinzentado de penas e à regulação da homeotermia e do metabolismo. O presente estudo melhora o conhecimento sobre CNVs no genoma de frango, especialmente sobre a distribuição de CNV herdadas, não herdadas e de novo, em duas populações experimentais de frango. Além disso, a associação genômica foi a primeira realizada na população de frangos de corte com características do músculo do peito, que são muito importantes para a avicultura. Os resultados do GWAS nos permitem compreender a provável relação entre alguns genes e CNVRs que foram significativamente associados às características do músculo do peito.

Copy number variation (CNV) is an important polymorphism that is associated with a wide range of traits in human, wild and livestock species. In chicken, an important source of animal protein and a developmental model organism, CNV is associated with several phenotypes and evolutionary footprints. However, identification and characterization of CNV inheritance on chicken genome lacks further investigation. We screened CNVs in chicken using two distinct populations with known pedigree. In 826 broilers we identified 25,819 CNVs (4,299 deletions and 21,520 duplications) of which 21,077 were inherited, 201 showed no inheritance and 4,541 were classified as de novo CNVs. In 514 F2 animals (layer and broiler cross) we identified 21,796 CNVs (2,254 deletions and 19,543 duplications) of which 18,230 were inherited, 587 not inherited and 2,979 were classified as de novo CNVs. After a strict filtering step to remove potential false positives and negative CNVs, only 220 (4.84%) and 430 (14.43%) de novo CNVs remained in the broiler and F2 populations, respectively. A total of 33.11% (50 out of 151) of the inherited CNVs identified in ten animals were validated by sequencing data. From the validated CNVs, 64% had more than 80% of their size (bp) validated. A total of 59% and 48.8% were classified as novel CNVs regions (CNVRs) in the broiler and F2, respectively. Considering the Bonferroni-corrected p-values for multiple testing and statistically significant p-values ≤ 0.01, we found two CNV segments significantly associated with breast weight, one with breast weight yield, six with breast meat weight, 18 CNV segments with breast meat yield, four with breast filet weight and two with breast yield. These CNV segments that were significantly associated overlapped with 181 protein-coding genes. The CNVseg 300, that was associated with all traits and encompass six CNVRs, overlapped a total of 26 protein-coding genes. Among these genes, the gene MYL1 (Myosin Light Chain 1) is expressed in the fast skeletal muscle fibers, and the genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) and RAB17 (Member RAS Oncogene Family), that were associated with the lavender phenotype (feather blue-grey color) and regulation of homeothermy and the metabolism. The present study improves our knowledge about CNV in the chicken genome and provides insight in the distribution and of different classes of CNVs, i.e. inherited and de novo CNVs, in two experimental chicken populations. In addition, the genome-wide association analyses were the first performed on broiler population with breast muscle traits, that are important characteristics for poultry production. The GWAS results allow us to understand the probably relationship between some genes and CNVRs that are significantly associated with breast muscle traits. / A variação de número de cópias (CNV) é um polimorfismo importante que está associado a uma ampla gama de características em seres humanos, espécies selvagens e domésticas. Em frango, que é uma importante fonte de proteína e considerado um modelo biológico, CNVs foram associados a vários fenótipos e passos evolutivos. No entanto, nenhum estudo foi realizado para a identificação e caracterização da herança da CNV no genoma da galinha. Identificamos as CNVs no genoma da galinha usando duas populações experimentais e com pedigree conhecido: uma população de frangos de corte e uma F2. Em 826 frangos de corte, identificamos 25.819 CNVs (4.299 deleções e 21.520 duplicações), dos quais 21.077 foram herdados, 201 não foram herdados e 4.541 foram CNVs denominados de novo. Em 514 animais F2, identificamos 21.796 CNVs (2.254 deleções e 19.543 duplicações) das quais 18.230 foram herdadas, 587 não foram herdadas e 2.979 foram de novo CNVs. Após a etapa de filtragem nos de novo CNVs, apenas 220 (4,84%) e 430 (14,43%) permaneceram nas populações de frango de corte e F2, respectivamente. Um total de 33,11% (50 de 151) das CNV identificadas por dados de genotipagem em dez animais foram validados por dados de sequenciamento. Dos validados, 64% tinham mais de 80% do tamanho (pb) validados. Um total de 59% e 48,8% foram classificados como novas regiões de CNVs (CNVRs) nas populações de frango de corte e F2, respectivamente. Considerando os p-values corrigidos por Bonferroni para testes múltiplos e estatisticamente significativos (≤ 0,01), encontramos dois segmentos de CNV significativamente associados ao peso do peito, um ao rendimento de peso de peito, seis ao peso de carne de peito, 18 ao rendimento de carne de peito, quatro ao peso de filé de peito e dois ao rendimento do filé de peito. Esses segmentos de CNV significativamente associados estão sobrepostos com 181 genes codificadores de proteínas. O CNVseg 300, que foi associado a todas as características e abrange seis CNVRs, foram sobrepostos a um total de 26 genes codificadores de proteínas. Entre estes genes, o gene MYL1 (Myosin Light Chain 1) é expresso nas fibras rápidas do músculo esquelético, e os genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) e RAB17 (Member RAS Oncogene Family), que foram anteiromente associados ao fenótipo de cor azul acinzentado de penas e à regulação da homeotermia e do metabolismo. O presente estudo melhora o conhecimento sobre CNVs no genoma de frango, especialmente sobre a distribuição de CNV herdadas, não herdadas e de novo, em duas populações experimentais de frango. Além disso, a associação genômica foi a primeira realizada na população de frangos de corte com características do músculo do peito, que são muito importantes para a avicultura. Os resultados do GWAS nos permitem compreender a provável relação entre alguns genes e CNVRs que foram significativamente associados às características do músculo do peito.

Major Depressive Disorder (MDD) is a complex and chronic disease that ranks fourth as cause of disability worldwide. Thirteen to 14 million adults in the U.S. are believed to have MDD and an estimated 75% attempt suicide making MDD a major public health problem. Recently several genome-wide association (GWA) studies of MDD have been reported; however, few GWA studies focus on the analysis for MDD related phenotypes such as neuroticism and age at onset of MDD. The purpose of this study is to determine risk factors for MDD, identify genome-wide genetic variants affecting neuroticism and age at onset as quantitative traits, and detect gender differences influencing neuroticism. Bivariate and multiple logistic regression analyses were performed on 1,738 MDD cases and 1,618 non-MDD controls to determine phenotypic risk factors for MDD. Multiple linear regression analyses in PLINK software were used for GWA analyses for neuroticism and age at onset of MDD with 437,547 Single Nucleotide Polymorphisms (SNPs). Gender (OR: 1.43; 95% CI: 1.24 - 1.64) and a family history (OR: 2.88; 95% CI: 2.48 - 3.35) were significantly associated with an increased risk of MDD, which supports the findings of prior studies. Through GWA analysis 34 SNPs were identified to be associated with neuroticism (p < 10-4). The best SNP was rs4806846 within the TMPRSS9 gene (p = 7.79 x10-6). Furthermore, 46 SNPs were found showing significant gene x gender interactions for neuroticism with p<10-4. The best SNP showing gene x gender interaction was rs2430132 (p = 5.37x10-6) in HMCN1 gene. In addition, GWA analysis showed that several SNPs within 4 genes (GPR143, ASS1P4, MXRA5 and MAGEC1/2) were significantly associated with age at onset of MDD (p < 5x10-7). This study confirmed previous findings that MDD is associated with an increased prevalence in women (about 43% more compared to men) and is highly heritable among first degree relatives. Several novel genetic loci were identified to be associated with neuroticism and age at onset. Gender differences were found in genetic influence of neuroticism. These findings offer the potential for new insights into the pathogenesis of MDD.

The purpose of this dissertation is to develop new statistical tools for mining big data in plant sciences. In particular, the dissertation consists of four inter-related projects to address various methodological and computational challenges in phylogenetic methods. Project 1 aims to systematically test different optimization tools and provide useful strategies to improve optimization in practice. Project 2 develops a new R package rPlant, which provides a friendly and convenient toolbox for users of iPlant. Project 3 presents a fast and effective group-screening method to identify important genetic factors in GWAS, with theoretical justifications and nice asymptotic properties. Project 4 develops a new statistical tool to identify gene-gene interactions, with the ability of handling the interactions between groups of covariates.

Despite many years of effort, linkage and candidate gene association studies have yielded disappointingly few risk loci for common human diseases such as diabetes, auto-immune disorders and cancers. Large sample sizes, increased understanding of the patterns of correlation in genetic variation, and plunging genotyping costs have enabled genome-wide association studies, which have good power to detect common risk alleles of modest effect. I present an evaluation of SNP choice in study design and show that overall, despite substantial differences in genotyping technologies, marker selection strategies and number of markers assayed, the first generation platforms all offer good levels of genome coverage (∼ 70%). I next describe the largest such project undertaken to date, the Wellcome Trust Case Control Consortium, which consisted of 2000 cases from each of seven common diseases and 3000 shared controls. It identified nearly two dozen new associations. I demonstrate the importance of careful data quality control, including both standard and unorthodox analyses. I next focus on the association results therein for Crohn’s disease. I present a replication experiment in over 1000 additional Crohn’s patients which unambiguously confirmed six previously published loci and four new loci. Next I describe, in a general context, several issues impeding the combination of genome-wide scans, including data annotation, population structure and differences in genotyping platform. Each of these problems is shown to be tractable with available methods, provided that these methods are applied prudently. I present the results of a meta-analysis of three genome-wide scans for Crohn’s disease. The data showed a striking excess of significant associations, and a replication experiment involving over 4000 independent Crohn’s patients verified twenty new risk loci. Finally, I discuss the early success of genome-wide association and its consequences for further understanding the biology of human disease.

Of central importance in the dissection of the components that govern complex traits is understanding the architecture of natural genetic variation. Genetic interaction, or epistasis, constitutes one aspect of this, but epistatic analysis has been largely avoided in genome wide association studies because of statistical and computational difficulties. This thesis explores both issues in the context of two-locus interactions. Initially, through simulation and deterministic calculations it was demonstrated that not only can epistasis maintain deleterious mutations at intermediate frequencies when under selection, but that it may also have a role in the maintenance of additive variance. Based on the epistatic patterns that are evolutionarily persistent, and the frequencies at which they are maintained, it was shown that exhaustive two dimensional search strategies are the most powerful approaches for uncovering both additive variance and the other genetic variance components that are co-precipitated. However, while these simulations demonstrate encouraging statistical benefits, two dimensional searches are often computationally prohibitive, particularly with the marker densities and sample sizes that are typical of genome wide association studies. To address this issue different software implementations were developed to parallelise the two dimensional triangular search grid across various types of high performance computing hardware. Of these, particularly effective was using the massively-multi-core architecture of consumer level graphics cards. While the performance will continue to improve as hardware improves, at the time of testing the speed was 2-3 orders of magnitude faster than CPU based software solutions that are in current use. Not only does this software enable epistatic scans to be performed routinely at minimal cost, but it is now feasible to empirically explore the false discovery rates introduced by the high dimensionality of multiple testing. Through permutation analysis it was shown that the significance threshold for epistatic searches is a function of both marker density and population sample size, and that because of the correlation structure that exists between tests the threshold estimates currently used are overly stringent. Although the relaxed threshold estimates constitute an improvement in the power of two dimensional searches, detection is still most likely limited to relatively large genetic effects. Through direct calculation it was shown that, in contrast to the additive case where the decay of estimated genetic variance was proportional to falling linkage disequilibrium between causal variants and observed markers, for epistasis this decay was exponential. One way to rescue poorly captured causal variants is to parameterise association tests using haplotypes rather than single markers. A novel statistical method that uses a regularised parameter selection procedure on two locus haplotypes was developed, and through extensive simulations it can be shown that it delivers a substantial gain in power over single marker based tests. Ultimately, this thesis seeks to demonstrate that many of the obstacles in epistatic analysis can be ameliorated, and with the current abundance of genomic data gathered by the scientific community direct search may be a viable method to qualify the importance of epistasis.