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dc.contributor.advisorJohnson, Gary S., 1943-eng
dc.contributor.authorGilliam, Douglas H., Jr., 1983-eng
dc.date.issued2016eng
dc.date.submitted2016 Springeng
dc.descriptionDissertation supervisor: Dr. Gary S. Johnson.eng
dc.descriptionIncludes vita.eng
dc.description.abstractThe genome of Canis lupus familiaris, the domestic dog, is an ideal tool for the study of inherited diseases. Its genome is uniquely suited for the mapping of genes that cause disease, and its reference genome has been published since 2008. Dogs and humans share many of the same diseases, making them an ideal model for the study of comparative genetics. The identification of disease-causing genes in dogs has relevance to human health. We here describe the identification of causal mutations for three different canine diseases in orthologs of genes with orthologous human diseases. We used Next Generation Sequencing in order to generate Whole Genome Sequences for 145 dogs from 69 different breeds with various inherited canine diseases, most of which were suspected to be inherited recessively. We here report the discovery of the causes of; Spinocerebellar Ataxia with Myokymia and Seizures in Russell-Group Terriers, Neuronal Ceroid Lipofuscinosis in Golden Retrievers and Dilated Cardiomyopathy in Standard Schnauzers. While all three were identified via whole genome sequencing, different methodologies and techniques were used to discover and validate the findings. We discuss the importance of these studies and suggest future studies for the projects as well as possible other means of discovering potential canine disease models via Whole Genome Sequencing. Summaries of these three findings are provided in the next three paragraphs. Juvenile-onset spinocerebellar ataxia has been recognized in Jack Russell Terriers and related Russell group terriers (RGTs) for over 40 years. Ataxia occurs with varying combinations of myokymia, seizures and other signs, thus more than one form of the disease has been suspected. Our objective was to identify the mutation causing the spinocerebellar ataxia associated with myokymia and/or seizures and distinguish the phenotype from other ataxias seen in the RGTs. We collected DNA samples from 16 RGTs with signs of spinocerebellar ataxia beginning from 2-to-12 months of age, 640 control RGTs, and 383 dogs from 144 other breeds along with the medical records of affected dogs in order to elucidate the genetic cause of this disease. Whole-genome sequencing was performed on one RGT with ataxia and myokymia. Unique, homozygous variants were identified in this dog by comparing its sequence to whole-genome sequences from 81 other canids. We found a missense mutation in the gene coding for the inward rectifying potassium channel Kir4.1 (KCNJ10:c.627C>G) that was significantly associated with the disease. Dogs homozygous for the mutant allele all showed spinocerebellar ataxia with varying combinations of myokymia, seizures and other signs. The identification of a mutation in KCNJ10 in dogs with spinocerebellar ataxia with myokymia and/or seizures (SAMS) clarifies the multiple forms of ataxia seen in these breeds and provides a DNA test to identify carriers. Understanding the role the Kir4.1 channel plays in extracellular potassium buffering by astrocytes could shed light on other conditions characterized by excessive neuronal membrane excitability such as other forms of ataxia, epilepsy and myokymia. We studied a recessive, progressive neurodegenerative disease occurring in Golden Retriever siblings with an onset of signs at 15 months of age. As the disease progressed these signs included ataxia, anxiety, pacing and circling, tremors, aggression, visual impairment and localized and generalized seizures. A whole genome sequence, generated with DNA from one affected dog, contained a plausibly causal homozygous mutation: CLN5:c.934_935delAG. This mutation was predicted to produce a frameshift and premature termination codon and encode a protein variant, CLN5:p.E312Vfs*6, which would lack 39 C-terminal amino acids. Eighteen DNA samples from the Golden Retriever family members were genotyped at CLN5:c.934_935delAG. Three clinically affected dogs were homozygous for the deletion allele; whereas, the clinically normal family members were either heterozygotes (n = 11) or homozygous for the reference allele (n = 4). Among archived Golden Retrievers DNA samples with incomplete clinical records that were also genotyped at the CLN5:c.934_935delAG variant, 1053 of 1062 were homozygous for the reference allele, 8 were heterozygotes and one was a deletion-allele homozygote. When contacted, the owner of this homozygote indicated that that their dog had been euthanized because of a neurologic disease that progressed similarly to that of the affected Golden Retriever siblings. We have collected and stored semen from a heterozygous Golden Retriever, thereby preserving an opportunity for us or others to establish a colony of CLN5-deficient dogs. Young-adult onset dilated cardiomyopathy (DCM) segregates in the Standard Schnauzer dog breed in a pattern consistent with an autosomal recessive mode of inheritance. To identify the molecular genetic cause of Standard Schnauzer DCM, DNA from an affected dog was used to generate a whole genome sequence with 31-fold average coverage. Among the sequence variants in this whole genome sequence was a 22-bp deletion and frameshift in RBM20, the canine ortholog of a gene previously associated with human DCM. The RBM20 deletion allele was homozygous in the whole genome sequence of the affected Schnauzer, but absent from 101 whole genome sequences of normal canids or dogs with other diseases. An additional 753 Standard Schnauzers, including 21 with DCM, were genotyped for the RBM20 deletion. In this cohort, all 20 of the deletion-allele homozygotes had DCM, only one of the 183 heterozygous dogs had DCM, and all of the reference-allele homozygotes were DCM free. RBM20 deficiency is known to alter exon-splicing patterns and produced aberrant titin isoforms in a rat model and in human DCM patients. To determine if the Standard Schnauzers with DCM had similar exon-splicing abnormalities, RNA prepared from their left ventricular walls was compared by RT-PCR to similarly prepared RNA from normal adult dogs. Titin transcripts with extensive exon skipping were detected in the normal RNA but not in the RNA from the dogs with DCM. Conversely, titin transcripts with retained exons were detected in the RNA from dogs with DCM but not in the normal-dog RNA. Thus, we have identified a canine model for human RBM20-associated DCM. While all three were identified via whole genome sequencing, different methodologies and techniques were used to discover and validate the findings. We discuss the importance of these studies and suggest future studies for the projects as well as possible other means of discovering potential canine disease models via Whole Genome Sequencing.eng
dc.description.bibrefIncludes bibliographical references (pages 77-92).eng
dc.format.extent1 online resource (x, 93 pages) : illustrationseng
dc.identifier.merlinb118797724eng
dc.identifier.oclc988340101eng
dc.identifier.urihttps://hdl.handle.net/10355/56525
dc.identifier.urihttps://doi.org/10.32469/10355/56525eng
dc.languageEnglisheng
dc.publisherUniversity of Missouri--Columbiaeng
dc.relation.ispartofcommunityUniversity of Missouri--Columbia. Graduate School. Theses and Dissertationseng
dc.rightsOpenAccess.eng
dc.rights.licenseThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.eng
dc.subject.FASTDogs -- Diseases -- Genetic aspectseng
dc.subject.FASTDiseases -- Animal modelseng
dc.titleMolecular genetic studies of canine inherited diseases including SAMS, neuronal ceroid lipofuscinosis and dilated cardiomyopathyeng
dc.title.alternativeMolecular genetic studies of canine inherited diseases including spinocerebellar ataxia with myokymia and seizures, neuronal ceroid lipofuscinosis and dilated cardiomyopathyeng
dc.typeThesiseng
thesis.degree.disciplineGenetic area program (MU)eng
thesis.degree.grantorUniversity of Missouri--Columbiaeng
thesis.degree.levelDoctoraleng
thesis.degree.namePh. D.eng


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