Way, MJ; (2017) The genetics of alcohol-related liver disease. Doctoral thesis , UCL (University College London).

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Abstract

The presence of cirrhosis is associated with significant morbidity and mortality. The prolonged heavy consumption of alcohol results in the development of cirrhosis in approximately 20% of people. This variance in risk is attributed to both genetic and environmental risk factors. To date there have been no systematic genome-wide association studies to investigate the contribution of common genetic variants to alcohol-related liver disease risk despite the success of these type of analyses in cirrhosis of other aetiologies. The work presented in the first half of this thesis describes the first genome-wide association study of alcohol-related liver disease identifying three highly replicable risk variants in the candidate loci: PNPLA3, TM6SF2 and MBOAT7. Subsequent to this, an extended GWAS was performed including over three-hundred additional cases, resulting in the identification of several novel loci. Of all the identified susceptibility loci, genetic variation in PNPLA3 is by far the most significant contributor to overall risk. The primary genetic variant in this locus is rs738409 and has the largest effect on cirrhosis risk of any genetic variants identified. It encodes a non-synonymous amino acid substitution (Ile148Met) in the PNPLA3 protein and is thought to be functional. This variant was genotyped in a UCL cohort of phenotypically well-characterized patients with cirrhosis and appropriate controls and investigated with regards to disease outcome and prognosis using time-to-event analysis. However, there is considerable debate about the functional significance of the PNPLA3 variant and to date little, if any, information about its structure. This information would provide insight into the pathogenesis of alcohol-related liver injury and identify possible targets for therapy. In the second half of the thesis the work undertaken to obtain the crystal structure of PNPLA3 is described. Initial computational structural modelling of the PNPLA3 protein was undertaken to characterize predicted structural features of the full-length protein to aid understanding of protein and crystallography process. A range of recombinant protein expression vectors were then developed for the purification of the protein in multiple expression systems. This led to the development of a high-yield expression protocol in E. coli obtaining milligram amounts of protein. Work to functionally characterize this protein is on-going.

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