Canine Genetics The tremendous phenotypic diversity of modern dog breeds represents the end point of a >15,000-year experiment. Each breed has undergone strong artificial selection in which dog fanciers selected for many traits including body size, fur color, skull shape, and even behavior, to create novel breeds. As a result, there is strong phenotypic homogeneity within breeds including breed-associated genetic disease. These are explored below. Canine Cancer Canine transmissible venereal tumor (CTVT) is a parasitic cancer clone that has propagated for thousands of years via sexual transfer of malignant cells (Ostrander et al., 2016). Because little is known about the mechanisms that converted an ancient tumor into the world's oldest continuously propagating somatic cell lineage, Ostrander lab postdoc Dr. Brian Davis created the largest existing catalog of canine genome-wide variation using over 400 whole genome sequences (WGS), to capture > 99% of domestic dog variation. Ostrander lab MD/PhD. student Brennan Decker utilized the catalog to analyze two CTVT by WGS. He showed that CTVT has undergone continuous adaptation to its transmissible allograft niche, with overlapping mutations at every step of immunosurveillance, particularly self-antigen presentation and apoptosis. We also identified chronologically early somatic mutations that may represent key initiators of clonal transmissibility. Thus, we provide the first insights into the specific genomic aberrations that underlie CTVT's perseverance in canids around the world (Decker et al., 2015a). Led by Brennan Decker and Dr. Heidi Parker we also tackled canine invasive transitional cell carcinoma of the bladder (InvTCC,) a common, naturally occurring malignancy that shares features with human muscle invasive bladder cancer (Decker et al., 2015b). To identify somatic drivers of canine InvTCC we determined the complete transcriptome for multiple tumors using RNAseq. We found that all tumors harbored a somatic mutation homologous to the human BRAF(V600E) mutation, and the mutation was present in 87% of InvTCC tumors. The mutation was also detectable in the urine sediment of all dogs tested with mutation-positive tumors, suggesting a diagnostic marker. Cell lines with the mutation have elevated levels of phosphorylated MEK compared to those that do not. This effect can be diminished through application of the BRAF(V600E) inhibitor vemurafenib. These findings set the stage for canine InvTCC as a powerful system to evaluate BRAF-targeted therapies in humans. Canine Genomics We have amassed a catalog of over 400 WGS and Drs. Davis and Jaemin Kim are analyzing domestic dog genome architecture. Inherent to our work is the problem that for most breeds one or at the most a few dogs per breed are sequenced. However, for most breeds several dogs have undergone SNP chip analysis with the Illumina 170,000 Chip. Led by Ostrander lab postdoc Dr. Dayna Dreger we showed that it is essential to consider the unique demography of each population/breed, including estimation of effective population size and timing of population bottlenecks, in GWAS study design. Studying WGS data for 90 dogs, and SNP data from 800 dogs/80 breeds, we demonstrate that each GWAS study should utilize cohort sizes that reflect breed-specific decay rates and levels of homozygosity listed in the paper. For sequence-based studies, most variant data is generated from deep sequencing of two unrelated individuals, and sequencing of a third is generally not economical. Publication of these data, which capture the distinctive and dynamic homozygosity landscape for over 100 pure dog breeds, will serve as a platform upon which future research initiatives can be modeled. Canine Origins To better understand demographically based evaluation of genomic regions under selection in domestic dogs, Ostrander lab postdocs Drs. Brian Davis and Heidi Parker collaborated with several investigators (Freedman et al., 2014) to investigate positive selection on canids early in domestication. The study identified 349 outlier regions consistent with positive selection at a low false discovery rate, which was computed using an inferred demographic model. The strongest signals focused on genes important in brain function and behavior, an expanded the set of neurobehavioral candidate genes, and genes related to lipid metabolism. This type of methods development represents advances in our ability to study selection in human populations (Freedman et al., 2016). Sardinian Dog Breeds We have become interested in the degree to which the development of dog breeds recapitulates the migration and settlement of humans to various geographic areas and focused our study on the island of Sardinia (Dreger et al., 2016). Dr. Dreger therefore studied the Fonni's Dog, a niche population on Sardinia which has not been subjected to intensive artificial selection, but rather has developed alongside the human population. It was influenced only by geographic isolation and aptitude for owner-desired behaviors. We analyzed 28 other Italian and Mediterranean breeds. Population and demographic analyses provided spatial identity of Fonnis Dog to other Mediterranean breeds. We show that the genetic history of Fonnis Dog parallels demographic events in local human populations. Specifically, humans in Sardinia show greatest genetic identity with populations from the Eastern Mediterranean and North Africa including Hungary, Egypt, Israel, and Jordan. These patterns are clearly reflected in the Fonni's Dog, which we show originated in the Middle East and North Africa as evidenced by its relationship to the Komondor, Sloughi, Saluki, etc. It also shares commonalities with Italian breeds such as the Cane Corso, Cane Paratore, etc. (Dreger et al., 2016). This work sets the stage for studies of other isolated human populations through the use of canine breed history. Smithsonian Collaboration The Smithsonian Museum holds a wealth of historical data and with it an untold number of samples. In the past year we have worked with the Smithsonian to produce DNA sequence from canine bones that are thousands of years old. This represents our first study of ancient DNA and we are actively collecting samples for more studies. Morphology After exploring the genetics of several morphologic traits, we have returned to studies of body size. We show that programming of large and small size occurs independently in the dog. Our studies thus far have identified most major genes associated with small size. Only a few of these contribute to gigantism, and that is at a low level. Led by Ostrander lab fellows Drs. Jocelyn Plassais and Falina Williams we have utilized WGS and SNP chip data to localize two new size genes and the relevant mutations on the X chromosome. Both contribute to large size. This work will be submitted for publication shortly. Finally, we undertook a study of hairlessness in the American Hairless Terrier (AHT), as it is a major trait of interest to humans (Parker et al., 2016). In work led by Ostrander lab Staff Scientist Dr. Heidi Parker, we used WGS and SNP analysis to identify a large region of homozygosity in 11 hairless terriers. We extensively evaluated all possible candidates in the 4.8 MB region and found that a deletion, which removes over half of the serum/glucocorticoid regulated kinase family member 3 gene, causes the phenotype. Interestingly, earlier work in mice has shown that a 30 amino acid deletion produced a milder phenotype with sparse, malformed hair shafts. Our more extensive mutation resulted in total baldness after 4-5 weeks. Other than the inherent obvious interest in baldness, the comparison of mutations and phenotypes between human, mouse, and dog can both validate the role of candidate genes, but also highlight the specific roles for portions o
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