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CCaanniinnee DDiisseeaassee GGeennee IIddeennttiiffiiccaattiioonn by Jeremy R. Shearman The University of New South Wales 2011 School of Biotechnology and Biomolecular Sciences University of New South Wales THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Shearman First name: Jeremy Other name/s: Ross Abbreviation for degree as given in the University calendar: PhD School: BABS Faculty: Science Title: Canine disease gene identification Abstract 350 words maximum: (PLEASE TYPE) The dog (Canis familiaris) was domesticated from wolves around 15,000 years ago in multiple locations in the northern hemisphere. Most modern dogs were developed from domestic dogs in the past 200 years in Europe. This breed development resulted in genetically isolated, highly inbred populations segregating diseases. Two autosomal recessive diseases are Trapped Neutrophil Syndrome (TNS) in Border collies and cerebellar abiotrophy in Australian Kelpies. TNS is an inherited neutropenia resulting in a compromised immune system. TNS was mapped to VPS13B using a candidate gene approach and linkage analysis. Sequencing of the gene in affected and control dogs identified a 4 bp deletion in exon 19 causing frame shift and premature truncation. Alternate transcripts of VPS13B are expressed in the brain of humans but not mice. Sequencing of cDNA from healthy dogs revealed that dogs also express alternate transcripts in the brain. Cerebellar abiotrophy in the Australian Kelpie results in an ataxia. Affymetrix SNP array v2 was used to perform whole genome mapping in twelve affecteds and twenty control Kelpies. Association analysis failed to identify the disease region. Homozygosity analysis identified a five megabase region where all affecteds were homozygous for a common haplotype. This region was enriched for two affecteds and one control using Nimblegen sequence capture arrays and sequenced on a 454 using titanium chemistry and multiplex identifiers. A total of 2019 differences were identified homozygous in the affecteds compared to controls, 682 of those were in genic regions, 25 were in exons and 8 changed an amino acid. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). …………………………………………… ……………………………………..……… ……….……………...…….… Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………………... Date …………………………………………………... Acknowledgements I would like to thank Alan Wilton first and foremost for taking me on as a student despite not having a scholarship. I was able to survive thanks to Angela Higgins from the UNSW sequencing centre employing me as a casual research assistant two days a week for most of my PhD. The sequencing centre then became a part of the Ramaciotti Centre for Gene Function Analysis, which fortunately kept me on as a casual employee – Thanks to Ian Dawes, Helen Speirs and Jason Koval. Thanks to all the past and current member of the Wilton lab: Louise, Carol, Scott, Natsuki, Pete, Julia, Yosh, Claire, Auda, Zi, Paulina, Pranoy, Mathew and Annie (roughly in order of appearance). Also Thanks to my co-supervisor: Peter Little and his lab group: Rohan, Mark, Oscar and Michael who occupied the other end of the lab. The next generation of occupants of the other end of the lab (my new co-supervisor) Bill Ballard and his group: Rich, Innes, Jonci, Pann Pann, Lou, Carolina and Kylie (there is something about that side of the lab that attracts head-of-school type PI’s). On the note of thanking all of those people (most of which have left UNSW already) it is good to be the one leaving for a change. Thanks to all my friends: Alex, Paul, Kellie, Kevin, Frances, Kye, Jackie, Grace, Alex, Richard, Michael, Claire, Auda, Eser, Paulina, Zi, Jonci, Pranoy, Natsuki, Lee- Anne, Clare, Emily, Michael, Allan, Graham, Aisha, Adam and Ken. Thanks to my family, especially my brothers who find it quite amusing that after 9 years of university, I will still be learning less than their truck driver wage. Special thanks to Auda for printing, binding and submitting my thesis for me while I lived it up it Thailand. Most special thanks to Kittiya - the reason that I am in Thailand. Abstract The dog (Canis familiaris) was domesticated from wolves around 15,000 years ago in multiple locations in the northern hemisphere. Most modern dogs were developed from domestic dogs in the past 200 years in Europe. This breed development resulted in genetically isolated, highly inbred populations segregating diseases. Two autosomal recessive diseases are Trapped Neutrophil Syndrome (TNS) in Border collies and cerebellar abiotrophy in Australian Kelpies. TNS is an inherited neutropenia resulting in a compromised immune system. TNS was mapped to VPS13B using a candidate gene approach and linkage analysis. Sequencing of the gene in affected and control dogs identified a 4 bp deletion in exon 19 causing frame shift and premature truncation. Alternate transcripts of VPS13B are expressed in the brain of humans but not mice. Sequencing of cDNA from healthy dogs revealed that dogs also express alternate transcripts in the brain. Cerebellar abiotrophy in the Australian Kelpie results in an ataxia. Affymetrix SNP array v2 was used to perform whole genome mapping in twelve affecteds and twenty control Kelpies. Association analysis failed to identify the disease region. Homozygosity analysis identified a five megabase region where all affecteds were homozygous for a common haplotype. This region was enriched for two affecteds and one control using Nimblegen sequence capture arrays and sequenced on a 454 using titanium chemistry and multiplex identifiers. A total of 2019 differences were identified homozygous in the affecteds compared to controls, 682 of those were in genic regions, 25 were in exons and 8 changed an amino acid. List of Publications Shearman JR and Wilton AN. (2011) Mapping Cerebellar Abiotrophy in Australian Kelpies. Animal Genetics. doi:10.1111/j.1365-2052.2011.02199.x Shearman JR and Wilton AN. (2011) The effects of inbreeding on the incidence of disease in a pedigree population. Animal Genetics. In prep Shearman JR and Wilton AN. (2011) A Canine Model of Cohen Syndrome: Trapped Neutrophil Syndrome. BMC Genomics. doi:10.1186/1471-2164-12- 258 Shearman JR and Wilton AN. (2011) Origins of the domestic dog and the rich potential for gene mapping. Genetics Research International. 2011: 1-6. Vonholdt BM, Pollinger JP, Lohmueller KE, Han E, Parker HG, Quignon P, Degenhardt JD, Boyko AR, Earl DA, Auton A, Reynolds A, Bryc K, Brisbin A, Knowles JC, Mosher DS, Spady TC, Elkahloun A, Geffen E, Pilot M, Jedrzejewski W, Greco C, Randi E, Bannasch D, Wilton A, Shearman J, Musiani M, Cargill M, Jones PG, Qian Z, Huang W, Ding ZL, Zhang YP, Bustamante CD, Ostrander EA, Novembre J and Wayne RK. (2010) Genome- wide SNP and haplotype analyses reveal a rich history underlying dog domestication. Nature. 464: 898-902. Shearman JR, Lau VM and Wilton AN. (2008) Elimination of SETX, SYNE1 and ATCAY as the cause of Cerebellar Abiotrophy in Australian Kelpies. Animal Genetics. 39: 573. Shearman JR and Wilton AN. (2007) Elimination of neutrophil elastase and the genes for adaptor protein complex 3 subunits as the cause of trapped neutrophil syndrome in Border collies. Animal Genetics. 38: 188-189. Shearman JR, Zhang QY and Wilton AN. (2006) Exclusion of CXCR4 as the cause of Trapped Neutrophil Syndrome in Border Collies using five microsatellites on canine chromosome 19. Animal Genetics. 37: 89. TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................IV LIST OF TABLES .....................................................................................................................
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