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Non-Alcoholic Fatty Liver Disease (NAFLD) is a chronic and dangerous condition which has grown in prevalence over recent decades due to increasing rates of obesity, affecting roughly a quarter of adults globally. This thesis develops a useable NAFLD definition which can be applied to large retrospective data sets consisting of medical records for selected cohorts. The morbidity and mortality associated with NAFLD is investigated, especially related to extrahepatic cancer. This thesis also aims to identify genetic modifiers of NAFLD risk in Scottish and South Indian populations. Data from three retrospective Scottish cohorts with electronic health records (EHRs) were analysed in the current thesis. These were the GoDARTS, SHARE and Tayside and Fife Diabetics cohorts. Genotypic data was available for a number of patients in GoDARTS and SHARE. Data from the Dr Mohan’s Diabetes Speciality Clinic (DMDSC) were also analysed, which consisted of clinical measurements from clinic visits, and genotypic data. An accurate and practical NAFLD definition based on two raised ALT levels was developed, which had a sensitivity of 97.4% in the GoDARTS cohort. This definition was used for subsequent NAFLD analyses. Patients with NAFLD experienced significantly more hospital admissions, and earlier death than those without NAFLD. We found NAFLD is associated with increased risk of cancer incidence, and cancer death, which accounts for a large portion of the excess morbidity and mortality seen in NAFLD patients. GWAS analyses revealed that PNPLA3 rs738409 is a major genetic risk factor in both Scottish and South Indian populations. Candidate gene studies revealed variants associated with endothelin function and with GLP1 receptors had significant effects on NAFLD. This thesis applies an accurate and novel NAFLD definition to retrospective cohorts with EHRs, and found increased morbidity and mortality in individuals with this phenotype. A large proportion of this is explained by increased cancer incidence and cancer death seen in these individuals. A number of genetic risk factors for NAFLD are described, including novel loci in endothelin and GLP1R related genes.

Primary sclerosing cholangitis: from genetic risk to disease biology Elizabeth Claire Goode One in 10,000 people in the Western world lives with Primary Sclerosing Cholangitis (PSC), an immune-mediated, inflammatory disease of the bile ducts that is highly co-morbid with inflammatory bowel disease (IBD). PSC confers risk of serious disease sequelae including hepatobiliary malignancy and progression to end-stage liver failure, for which the only treatment option is liver transplantation. The absence of effective medical therapies for PSC reflects our current limited understanding of the disease's aetiology and pathogenesis. Our DNA, laid down at conception, gives us an unrivalled opportunity to understand the underlying causal biology of disease. This is because the genetic variants associated with disease susceptibility perturb genes and biological pathways that contribute to disease causality. Twenty-two regions of the genome, outside of the HLA, have been associated with PSC risk. These loci offer the potential for huge insight into the causal biology of PSC, if only we can robustly identify the true causal variants driving these loci and the genes they perturb. However, this is complicated by several scientific challenges. Firstly, the majority of disease-associated risk loci occur within non-coding regions of the genome. Secondly, patterns of correlation between variants within a risk locus means that the true causal variant driving the signal could be any of those highly correlated with the variant with the smallest p-value. In this thesis, I present analyses aimed at identifying the true genes and causal variants underlying each of the twenty-two PSC risk loci. Many non-coding risk variants associated with complex disease exert a quantitative affect upon gene expression i.e. are expression quantitative trait loci (eQTLs). Colocalisation assesses the evidence that a single shared causal variant is responsible for driving PSC risk and gene expression via an eQTL. In order to assign dysregulated genes to PSC risk loci, I perform colocalisation with eQTLs mapped in multiple cell-types and tissues mechanistically relevant to PSC. Because PSC is rare, eQTLs have not previously been mapped in all cell-types most relevant to this disease. In addition, I therefore map eQTLs in six peripheral blood T-cell subsets (including the rare CCR9+ gut-homing T-cells) from 80 patients with PSC and IBD. With colocalisation, I assign causal genes to five PSC risk loci, and assign other epigenetic regulatory features including methylation or histone modification, to six risk loci. Statistical fine-mapping of each risk locus in both the GWAS and eQTL data enables me to resolve three PSC risk loci to a single causal variant and nine loci to 95% credible sets containing ten or fewer variants. The results presented in this thesis identify three genes (PRKD2, ETS2 and UBASH3A), causal in the pathogenesis of PSC, which are currently the target of existing or experimental therapeutic agents. Firstly, reduced expression of PRKD2 causes excessive cell-autonomous T-follicular helper cell development and B-cell activation, and is associated with increased risk of PSC. Several studies are investigating the therapeutic effects of increasing the kinase activity of PRKD2. ETS2 is involved in the induction of pro-inflammatory cytokine release from macrophages and IL-2 regulation in Th to Th0 transition. ETS2 inhibitors are currently the subject of early therapeutic trials. Finally, UBASH3A attenuates the NF-kB/I-KKb pathway, an inflammatory pathway that is already targeted by proteasome inhibitors and acetylsalicylic acid, both of which could be potentially therapeutic in PSC. PSC is a debilitating disease with serious disease sequelae, for which new therapeutic options are urgently needed. In this thesis, I elucidate multiple genes with a causal role in PSC pathogenesis, several of which are potential candidates for future therapeutic target.

Non-alcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease in the developed world, afflicting approximately one in four adults globally. NAFLD is defined by the accumulation of fat within the liver which ranges in severity from simple steatosis to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. Until recently, NAFLD has been considered to be a consequence of metabolic syndrome; however, recent studies suggest that genetic factors may also influence disease onset and progression. Accordingly, genome wide association studies have linked the I148M variant in the gene coding for Patatin-like phospholipase domain-containing protein 3 (PNPLA3) with NAFLD aggravation without underlying metabolic disease. However, despite over a decade of research, the function of PNPLA3 and its role in the pathogenesis of NAFLD remains largely obscure. The lack of clarity regarding the function and disease associations of PNPLA3 is due in large part to the lack of a comprehensive human model of PNPLA3-associated NAFLD. In order to address this need, we have developed an in vitro model that takes advantage of the unique properties of human induced pluripotent stem cells (hiPSC) and the CRISPR/CAS9 gene editing technology. We first used CRISPR/CAS9 to generate hiPSC lines with either a knock-out (PNPLA3KO) of the PNPLA3 gene or with the I148M variant knocked in (PNPLA3I148M). The resulting cells were then differentiated into hepatocytes and grown in 3D culture conditions to improve their maturity and functionality. The differentiated cells were treated with either monounsaturated or saturated free fatty acids to induce NAFLD-like phenotypes (lipid accumulation and lipid toxicity, respectively) and characterized by various functional, genomic, and lipidomic assays. The genetically edited sublines showed similar differentiation efficiency toward hepatocytes as untargeted cells indicating that changes in PNPLA3 activity does not affect hepatic development. Following treatment with free fatty acids, PNPLA3KO cells showed higher lipid accumulation than untargeted cells as well as an altered pattern of response to lipid-induced stress. Indeed, PNPLA3KO cells were resistant to saturated fatty acid-induced lipotoxicity. Furthermore, lipidomic analyses suggest that the PNPLA3 edited cells may be avoiding cell death by shuttling the saturated fatty acids into triglycerides rather than other metabolic pathways. These findings were initially incongruent with the human disease; however, despite their resistance to lipid induced stress, the PNPLA3KO cells downregulated all phases of drug metabolizing enzymes which made them more susceptible to other forms of hepatotoxicity such as ethanol. The PNPLA3I148M cells exhibited an intermediate phenotype between untargeted and PNPLA3KO cells. These results demonstrate for the first time in a fully human model that the I148M variant in PNPLA3 is a loss of function variant. This loss of PNPLA3 activity leads to the global downregulation of metabolic pathways likely due to the sequestration of fatty acids in triglycerides caused by reduced lipid droplet remodelling capacity in these hepatocytes. Our results indicate that patients carrying the I148M variant have lower hepatic metabolic activity which causes steatosis, reduced susceptibility to lipotoxicity, and increased susceptibility to other forms of hepatotoxicity which may contribute to NAFLD progression. This novel system provides the first opportunity to study the role of PNPLA3 in the development and progression of human NAFLD in vitro.