Pathogenicity and selective constraint in the non-coding genome Patrick J. Short St. Edmund’s College Submitted: September, 2018 Supervisor: Dr. Matthew Hurles This dissertation is submitted in accordance with the requirements of the University of Cambridge for the degree of Doctor of Philosophy. Acknowledgements I would like to thank all of the members of the Hurles team and the DDD project for four wonderful years of research and for being such great scientists and friends. To Jeremy, Sebastian, and Jeff especially, thank you for all of your mentorship. To my supervisor Matt, I am incredibly thankful to have a supervisor that is both a rigorous, visionary scientist, and such a down-to-earth person. Thank you so much for all of your support. To the Wellcome Trust and the Mathematical Genomics and Medicine PhD programme, thank you for providing the structure and financial support to make all of this work possible. To my parents, Jack and Denice, my brother John, and my fiancée Timarie, thank you so much for supporting me, even as I moved ‘across the pond’ to pursue this dream. I consider myself so lucky to have a family like you. Preface This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text It does not exceed the prescribed word limit for the School of Biological Sciences. Table of Contents CHAPTER 1: INTRODUCTION ..................................................................................................... 1 SECTION 1.1: THE FUNCTION OF NON-CODING DNA IN THE HUMAN GENOME ...................................... 1 SECTION 1.1.1. FUNCTIONAL ROLES OF NON-CODING ELEMENTS IN THE HUMAN GENOME ....................... 1 SECTION 1.1.2. CHARACTERISING NON-CODING ELEMENTS IN THE HUMAN GENOME .............................. 3 SECTION 1.2: THE ROLE OF NON-CODING VARIATION IN MENDELIAN DISEASE ....................................... 9 SECTION 1.2.1. THE CONTRIBUTION OF PROTEIN-CODING VARIATION TO MENDELIAN DISEASE .................. 9 SECTION 1.2.2. REGULATORY VARIATION IN MENDELIAN PHENOTYPES ............................................. 11 SECTION 1.2.3 THE ROLE OF REGULATORY VARIATION IN CANCER AND NEURODEVELOPMENTAL DISORDERS 13 CHAPTER 2: DE NOVO MUTATIONS IN REGULATORY ELEMENTS CONTRIBUTE TO SEVERE NEURODEVELOPMENTAL DISORDERS............................................................................... 21 INTRODUCTION ......................................................................................................... 21 METHODS ............................................................................................................... 25 SEQUENCING IN THE DDD COHORT ............................................................................... 25 ALIGNMENT, VARIANT CALLING, AND QUALITY CONTROL ................................................... 25 RESULTS SECTION 2.1: ASSESSING THE ROLE OF DE NOVO MUTATIONS IN REGULATORY ELEMENTS IN SEVERE DD ........................................................................................................................ 33 RESULTS SECTION 2.1.1. MODELLING THE GERMLINE MUTATION RATE IN THE NON-CODING GENOME 33 RESULTS SECTION 2.1.2. FETAL BRAIN ACTIVE ULTRA-CONSERVED NON-CODING ELEMENTS ARE ENRICHED FOR MUTATIONS IN EXOME-NEGATIVE PROBANDS WITH NEURODEVELOPMENTAL PHENOTYPES ..... 37 RESULTS SECTION 2.1.3. RECURRENTLY MUTATED REGULATORY ELEMENTS ............................. 46 RESULTS SECTION 2.2: EXTRAPOLATING RESULTS FROM TARGETED REGULATORY ELEMENTS TO GENOME-WIDE ESTIMATES ............................................................................................................... 49 RESULTS SECTION 2.2.1. ESTIMATE OF PROPORTION OF PROBANDS CARRYING A PATHOGENIC MUTATION IN A REGULATORY ELEMENT GENOME-WIDE. .......................................................................... 49 RESULTS SECTION 2.2.2. POWER CALCULATIONS AND ESTIMATION OF FRACTION OF BASES CONTRIBUTING TO SEVERE DD WHEN MUTATED ........................................................................................ 50 DISCUSSION ............................................................................................................. 53 CHAPTER 3: MUTATION RATE AND SELECTIVE CONSTRAINT IN THE NON-CODING GENOME ...................... 59 INTRODUCTION .................................................................................................................... 59 HETEROGENEITY IN THE HUMAN GERMLINE MUTATION RATE ........................................................... 59 MEASURING SELECTIVE CONSTRAINT IN THE HUMAN GENOME ......................................................... 61 METHODS .......................................................................................................................... 63 RESULTS SECTION 3.1: MODELLING THE HUMAN GERMLINE MUTATION RATE ..................................... 69 RESULTS SECTION 3.1.1. IMPROVED MODELLING OF THE HUMAN GERMLINE MUTATION RATE................. 69 RESULTS SECTION 3.1.2. GENOMIC FEATURES ASSOCIATED WITH HYPERMUTABILITY ............................ 74 RESULTS SECTION 3.2: MEASURING SELECTIVE CONSTRAINT IN REGULATORY ELEMENTS GENOME-WIDE .... 79 RESULTS SECTION 3.2.1. PATTERNS OF PURIFYING SELECTION IN NON-CODING ELEMENTS GENOME-WIDE . 79 RESULTS SECTION 3.2.2. NUCLEOTIDE-LEVEL CONSERVATION SCORES ARE MORE INFORMATIVE OF SELECTIVE CONSTRAINT THAN LOCUS-LEVEL SCORES .................................................................................... 87 RESULTS SECTION 3.2.3. DOMINANCE AND SELECTION IN THE NON-CODING GENOME .......................... 93 DISCUSSION ........................................................................................................................ 95 CHAPTER 4: FUNCTIONAL CHARACTERISATION OF MUTATIONS IN ULTRA-CONSERVED ELEMENTS ASSOCIATED WITH SEVERE DEVELOPMENTAL DISORDERS ............................................................................... 103 INTRODUCTION .................................................................................................................. 103 METHODS FOR ASSESSING ENHANCER ACTIVITY USING REPORTER CONSTRUCTS .................................. 103 MASSIVELY PARALLEL REPORTER ASSAYS METHODS AND APPLICATIONS ............................................ 104 METHODS ........................................................................................................................ 107 RESULTS SECTION 4.1: ASSESSING DISEASE-ASSOCIATED ENHANCER ACTIVITY USING MASSIVELY PARALLEL REPORTER ASSAYS............................................................................................................... 114 RESULTS SECTION 4.1.2. IMPACT OF SNVS AND INDELS ON ULTRA-CONSERVED ELEMENT FUNCTION ...... 125 RESULTS SECTION 4.1.3. ASSESSING THE IMPACT OF PATIENT MUTATIONS USING MPRAS ................... 127 RESULTS SECTION 4.2: ASSESSING DISEASE-ASSOCIATED ENHANCER ACTIVITY USING MOUSE TRANSGENESIS ASSAYS ............................................................................................................................ 129 RESULTS SECTION 4.2.1. MOUSE TRANSGENESIS ASSAYS TO IDENTIFY PUTATIVE DEVELOPMENTAL ENHANCERS ..................................................................................................................................... 129 DISCUSSION ...................................................................................................................... 132 CHAPTER 5: DISCUSSION........................................................................................... 137 APPENDIX 1 ........................................................................................................... 151 Abbreviations 3C – Chromosome Conformation Capture ASD – autism spectrum disorder ASE – allele specific expression BWA – Burrows-Wheeler Aligner CADD – combined annotation dependent depletion CDTS – context dependent tolerance score CNEs – conserved non-coding elements CNN – convolutional neural network CNVs – copy number variations CRISPRa – CRISPR activation CRISPRi – CRISPR inactivation DDD – Deciphering Developmental Disorders DD – Developmental Disorders DHS – DNase I hypersensitive site DNA – deoxy ribonucleic acid DNM – de novo mutation DSB – double strand break ENCODE – Encyclopedia of DNA Elements eQTL – expression quantitative trait loci ESCs – embryonic stem cells ExAC – exome aggregation consortium FDR – false discovery rate GAM - Genome Architecture Mapping GATK – Genome Analysis Toolkit GFP – green fluorescent protein gnomAD – genome aggregation database HARs – human accelerated regions HGEs – human gained enhancers Indels – insertions/deletions iPSCs – induced pluripotent stem cells LCLs – Lymphoblastoid Cell Lines lncRNAs – long non-coding RNAs MAPS – mutability adjusted proportion of singletons MAVEs – multiplexed assays of variant effect MPRAs – massively parallel reporter assays NHEJ – non-homologous end-joining