DOCTOR OF PHILOSOPHY Next Generation Sequencing and Genome-Wide Association Studies to Identify Mitochondrial Genomic Features Associated with Diabetic Kidney Disease Skelly, Ryan Award date: 2020 Awarding institution: Queen's University Belfast Link to publication Terms of use All those accessing thesis content in Queen’s University Belfast Research Portal are subject to the following terms and conditions of use • Copyright is subject to the Copyright, Designs and Patent Act 1988, or as modified by any successor legislation • Copyright and moral rights for thesis content are retained by the author and/or other copyright owners • A copy of a thesis may be downloaded for personal non-commercial research/study without the need for permission or charge • Distribution or reproduction of thesis content in any format is not permitted without the permission of the copyright holder • When citing this work, full bibliographic details should be supplied, including the author, title, awarding institution and date of thesis Take down policy A thesis can be removed from the Research Portal if there has been a breach of copyright, or a similarly robust reason. 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Oct. 2021 Next Generation Sequencing and Genome-Wide Association Studies to Identify Mitochondrial Genomic Features Associated with Diabetic Kidney Disease A thesis submitted to the School of Medicine, Dentistry and Biomedical Sciences of the Queen’s University of Belfast for the degree of: Doctor of Philosophy (PhD) 30th September 2019 Ryan Skelly B.Sc. (Hons) Human Biology (QUB, 2013) M.Sc. Bioinformatics and Computational Genomics (QUB, 2015) 1 Preface The research described in this thesis was carried out within the Nephrology Research Group at the Belfast City Hospital and the Centre of Public Health, Queen’s University Belfast between September 2016 and September 2019. Illumina sequencing was performed by the Genomics Core Technology Unit, Queen’s University Belfast. Funding was provided by the Department for the Economy. For any Tables and Figures reproduced or adapted from other sources suitable permissions have been requested and relevant references provided in the bibliography. 2 Acknowledgements I would like to thank my supervisors, Dr Amy Jayne McKnight and Professor Peter Maxwell, for their guidance and support throughout this project. Thank you for giving me the opportunity to carry out this research and your encouragement. I would also like to thank Jill Kilner and Christopher Wooster for providing support with lab work as well as Dr Laura Smyth and Dr Marisa Cañadas Garre for their help and guidance at various stages of the project. To everyone else within the NephRes group I offer my sincere thanks and gratitude and I am pleased to have been a part of this research team. I would also like to thank my family and friends for their support and understanding over the past three years. 3 Publications and Presentations from Work in this Thesis Genetic and epigenetic analysis in genes affecting mitochondrial function are associated with chronic kidney disease in an older population Cappa, R., Smyth, L., Cañadas-Garre, M., Polpo de Campos, C., Skelly, R., Cruise, S., Kee, F., McGuinness, B., Godson, C., Maxwell, A. & McKnight, A., 2018, (Submitted). Research output: Contribution to conference › Abstract Genetic susceptibility to chronic kidney disease – some more pieces for the heritability puzzle: Genetic predisposition to kidney disease Garre, M. C., Anderson, K., Cappa, R., Skelly, R., Smyth, L., McKnight, A. J. & Maxwell, A. P., 31 May 2019, In : Frontiers in Genetics. Research output: Contribution to journal › Review article Mitochondria and chronic kidney disease: a molecular update Skelly, R., Maxwell, A. & McKnight, A., 01 May 2019, In : SPG Biomed. Research output: Contribution to journal › Article Genetic risk factors in mitochondrial DNA associated with diabetic kidney disease – GWAS discovery and meta-analysis Skelly, R., Smyth, L., Cole, J., Maxwell, A., GENIE Consortium, DNCRI Consortium & McKnight, A., 2019, (Accepted). Research output: Contribution to conference › Poster 4 Abbreviations Used in this Thesis Abbreviation Long form 11βHSD1 11β-hydroxysteroid dehydrogenase 1 2-h PG one-off random plasma glucose concentration ACE angiotensin-converting enzyme ACE2 angiotensin-converting enzyme 2 ACR albumin to creatinine ratio AGEs advanced glycation end products AKI acute kidney injury AMPK 5' adenosine monophosphate-activated protein kinase AT1 angiotensin II receptor type 1 AT2 angiotensin II receptor type 2 ATP Adenosine Triphosphate AusDiane Austrian Diabetic Nephropathy Study BCH Belfast City Hospital BCL binary base call format BRTx Belfast renal transplant BST-DNA bisulfite-treated DNA CACTI Coronary Artery Calcification in Type 1 Diabetes cAMP cyclic adenosine monophosphate CD95L CD95 ligand CD-CV common disease/common variant CKD chronic kidney disease CVD cardiovascular disease DCCT/EDIC Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications DKD diabetic kidney disease D-loop displacement-loop DM diabetes mellitus DNA deoxyribonucleic acid DNMT DNA methyltransferase DNCRI Diabetic Nephropathy Collaborative Research Initiative DPP-4i dipeptidyl peptidase-4 inhibitors 5 ECM extra cellular matrix EDC Pittsburgh Epidemiology of Diabetes Complications Study eGFR estimated glomerular filtration rate EGFR epidermal growth factor receptor ESRD end-stage renal disease ET-1 endothelin-1 ETC electron transfer chain FFA free fatty acid FGF21 fibroblast growth factor 21 FIND Family Investigation of Nephropathy and Diabetes FinnDiane Finnish Diabetic Nephropathy Study FPG fasting plasma glucose GAD glutamic acid decarboxylase 65 GADPH glyceraldehyde-3-phosphate dehydrogenase GBM glomerular basement membrane GCTU Genomics Core Technology Unit GDM gestational diabetes mellitus GENEDIAB French and Belgian subjects from the Genetics of Diabetic Nephropathy GENIE Genetics of Nephropathy – an International Effort GFR glomerular filtration rate GLP1 glucagon-like peptide 1 GoKinD Genetics of Kidneys in Diabetes GoKinD Genetics of Kidneys in Diabetes US Study GSH reduced glutathione GWAS genome-wide association studies HapMap International Haplotype Map HbA1c haemoglobin A1c HGNC HUGO Gene Nomenclature Committee HLA human leucocyte antigens HSP heavy strand promoter H-Strand heavy strand iDCs immature dendritic cells 6 IDF International Diabetes Federation IFG impaired fasting glucose IFNγ interferon-γ IGT impaired glucose tolerance IR insulin resistance JAK2 janus kinase-2 JOSLIN Joslin Kidney Study LADA latent autoimmune diabetes of adults LatDiane Latvian Diabetic Nephropathy Study LD linkage disequilibrium LitDiane Lithuanian Diabetic Nephropathy Study lncRNA long ncRNA LSP Light Strand Promoter L-Strand Light Strand MAF minor allele frequency MHC major histocompatibility complex miRNA micro RNA mmol/L millimoles per litre MODY maturity onset diabetes of the young mRNA messenger RNA mtDNA mitochondrial DNA mtSSB mitochondrial single-stranded DNA-binding protein NADH reduced nicotinamide adenine dinucleotide NCR non-coding region ncRNA non-coding RNAs nDNA nuclear DNA NEMGs nuclear encoded mitochondrial genes NephRes QUB Nephrology Research Group NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NGS next-generation sequencing NICOLA Northern Ireland Cohort for the Longitudinal study of Ageing NOD non-obese diabetic 7 NP natriuretic peptide OH origin of replication for the H-strand OR odds ratio OXPHOS oxidative phosphorylation PCR polymerase chain reaction PIF1 petite integration frequency 1 piRNA piwi-interacting RNA PKC protein kinase C POLRMT mitochondrial RNA polymerase POLγ DNA polymerase γ POLγB DNA polymerase γ processivity factor PPARγ peroxisome proliferator-activated receptor gamma PTM post-translational modification QC quality control QUB Queen's University Belfast RAAS renin-angiotensin-aldosterone system RNA ribonucleic acid RNaseH1 ribonuclease H1 ROCK1 Rho-associated coiled coil-containing protein kinase 1 RomDiane Romanian Diabetic Nephropathy Study ROS reactive oxygen species rRNA Ribosomal RNA RRT renal replacement therapy SBS sequencing by synthesis SDM strand-displacement model SDRNT1BIO Scottish Diabetes Research Network Type 1 Bioresource SGLT2 sodium/glucose co-transporter 2 siRNA small interfering RNA SNP single nucleotide polymorphism snRNA small nuclear RNA SOD superoxide dismutase SOLiD Sequencing by Oligonucleotide Ligation and Detection 8 SPPARM selective peroxisome proliferator-activated receptor modulator SUMMIT Surrogate markers for Micro- and Macro-vascular har d endpoints for Innovative diabetes Tools SUV3 suppressor of Var1 3-Like Protein 1 T1DM type 1 diabetes mellitus T2DM type 2 diabetes mellitus TFAM mitochondrial transcription factor A TFBM mitochondrial transcription factor B TGF-β