Cancer Genetics and Comparative Genomics

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Canine Genetics The tremendous phenotypic diversity of modern dog breeds represents the end point of a 20,000-year experiment. Each breed has undergone strong artificial selection for morphologic and behavioral traits to create populations that are unique in size, shape, behavior and disease susceptibility. Our multi-pronged approach to studying canine genetics demonstrates the utility of the canine system for developing a better genetic understanding of humans and their pets. Canine Whole Genome Sequence We have continued our leadership role with Dog10K (Ostrander et al., Natl Sci Rev, 2019), an International Consortium involving 18 labs that aims to generate whole genome sequence (WGS) across the Canidae. Thus far, about 4000 canines have been sequenced by the community, with nearly 2000 done by Dog10K (Meadows, Kidd et al., Submitted). The analysis includes in depth studies on the demography of breeds, single nucleotide and structural variants, analysis of mitochondrial DNA, and selection analysis. We generate the largest and most comprehensive analysis of the canine genome to date, providing the information for detailed studies of domestication, mapping morphologic and behavioral traits, and studies of disease susceptibility. Phylogenetic Studies Using genetic distance measures and genome-wide haplotype sharing we previously published evidence of phenotypic, behavioral, and geographic patterns of breed development (Parker et al., Cell Reports, 2017). We now expand our studies to focus on geographically isolated populations e.g., Alaska (Ali et al., J Heredity, 2023) and Patagonia (Barrios et al., PLOS Genetics, 2022). We know that where humans travel they bring their dogs, providing insight into early human migration patterns and, eventually, origins of disease. Ongoing studies focus on Greenland Sled Dogs, ancient dogs from the Americas, and modern dogs from the Galapagos Islands. Recent studies also address the genetic differences between European and American dogs of the same breed, e.g., the cancer-prone Bernese Mountain Dog, highlighting the importance of accurate ancestry information in genetic studies (Letko et al., Genes, 2023). Morphology We previously documented over 91 million canine genome variants for studies of breed morphology (Plassais et al., Nature Comm., 2019). We identify strong impact variants associated with 16 phenotypes, including body weight variation, highlighting genes that when mutated in humans contribute to fatty acid metabolism, obesity and metabolic syndrome. In our most recent study, we discovered the much sought after functional variants accounting for the association between the IGF1 gene and body size. IGF1 encodes a hormone related to normal and abnormal bone and tissue growth. Analyzing 1,431 genomes from ancient and modern canids we identified, for the first time, a single variant in an antisense long non-coding RNA (IGF1-AS) that interacts with IGF1, affecting protein production (Plassais et al., Current Biology, 2022). While the derived mutation predominates in both modern gray wolves and large domestic breeds, the ancestral allele, which predisposes to small size, was common in small-sized breeds and smaller wild canids. Our analyses demonstrate that this major regulator of canid body size nearly vanished in Pleistocene wolves, before its recent resurgence resulting from human-imposed selection for small-sized breed dogs. Invasive Urothelial Cancer (iUC) We have worked with clinical collaborators to determine the suitability of dogs with high breed-associated risk for naturally-occurring iUC to serve as models for early detection and intervention (Dhawan et al., Front Oncol, 2022). Scottish terriers (STs) 6 years old with no outward evidence of urinary disease were screened at 6-month intervals for three years by physical exam, ultrasonography, and urinalysis, by colleagues at Purdue School of Veterinary Medicine. Biopsy-confirmed bladder cancer was detected in 27% of STs, including 29 with iUC. Transcriptomic signatures, including druggable targets, such as EGFR and the PI3K-AKT-mTOR pathway, were very similar between canine and human iUC, and marked transcriptomic differences were noted between early and later canine tumors. The study suggests that breed dogs are important experimental systems for studies of iUC. Our current studies focus on susceptibility genes and variants in Shetland Sheepdogs. Dogs of Chernobyl Our Chernobyl study is the first of its kind, characterizing the genetic structure of three free-roaming dog populations (now over 350 dogs) living 15-45 km from the 1986 nuclear disaster site, including dogs living within the nuclear power plant (NPP) itself. We compared the genomes of Chernobyl dogs to several hundred breed dogs, as well as village dogs from adjacent countries (Spatola et al., Science Advances, 2023). We show that individuals from NPP and Chernobyl City (15 Km apart) are genetically distinct, with the former displaying increased intrapopulation genetic similarity and differentiation. Analysis of shared ancestral genome segments highlights differences in the extent and timing of western breed introgression. Kinship analysis reveals 15 families, with the largest spanning all collection sites within the radioactive exclusion zone. This study is the first characterization of a domestic species in Chernobyl, establishing the importance of dogs for genetic studies into the effects of exposure to long-term, low-dose ionizing radiation. Aging Epigenetic estimators of age (known as clocks) may permit cross-species interventions that slow or reverse aging. Previous epigenetic clocks only applied to a single species at a time. In work with colleagues this year we describe reliable and highly accurate epigenetic clocks that apply to 93 breeds (Horvath et al., Proc Natl Acad Sci USA, 2022). The methylation profiles were generated using an established mammalian methylation array utilizing mammalian-species conserved sequences. Epigenetic clocks were constructed to estimate age and also average time to death. We also present two highly accurate human-dog dual species epigenetic clocks (R = 0.97), which we hypothesize may facilitate the ready translation from canine to human use (or vice versa) of anti-aging treatments being developed for longevity and preventive medicine. Finally, our studies reveal individual methylation sites that may underlie the inverse relationship between breed weight and lifespan. We then worked with additional collaborators (Rubbi et al., Epigenetics, 2022) using targeted bisulphite sequencing to identify novel associations between methylomes and sex, weight, and sterilization status. We also show that several factors moderate the relationship between epigenetic age and real age, such as body weight, which increases epigenetic aging. Behavior Our recent experiments (Dutrow et al., Cell, 2022) overcome longstanding difficulties in identifying genetic drivers of canine behavior by developing a framework for understanding relationships between breeds and the behaviors that define them. We utilized genetic data for over 4,000 domestic, semi-feral, and wild canids, and behavioral survey data for over 46,000 dogs. We identified ten major canine genetic lineages and their behavioral correlates, and we show that breed diversification is predominantly driven by non-coding regulatory variation. We determine that lineage-associated genes converge in neurodevelopmental co-expression networks, identifying a sheepdog-associated enrichment for interrelated axon guidance functions. This work presents a scaffold for canine diversification that positions the domestic dog as an unparalleled system for revealing the genetic origins of behavioral diversity. Building on this data we are now studying breed-specific and anomalous behaviors in herding-type