UNIVERSITY OF CALIFORNIA Los Angeles Applications of Next-Generation DNA Sequencing to the Identification of Rare Variants in Congenital DisorDers of the Intestine anD Brain A Dissertation submitteD in partial satisfaction of the requirements for the Degree of Doctor of Philosophy in Human Genetics by Michael Yourshaw 2014 © Copyright by Michael Yourshaw 2014 ABSTRACT OF THE DISSERTATION Applications of Next-Generation DNA Sequencing to the Identification of Rare Variants in Congenital DisorDers of the Intestine anD Brain by Michael Yourshaw Doctor of Philosophy in Human Genetics University of California, Los Angeles, 2014 Professor Stanley F. Nelson, Chair High throughput, massively parallel DNA sequencing proviDes a powerful technology to stuDy the human genome anD to iDentify variations in DNA that cause disease. Sequencing the protein coDing region of the genome (‘whole-exome sequencing’) is a cost effective methoD to search the part of the genome that is most likely to harbor Disease relateD mutations. We DevelopeD software methoDs to process sequencing data and to annotate variants with Data on genes, function, conservation, expression, Diseases, pathways, anD protein structure. We applieD whole-exome sequencing to search for the molecular basis of Disease in three projects: 1) a cohort of patients with congenital diarrheal disorders (CDDs); 2) a cohort of patients with congenital chronic intestinal pseuDo-obstruction (CIPO) or the relateD Disease, megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIH); anD 3) four siblings with infantile pontocerebellar hypoplasia and spinal motor neuron Degeneration. ii We sequenceD 45 probanDs from Diverse ethnic backgrounDs who were DiagnoseD with a variety of CDDs of probable, but unknown genetic cause. Patients had been diagnosed with generalizeD malabsorptive Diarrhea, selective nutrient malabsorption, secretory diarrhea, anD infantile IBD. We founD homozygous or compound heterozygous mutations, 25 of them novel, in genes known to be associated with CDDs in 27 cases (60%). The genes implicateD were ADAM17, DGAT1, EPCAM, IL10RA, MALT1, MYO5B, NEUROG3, PCSK1, SI, SKIV2L, SLC26A3, anD SLC5A. With whole-exome sequencing in a cohort of 20 patients with congenital CIPO or MMIH, we iDentifieD a subset of 10 cases with potentially Damaging De-novo dominant acting mutations at highly conserveD loci in the ACTG2 gene, encoding actin, gamma-enteric smooth muscle precursor, a protein essential to the functioning of muscle cells in the intestinal wall. By exome sequencing, we Discovered rare recessive mutations in EXOSC3 (encoDing exosome component 3) that were responsible for pontocerebellar hypoplasia and spinal motor neuron degeneration in the four probanDs, anD identifieD identical anD aDDitional novel mutations in a large percentage of other chilDren with the same DisorDer. In conclusion, we DemonstrateD that whole-exome sequencing is an effective approach for the identification of casual mutations in that may escape Detection with stanDarD practice involving a complex Diagnostic workup and targeted gene sequencing. iii The Dissertation of Michael Yourshaw is approveD. Rita M. Cantor Lars Dreier J. AlDons Lusis Stanley F. Nelson, Committee Chair University of California, Los Angeles 2014 iv Dedicated to my parents Mike and Elizabeth my children Ivan, Erik, Alexis, Amanda, and Christopher and my grandchildren Sarah, Thunder, Steel, Heaven, Jewel, Love, and Mercy v TABLE OF CONTENTS ABSTRACT OF THE DISSERTATION ......................................................................................................................... ii LIST OF FIGURES .......................................................................................................................................................... viii LIST OF TABLES ................................................................................................................................................................ x ACKNOWLEDGMENTS .................................................................................................................................................. xi Curriculum Vitae ........................................................................................................................................................... xiv CHAPTER ONE IntroDuction ............................................................................................................................................ 1 References ................................................................................................................................................................... 22 CHAPTER TWO Rich annotation of DNA sequencing variants by leveraging the Ensembl Variant Effect PreDictor with plugins .................................................................................................................................... 37 References ................................................................................................................................................................... 58 CHAPTER THREE Whole-exome sequencing for the iDentification of casual mutations in congenital Diarrheal DisorDers ................................................................................................................................ 61 References ................................................................................................................................................................... 82 CHAPTER FOUR Exome sequencing finds a novel PCSK1 mutation in a chilD with generalizeD malabsorptive Diarrhea anD Diabetes insipiDus .............................................................................................. 88 CHAPTER FIVE Functional consequences of a novel variant of PCSK1 ...................................................... 99 CHAPTER SIX Mutations in ACTG2 are associateD with sporadic congenital chronic intestinal pseudo-obstruction anD megacystis-microcolon-intestinal hypoperistalsis syndrome ............. 108 References ................................................................................................................................................................ 121 vi CHAPTER SEVEN Mutations in the RNA exosome component gene EXOSC3 cause pontocerebellar hypoplasia and spinal motor neuron degeneration .................................................................................... 128 CHAPTER EIGHT Conclusions .................................................................................................................................... 149 References ................................................................................................................................................................ 157 vii LIST OF FIGURES Chapter 2 Figure 1. Overview of DNA sequencing and annotation .................................................................................. 53 Text box 1. ProteinSeq plugin ..................................................................................................................................... 54 Text box 2. Mito plugin ................................................................................................................................................... 55 Chapter4 Figure 1. Exome sequencing results ......................................................................................................................... 93 Figure 2. Functional characterization and visualization of wilD-type (WT) prohormone convertase 1/3 (PC1/3) complementary DNA anD PC1/3 containing the Y343X nonsense mutation ............................................................................................................................................................................... 94 Figure 3. Absence of prohormone convertase 1/3 (PC1/3)-positive enteroendocrine cells in small bowel mucosa ........................................................................................................................................................ 95 Table S1. Potentially harmful mutations in PCSK1 found in public Datasets ......................................... 98 Chapter 5 Figure 1. Domain structure and SNP locations within preproPC1/3 ..................................................... 101 Figure 2. Direct Sanger sequencing of genomic DNA from a subject bearing the Arg80Gln variant ................................................................................................................................................................................ 103 Figure 3. Western blotting of wilD-type anD variant PC1/3 proteins expresseD in HEK cells ..... 104 Figure 4. Specific activities of wilD-type anD variant PC1/3 proteins, expresseD in HEK cells ... 104 Figure 5. Western blotting of wilD-type anD novel R80Q (rs1799904) variant PC1/3s, expressed in Neuro-2A cells ..................................................................................................................................... 105 Chapter 7 Figure 1. Neuroimaging, neuromuscular anD pathological features in family 1 ................................ 130 Figure 2. EXOSC3 mutations in PCH1 .................................................................................................................... 131 Figure 3 KnockDown of exosc3 in zebrafish embryos Disrupts normal Development ..................... 132 Supplementary Figure 1. Genome-wiDe SNP Genotyping & Linkage Analysis ................................... 143 Supplementary