Whole genome sequence-based analysis of Thoroughbred stallions with impaired acrosomal exocytosis and subfertility

An idiopathic form of subfertility in Thoroughbred stallions with normal physical and semen parameters has been attributed to impaired acrosomal exocytosis (IAE) (see Hernández-Avilés et al 2023 PMID: 36413868). This impairment is significantly associated with a double-homozygous A/A-A/A genotype in FKBP6 exon5 on horse chromosome 13 (Raudsepp et al 2012 PMCID: PMC3527208; Castaneda et al 2021 PMID: 34610162). While this genotype is present in 4% of global horse population, it is associated with subfertility only in Thoroughbreds, suggesting FKBP6 tags Thoroughbred case-specific haplotypes containing causative variants. To find these variants, we designed a TaqMan genotyping assay for FKBP6 exon5 and identified 26 subfertile Thoroughbred stallions with the A/A-A/A susceptibility genotype. Whole genome sequencing short-read data (Illumina) was generated for 25 stallions and long-read data (PacBio Hi-Fi) for four stallions. Sequences of all Thoroughbred cases were aligned to EquCab3 reference and the recently available Telomere-to-Telomere (T2T) Thoroughbred genome in combination with over 200 other horse genomes. Comparison of sequence variant landscape between Thoroughbred cases and non-case horses, identified a 1,447,268 bp haplotype region around the FKBP6 gene that was specific to Thoroughbred cases. The region contains 372 unique or rare single nucleotide variants and several deletions of interest. For example, a 66 bp deletion in the BAZ1B gene was in complete linkage disequilibrium with the FKBP6 exon5 A/A-A/A genotype in case Thoroughbreds only. Functional relevance of this and other variants is under investigation. In addition, we are conducting sequence-based whole genome association study to identify additional genomic regions that could be associated with the subfertility phenotype in Thoroughbred cases. While molecular studies on Thoroughbred stallion subfertility due to impaired acrosome exocytosis are still ongoing, the generated short-read and long-read whole genome sequence data contribute to the Equine Genome Variant Database and the Equine Pangenome Project, respectively.