Idiopathic epilepsy (IE) is a neurological disease that causes seizures in a variety of dog breeds. Belgian shepherds are disproportionately affected, with Belgian Tervuren having a prevalence estimate of 27%. These dogs suffer from chronic convulsions, loss of consciousness, and atypical behavior, placing stress and confusion on both the dog and owner. Due to their high disease prevalence, Belgian shepherds have been the subject of multiple studies to determine the mechanism of disease. Initial research suggested genetic transmission of IE with incomplete penetrance. Genome-wide association studies highlighted multiple variants near genes with known neurological function, including KLF7, ADAM23, and RAPGEF5. These risk markers have not yet led to definitive causative variation, nor do they fully account for disease risk, prompting further investigation into IE etiology.With dog breeders identifying IE in their lines and the risk variants near the three candidate genes publicly available, efforts to breed away from the phenotype and markers were expected to reduce risk-allele frequency and IE prevalence. However, risk-allele frequency either remained unchanged or increased among control Belgian Tervuren over a thirty-year span. Simultaneously, IE dogs had reduced risk-allele frequency, suggesting increasingly heterogenic causes of IE have emerged over time. Over that same period, IE prevalence has increased by 59% (from 17% to 27%), suggesting unsuccessful selection against the disease. As the published research did not work to control the spread of IE in Belgian shepherds, additional research with novel methods and technology was required.
Genetic and transcriptomic research heavily rely on reference genomes for accurate variant calling and transcript quantification. CanFam3.1., a highly utilized reference genome, was shown to pose multiple challenges for disease research. Incorrect and missing segments from the reference resulted in sequence reads repeatedly aligning to incorrect and/or multiple loci, leading to misleading variant calls. In the context of transcriptomic data, this would result in inaccurate expression estimates. Modern references generated using long-read sequencing technology were shown to mitigate some of these issues, allowing for more reliable sequencing pipelines. These results prompted future work to be conducted using Dog10K_Boxer_Tasha, a modernized version of CanFam3.1, resequenced using long-read technology.
Gene expression levels were evaluated in blood samples from healthy and IE Belgian shepherds to identify pathways affected by the disease. These dogs were additionally evaluated for differential expression based on their RAPGEF5 haplotype. Two genes previously associated with seizures were upregulated in IE dogs, MFSD2A (punadjusted = 0.06, log2FC = 1.59) and RPL19 (punadjusted = 0.04, log2FC = 1.72). Downregulated genes participated in interferon signaling, indicating that IE may be related to immunity and/or retinol metabolism. Variants associated with IE were identified near differentially expressed genes in regions known to regulate GAB2, NARS2, KLF6, ELF3, and SPDEF, genes with neurological or interferon-signaling functions. When comparing expression based on RAPGEF5 haplotype, IE dogs had upregulation of CLIC1 (p A< 0.01, log2FC = 18.85), ACE2 (p = 0.02, log2FC = 6.44), and PIGN (p = 0.02, log2FC = 0.81) and downregulation of EPDR1 (p = 0.10, log2FC = -1.37). These genes have been implicated in epilepsy or the Wnt/β-catenin signaling pathway, which is regulated by RAPGEF5.
