Over the course of this study, we collected DNA samples from hundreds of affected and unaffected PWDs. We performed high-coverage whole-genome sequencing of 16 PWDs (and used publicly available resources to acquire an additional 20 high-coverage samples); low-pass whole-genome sequencing of 101 PWDs, and class II MHC genotyping of the same 101 PWDs. We focused our principal analyses on the 101 low-pass/MHC PWDs because these dogs formed the least-related population out of the hundreds of DNA samples collected.
One of the main goals of this study was to perform a genome-wide association study to look for regions of the genome associated with Addison’s disease in the breed. We performed this GWAS using a variety of approaches with the imputed low-pass data, but unfortunately, we were unable to find a genomic region significantly associated with Addison’s disease. This implies that within PWDs, Addison’s may be: (1) fixed, meaning all PWDs are equally predisposed; (2) highly polygenic, meaning that many genomic regions are responsible for Addison’s disease, however our study did not have enough samples to identify these multiple regions; (3) caused by mutations that are not easily detectable by the methods we used, such as structural genomic variants; (4) caused by a combination of these factors.
We also thoroughly interrogated various major histocompatibility complex genes, which are important and diverse genes associated with the immune system. We found one group of MHC genes are associated with Addison’s in the breed, and another group of genes appears to be protective. These findings reinforce (but do not prove) the idea that Addison’s disease has an immune-mediated component in PWDs. That said, these associations have a small effect size, meaning that they only increase or decrease the risk of developing Addison’s disease by a small amount. This suggests that other genetic or environmental factors are at play.
To interrogate the potential for fixed effects, we are in the process of undertaking several different analyses on the data we have already collected. However, it is difficult to definitively prove the impact of fixed effects because, in this scenario, all dogs have the potential to carry deleterious genetic variants. These analyses are actively underway and will continue despite the conclusion of this grant. We are also continuing to collect DNA samples that are minimally related to our existing study population from around the world, and we are working to better understand larger structural variants that may be associated with Addison’s disease in PWDs. We hope to have a publication submitted regarding our findings within the next year.