Whole Genome Sequencing to Explore Genetic Risk Factors in Canine Myxomatous Mitral Valve Disease

Canine myxomatous mitral valve disease (MMVD) is the most common acquired cardiac disease in dogs, with cavalier King Charles spaniel (CKCS) being particularly overrepresented. Almost all CKCS >10 years of age are affected, suggesting that genetic risk factors may be ‘fixed’ in this breed. MMVD is believed to be a polygenic trait, and previous genetic work in canine MMVD has employed a case-control design within individual breeds. However, this design precludes the identification of disease-associated variants that are ‘fixed’ within high risk breeds and may result in only minimal genetic differences between cases and controls being identified.   The aim of the present study was to identify new canine MMVD genes as potential future targets for prevention or treatment of disease. This was achieved by applying a new model for identification of genetic risk variants in complex disease, using whole genome sequencing (WGS) of breeds at relatively high and low risk of disease. Additionally, genetic differences between CKCS with early-onset MMVD that died due to congestive heart failure (group C) and CKCS with late-onset MMVD that died due to non-cardiac disease (group NC) were explored.   Whole genome sequencing (WGS) at 30X coverage was undertaken on 12 CKCS, 6 in group C and 6 in group NC. Results were compared with WGS from 48 non-CKCS breed dogs, mainly sequenced as part of the Canine Diabetes Genetics Partnership initiative. DNA was extracted from blood samples that were surplus to requirements for clinical purposes. A custom bioinformatics pipeline was used to call, annotate and prioritise variants for follow-up, based on the Genome Analysis ToolKit, using CanFam3.1 as a reference. Variants were prioritised according to a range of criteria, including their alternate allele frequency (by breed or disease severity status), predicted impact on gene function, or location near a region with a plausible role in cardiac disease.   In CKCS, 1993 breed-unique high or moderate impact variants were identified, 1393 of which were present in more than one CKCS, and 43 of which were found exclusively in group C. A total of 103 variants of high or moderate impact were identified in or near genes with a plausible role in cardiac disease and at a high frequency in all CKCS. A further 539 similar variants of potential cardiac relevance were at a higher frequency in group C compared to group NC.  A follow-up genotyping study is in progress using a larger cohort and more breeds. This work demonstrates that WGS offers a promising route for investigation of complex diseases where genetic risk may be fixed within a breed.