Translational Genomics, Transcriptomics and Metabolomics Analyses of the Metabolic Effects of Chronic Hepatitis C Infection and Their Clinical Implications
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Translational genomics, transcriptomics and metabolomics analyses of the metabolic effects of chronic hepatitis C infection and their clinical implications. Dr Paul J Clark MBBS (Hons I), MPH&TM, FRACP A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy Kirby Institute for Infection and Immunity in Society Faculty of Medicine The University of New South Wales, Sydney, Australia August 2012 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: CLARK First name: Paul Other name/s: James Abbreviation for degree as given in the University calendar: PhD School: The Kirby Institute Faculty: Medicine Title: Translational genomics, transcriptomics and metabolomics analyses of the metabolic effects of chronic hepatitis C infection and their clinical implications. Abstract 350 words maximum: (PLEASE TYPE) Background: The hepatitis C virus (HCV) relies on host lipid pathways for its life cycle, leading to metabolic consequences including hypocholesterolemia, insulin resistance and hepatic steatosis. These sequelae have varying clinical implications, including poorer sustained viral response (SVR) to pegylated interferon/ribavirin (PEG/RBV) therapy, increased risk of hepatic fibrosis and residual risk of liver disease (eg fibrosis, steatosis) and complications (eg hepatocellular carcinoma), even after SVR. Aims: To define host genomic and metabolomic profiles associated with hypocholesterolemia, hepatic steatosis, hepatic fibrosis and hepatic inflammation in chronic HCV infection, and to assess their clinical impacts. Methods: Genome wide association studies, candidate gene studies, microarray based mRNA transcription profiling, liquid chromatography/mass spectrometry metabolomic assays, multivariable regression models (MVR). Results: In HCV genotype 1 (G1), IL28B genotype is the only common variant associated with LDL-C levels. LDL-C levels remain significant predictors of SVR. Host IL28B and PNPLA3 polymorphism are significant host determinants of susceptibility to hepatic steatosis in chronic HCV G1 infection. Lower PNPLA3 genotype frequency in African American (AA) patients may explain their lower frequency of hepatic steatosis relative to Caucasian Americans, despite greater metabolic risk factors in AA patients. PNPLA3 was associated with Mallory bodies and lobular inflammation (a pattern seen in non-alcoholic fatty liver disease) but not fibrosis. Gene expression enrichment associated with PNPLA3 genotype and varied phenotypes of histopathological injury include a number of novel genetic associations as well as transcripts previously identified which warrant further investigation. HCV perturbs the distal cholesterol synthesis pathway in a HCV genotype-specific manner but the levels of the proximal sterol lanosterol, which has important cholesterol regulation effects, is preserved. Conclusions: Taken together, these translational genomics and metabolics studies provide insights into the nature of host-virus metabolic interactions in chronic HCV and their clinical sequelae. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). 17 August 2012 ……………………………………… ……….……………………...… Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS ii Originality Statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed ... ...................................................... Date 22nd August 2012 iii (a) Copyright Statement ‘I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or hereafter known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the abstract of my thesis in Dissertations Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.’ Signed ............................................................ 20th August 2012 Date ............................................................ (b) Authenticity Statement ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ............................................................ 20th August 2012 Date ............................................................ iv Contents Thesis Abstract xiv Acknowledgements xvii i Abbreviations xxi Publications arising from PhD xxii Additional PhD-related publications xxii i Chapter 1. Hepatitis C virus dependency on host lipid metabolism and its clinical implications. 1.1 Introduction 1 1.2 HCV biology and dependency on host lipid metabolism 3 1.2.1 Entry 3 1.2.2 Replication 4 1.2.3 Assembly 4 1.2.4 Secretion and egress 6 1.3 Chronic Hepatitis C therapy and the clinical implications of host- HCV metabolic interaction 7 1.3.1 HCV treatment regimens, definitions of sustained viral response (SVR), and the need for pre-treatment prediction of SVR. 7 1.3.2 Current pre-treatment viral predictors of interferon sensitivity and SVR to PEG/RBV. 8 1.3.3 Current pre-treatment host clinical and metabolic predictors of response. 8 1.4 Summary 11 1.5 References 12 v Contents (continued) Chapter 2. Overview of translational methodological approaches to assess clinical phenotypes and a review of host genomics applied to chronic HCV infection and HCV treatment response. 2.1 Background: Human Genetic Variation and Clinical Associations 19 2.1.1 Genome-wide association studies and candidate gene 19 approaches 2.1.2 Transcriptomics/Gene Expression Studies 21 2.1.3 Metabolomics 23 2.2 Genomic Variation in IL28B and Hepatitis C 24 2.2.1 Sustained viral response (SVR) 24 2.2.2 Ethnicity, IL28B and SVR 26 2.2.3 IL28B and Biological Mechanisms 26 2.3 Genomic Variation PNPLA3 and Hepatitis C infection 28 2.4 Summary 29 2.5 References 31 Chapter 3. A genome-wide association study of host cholesterol and triglyceride levels in genotype-1 chronic hepatitis C virus (HCV) infection and relationships to HCV treatment response. 3.1 Summary 36 3.2 Introduction 37 3.3 Methods 37 3.3.1 Patients 37 3.3.2 Genetic analysis 39 3.3.3 Clinical phenotypes 40 3.3.4 Statistical analyses 40 3.4 Results 41 3.4.1 Serum lipid and triglyceride genome-wide association studies 41 3.4.2 Defining the role of LDL-C and IL28B in predicting SVR 44 3.5 Discussion 48 vi Contents (continued) 3.6 Conclusions 51 3.7 References 52 3.8 Supplementary Materials 55 Chapter 4. A candidate-gene study of interleukin-28B (IL28B) and patatin-like phospholipase domain-containing 3 (PNPLA3) polymorphisms in association with hepatic steatosis in genotype 1 chronic Hepatitis C infection. 4.1 Summary 69 4.2 Introduction 70 4.3 Methods 72 4.3.1 Patients 72 4.3.2 Genetic analysis 73 4.3.3 Phenotype definition of hepatic steatosis 73 4.4 Results 74 4.4.1 IL28B and PNPLA3 variants are associated with genotype- 1 HCV-induced hepatic steatosis prevalence and steatosis severity. 74 4.4.2 Host metabolic risk factors are the major determinants of hepatic steatosis 79 4.4.3. IL28B attenuates the impact of hepatic steatosis on SVR but PNPLA3 has no significant association with SVR directly, or indirectly through steatosis. 80 4.5 Discussion