Genetic association studies have successfully identified many genetic markers associated with complex human diseases and related quantitative traits. However, for most complex diseases and quantitative traits, all associated genetic markers identified to date only explain a small proportion of heritability. Thus, exploring the unexplained heritability in these traits will help us discover novel genetic determinants for these traits and better understand disease etiology and pathophysiology. Due to limited sample size, a single cohort study may not have sufficient power to identify novel genetic association with a small effect size, and meta-analysis approaches have been proposed and applied to combine results from multiple cohorts in large consortia, increasing the sample size and statistical power. Rare genetic variants and gene by environment interaction may both play a role in genetic association studies. In this dissertation, we develop statistical methods in meta-analysis, rare genetic variants analysis and gene by environment interaction analysis, conduct extensive simulation studies, and apply these methods in real data examples. First, we develop a method of moments estimator for the between-study covariance matrix in random effects model multivariate meta-analysis. Our estimator is the first such estimator in matrix form, and holds the invariance property to linear transformations. It has similar performance with existing methods in simulation studies and real data analysis. Next, we extend the Sequence Kernel Association Test (SKAT), a rare genetic variants analysis approach for unrelated individuals, to be applicable in family samples for quantitative traits. The extension is necessary, as the original test has inflated type I error when directly applied to related individuals, and selecting an unrelated subset from family samples reduces the sample size and power. Finally, we derive methods for rare genetic variants analysis in detecting gene by environment interaction on quantitative traits, in the context of univariate test on the interaction term parameter. We develop statistical tests in the settings of both burden test and SKAT, for both unrelated and related individuals. Our methods are relevant to genetic association studies, and we hope that they can facilitate research in this field and beyond.

Immune thrombocytopenia (ITP) is the consequence of a complex, still incompletely understood immunological dysregulation. Proposed mechanisms include autoantibody-induced platelet destruction, impaired platelet production as well as abnormalities in T-cell immunity, such as T helper cells (Th1) polarization, a high proportion of Th17 cells, and a reduced number of regulatory T cells. Although the etiology of ITP is incompletely understood and considered multifactorial in most cases, genetic variants are thought to play a key role in susceptibility to ITP, especially in persistent or chronic ITP. Efforts are currently underway to uncover possible predisposing genetic factors for the development of ITP. Single-nucleotide polymorphisms and copy number variations have been identified in several immune-related genes, such as cytokine genes, Fcγ receptor genes or T-cell costimulation genes, and have been associated with patients’ susceptibility to ITP. However, because of the clinical heterogeneity and low incidence of ITP it remains challenging to perform genetic analyses with sufficiently large sample size within informative patient populations, highlighting the need for collection of well-annotated biomaterials in clinical trials or registry projects. Another significant challenge is to go beyond performing association studies alone and to establish genotype-phenotype associations, thus proving causality between a genetic alteration and ITP pathogenesis. This review summarizes our current knowledge on genetic alterations identified as potential predisposing factors for the development of ITP in adults, thereby addressing signaling pathways considered critical for ITP pathogenesis.

Introdução: Em diversos países, inclusive no Brasil, a fortificação de alimentos com ácido fólico (AF) foi adotada como política pública de prevenção e combate à deficiência nutricional da vitamina, motivados principalmente pela redução da incidência dos defeitos do tubo neural. No período pós-fortificação observa-se tanto a evolução positiva do consumo e nível sérico da vitamina quanto a diminuição da concentração plasmática de homocisteína, e ainda, o aumento do ácido fólico não metabolizado na maioria desses países. Não se conhece ainda os efeitos biológicos do AFNM, no entanto, considera-se que o AFNM pode ser um fator relevante nas questões de segurança associadas com alto consumo de AF. Objetivo: Avaliar o consumo dietético e nível de folato, homocisteína e ácido fólico não metabolizado após a fortificação mandatória de alimentos com ácido fólico, e a interação com os polimorfismos envolvidos no metabolismo do folato e homocisteína. Metodologia: Os dados foram oriundos do estudo transversal de base populacional ISA Capital 2008 conduzido em uma amostra representativa de residentes do município de São Paulo, de ambos os sexos, e com idade acima de 14 anos. Coletou-se recordatórios alimentares de 24 horas e amostra de sangue em jejum de 12 horas para análises bioquímicas e moleculares. As análises estatísticas foram realizadas no programa STATA®, versão 13.0, com nível de significância de 5 por cento . Resultados: O estudo foi conduzido em 750 indivíduos, sendo 53,1 por cento mulheres e média de idade 40,7 (IC95 por cento 38,8-42,5) anos. A média de consumo e nível de folato foi de 375,8 (IC95 por cento 363,0-388,6) mcg/dia e 13 (IC95 por cento 12,0-13,9) ng/ml, respectivamente. Apenas 1,76 por cento da população apresentou deficiência de folato (<3ng/ml). Foi 5 detectado AFNM em 79,8 por cento da população com média plasmática de 2,4 (IC95 por cento 2,1-2,7) ng/ml. No modelo linear generalizado para previsão de AFNM, nível de folato (p<0,001), idade (p<0,001), tabagismo (p=0,002), raça (p<0,001) e concentrações de B6 (p<0,001), além disso, a interação de homozigotos e heterozigotos para a deleção de pares de base da DHFR e nível de folato (p=0,003) foram efeitos significantes. Tem-se um aumento relativo esperado de 3 por cento no AFNM se aumentarmos em 1 ng/ml o nível de folato médio, considerando fixas as demais variáveis do modelo. Conclusão: Nota-se baixa prevalência da deficiência da vitamina e alta prevalência dos níveis detectáveis de AFNM na população decorrente do aumento do nível de folato. Todavia, apesar do aparente sucesso da fortificação de alimentos com ácido fólico, a comunidade científica não é unânime na aprovação de políticas de fortificação mandatória e do uso indiscriminado de suplementos. Recomenda-se um monitoramento constante para evitar que a população seja exposta a altas doses da vitamina, e consequentemente, efeitos adversos à saúde. / Introduction: Food fortification is an important strategy in public health policy for controlling micronutrient malnutrition, and a major contributory factor in the eradication of micronutrients deficiencies. Motivated by the reduction in the occurrence of neural tube defects, countries worldwide, including Brazil, adopted food fortification with folic acid (FA). Folic acid fortification has increased dietary intakes of folic acid and folate status, but it is also associated with the presence of circulating FA. Although the metabolism and biological effects of circulating of folic acid are not well known, it may be a contributing factor in safety concerns associated with high oral doses of folic acid. Objective: To assess the folate intake and status, homocysteine and circulating FA after mandatory fortification with folic acid, and interaction with polymorphisms involved in 1-carbon metabolism. Material and Methods: Data were from 750 individuals aged > 14 years old who participated of a cross-sectional population-based survey in Sao Paulo City-Brazil. Fasting blood samples and information about food intake based on two measures of 24 hour food recall were collected. All analyses were carried out using STATA (version 13.0) and p-value <.05 was considered to be statistically significant in all tests. Results: Results were from 750 individuals. Women accounted for 53.1 per cent of the population and average age was 40.7 (IC95 per cent 38.8-42.5) years. The overall mean folate intake and folate concentration were 375.8 (95 per cent CI: 363.0-388.6) mcg/day of DFE and 13 (95 per cent CI: 12-14) ng/mL, respectively. Only 1.76 per cent of 7 population had folate deficiency (<3 ng/mL). Circulating FA was detected in 79.8 per cent of the population with a mean concentration of 2.4 (95 per cent CI: 2.1-2.7) pmol/ml. Effects of total folate concentration (p<0.001), age (p<0.001), current smoker (p=0.002), race (p<0.000) and vitamin B6 (p<0.001) as well as interaction between folate concentration and 19-base pair deletion polymorphism in DHFR (p=0.003) were found in the model to predict the circulating FA. An increase of one ng/mL in folate concentrate was associated with increased of 3 per cent in circulating FA. Conclusions: There is low prevalence of folate deficiency and high prevalence of detectable levels of circulating FA the population. The fortification effectively increased folate status and folic acid intake, and had a notable influence on rankings of food contributors of folate intake. Although the success of folic acid fortification was observed in many countries, the scientific community is not unanimous in approval of mandatory fortification policies and the indiscriminate use of supplements. The monitoring is recommended to guarantee the safety of exposure to folic acid, and avoiding adverse health effects.

De façon physiologique, la voie alterne (VA) du complément est activée en permanence. Elle doit donc être finement régulée à tous les niveaux de la cascade afin de ne pas être délétère pour l'hôte. Chez l'homme, deux pathologies rénales sont associées à une activation non contrôlée de la VA du complément: le syndrome hémolytique et urémique atypique et la glomérulopathie à dépôts de C3 (GP-C3). Pathologie du sujet jeune essentiellement, la GP-C3 regroupe deux entités, la maladie des dépôts denses (GN-DD) et la glomérulonéphrite à dépôts de C3 (GN-C3), d'expressions clinique et histologique hétérogènes et toutes les deux de pronostic rénal réservé en l’absence de traitement efficace disponible (médiane de survie rénale de l’ordre de 8 à 10 ans). Les anomalies de la VA du complément mises en évidence dans la GP-C3 sont essentiellement acquises en rapport avec la présence d'auto anticorps (Ac) dirigés contre la C3 convertase alterne, le C3NeF, ou contre le FH, une protéine régulatrice essentielle de la VA. Dans 15 à 20% des cas seulement, les anomalies sont génétiques en rapport avec des mutations du FH, du FI. Les approches in silico de modélisation moléculaire des protéines mutées apportent des éléments de réponse quant à la responsabilité potentielle d'une mutation dans l’activation non contrôlée de la VA, néanmoins, l'étude des conséquences fonctionnelles est indispensable pour relier l'anomalie à la survenue de la pathologie rénale mais aussi pour comprendre les mécanismes précis impliqués dans le déterminisme des lésions rénales, de GN-DD ou de GN-C3. J'ai dans un premier temps étudié les conséquences fonctionnelles de la première mutation de C3 identifiée dans une forme familiale de GN-C3. In silico, cette mutation I734T est située au niveau d'un site impliqué dans la liaison de C3 avec deux protéines régulatrices, le FH et CR1 et à proximité du site d’interaction avec le FB. In vitro, j'ai pu confirmer que la mutation était responsable d'une activation de la VA au niveau tissulaire en rapport avec un défaut induit de régulation par le FH mais surtout CR1, une protéine régulatrice membranaire, identifié pour la première fois comme acteur dans la physiopathologie de la GP-C3. Par ailleurs, en raison de l'expression exclusivement podocytaire de CR1 au niveau glomérulaire, nous avons émis l'hypothèse que les régulateurs pouvaient jouer un rôle déterminant dans la localisation préférentielle des dépôts de C3 au niveau glomérulaire dans cette pathologie. Dans une deuxième partie, à la fois clinique et expérimentale, j'ai travaillé sur les formes acquises de GP-C3 en étudiant un sous-groupe particulier de patients de la cohorte française de GP-C3, âgés de plus de 50 ans et présentant une gammapathie monoclonale. La partie clinique de ce travail a permis de démontrer que la fréquence des gammapathies monoclonales est très nettement augmentée dans la GP-C3 chez les patients âgés de plus de 50 ans comparée à la population générale, que le pronostic rénal est particulièrement sombre avec une médiane de survie rénale de l’ordre de 2 ans et demi mais que le traitement efficace du clone B sous-jacent permet d'améliorer significativement la survie rénale. Les données de l’étude clinique suggèrent indirectement qu’il existe un lien entre l'immunoglobuline (Ig) monoclonale et l'activation de la VA responsable de l'apparition des lésions rénales. Dans la partie expérimentale, j'ai étudié les mécanismes d'activation de la VA dans ce contexte de gammapathie afin de confronter les données de l’étude clinique. L'étude des biomarqueurs d'activation de la C3/C5 convertase et le démembrement des mécanismes d'activation ont permis de conclure que la GP-C3 associée aux gammapathies monoclonales est caractérisée par une activation tissulaire de la VA impliquant la C3 convertase mais aussi et surtout la C5 convertase. (...) / Complement alternative pathway is physiologically activated. It need to be tightly regulated to avaoid uncontrolled deleterious overactivation on host cell surface. In human, two renal diseaes are associated with uncontrolled AP activation, hemolytic uremic syndrom atypical (aHUS) and C3 glomerulopathy (C3G). C3G occures mainly in children and young adults and regoups two distinct histopathological entities, dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). Renal outcomes in C3G is poor since up to 50% of patients reach end stage renal disease 8 to 10 years after diagnosis. Complement abnormalities in C3G are mainly acquired induced by the presence of C3 Nephritic Factor (C3NeF): an autoantibdy targeting the AP C3 convertase. Less frequently C3G patients have anti-FH autoantibodies (Ref) or genetic abnormalities (variants in the FH, FI or CFHR5 genes. In silico analysis of mutated proteins give information about the role of mutation on AP overactivation. However, characterization of functional consequences of these mutations is required to proved the direct link between the abnormality and the occurrence of C3G. I first studied the functional consequences of the first C3 mutation, C3I734T, identified in a familial C3GN. In silico analysis revealed that the mutated residue, T734, is located on the C3 and C3b protein surface and that the substitution I734T may not be associated with major structural changes. The mutated amino acid is located on interaction site between C3b and complement regulatory proteins, FH and CR1. In vitro, the major defect of C3I734T was a decrease in binding to CR1, resulting in lower CR1 dependent cleavage of C3b by FI. These results provide evidence for a CR1 functional deficiency being responsible for deficient complement regulation. Binding of C3I734T to Factor H (FH) was normal, but C3I734T was less efficiently cleaved by Factor I, leading to enhanced C3 fragments binding on glomerular cells. In the second part of my work, I studied acquired C3G in patients with concomitant monoclonal gammopathy. In the clinical part of this study, we demonstrated The high prevalence of monoclonal gammopathy in C3G patients aged over 50, reaching 65% in the French C3G national cohort, strongly suggests a pathogenic link between the two conditions. Next, we demonstrated that renal outcomes is significantly worser in patients with monoclonal gammopathy compared to patients without monoclonal gammopathy but that efficient chemotherapy resulted in higher renal response rate and longer renal survival than conservative or immunosuppressive therapy. Results of the clinical part of the study strongly suggest a link betwwen the monoclonal gammopathy and the occurrence of C3G. In the experimental part of this work, I studied the mechanisms of complement AP activation in these population. Biomarkers of C3 and C5 convertase activation were present 40% and 81% of patients respectively. Anti-complement protein antibodies were found in 23/41 (56%) patients, including in most of patients, anti-FH and anti-CR1 antibodies. I found new antigenic target, C5 and properdin in few cases. The anti-FH and anti-CR1 antibodies were associated with clear functional consequences. Nevertheless, the anti-complement proteins reactivity was not carried out by the MIg in 75% of the cases. I discovered that the MIg induced a direct AP C3 convertase overactivation in 23/34 (67%) patients responsaible for a C5 convertase overactivation in presence of patients’ Ig, in a properdin dependant manner. Our results suggest that MIg and polyclonal autoantibodies could act in synergy: AP overactivation induced by the MIg could be amplified by the inefficient complement regulation, caused by the polyclonal anti-complement autoantibodies. All my results allow to better understand the pathophysiological mechanisms involved in C3G and open up reflection on therapeutic approaches for C3G associated with monoclonal gammopathy.

Major depressive disorder (MDD) is a heritable and highly debilitating condition with antidepressants, first-line treatment, demonstrating low to modest response rates. No current biological mechanism substantially explains MDD but both neurostructural and neurochemical pathways have been suggested. Further explication of these may aid in identifying subgroups of MDD that are better defined by their aetiology. Specifically, genetic stratification provides an array of tools to do this, including the intermediate phenotype approach which was applied in this thesis. This thesis explores genetic overlap with regional brain volume and MDD and the genetic and non-genetic components of antidepressant response. The first study utilised the most recent published data from ENIGMA (Enhancing Neuroimaging Genetics through Meta-analysis) Consortium's genome-wide association study (GWAS) of regional brain volume to examine shared genetic architecture between seven subcortical brain volumes and intracranial volume (ICV) and MDD. This was explored using linkage disequilibrium score regression (LDSC), polygenic risk scoring (PRS) techniques, Mendelian randomisation (MR) analysis and BUHMBOX (Breaking Up Heterogeneous Mixture Based On Cross-locus correlations). Results indicated that hippocampal volume was positively genetically correlated with MDD (rg= 0.46, P= 0.02), although this did not survive multiple comparison testing. Additionally, there was evidence for genetic subgrouping in Generation Scotland: Scottish Family Health Study (GS:SFHS) MDD cases (P=0.00281), however, this was not replicated in two other independent samples. This study does not support a shared architecture for regional brain volumes and MDD, however, provided some evidence that hippocampal volume and MDD may share genetic architecture in a subgroup of individuals, albeit the genetic correlation did not survive multiple testing correction and genetic subgroup heterogeneity was not replicated. To explore antidepressant treatment resistance, the second study utilised prescription data in (GS:SFHS) to define a measure of (a) treatment resistance (TR) and (b) stages of resistance (SR) by inferring antidepressant switching as non-response. GWAS were conducted separately for TR in GS:SFHS and the GENDEP (Genome-based Therapeutic Drugs for Depression) study and then meta-analysed (meta-analysis n=4,213, cases=358). For SR, a GWAS on GS:SFHS only was performed (n=3,452). Additionally, gene-set enrichment, polygenic risk scoring (PRS) and genetic correlation analysis were conducted. No significant locus, gene or gene-set was associated with TR or SR, however power analysis indicated that this analysis was underpowered. Pedigree-based correlations identified genetic overlap with psychological distress, schizotypy and mood disorder traits. Finally, the role of neuroticism, psychological resilience and coping styles in antidepressant resistance was investigated. Univariate, moderation and mediation models were applied using logistic regression and structural equation modelling techniques. In univariate models, neuroticism and emotion-orientated coping demonstrated significant negative association with antidepressant resistance, whereas resilience, task-orientated and avoidance-orientated coping demonstrated significant positive association. No moderation of the association between neuroticism and TR was detected and no mediating effect of coping styles was found. However, resilience was found to partially mediate the association between neuroticism and TR. Whilst the first study does not indicate a genetic overlap between regional brain volumes and MDD, it demonstrates the utility of the intermediate approach in complex disease. Antidepressant resistance was associated with neuroticism both genetically and phenotypically, indicating its role as an intermediate phenotype. Nonetheless, larger sample sizes are needed to adequately address the components of antidepressant resistance. Further work in antidepressant non-response may help to identify biological mechanisms responsible in MDD pathology and help stratify individuals into more tractable groups.

No description available.

Introdução: Em diversos países, inclusive no Brasil, a fortificação de alimentos com ácido fólico (AF) foi adotada como política pública de prevenção e combate à deficiência nutricional da vitamina, motivados principalmente pela redução da incidência dos defeitos do tubo neural. No período pós-fortificação observa-se tanto a evolução positiva do consumo e nível sérico da vitamina quanto a diminuição da concentração plasmática de homocisteína, e ainda, o aumento do ácido fólico não metabolizado na maioria desses países. Não se conhece ainda os efeitos biológicos do AFNM, no entanto, considera-se que o AFNM pode ser um fator relevante nas questões de segurança associadas com alto consumo de AF. Objetivo: Avaliar o consumo dietético e nível de folato, homocisteína e ácido fólico não metabolizado após a fortificação mandatória de alimentos com ácido fólico, e a interação com os polimorfismos envolvidos no metabolismo do folato e homocisteína. Metodologia: Os dados foram oriundos do estudo transversal de base populacional ISA Capital 2008 conduzido em uma amostra representativa de residentes do município de São Paulo, de ambos os sexos, e com idade acima de 14 anos. Coletou-se recordatórios alimentares de 24 horas e amostra de sangue em jejum de 12 horas para análises bioquímicas e moleculares. As análises estatísticas foram realizadas no programa STATA®, versão 13.0, com nível de significância de 5 por cento . Resultados: O estudo foi conduzido em 750 indivíduos, sendo 53,1 por cento mulheres e média de idade 40,7 (IC95 por cento 38,8-42,5) anos. A média de consumo e nível de folato foi de 375,8 (IC95 por cento 363,0-388,6) mcg/dia e 13 (IC95 por cento 12,0-13,9) ng/ml, respectivamente. Apenas 1,76 por cento da população apresentou deficiência de folato (<3ng/ml). Foi 5 detectado AFNM em 79,8 por cento da população com média plasmática de 2,4 (IC95 por cento 2,1-2,7) ng/ml. No modelo linear generalizado para previsão de AFNM, nível de folato (p<0,001), idade (p<0,001), tabagismo (p=0,002), raça (p<0,001) e concentrações de B6 (p<0,001), além disso, a interação de homozigotos e heterozigotos para a deleção de pares de base da DHFR e nível de folato (p=0,003) foram efeitos significantes. Tem-se um aumento relativo esperado de 3 por cento no AFNM se aumentarmos em 1 ng/ml o nível de folato médio, considerando fixas as demais variáveis do modelo. Conclusão: Nota-se baixa prevalência da deficiência da vitamina e alta prevalência dos níveis detectáveis de AFNM na população decorrente do aumento do nível de folato. Todavia, apesar do aparente sucesso da fortificação de alimentos com ácido fólico, a comunidade científica não é unânime na aprovação de políticas de fortificação mandatória e do uso indiscriminado de suplementos. Recomenda-se um monitoramento constante para evitar que a população seja exposta a altas doses da vitamina, e consequentemente, efeitos adversos à saúde. / Introduction: Food fortification is an important strategy in public health policy for controlling micronutrient malnutrition, and a major contributory factor in the eradication of micronutrients deficiencies. Motivated by the reduction in the occurrence of neural tube defects, countries worldwide, including Brazil, adopted food fortification with folic acid (FA). Folic acid fortification has increased dietary intakes of folic acid and folate status, but it is also associated with the presence of circulating FA. Although the metabolism and biological effects of circulating of folic acid are not well known, it may be a contributing factor in safety concerns associated with high oral doses of folic acid. Objective: To assess the folate intake and status, homocysteine and circulating FA after mandatory fortification with folic acid, and interaction with polymorphisms involved in 1-carbon metabolism. Material and Methods: Data were from 750 individuals aged > 14 years old who participated of a cross-sectional population-based survey in Sao Paulo City-Brazil. Fasting blood samples and information about food intake based on two measures of 24 hour food recall were collected. All analyses were carried out using STATA (version 13.0) and p-value <.05 was considered to be statistically significant in all tests. Results: Results were from 750 individuals. Women accounted for 53.1 per cent of the population and average age was 40.7 (IC95 per cent 38.8-42.5) years. The overall mean folate intake and folate concentration were 375.8 (95 per cent CI: 363.0-388.6) mcg/day of DFE and 13 (95 per cent CI: 12-14) ng/mL, respectively. Only 1.76 per cent of 7 population had folate deficiency (<3 ng/mL). Circulating FA was detected in 79.8 per cent of the population with a mean concentration of 2.4 (95 per cent CI: 2.1-2.7) pmol/ml. Effects of total folate concentration (p<0.001), age (p<0.001), current smoker (p=0.002), race (p<0.000) and vitamin B6 (p<0.001) as well as interaction between folate concentration and 19-base pair deletion polymorphism in DHFR (p=0.003) were found in the model to predict the circulating FA. An increase of one ng/mL in folate concentrate was associated with increased of 3 per cent in circulating FA. Conclusions: There is low prevalence of folate deficiency and high prevalence of detectable levels of circulating FA the population. The fortification effectively increased folate status and folic acid intake, and had a notable influence on rankings of food contributors of folate intake. Although the success of folic acid fortification was observed in many countries, the scientific community is not unanimous in approval of mandatory fortification policies and the indiscriminate use of supplements. The monitoring is recommended to guarantee the safety of exposure to folic acid, and avoiding adverse health effects.

This thesis work describes the role of Rab proteins in amyloid processing and clearance in different cell pathways. It also describes an experimental approach used to analyze the expression effects of Rab10 in amyloid beta production. Since the main theory behind neurodegeneration in Alzheimer's disease claims that high levels of amyloid beta 42 (Aβ42) molecules trigger widespread neuronal death, control of Aβ42 has been a main target in Alzheimer's disease research. In addition, several studies show increased levels of particular Rab proteins in Alzheimer's pathogenesis. However, no review consolidates current findings in neurodegeneration of Alzheimer's with Rab protein dysfunction. The first chapter of this thesis aims to address this need by providing a current review of Rab proteins associated with APP and neurodegeneration. The second chapter constitutes an experimental approach used to characterize the effects of Rab10 and Sar1A GTPases in APP and amyloid processing. We found that Rab10 expression does not affect APP production but significantly changes Aβ generation, particularly the toxic Aβ42 and Aβ42:40 ratio. On the other hand, we found no significant effect of Sar1A expression on either APP or amyloid beta generation. These findings partially confirm the work done by Kauwe et al (2015) and provide preliminary evidence for two potential targets for protective effects in neurodegeneration.

Obsessive-compulsive disorder (OCD) and the spectrum of associated conditions, affect 2-4% of the population worldwide. Although heritability studies in OCD have shown a 3 - 12 times increased risk for first degree relatives, the identification of the underlying risk-conferring genetic variation using classic genetic association studies has proven to be difficult. The possibility of a larger contribution of rare genetic variants to the risk of psychiatric disorder has been suggested by several successful studies. We expect that a spectrum of risk allele frequencies exists, which includes not only common variation but also a substantial amount of rare genetic variants that contribute to OCD. This thesis is aimed at identifying and functionally characterizing rare genetic variation in the OCD spectrum. Identified statistically significant variants were scrutinized for changes related to synaptic function using high content screening and subsequent functional analyses. Identifying the genetic profile of rare variants found in the OCD spectrum cohort combined with the functional impact that these variants have has provided insight into the etiology of the OCD spectrum. With these approaches a foundation can be laid for the development of a predictive model of the OCD spectrum.

Malignant hyperthermia (MH) is a dominant skeletal muscle disorder caused by mutations in the ryanodine receptor skeletal muscle calcium release channel (RyR1). Allele-specific differences in RyR1 expression levels might provide insight into the observed incomplete penetrance and variations in MH phenotypes between individuals. Firstly, an H4833Y allele-specific PCR (AS-PCR) assay was designed that allowed for the relative quantification of the two RYR1 mRNA alleles in heterozygous samples. In four MHS skeletal muscle samples and two lymphoblastoid cell lines (LCLs), the wild type allele was found to be expressed at higher levels than the mutant RyR1 allele. These differences were not caused by variations in RYR1 mRNA stabilities. Secondly, high-throughput amplicon sequencing was employed for the quantification of both the T4826I and H4833Y causative MH mutations in heterozygous MHS samples. With the exception of one, all detected H4833Y and T4826I mutation frequencies were about 50%. This included a control, which was constructed and proven to have a 3:1 ratio of the wild type (H4833) versus the mutant (Y4833) RYR1 allele. This suggested that that the high-throughput amplicon sequencing approach as used here, was not suitable for accurate quantification of the two RyR1 alleles in heterozygous H4833Y MHS samples. To detect possible variations in RyR1 alleles at the protein level, the RyR1 was to be isolated from microsomes prepared from a H4833Y MHS frozen skeletal muscle tissue. Microsomes isolated from MHS skeletal muscle tissues lacked the immunoreactive band that was believed to be the full length RyR1. Poor muscle quality, due to long term storage was believed to be the main cause of RyR1 depletion. Faster and less expensive screening methodologies are required for the identification of genetic variants in MH research. Thus, in an additional project inexpensive and high-throughput high-resolution melting (HRM) assays were developed to allow screening of the RYR1 gene, for mutations associated with MH and/or central core disease (CCD).