Genome-wide association studies (GWAS) have made substantial progress in implicating genomic regions in type 2 diabetes (T2D) susceptibility. Whilst attributing causal mechanisms to loci has proved non trivial, these studies have provided insights into the genetic architecture underlying the disease. GWAS findings indicate a causal role for gene regulatory processes, and suggest that pancreatic beta-cells play a pivotal role in mediating common T2D association. Work presented in this thesis therefore sought to generate novel regulatory annotations from human islets, and to assess whether T2D-associated loci can be accurately fine-mapped using statistical approaches, with the aim of improving understanding of causal mechanisms underlying these associations through integration of the two approaches. Using small RNA sequencing in human islets and enriched beta-cell populations (both n=3) and mRNA sequencing in a large number of human islets (n=130), I increased the number of available human islet annotations. These studies identified high or islet-specific expression in many micro RNAs (miRNAs) without previously known roles in human islets. It also provided the largest study of quantitative trait loci (eQTLs) and allele-specific expression (ASE) in human islets to date, identifying significant eQTLs for 1,636 genes and significant ASE at 8,754 genes. There was enrichment of active islet chromatin, compared to other tissues, at the best eQTL variant for each gene, but also substantial sharing of significant eQTLs between islets and other tissues. Simulations were used to assess the utility of fine-mapping approaches for refining common disease-associated loci to smaller intervals or sets of variants likely to include the causal variant. The results demonstrated that fine-mapping can indeed refine these loci to sets or intervals of a size more amenable to functional follow-up or focussed intersection with high quality annotations. Furthermore, using an approximated Bayesian approach, I was able to refine twenty-one of the known common T2D-associated loci. Finally, using the newly generated annotations, I demonstrated enrichment of T2D association signal for regulatory RNA annotations (islet lncRNAs and miRNA target gene sets). I also identified examples in which these types of annotation overlap common and rare variation suggestive of a role in T2D pathogenesis. Using further islet annotations, I also uncovered potential causal mechanisms at four of the twentyone fine-mapped common T2D loci. These data therefore provide many novel islet regulatory annotations that can be intersected with T2D genetics, and provide a first example of how such an approach can lead to novel potential causal mechanisms underlying association loci.

The tumour suppressor p53 is at the centre of the signalling pathway that controls cellular processes crucial in tumourogenesis, cancer progression and tumour clearance. Alterations in the p53 pathway that lead to cancer progression can be good candidates for molecular biomarkers that would assist in the identification of patients with different prognoses, but also serve as good predictors of appropriate targeted therapies. Patient cohorts and cancer cell panels are utilised to seek associations with the attenuation of the p53 pathway and cancer progression. Firstly, the alternatively spliced transcript of the p53 inhibitor HDMX, which is frequently found in tumours with poor prognosis, is studied. The high ratio of the alternatively spliced HDMX-S transcript over the full-length HDMX-FL transcript (HDMX-S/FL) is demonstrated to associate with p53 pathway attenuation in cancer cells and breast carcinomas, and with faster metastatic progression of osteosarcoma and breast cancer patients. Secondly, inherited polymorphism in the HDMX gene is investigated and demonstrated as a unique and highly reproducible eQTL, which identifies patients with different prognoses for metastatic disease in breast cancer and melanoma cohorts. Lastly, a screening approach to identify novel inherited polymorphisms in the p53 pathway genes that associate with metastatic progression of melanoma is developed and implemented, and subsequently in silico and in vitro functional analyses are performed to investigate a mechanism behind the FOXO3 SNP, identified as the strongest candidate, whereby the experimental evidence demonstrate that the causal SNP in the FOXO3 haplotype is controlled by the GATA3 transcription factor. Together, the work presented in this thesis provides strong support for the role of the p53 pathway in the metastatic progression of cancer, and suggests that molecular biomarkers that can detect changes in the activity of p53 pathway genes could offer a robust set of biomarkers for cancer progression applicable to different types of cancer.

Type 2 diabetes (T2D) and fasting plasma glucose (fpg) levels have distinct genetic components which are as yet only modestly understood. Understanding the genetics of this complex disorder and its related traits is likely to be of significant benefit to the field. Not only will it shed light on critical genes, pathways and mechanisms of regulation, but it may also contribute to the development of pharmaceutical anti-hyperglycaemic agents via the identification of key therapeutic targets. Therefore the aim of this thesis was to utilise a broad, multidisciplinary approach to study the genetics of insulin secretion and signalling. Traditionally genes which harbour rare variants causing monogenic beta-cell dysfunction have also been found to harbour common variants associated with T2D and fpg. As genome-wide association studies (GWAS) identify an increasing number of common variants and genes, they also increase the number of genes which act as monogenic candidates. I screened G6PC2, a novel fpg associated gene, in patients with monogenic forms of beta-cell dysfunction and demonstrated that rare variants in this gene are unlikely to be a common cause of monogenic beta-cell dysfunction. Although GWAS have been of considerable benefit to our understanding of complex disease genetics, they are not without their own limitations, primarily concerning signal refinement. To try to overcome this barrier for T2D and fpg signals I established a pipeline for fluorescence activated cell sorting of human islets to obtain pure beta-cells. In these cells, I performed transcript profiling of genes falling within T2D and fpg associated loci, demonstrating how this approach, alongside physiological analysis, can be of benefit for GWAS researchers and provide a starting point for fine mapping. Access to human beta-cells also enabled me to follow up one novel fpg association signal, SLC2A2. Through analysis within this metabolically relevant tissue I was able to establish that the mechanism for increased fpg levels is unlikely to be mediated via a beta-cell pathway. Although GWAS have highlighted a number of key genes associated with beta-cell dysfunction; they have been far less successful at identifying genes associated with insulin resistance, another key component of T2D pathogenesis. Additional approaches, including the study of rodent models, may be required to study this aspect of T2D. PTEN is known to negatively regulate the insulin signalling pathway and adipose tissue specific Pten-/- animals were shown to be markedly insulin sensitive. To assess the role of PTEN in human insulin sensitivity I performed mRNA expression profiling of PTEN in human adipose tissue biopsies from subjects with T2D and matched controls, demonstrating that PTEN is significantly reduced in the subcutaneous adipose tissue of the former. This response is likely to be a compensatory mechanism to counteract muscular insulin resistance although further investigation needs to be performed to determine the mechanism of compensatory downregulation. These data provide insights into a number of aspects of T2D genetics, and demonstrate how a multidisciplinary approach is of benefit to T2D genetic research.

Genome wide association studies (GWAS) and their subsequent meta-analysis have identified a large number of susceptibility variants for Type 2 diabetes (T2D) risk. However, the familial aggregation seen in this disease is not yet fully explained. The sibling relative risk (λ_s) due to all known variants is ~1.104 which is well below the epidemiological estimates of λ_s of ~3.0. There has therefore been great interest in the potential role of variants that would have been largely invisible to the initial wave of GWAS and linkage approaches. Low frequency (minor allele frequency 1-5%), incompletely penetrant (odds ratio 2-4) variants (LFIP), are one such group of potential susceptibility variants. The overall objective of this project (designed and implemented in 2007-2010) was to evaluate the contribution of LFIP variants to the inherited susceptibility to T2D. I tested the specific hypothesis that genes already-implicated in diabetes pathogenesis (due to an established role in monogenic or multifactorial disease) also harbour LFIP variants, and that those variants may contribute appreciably to the prediction of disease risk. Mutations in exons only encoding isoform-A of HNF1A have been demonstrated to lead to a later age of diagnosis of HNF1A-MODY. This region was therefore felt to be auspicious for harbouring LFIP variants impacting on T2D risk. I have demonstrated that such variants impacting on T2D risk are unlikely to be present in this region by use of Sanger sequencing in a sample enriched for young onset, familial T2D. The role in T2D risk of candidate LFIP variants across 5 genes (HNF1A, HNF4A, PDX1, KCNJ15 and LARS2), was evaluated by large scale association studies. For one variant, T130I of HNF4A, a modest association (p=5x10^-4) with T2D was seen in UK samples and the strength of association was marginally improved by incorporation of all previous studies of this variant in T2D in a meta-analysis (p=2.1x10^-5). This study demonstrated the difficulties encountered in confirming the association of low frequency variants to complex diseases, especially for those with modest effect sizes. At the time of project design and inception “next-generation” sequencing platforms were in their infancy and the study design I planned (that of pooled, targeted sequencing) had not been widely applied. It was therefore necessary to design and optimise protocols for sample preparation for sequencing on this platform. I used the Genome Analyzer II platform to sequence ten genes previously implicated in T2D or monogenic diabetes pathogenesis in pooled DNA samples. This approach yielded in excess of 2900 variants, a large portion being novel. As part of this project I have highlighted heuristics that can be used in the follow-up of potential susceptibility variants discovered using high throughput sequencing. I have also established protocols and pathways for sample preparation that can be utilised across several next generation sequencing platforms for future studies in the host institution and beyond.

Paired receptor families, consisting of multiple genetically and structurally similar but functionally opposite activating and inhibitory cell surface receptors, are among the fine tuners of the immune regulation. Recent studies on the evolutionary origin of these receptor families have suggested links to pathogen driven diversification, according to which activating receptors continuously evolve in order to counterbalance pathogens that try to subvert the immune response by stimulating the inhibitory receptor through their virulence factors. This thesis is about the CD200R paired receptor family. This family consists of an inhibitory receptor CD200R which is expressed on various leukocytes and delivers inhibitory signals upon engagement with its ligand CD200. In this study, the possibility that the activating members of the family evolved under pathogen pressure was investigated. Genomic DNA from twenty two different mice strains was screened for the presence of members of CD200R family. The number of activating receptors varied, CD200RLe and CD200RLc were found to be mutually exclusive and three strains possessed previously unknown members of CD200R family. In addition, the possibility that CD200R family members and other paired receptors interacted directly with bacteria was tested with a new assay but only the interaction of PIR-A1 with <em)S. aureus was found as previously reported. The rabbit CD200R family has been characterized and ligand receptor interaction between rabbit CD200 and rabbit CD200R has been demonstrated. However, no interaction between rabbit CD200R and a candidate viral CD200 homologue, the M141R protein of myxoma viruses, could be shown. This finding suggested a CD200R independent role for M141R molecule and possibly other homologues in pox viruses. Finally, two novel antibodies (OX131 and OX132) were characterized together with formerly generated antibodies against mouse CD200R family. The binding specificities and their effects on the CD200-CD200R interaction have been shown. This will help usage of these antibodies in various studies on the functionality and distribution of these receptors.

Whole genome sequencing (WGS) has transformed molecular infectious diseases epidemiology in the last five years, and represents a high resolution means by which to catalogue the genetic content and variation in bacterial pathogens. This thesis utilises WGS to enhance our understanding of antimicrobial resistance in two clinically important members of the Enterobacteriaceae family of bacteria, namely Escherichia coli and Klebsiella pneumoniae. These organisms cause a range of clinical infections globally, and are increasing in incidence. The rapid emergence of multi-drug resistance in association with infections caused by them represents a major threat to the effective management of a range of clinical conditions. The reliability of sequencing and bioinformatic methods in the analysis of E. coli and K. pneumoniae sequence data is assessed in chapter 4, and provides a context for the subsequent study chapters, investigating resistance genotype prediction, outbreak epidemiology in two different contexts, and population structure of an important global drug-resistant E. coli lineage, ST131 (5-8). In these, the advantages (and limitations) of short-read, high-throughput, WGS in defining resistance gene content, associated mobile genetic elements and host bacterial strains, and the relationships between them, are discussed. The overarching conclusion is that the dynamic between all the components of the genetic hierarchy involved in the transmission of important antimicrobial resistance elements is extremely complicated, and encompasses almost every imaginable scenario. Complete/near-complete assessment of the genetic content of both chromosomal and episomal components will be a prerequisite to understanding the evolution and spread of antimicrobial resistance in these organisms.

The Major Histocompatibility Complex (MHC) is a highly polymorphic region of the genome located on chromosome 6p21 in which genetic diversity has been associated with susceptibility to many autoimmune, infectious and other common diseases. Despite strong associations between disease and variation in the MHC that have been identified initially from serological testing and more recently by genome-wide association studies, functional insights into how specific variants may be altering disease susceptibility remain poorly understood in most cases. It is predicted that gene expression will play a significant role in the modulation of disease susceptibility and so further understanding of allele specific gene expression in the MHC will be necessary to help define the function of disease associated variants in this region. This thesis aimed to define allele specific gene expression in the MHC by characterising specific candidate genes together locus-wide approaches in order to try and resolve functional variants. Gene expression was analysed in both lymphoblastoid cell lines (LCLs) and primary human peripheral blood mononuclear cells (PBMCs). Data is presented validating a novel haplotype-specific MHC microarray and fine mapping putative local, likely cis-acting, regulatory variants. This was done by expression quantitative trait mapping for two cohorts of healthy volunteers. A transcription factor ZFP57, encoded in the MHC, was found to show significant differential allelic expression relating to specific single nucleotide polymorphisms (SNPs) and possession of HLA-type. This provided new insights into reported disease associations, notably HIV-1 infection and cancer. The function of ZFP57 was further investigated in terms of genome-wide DNA binding sites by ChIP-seq together with its binding co-factor KAP1. Allele-specific gene expression was also demonstrated for several classical HLA genes including the HLA-C and HLA-DQ genes, fine mapping specific putative regulatory variants. This provided new insights into disease association, notably variants of HLA-DQB1 and susceptibility to leprosy. The applicability and sensitivity of the technique of RNA sequencing (mRNA-seq) for allele-specific quantification of gene expression was investigated for different allelic ratios of RNA from LCLs homozygous for sequence across the MHC. Significant challenges were identified in successful application of this technique to MHC genes while high levels of accuracy were observed dependent on read depth in non-MHC genes. This thesis provides new insights into the extent and nature of allele-specific gene expression in the MHC, experimental approaches that can be used and insights gained into disease susceptibility for this important genomic region.

The production of adult blood initiates from the haematopoietic stem cell (HSC). This clinically important cell has the capacity to maintain all blood lineages throughout the lifetime of an organism. HSCs emerge de novo from the haemogenic endothelium in the ventral wall of the embryonic dorsal aorta, from where they go on to seed adult sites of haematopoiesis. We have shown that Lmo4a is required for the emergence of HSCs in the zebrafish, and go on to demonstrate that Lmo4a regulates expression of the critical transcription factor, gata2a. Strikingly, both over- and under-expression of gata2a in the dorsal aorta severely diminishes HSC production. The LIM-only domain protein Lmo4 has previously been shown to interact with the known haematopoietic regulator, Ldb1. Together with our collaborators, we have identified novel binding partners of Lmo4 in mouse erythroleukaemic cells. Our functional analysis shows that many of these partners are also necessary for HSC emergence, thus revealing several new potential regulators of HSC formation. Given that these proteins were identified in an in vitro model of definitive erythropoiesis, it is remarkable that they also appear to act together in vivo at the level of HSC formation, and our data suggests that a transcriptional complex containing Lmo4 and these partners may directly repress gata2a. The related protein Lmo2 is also known to bind Ldb1. Together with Scl, Lmo2 is a master regulator of the haemangioblast programme. We have been utilising this activity, together with recent structural studies, to identify functionally important residues in the Lmo2 molecule. As a cell’s transcriptional programme drives both normal and pathological development, and misexpression of both Lmo2 and Lmo4 is involved in a variety of oncogenic states, the work presented in this thesis is likely to inform efforts to develop therapeutically relevant reagents.

The myeloid cell lineage is a fundamental element of the immune system and it can give rise to a diverse set of terminally differentiated cells, such as macrophages or osteoclasts among many others. Mutations or misregulation of gene expression may lead to severe clinical conditions, such as arthritis, osteoporosis or cancers. Epigenetics, the regulation of gene expression and chromatin remodelling, is implicated in cell differentiation, function and disease, and hence it is a promising new area to explore in order to explain underlying cellular mechanisms. Firstly, human macrophage subtypes were studied. Chemokine (C-C motif) ligand (CCL) 1 and mannose receptor were validated to be granulocyte macrophage (GM) colony stimulating factor (CSF) induced macrophage markers, while CCL₂ was specifically expressed in macrophage CSF (MCSF) macrophage population. By utilising publicly available high-throughput sequencing data, new biomarkers dehydrogenase/reductase (SDR family) member 2 and CCL₂₆ were discovered to be MCSF-macrophage specific while guanylate binding protein 5 and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A were highly up-regulated in GMCSF cells. Secondly, a range of gene knock-down techniques for the myeloid cell lineage were optimised and established. Lentiviral short-hairpin RNA (shRNA) delivery methods were shown to induce an undesirable pro-inflammatory response in macrophages. Furthermore, the frequently utilised cytomegalovirus promoter for gene expression was shown to be completely silenced in macrophage populations. Locked nucleic acids were selected as a suitable alternative to shRNA knock-down and by employing this new tool it was shown that a histone demethylase lysine (K)-specific demethylase (KDM) 6B is fundamental for macrophage differentiation. Finally, a small molecule GSK-J₄, a potent inhibitor of histone demethylases KDM6A, KDM6B and KDM₅B specific for H₃K_27me3 and H₃K_4me3, respectively, was used to dissect epigenetic signalling in osteoclasts and multiple myeloma. In osteoclasts it was shown to act mainly by inhibiting transcriptional changes required for osteoclastogenesis when MCSF-macrophages are stimulated with Receptor Activator Of Nuclear Factor Kappa-B Ligand (RANKL), as indicated by the differential increase in H₃K_27me3 marks, leading to inhibition of c-Jun and potentially abolition of transcription factor AP-1, required for the transcriptional initiation of nuclear factor of activated T-cells 1 (NFATc1). In multiple myeloma cells, GSK-J₄ causes a dramatic increase in expression, further supported by the build-up of global H₃K_4me3 marks, which results in the upregulation of the unfolded protein response pathway. In both cell systems, there is an early upregulation of metallothionein genes, which in multiple myeloma was shown to increase potentially due to rapid influx of zinc ions within the first 30 minutes, and as such may cause induction of apoptosis in multiple myeloma and may inhibit differentiation of osteoclasts.

During the course of infection with chronic pathogens such as Hepatitis C virus (HCV), Hepatitis B virus (HBV) and HIV, virus-specific CD8^&plus; T cells differentiate into heterogeneous dysfunctional subpopulations. Advances in multi-parameter flow cytometry have allowed these subpopulations to be further classified into classes of exhausted T cells, primarily by their expression of multiple inhibitory receptors. However, the exact phenotype of CD8^&plus; T cells during exhaustion is an area of great interest as many inhibitory receptors are also expressed on functional CD8^&plus; T cells. Discovering novel and specific markers of T cell exhaustion is fundamental in developing strategies to restore CD8^&plus; T cell function in chronic viral infections. Recently, genome wide expression profiles have identified broad molecular phenotypes in exhausted T cells that could not have been discovered by flow cytometry alone. I show how similar genomic approaches identify and further characterise the ectonucleotidase CD39 as a novel marker of CD8^&plus; T cell exhaustion in chronic viral infection. I show that CD39 is highly expressed in HCV and HIV-specific CD8^&plus; T cells and that CD39^&plus; CD8^&plus; T cells are enriched with gene signatures of exhaustion. CD39 is highly co-expressed with multiple inhibitory receptors including PD-1, enzymatically active on CD8^&plus; T cells in HCV infection and positively correlated with viral load in both HCV and HIV. I also demonstrate the discovery of a novel CD39^High population of cells in the mouse model of chronic Lymphocytic Choriomenigitis Virus (LCMV) infection, which express the highest degrees of PD-1, LAG3 and 2B4 in the CD39^&plus; fraction. Thus, CD39 is a novel and specific marker of severe CD8^&plus; T cell exhaustion in human and animal models of chronic viral infection.