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Discovering Disease Causing Variants in Dogs Through Whole Genome Sequencing

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Discovering Disease Causing Variants in Dogs Through Whole Genome Sequencing DISCOVERING DISEASE CAUSING VARIANTS IN DOGS THROUGH WHOLE GENOME SEQUENCING A Dissertation presented to the Faculty of the Graduate School University of Missouri In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy in Veterinary Pathobiology by Ana Leticia Kolicheski Dr. Gary S. Johnson, Dissertation Supervisor July 2017 The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled DISCOVERING DISEASE CAUSING VARIANTS IN DOGS THROUGH WHOLE GENOME SEQUENCING, presented by Ana Leticia Kolicheski a candidate for the degree of Doctor of Philosophy in Pathobiology and hereby certify that in their opinion it is worthy of acceptance. Dr. Gary S. Johnson Dr. Bimal K. Ray Dr. Leslie A. Lyons Dr. Christine G. Elsik Dr. Robert D. Schnabel Acknowledgements First and foremost I would like to thank Dr. Gary S. Johnson for giving me the opportunity to work in his lab and guiding me through this PhD program. Dr. Johnson is an amazing mentor, not only for the free meals and frogs, but he made a permanent mark in my life with his kind nature, quirky and dry sense of humor, patience, hard work and perseverance. I would like to express my gratitude to my dissertation committee, Dr. Bimal K. Ray, Dr. Leslie Lyons, Dr. Christine G. Elsik, and Dr. Robert D. Schnabel for all the helpful comments and guidance throughout my program. Each of you has had a very positive impact on me. I am thankful to have learned so much from you and your different areas of expertise; those are useful skills that I will carry throughout my professional life. I would also like to thank all the lab crew: Dr. Tendai Mutangadura, Dr. Juyuan Guo, Liz Hansen, Peggy Ann Eichen, Natalie Villani, and the so many other members that have come and gone throughout the years. I also acknowledge Dr. Dennis O’Brien, Dr. Martin Katz, Dr. Joan Coates, and the many other research collaborators. I am also extremely thankful for all the support and encouragement I received from all of my family. My family was always there for me and helped to motivate me through the hard times, even from far away. A special thank you to my lovely boyfriend Thomas Stauffer, and to my adoptive American family: Donna and Don Stauffer, and Brian and Alexandria Stauffer, who were always supportive and believed in me. Last but not least, I would like to thank my friends in Columbia, Guilherme Hosotani, Mariana Masiero, Estela Kobashigawa, Francine Russo, Brock Duck, Tim Poindexter, Sidharth Sen, Barbara Gandolfi and Erica Creighton. And my friends who are far away and still being supportive, Nathaly Gasparin, Mieke Stevens, Stefan Norenius, Brian Davis and Fabiana Farias. ii Table of Contents Acknowledgements ...............................................................................................ii Abstract ................................................................................................................ v Chapter 1 .............................................................................................................. 1 Introduction ....................................................................................................... 1 Chapter 2 ............................................................................................................ 16 A homozygous PIGN missense mutation in Soft Coated Wheaten Terriers with a canine paroxysmal non-kinesigenic dyskinesia ................................. 16 Abstract ........................................................................................................ 16 Introduction .................................................................................................. 17 Methods ....................................................................................................... 19 Results ......................................................................................................... 22 Discussion .................................................................................................... 27 Chapter 3 ............................................................................................................ 42 Australian Cattle Dogs with Neuronal Ceroid Lipofuscinosis are Homozygous for a CLN5 Nonsense mutation Previously Identified in Border Collies ........... 42 Abstract ........................................................................................................ 42 Introduction .................................................................................................. 43 Materials and Methods ................................................................................. 45 Results ......................................................................................................... 54 Discussion .................................................................................................... 61 Footnotes ..................................................................................................... 65 Chapter 4 ............................................................................................................ 66 A homozygous PPT1 splice donor mutation in a Cane Corso Dog with neuronal ceroid lipofuscinosis ........................................................................................ 66 Abstract ........................................................................................................ 66 Case Report ................................................................................................. 67 Supplemental material ................................................................................. 85 Footnotes ..................................................................................................... 88 Chapter 5 ............................................................................................................ 89 iii GM2 gangliosidosis in Shiba Inu dogs with an inframe deletion in HEXB ....... 89 Abstract ........................................................................................................ 89 Introduction .................................................................................................. 90 Methods ....................................................................................................... 91 Clinical Findings ........................................................................................... 91 Molecular-genetic diagnosis ......................................................................... 94 Biochemical and electron microscopic confirmation ..................................... 95 Autofluorescent storage bodies in NCL and other diseases....................... 101 Comparison with other gangliosidoses in dogs .......................................... 103 Conclusions ............................................................................................... 103 Supplemental Material ............................................................................... 105 Chapter 6 .......................................................................................................... 110 Discussion and Conclusion ........................................................................... 110 References ....................................................................................................... 127 VITA ................................................................................................................. 151 iv Abstract This dissertation focuses on the use of whole genome sequencing (WGS) for the identification of disease causing variants in canine genomes. A brief review on the historical milestones of genetics, the creation and popularization of the fast throughput DNA sequencing technologies and their advantages and potential problems and biases, the importance of the study of canine genetics and the current state of the canine genome assembly is presented. Our lab sequenced ~100 dogs in the attempt to discover disease-causing variants. So far 20 such variants have been identified. This dissertation contains detailed accounts of the discovery variants likely to be responsible for four canine diseases. Those diseases are: Paroxysmal dyskinesia in Soft Coated Wheaten Terriers that is associated to the missense mutation PIGN:c.398C>T; two different forms of neuronal ceroid lipofuscinosis, one in Australian Cattle dogs caused by CLN5:c.619C>T, and one in the Cane Corso caused by the splice site mutation PPT1c.124+1G>A; and a Shiba Inu GM2 gangliosidosis caused by HEXB.c:948_950delCCT. Furthermore, examples of not so successful attempts, possible reasons for failures and suggestions to successfully conclude other ongoing investigations. v Chapter 1 Introduction Next-generation sequencing (NGS) is one of the current technologies used for genetic variation screening and novel variant discovery for personalized medicine. Due to decreased costs and fast turnaround, NGS is also becoming viable option for personalized veterinary medicine. This dissertation focuses on the use of whole genome sequencing (WGS) for the identification of disease-causing variants in canine genomes. This introductory section reviews important milestones in the history of genetics and sequencing technologies. In addition it describes how the newer technologies have contributed to the establishment of personalized medicine and how WGS is contributing to the study of canine genetics and inherited canine diseases. Genetics as a science and formal discipline is relatively young historically.
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