University of Dublin, Trinity College School of Medicine, Department of Medicine Investigation of the monogenic causes of chronic kidney disease PhD Thesis April 2020 Dervla Connaughton Supervisor: Professor Mark Little Co-Supervisors: Professor Friedhelm Hildebrandt and Professor Peter Conlon 1 DECLARATION I declare that this thesis has not been submitted as an exercise for a degree at this or any other university and it is entirely my own work. This work was funding by the Health Research Board, Ireland (HPF-206-674), the International Pediatric Research Foundation Early Investigators’ Exchange Program and the Amgen® Irish Nephrology Society Specialist Registrar Bursary. I agree to deposit this thesis in the University’s open access institutional repository or allow the Library to do so on my behalf, subject to Irish Copyright Legislation and Trinity College Library conditions of use and acknowledgement. I consent to the examiner retaining a copy of the thesis beyond the examining period, should they so wish (EU GDPR May 2018). _____________________ Dervla Connaughton 2 SUMMARY Chapter 1 provides an introduction to the topic while Chapter 2 provides details of the methods employed in this work. In Chapter 3 I provide an overview of the currently known monogenic causes for human chronic kidney disease (CKD). I also describe how next- generation sequencing can facilitate molecular genetic diagnostics in individuals with suspected genetic kidney disease. Chapter 4 details the findings of a multi-centre, cross-sectional study of patients with CKD in the Republic of Ireland. The primary aim of this study (the Irish Kidney Gene Project) was to describe the prevalence of reporting a positive family history of CKD among a representation sample of the CKD population. Of the 1,840 adult patients with CKD recruited, 629 (34%) reported a positive family history of CKD. Excluding autosomal dominant polycystic kidney disease (134 of 629), a positive family history was reported by 495 participants (27%). CKD of unknown aetiology was the commonest aetiology in the non-polycystic kidney disease group with a positive family history (67 of 629, 11%). Using family history of CKD as a surrogate marker of genetically inherited CKD, I then proceed to study the frequency of monogenic mutations in adults with CKD. In Chapter 5, I describe how whole exome sequencing (WES) was performed in a multi-centre, patient cohort of 114 families including 138 affected individuals with CKD. Following WES, I detected a mutation in a known CKD gene in 42 of 114 families (37%). Among the 42 families in whom a monogenic mutations in a gene known to cause CKD was identified, WES confirmed the clinical diagnosis in 17 (40%), corrected the clinical diagnosis in 9 (22%), and established a diagnosis for the first time in 16 families referred with CKD of unknown aetiology (38%). Finally, in Chapter 6, I focus on the prevalence of monogenic mutations in a paediatric cohort with congenital anomalies of the kidney and urinary tract (CAKUT). Following WES analysis in 232 families with CAKUT, I identified monogenic mutations in known human CAKUT gene or CAKUT phenocopy gene in 14% of the families with CAKUT. Interestingly, an additional search for novel monogenic causes of CAKUT in consanguineous and multiplex families revealed a potential single, novel monogenic CAKUT gene in 24 of 232 families (10%). These findings demonstrate that WES can reveal monogenic mutations in a high fraction of patients with CAKUT and will provide a new basis for the mechanistic understanding of CAKUT. 3 Table of Contents DISSEMINATION OF THESIS ................................................................................... 12 ACKNOWLEDGEMENTS ......................................................................................... 14 ABBREVIATIONS ................................................................................................... 15 GLOSSARY OF TERMS ............................................................................................ 18 ABSTRACT ............................................................................................................. 21 CHAPTER 1 INTRODUCTION .................................................................................. 23 1.1 Chronic Kidney Disease (CKD) .................................................................................................. 23 1.2 Genetic Kidney Disease (GKD) ................................................................................................. 23 1.3 Whole Exome Sequencing (WES) ............................................................................................. 24 1.4 Familial Kidney Disease ............................................................................................................ 24 1.5 The Genetic Basis of CKD ......................................................................................................... 25 1.6 Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) ............................................. 25 1.7 STUDY HYPOTHESES ............................................................................................... 26 1.8 AIMS ...................................................................................................................... 26 CHAPTER 2 METHODS ........................................................................................... 27 2.1 Isolation of genomic DNA From biological specimens ............................................................. 27 2.2 Whole Exome Sequencing ....................................................................................................... 29 2.3 Web Resources ........................................................................................................................ 35 CHAPTER 3 Personalized medicine in chronic kidney disease by detection of monogenic mutations ........................................................................................... 36 3.0 SUMMARY .............................................................................................................. 36 3.1 INTRODUCTION ...................................................................................................... 37 3.11 Chronic Kidney Disease (CKD) ................................................................................................ 37 3.12 Genetic Kidney Disease (GKD) ............................................................................................... 37 3.13 Monogenic kidney disease ..................................................................................................... 40 3.14 Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) ........................................... 43 3.15 Steroid Resistant Nephrotic Syndrome (SRNS) ...................................................................... 43 3.16 Chronic glomerulopathies ...................................................................................................... 44 3.17 Cystic kidney disease ............................................................................................................. 45 3.18 Autosomal Dominant Tubulo-Interstitial Kidney Disease (ADTKD) ........................................ 46 4 3.19 Nephrolithiasis and Nephrocalcinosis (NLNC) ....................................................................... 46 3.20 Renal tubulopathies ............................................................................................................... 47 3.21 CKD of unknown aetiology (CKDu) ........................................................................................ 47 CHAPTER 4 The Irish Kidney Gene Project - Prevalence of Family History in Patients with Chronic Kidney Disease in Ireland ................................................................. 49 4.0 SUMMARY .............................................................................................................. 49 4.1 INTRODUCTION ....................................................................................................... 50 4.2 METHODS ............................................................................................................... 51 4.21 Human subjects ..................................................................................................................... 51 4.22 Data collection ....................................................................................................................... 52 4.23 Establishing the aetiology of CKD .......................................................................................... 53 4.24 Statistical analysis .................................................................................................................. 54 4.3 RESULTS .................................................................................................................. 55 4.31 Baseline demographics .......................................................................................................... 55 4.32 Prevalence oF Familial CKD ..................................................................................................... 56 4.33 Characteristic of patients With Familial CKD .......................................................................... 56 4.34 Factors associated With Familial CKD ..................................................................................... 58 4.4 DISCUSSION ...........................................................................................................