Genetic Analysis of Retinopathy in Type 1 Diabetes by Sayed Mohsen Hosseini A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by S. Mohsen Hosseini 2014 Genetic Analysis of Retinopathy in Type 1 Diabetes Sayed Mohsen Hosseini Doctor of Philosophy Institute of Medical Science University of Toronto 2014 Abstract Diabetic retinopathy (DR) is a leading cause of blindness worldwide. Several lines of evidence suggest a genetic contribution to the risk of DR; however, no genetic variant has shown convincing association with DR in genome-wide association studies (GWAS). To identify common polymorphisms associated with DR, meta-GWAS were performed in three type 1 diabetes cohorts of White subjects: Diabetes Complications and Control Trial (DCCT, n=1304), Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR, n=603) and Renin-Angiotensin System Study (RASS, n=239). Severe (SDR) and mild (MDR) retinopathy outcomes were defined based on repeated fundus photographs in each study graded for retinopathy severity on the Early Treatment Diabetic Retinopathy Study (ETDRS) scale. Multivariable models accounted for glycemia (measured by A1C), diabetes duration and other relevant covariates in the association analyses of additive genotypes with SDR and MDR. Fixed-effects meta- analysis was used to combine the results of GWAS performed separately in WESDR, ii RASS and subgroups of DCCT, defined by cohort and treatment group. Top association signals were prioritized for replication, based on previous supporting knowledge from the literature, followed by replication in three independent white T1D studies: Genesis-GeneDiab (n=502), Steno (n=936) and FinnDiane (n=2194). No SNP reached genome-wide significance in survival meta-GWAS for SDR. In a case- control meta-GWAS, however, SNPs in DPP10 showed close to genome-wide significant association with SDR. Although, this association could not be replicated in three other studies (P>0.05), the direction of effect remained consistent in all but one of the examined populations. Among the top hits for SDR short of genome-wide significance, SNPs near NLPR3 and AKR1E2 were replicated, after accounting for multiple testing. These signals and other top signals in the meta-GWAS of SDR generally fall in proximity to strong functional candidate genes. In survival and case-control meta-GWAS for MDR, no SNP reached genome-wide significance. Consistently, our estimation of common additive heritability suggests a stronger genetic component for SDR compared to MDR. iii Acknowledgments First and foremost, I am grateful to all the patients who, despite their pain and suffering, selflessly took the time and effort to participate in long term studies of DCCT/EDIC, WESDR and RASS. You inspire us, teach us and help us be useful. I really hope that the results of my research be a step in the right direction and translate to useful measures to help patients suffering from diabetes in the future. I am truly grateful to my mentor, Dr. Andrew Paterson, for his unwavering support and great patience over years, giving me the opportunity to start and complete this work. Your direction and understanding have been indispensable. I am thankful to members of Paterson and Bull lab, past and present, for their assistance and friendships: Daryl Waggott, Charlie “Zhijian” Chen, Enqing Shen. I would also like to thank everyone from our “Friday meetings” for stimulating discussions, great insights and support, in particular Drs. Shelley Bull, Andrew Boright, Lei Sun and Angelo Canty. Thanks to Dr. Karen Eny for reviewing introduction of the thesis and providing helpful suggestions. I also thank Dr. Jerald Lawless for his instrumental advice for developing time-to event models. I am thankful to the members of my advisory committee, Dr. Thomas Hudson and Dr. George Fantus, for their input, guidance and advice during my PhD work. My doctorate study would not have been possible without graduate scholarships and research assistantships from the Hospital for Sick Children, Vision Science Research Program, Peterborough K.M. Hunter Foundation, University of Toronto and Juvenile Diabetes Research Foundation Canada. Travel awards from Banting and Best Diabetes Centre have certainly enriched my learning experience. iv On a personal note, I am thankful to my parents, brother and sisters for their consistent faith in me and for their continued love, support and encouragement. You are the reason I keep going on. I am grateful to my friends, especially Afshin, Vahid and Vahideh, who stood by my side in difficult times. Finally, I am thankful to the ones who hurt me; you helped me improve, made me stronger and reminded me that: “Au milieu de l'hiver, j'ai découvert en moi un invincible été.” v List of Abbreviations A1C Glycated hemoglobin ACACB acetyl-CoA carboxylase beta ACE Angiotensin I Converting Enzyme ACN9 ACN9 homolog ACO1 aconitase 1 ACP6 acid phosphatase 6, lysophosphatidic ACR Albumin Creatinine Ratio ADCK4 aarF domain containing kinase 4 AER Albumin Excretion Ratio AFF3 AF4/FMR2 family, member 3 AGE Advanced Glycation End product AGER Advanced Glycosylation End product-specific Receptor AGT angiotensinogen AKR1B1 aldo-keto reductase family 1, member B1 AKR1E2 aldo-keto reductase family 1, member E2 ARHGAP22 Rho GTPase activating protein 22 ARL4C ADP-ribosylation factor-like 4C ASAP2 ArfGAP with SH3 domain, ankyrin repeat and PH domain 2 ASB3 ankyrin repeat and SOCS box containing 3 BBS5 Bardet-Biedl syndrome 5 bFGF basic Fibroblast Growth Factor BMI Body Mass Index BP Blood Pressure BRB Blood-Retina Barrier CACNA1E calcium channel, voltage-dependent, R type, alpha 1E subunit CCBP2 chemokine binding protein 2 CCNE1 cyclin E1 CD300A CD300a molecule CHN2 chimerin 2 CI Confidence Interval CLOGLOG Complementary Log Log COMMD6 COMM domain containing 6 COX7A2 cytochrome c oxidase subunit VIIa polypeptide 2 CPNE4 copine IV CPVL carboxypeptidase, vitellogenic-like CSME Clinically Significant Macular Edema vi CVD Cardiovascular Disease DAG diacylglycerol DBC1 deleted in bladder cancer 1 DBP Diastolic Blood Pressure DCCT Diabetes Complications and Control Trial DDX5 DEAD (Asp-Glu-Ala-Asp) box helicase 5 DM Diabetes Mellitus DME Diabetes Macular Edema DN Diabetic Nephropathy DPP10 dipeptidyl-peptidase 10 DR Diabetic Retinopathy dur Duration of Diabetes DZ Di-Zygotic EDIC Epidemiology of Diabetes Interventions and Complications EFNB2 ephrin B2 EFNB2 ephrin B2 EPO Erythropoietin ESRD End Stage Renal Disease ETDRS Early Treatment Diabetic Retinopathy Study FAM107B family with sequence similarity 107, member B FAM198A family with sequence similarity 198, member A FDR False Discovery Rate FGFR1 fibroblast growth factor receptor 1 FinnDiane Finnish Diabetic Nephropathy Study FSTL5 follistatin-like 5 FTO fat mass and obesity associated GAPDH glyceraldehyde-3-phosphate dehydrogenase GBE1 glucan (1,4-alpha-), branching enzyme 1 GeneDiab Génétique de la Néphropathie Diabétique GFR Glomerular Filteration Rate GH Growth Hormone GJA5 gap junction protein, alpha 5 GoKinD Genetics of Kidney in Diabetes (Study) GUSBP10 glucuronidase, beta pseudogene 10 GWAS Genome-Wide Association Study HAND2 heart and neural crest derivatives expressed transcript 2 HbA1c Glycated hemoglobin HDL High Density Lipoprotein HGF Hepatocyte Growth Factor vii HMGB1 high mobility group box 1 HR Hazard Ratio HS6ST3 heparan sulfate 6-O-sulfotransferase 3 HWE Hardy-Weinberg Equilibrium IBD Identity-By-Descent IBS Identity by State ICAM1 intercellular adhesion molecule 1 ICC Intra-Class Correlation IGF-1 Insulin-like Growth Factor 1 IGSF21 immunoglobin superfamily, member 21 IRMA Intra-Retinal Microvascular Abnormalities IRS2 insulin receptor substrate 2 IRX4 iroquois homeobox 4 ITGA2 integrin, alpha 2 KCNIP4 Kv channel interacting protein 4 KCNN2 potassium intermediate/small conductance calcium-activated channel, subfamily N, member 2 KLF12 Kruppel-like factor 12 LD Linkage Disequilibrium LDL Low Density Lipoprotein LINC00426 long intergenic non-protein coding RNA 426 LINC00460 long intergenic non-protein coding RNA 460 LINC00523 long intergenic non-protein coding RNA 523 LINC01118 long intergenic non-protein coding RNA 1118 LMO7 LIM domain 7 LOC643441 uncharacterized LOC643441 LOXHD1 lipoxygenase homology domains 1 LPA lysophosphatidic acid LRP2 low density lipoprotein receptor-related protein 2 LUZP2 leucine zipper protein 2 MA MicroAneurysm MAF Minor Allele Frequency MAGI3 membrane associated guanylate kinase, WW and PDZ domain containing 3 MDR Mild Diabetic Retinopathy MDS Multidimensional Scaling MHC Major Histocompatibiltiy Complex MIR3924 microRNA 3924 MKI67 marker of proliferation Ki-67 MTHFR methylenetetrahydrofolate reductase viii MTUS1 microtubule associated tumor suppressor 1 MVCD MicroVascular Complications of Diabetes MYLIP myosin regulatory light chain interacting protein MYSM1 Myb-like, SWIRM and MPN domains 1 MZ Mono-Zygotic N6AMT2 N-6 adenine-specific DNA methyltransferase 2 NF - κB Nuclear Factor - kappa B NLRP3 NLR family, pyrin domain containing 3 NOS3 nitric oxide synthase 3 NPDR Non-Proliferative Diabetic Retinopathy ODF1 outer dense fiber of sperm tails 1 OR Odds Ratio OR4K17 olfactory receptor, family 4, subfamily K, member 17 PARP2 poly (ADP-ribose) polymerase 2 PC Principal Component PCA Principal Component Analysis PCSK2 proprotein convertase subtilisin/kexin type 2 PDR Proliferative Diabetic Retinopathy PECAM1 platelet/endothelial cell adhesion molecule 1