<p dir="ltr">Genome-wide association studies (GWAS) have uncovered numerous variants linked to a wide range of complex traits. However, understanding the mechanisms underlying these associations remains a challenge. To determine genetically regulated mechanisms, additional layers of gene regulation, such as transcriptome and proteome, need to be assayed. Transcriptome-wide association studies (TWAS) and Proteome-wide association studies (PWAS) offer a gene-centered approach to illuminate these mechanisms by examining how variants influence transcript expression and protein expression, thereby inferring their impact on complex traits. In the introductory chapter of this dissertation, I discuss the methodology of TWAS and PWAS, exploring the assumptions they make in estimating SNP-gene effect sizes, their applications, and their limitations. In Chapter 2, I undertake an integrative analysis of TWAS and PWAS using the largest cohort of individuals affected with Tourette’s Syndrome within the Psychiatric Genomics Consortium (PGC) – Tourette’s Syndrome working group. I identified genomic regions containing multiple TWAS and PWAS signals and integrated these results using the computational colocalization method to gain insights into genetically regulated genes implicated in the disorder. In Chapter 3, I conduct an extensive TWAS of the Myasthenia Gravis phenotype, uncovering novel genes associated with the disorder. Utilizing two distinct methodologies, I performed individual tissue-based and cross-tissue-based imputation to assess the genetic influence on transcript expression. A secondary TWAS analysis was conducted after removing SNPs from the major histocompatibility complex (MHC) region to identify significant genes outside this region. Finally, in Chapter 4, I present the conclusions drawn from both studies, offering a comprehensive understanding of the genetic architecture underlying these traits. I also discuss future directions aimed at advancing the mechanistic understanding of complex non-Mendelian disorders.</p>

Developmental language impairments are neurodevelopmental disorders in which the acquisition of language, a task which children typically perform with ease, is hindered or fraught with difficulty. This work focuses on specific language impairment (SLI), a common and highly heritable language impairment in which language development is abnormal while other developmental domains are normal. Additionally, a case-study of a child with a broader linguistic and behavioural phenotype is also presented. The work described in this thesis includes both genetic and functional investigations which were aimed at identifying candidate genes for language impairment and provide insight into the genetic mechanisms that underlie language development. I performed a genome-wide association study of SLI which included child genotype effects, maternal genotype effects, parent-of-origin effects, and maternal-foetal interaction effects. This study found significant paternal parent-of-origin effects with the gene NOP9 on chromosome 14, and suggestive maternal parent-of-origin effects with a region on chromosome 5 which had previously been implicated in autism and ADHD. Case-control and quantitative association analyses of HLA genes and SLI identified several risk alleles and protective alleles. A case-control association analysis for related individuals which used an isolated population affected by SLI identified a non-synonymous coding variant in the gene NFXL1 which was significantly more frequent in affected individuals than in unaffected individuals. High-throughput sequencing of the coding regions of NFXL1 and LD blocks surrounding associated variants in ATP2C2, CMIP and CNTNAP2 (as reported in previous studies) identified novel or rare non-synonymous coding variants in NFXL1 and ATP2C2 in SLI families as well as intronic variants in all four genes that were significantly more frequent in SLI probands than in population controls. I describe a functional study of NFXL1 examining its expression in various brain regions, the presence of different splice variants across several tissues, its effect on genes it potentially interacts with, and the subcellular localisation of the protein. Finally, I present the case-study of a child with language impairment who had chromosomal rearrangements which spanned the location of FOXP2. I examine the potential influence the chromosomal rearrangements had on FOXP2 expression and describe a lincRNA gene which was disrupted by the chromosomal inversion. In conclusion, this work identified new candidate genes for language impairment, provided further support for the involvement of previously-identified candidate genes in SLI and contributed to the understanding of the molecular function of a newly-identified candidate gene for SLI.

Synucleins are highly conserved presynaptic proteins with unknown function. &alpha;-synuclein plays a key role regulating dopamine homeostasis and is intimately involved in Parkinson’s disease (PD) pathogenesis. However, the normal/pathological role of &alpha;-synuclein remains unidentified. Studies exploring its function are limited as current transgenic mouse models do not fully recapitulate PD pathology. This thesis reports the functional characterisation of two novel synuclein-based mouse models. I report the molecular and functional characterisation of transgenic mouse lines with wild-type or A30P-mutant human &alpha;-synuclein genomic locus carried within a bacterial artificial chromosome. SNCA-A30P^&plus;Snca-/- mice exhibited a highly physiologically relevant expression pattern of the transgene, including expression in the substantia nigra pars compacta (SNpc) and a specific, age-related loss of TH^&plus; cells in the SNpc, the key region of preferential cell loss in PD, compared with non-transgenic Snca -/- littermate controls. Analysis of dopamine signalling using fast-scan cyclic voltammetry (FCV) showed young adult SNCA-A30P^&plus;Snca-/- mice had an approximately 20&percnt; lower evoked extracellular dopamine concentration ([DA]o) compared with non-transgenic Snca -/- littermate controls, a decrease specific to the dorsal striatum. This difference diminished with age and could not be attributed to changes in dopamine reuptake/content. I detail the behavioural and neurochemical phenotype in mice lacking all three synucleins (&alpha;/&beta;/&gamma;). Functional compensation between synucleins emphasises the importance of studying their effects by removing all three proteins simultaneously. Triple-null mice exhibited hyperactivity in a novel environment reminiscent of a hyperdopaminergic-like phenotype, but showed no phenotype in anxiety or motor related tests. FCV revealed synuclein triple-null mice had a two-fold increase in [DA]o, specific to the dorsal striatum and not attributable to changes in dopamine reuptake/content, changes in striatal nicotinic receptor activity nor calcium-dependent changes in dopamine exocytosis. Together, the analysis from these two novel mouse models reveal synucleins play an important role in altering synaptic function in the dorsal striatum (the region selectively affected in PD) and contributes to growing evidence suggesting synucleins are negative regulators of synaptic dopamine release.

L’autisme est un trouble envahissant du développement défini uniquement sur des critères comportementaux et l’âge d’apparition. Le X fragile est une pathologie d’origine monogénique dont 15-25% des patients présente le diagnostique complet de l’autisme et dont de nombreux symptômes chevauchent avec l’autisme. Une souris Fmr1 KO a été créée et validée comme modèle pour le X fragile. A l’instar de la variabilité des phénotypes du X fragile chez l’humain, les données préliminaires montrent que la mutation nulle Fmr1 chez la souris interagit avec l'arrière fond génétique. Les travaux présentés visaient à déterminer les caractéristiques autistiques exprimées par les souris Fmr1 KO, ainsi que l’interaction de la mutation nulle avec le fond génétique (souches C57BL/6J, FVB.129P2tm1Cgr /J et leurs hybrides). Les résultats de ces travaux montrent notamment que les souris Fmr1 KO présentent un évitement initial d’approche sociale, des altérations principalement qualitatives des vocalisations, de l’hyperactivité et une augmentation de l’activité diurne. La mutation interagit avec le fond génétique et les résultats actuels indiquent que les KO de fond FVB.129P2tm1Cgr /J ont le phénotype le plus marqué. / Autism is a pervasive developmental disorder defined by behavioural criteria and age of onset. Fragile X is a disorder due to the silencing of the Fmr1 gene. About 15-25% of Fragile X patients are diagnosed as autistic and many symptoms overlap between the two disorders. A mouse Fmr1 KO was created and validated as a model for Fragile X Syndrome. Preliminary data also show that the null mutation interacts with the genetic background. The work presented in this thesis aimed to determine the autistic features expressed in Fmr1 KO mice, as well as the influence of the genetic background (C57BL/6J and FVB.129P2tm1Cgr/J strains, and their reciprocal hybrids) on the expression of the Fmr1 mutation. Our results show an initial inhibition of social approach in Fmr1 KO mice and a qualitative alteration of ultrasonic vocalizations in isolated pups, as well as an increase in activity, especially during the diurnal period. The Fmr1 mutation interacts with the genetic background and the results indicate that KO on the FVB.129P2tm1Cgr/J background show the most marked phenotype.

No description available.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder characterized by loss of upper and lower motor neurons. TDP-43 was identified as a major protein component of the characteristic neuronal inclusions and it has been detected in 90% of ALS cases. Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, were found in both sporadic and familial ALS cases. The aim of this study is to investigate the molecular mechanisms of cellular dysfunction and ultimately death caused by TDP-43 mutations in human cells using established cell lines and human motor neurons derived from induced pluripotent stem cells (iPSCs). We generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying three ALS-specific mutations, A382T, M337V or Y374X. In site specific bacterial artificial chromosome (BAC) human stable cell lines, TDP M337V mislocalized to the cytoplasm more frequently than wild-type TDP-43 (TDP Ypet) and TDP-A382T, an effect potentiated by oxidative stress. Cytoplasmic mislocalization was significantly higher in TDP M337V cells compared to TDP-Ypet and correlated with cell death. Cells expressing the mislocalized TDP M337V mutant spontaneously developed cytoplasmic punctae, while for TDP-A382T punctae were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin (TG). Lowering Ca2+ concentration in the ER of TDP-Ypet cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca²⁺ dysregulation as a potential mediator of pathology. Ca²⁺ signaling from the ER was impaired in cells carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca²⁺ stores content and delayed Ca²⁺ release induced by carbachol compared to TDP-Ypet cells. The deficits in Ca²⁺ release correlated with upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored amplitude of Ca²⁺ oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 through Bcl-2 regulation of ER Ca²⁺ signalling. Preliminary work in iPSC-derived motor neurons transduced with genomic DNA expression TDP-43 vectors using Herpes Simplex Virus type 1 (HSV-1) amplicons showed cytoplasmic redistribution of TDP-43 under high oxidative stress, without significant differences between mutations and wild-type. TDP-43 mutations delivered by HSV-1 amplicons also did not affect survival of iPSC-derived motor neurons. In ALS patient-derived motor neurons carrying C9orf72 expansions, TDP-43 pathology was not detected. However, preliminary data indicate that C9orf72 MNs present ER Ca²⁺ dysregulation with significantly high intracellular Ca²⁺ concentration, which correlates with high protein levels of ER stress markers and low levels of Bcl 2. This work highlights a potentially pathogenic role for TDP-43 mutations in the dysregulation of Ca²⁺ homeostasis and explores the use of iPS technology to investigate the effects of ALS-associated mutations in healthy and patient-derived motor neurons.

Aggregations of fused in sarcoma (FUS), a multifunctional RNA processing protein, define a pathological subtype of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), whilst mutations in the FUS gene are causative for ALS. To model the impact of FUS mutations, expression vectors containing the entire genomic sequence of FUS, up and downstream regions, and native promoter sequences have been generated. The constructs have been tagged with an mCherry fluorescent tag, and three separate pathological mutations (R244C, R521C, and P525L) have been separately inserted. Transgenic mice have been generated using the WT and P525L FUS vectors to provide a highly physiological model of FUS in disease. Within transfected HEK293 cells, insertion of the P525L and R521C FUS mutations leads to relocalisation of FUS from the nucleus to the cytoplasm. R521C and P525L mutant FUS incorporates into cytoplasmic aggregations of untranslated mRNA and RNA binding proteins known as stress granules. The strong relocalisation seen with P525L-FUS is associated with a gain of cytotoxicity. Reversal of this cytoplasmic relocalisation by demethylation of FUS rescues this cytotoxicity, suggesting a toxic gain of cytoplasmic function in the majority of FUS mutations. By contrast, insertion of the R244C mutation leads to neither relocalisation, stress granule association, nor cytotoxicity. Notably the R244C mutation, located away from the nuclear localization domain in which the majority of FUS mutations are found, leads to the presence of smaller FUS fragments in western blot analyses. These fragments appear not to be due to splicing defects in FUS but rather are due to post-translational modifications or aberrant protein cleavage. These data suggest an alternative pathway for FUS toxicity based upon a nuclear loss of function.

Overexpression of the NR2B subunit of the NMDA receptor in the forebrain has been shown to improve learning and memory in mice (Tang et al 1999), which provides exciting implications for the enhancement of human cognition. However, it was first essential to establish replicability, and since the Tang et al (1999) study used only male mice we wished to investigate possible sex differences. On the hidden platform watermaze, we found a trend for male NR2BOE mice to learn the task more quickly than male wildtype mice (as observed by Tang et al. 1999), but the opposite trend in female mice; female NR2BOE mice were slower to reach the hidden platform than female wildtype mice. This pattern of results was also observed on the spatial reference Y memory task and open field task (for anxiety), although not on the spatial working memory T maze task (despite a sex difference). However, wildtype and NR2BOE mice performed at similar levels on the novel object recognition task, the spatial novelty preference task, visible platform watermaze and visual discrimination task. A battery of tests considering some species typical behaviours of mice demonstrated that wildtype and NR2BOE mice were comparable on tests of motor ability, strength, co-ordination, anxiety, burrowing and nesting. This suggests that our behavioural results are not due to a general impairment or enhancement of species typical behaviours. We considered the possibility that the difference between the results of Tang et al (1999) and those we observed may be caused by age differences; hence we attempted to replicate our results on the hidden platform watermaze, spatial reference Y maze and open field test in age matched mice. However, the second cohort of NR2BOE mice performed at similar levels to wildtype mice, and at significantly improved levels compared to the mice of the first cohort. We also considered the effects of knocking out the NR2B subunit on learning and memory, and NR1 subunit deletion within the hippocampus. On the spatial working memory T maze, these mouse strains performed similarly to their respective wildtype strains. Similarly, on a two beacon watermaze (with one indicating the platform position), mice lacking the NR2B subunit were able to locate the platform in a similar length of time. To ensure that the null results we had observed in the second cohort were not due to loss of the NR2B protein overexpression in the forebrain, we performed polymerase chain reactions (PCR), quantitative real-time PCR, and Western blots. We ascertained that the transgene was indeed present and that NR2B mRNA and protein levels were elevated in the hippocampi of the NR2BOE mice. In conclusion, it is unclear why the behaviours we observed in the NR2BOE mice are different to those published in the literature. It is possible that they may be due to differences in environmental enrichment, but the cause of the genotype by sex differences observed in the mice of cohort 1 is unclear. Nonetheless, we have advanced our knowledge of the effects of modifications in the levels of the NR2B subunit of the NMDA receptor on learning and behaviour.

The population level bias towards right-handedness in humans implies left-hemisphere dominance for fine motor control. Left-handedness and reduced cerebral asymmetry have been linked to neurodevelopmental disorders such as dyslexia. Understanding the biology of these traits at a genetic level is crucial for understanding the relationship between handedness and neurodevelopmental disorders. Here I present genome-wide association study (GWAS) meta-analyses for both relative hand skill (handedness, n = 728) and reading-related traits (n = 548) in individuals with dyslexia. I uncovered a genome-wide significant association in an intron of PCSK6 associated with relative hand skill. PCSK6 is a protease that cleaves NODAL proprotein into an active form, and NODAL determines the development of left/right (LR) asymmetry in bilaterians. I performed pathway analyses of the GWAS data that revealed handedness is determined in part by the mechanisms that establish left/right (LR) asymmetry early in development, such as NODAL signalling and ciliogenesis. This finding replicated in a general population cohort unaffected with neurodevelopmental disorders (n = 2,666). A key stage in LR asymmetry development is the rotation of cilia that creates a leftward flow of NODAL. Candidate genes for dyslexia are involved in both neuronal migration and ciliogenesis. Ciliopathies can cause not only LR body asymmetry phenotypes, but also cerebral midline phenotypes such as an absent corpus callosum. Furthermore, I identified a genome-wide significant association with non-word reading located in an intron of MAP1B, a gene involved in neuronal migration that causes an absent corpus callosum when disrupted in mice. However, this finding did not replicate in two independent cohorts with dyslexia (n = 156 & 199), or in the general population cohort (n = 2,359). Though these cohorts had inadequate reading measures and poorly matched ascertainment for dyslexia. I also performed copy number variation (CNV) pathway and burden analyses of 920 individuals with dyslexia and 1,366 unselected controls, but did not find that rare CNVs play a major role in the etiology of dyslexia. Based on these results I propose that common variants in genes responsible for ciliogenesis and corpus callosum development influence traits such as handedness and reading ability.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised clinically by rapidly progressive paralysis leading ultimately to death from respiratory failure. There is no cure for ALS and no definitive explanation for the onset and rapid progression of motor neuron degeneration. Genetics is a known risk factor for a portion of familial cases. However, the role of genetics in the commoner sporadic form of the disease is poorly understood, although numerous genes have been implicated. The primary aim of this thesis project is to uncover the genetic causes that underlie ALS. To accomplish this goal, the main focus of this thesis is to perform genome-wide association analysis of sporadic ALS using high density SNP arrays. This thesis describes the first and the largest genome-wide association studies of ALS to date. Results demonstrate that there is no single large effect susceptibility variant underlying a large proportion of ALS, such as ApoE in Alzheimer’s disease. However, the genotyping data has been made publically available and the digital nature of this data means that it is a resource that can grow with future studies. Beyond genome-wide association, this thesis describes work using linkage, haplotype and sequence analysis to investigate the genetic overlap between ALS and frontotemporal dementia. Lastly, this thesis presents a novel method for linkage analysis using high throughput SNP arrays. Ultimately, it is hoped that by uncovering the genes that cause ALS, such knowledge will shed light on the pathogenic mechanisms underlying motor neuron degeneration and potentially lead to new rational therapies effective in slowing or even halting disease progression.