Genomic studies of Thoroughbred stallions’ ‘idiopathic’ subfertility

Stallions presented for subfertility are evaluated for breeding soundness by detailed physical examination and semen analysis. Such analyses, in rare cases, fail to detect forms of idiopathic subfertility where physical and semen parameters are within normal limits. Some idiopathic cases of subfertility in Thoroughbred stallions are attributed to impaired acrosome exocytosis (IAE), which according to a genome-wide association study of 7 affected stallions, is significantly associated with a certain double-homozygous A/A-A/A genotype in exon5 of an autosomal gene FKBP6 in chr13. Recent comparison of breeding records of 150 Thoroughbred stallions with their FKBP6 genotype confirmed a significant association of the A/A-A/A genotype with subfertility, previously defined as less than 46% per-cycle pregnancy rate. The molecular causes of this association, however, are unknown. Development of a cost-effective and accurate TaqMan allelic discrimination assay for FKBP6 genotyping determined that the frequency of the A/A-A/A genotype in global horse populations and Thoroughbreds separately is 4%. While the A/A-A/A genotype is also found in other breeds, it is associated with subfertility only in Thoroughbreds, suggesting that FKBP6 is only tagging a Thoroughbred-specific haplotype and not the cause. The aim of this study was to identify this haplotype and search for likely candidate genes and variantsfor IAE. Using the TaqMan assay, we have detected the FKBP6 A/A-A/A genotype in 22 sub-fertile Thoroughbred stallions, of which 14 have been tested for acrosome exocytosis. We generated short-read Illumina whole genome sequence data for 9 case Thoroughbreds and aligned the data with our Equine Genome Variant Database (EGVD) comprising of 428 horses of 46 breed groups, including 55 Thoroughbreds. We found FKBP6 A/A-A/A genotype in 21 horses (9 cases, 1 EGVD Thoroughbred, and 11 horses of other breeds) and showed that despite the same genotype, the sequence variant landscape in a 110 kb region around FKBP6 is the same only across Thoroughbreds and is different in other breeds. Next, we inspected 8,447 single nucleotide variants (SNVs) in all Thoroughbreds (10 A/A-A/A and 54 other), determined a 171 kb haplotype block specific to A/A-A/A Thoroughbreds only, and identified 38 implicated SNVs in 5 genes (POM121C, FKBP6, TRIM50, BAZ1B, HIP1) for further investigation. Of these, a variant in POM121C is homozygous only in case Thoroughbreds and of low allele frequency (6%) in EGVD horses. These findings strongly support our hypothesis that the A/A-A/A genotype in FKBP6 exon5 is tagging Thoroughbred and case-specific haplotypes which are expected to contain genetic variants responsible for the subfertility phenotype and IAE. Ongoing studies involve PacBio and RNA sequencing for the discovery of candidate structural and regulatory variants.