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Mutational and Functional Analyses of Kabuki Syndrome Genes Demonstrate Critical Roles in Craniofacial, Heart and Brain Developm MUTATIONAL AND FUNCTIONAL ANALYSES OF KABUKI SYNDROME GENES DEMONSTRATE CRITICAL ROLES IN CRANIOFACIAL, HEART AND BRAIN DEVELOPMENT By PETER MARCEL VAN LAARHOVEN B.S., University of California, Los Angeles, 2008 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Human Medical Genetics and Genomics Program 2015 This thesis for the Doctor of Philosophy degree by Peter Marcel Van Laarhoven has been approved for the Human Medical Genetics and Genomics Program by Kristin B. Artinger, Chair Tamim H. Shaikh, Advisor Bruce H. Appel Paul C. Megee Jay R. Hesselberth Date _01/27/2015_ ii Van Laarhoven, Peter Marcel (Ph.D., Human Medical Genetics and Genomics) Mutational and Functional Analyses of Kabuki Syndrome Genes Demonstrate Critical Roles in Craniofacial, Heart, and Brain Development Thesis directed by Associate Professor Tamim H. Shaikh. ABSTRACT Kabuki syndrome (KS) is a rare multiple congenital anomaly syndrome characterized by distinctive facial features, global developmental delay, intellectual disability, and cardiovascular and musculoskeletal abnormalities. Mutations in KMT2D have been identified in a majority of KS patients, and mutations in KDM6A have been identified as a rare cause of KS. Fifty-seven individuals clinically diagnosed with KS were analyzed for mutations in KMT2D and KDM6A, 17 by the group that implicated KMT2D and forty by ourselves. Putative pathogenic mutations were detected in KMT2D in 27 subjects and KDM6A in 4 subjects. Observed mutations included single nucleotide variations and indels leading to frameshifts, nonsense, missense or splice site alterations in both genes. Custom target enrichment and whole exome sequencing were used to identify new candidate genes. Whole exome sequencing of five KMT2D and KDM6A mutation negative subjects identified the KMT2D paralog KMT2C as a possible candidate gene. To elucidate the functional roles of KMT2D and KDM6A, we knocked down the expression of their orthologs in zebrafish. Following knockdown of kmt2d and the two zebrafish paralogs kdm6a and kdm6al, we analyzed morphants for developmental abnormalities in tissues that are affected in individuals with KS, including craniofacial structures, heart and brain. The kmt2d morphants exhibited severe abnormalities in all tissues examined. Although the kdm6a and kdm6al morphants had similar brain abnormalities, they differed in their roles in craniofacial and cardiac development. The kdm6a morphants exhibited craniofacial abnormalities, while kdm6al morphants had prominent defects in heart development. Our iii results provide further support for the roles of KMT2D and KDM6A in KS by identifying additional cases with KMT2D and KDM6A mutations. Furthermore, we have used a vertebrate model organism to provide direct evidence for the role of KMT2D and KDM6A in the development of organs and tissues affected in KS patients. The form and content of this abstract are approved. I recommend its publication. Approved: Kristin B. Artinger iv ACKNOWLEDGEMENTS First and foremost, I would like to thank my amazing wife, Mary Van Laarhoven. Without your love and support I would have never gotten where I am today. You are the reason I have pushed through when things seemed impossible. You make me strive to be a better, smarter and stronger person. I would also like to thank Leif Neitzel, for his contributions to the zebrafish project and for sharing his knowledge in the field of developmental biology. I would also like to thank James Yu for all of his help with the next- generation sequencing project and for his advice. I want to thank Elizabeth Geiger for keeping everything running smoothly and for answering all of the hundreds of questions I have asked over the years. Thank you Tamim for pushing me to be a better scientist. v CONTENTS CHAPTER I. BACKGROUND ....................................................................................................................................... 1 Kabuki Syndrome ......................................................................................................................... 1 History and Phenotype .............................................................................................. 1 Genetics of Kabuki Syndrome ................................................................................. 6 Structure and Function of KS Genes ..................................................................................... 9 The KMT2 Gene Family of Histone Methyltransferases ............................... 9 KMT2D Structure and Function .............................................................................. 10 The KDM6 Gene Family of Demethylases .......................................................... 13 KDM6A Structure and Function .............................................................................. 13 The KMT2D Complex .................................................................................................. 15 The Role of KS Genes in Development ................................................................. 16 II. ANALYSIS OF THE GENETIC BASIS OF KABUKI SYNDROME .......................................... 17 Introduction ................................................................................................................................... 17 Background ..................................................................................................................... 17 Mutation Screen of KMT2D and KDM6A ............................................................. 18 Identification of KS Candidate Genes ................................................................... 19 Materials and Methods .............................................................................................................. 20 Sanger Sequencing of KMT2D and KDM6A in KS Subjects........................... 20 Custom Capture of Potential KS Candidate Genes and Next-Generation Sequencing ................................................................................... 21 Whole Exome Sequencing ........................................................................................ 24 Results .............................................................................................................................................. 25 Sanger Sequencing Analysis of KMT2D and KDM6A ...................................... 25 In-silico Analysis of Missense Variants in KMT2D and KDM6A ................. 27 vi Analysis Pipeline for Next-generation Sequencing Data ............................. 32 Custom Capture and Next-Generation Sequencing ........................................ 37 Exome Sequencing ....................................................................................................... 38 Discussion ....................................................................................................................................... 41 KMT2D and KDM6A Mutations in KS .................................................................... 41 KMT2D and KDM6A Mutations are Not Identified in All KS Subjects ..... 43 Custom Capture and Sequencing of Potential KS Candidate Genes ........ 43 Whole Exome Sequencing ........................................................................................ 44 Identification of a Novel KS Candidate Gene..................................................... 45 III. THE ROLES OF KMT2D AND KDM6A IN DEVELOPMENT ................................................ 48 Introduction ................................................................................................................................... 48 Known Roles of KMT2D and KDM6A Relevant to Development ............... 48 The Zebrafish as a Model Organism for Studying the Genetics of MCA Disorders ............................................................................................................... 49 Conservation of KS Genes in Zebrafish ............................................................... 49 Morpholino Knockdown of Genes Implicated in Kabuki Syndrome ....... 50 Stable Gene Knockout Strategies ........................................................................... 51 The Role of Retinoic Acid Signaling in Development .................................... 52 Materials and Methods .............................................................................................................. 53 Zebrafish Transgenic Strains and Husbandry .................................................. 53 In-situ Hybridization Probes.................................................................................... 53 Whole Mount In-situ Hybridization ...................................................................... 54 Antisense Morpholino and mRNA Injections ................................................... 55 Phenotyping of Morphants ....................................................................................... 56 Hematoxylin and Eosin Staining ............................................................................ 57 Immunofluorescence .................................................................................................. 57 vii Validation of Splice-site Morpholino Effects ..................................................... 58 Imaging ............................................................................................................................. 59 Zinc-finger Nucleases ................................................................................................
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