Horses belong to the order Perissodactyla and bear the majority of their weight on their third toe; therefore, tremendous force is applied to each hoof. An inherited disease characterized by a phenotype restricted to the dorsal hoof wall was identified in the Connemara pony. Hoof wall separation disease (HWSD) manifests clinically as separation of the dorsal hoof wall along the weight-bearing surface of the hoof during the first year of life. Parents of affected ponies appeared clinically normal, suggesting an autosomal recessive mode of inheritance. A case-control allelic genome wide association analysis was performed (n_(cases)= 15, n_(controls)= 24). Population stratification (λ = 1.48) was successfully improved by removing outliers (n_(controls)= 7) identified on a multidimensional scaling plot. A genome-wide significant association was detected on chromosome 8 (p_(raw)= 1.37x10~(-10), p_(genome)= 1.92x10~(-5)). A homozygous region identified in affected ponies spanned from 79,936,024-81,676,900 bp and contained a family of 13 annotated SERPINB genes. Whole genome next-generation sequencing at 6x coverage of two cases and two controls revealed 9,758 SNVs and 1,230 indels within the ~1.7-Mb haplotype, of which 17 and 5, respectively, segregated with the disease and were located within or adjacent to genes. Additional genotyping of these 22 putative functional variants in 369 Connemara ponies (n_(cases)= 23, n_(controls)= 346) and 169 horses of other breeds revealed segregation of three putative variants adjacent or within four SERPIN genes. Two of the variants were non-coding and one was an insertion within SERPINB11 that introduced a frameshift resulting in a premature stop codon. Evaluation of mRNA levels at the proximal hoof capsule (n_(cases)= 4, n_(controls)= 4) revealed that SERPINB11 expression was significantly reduced in affected ponies (p<0.001). Carrier frequency was estimated at 14.8%. This study describes the first genetic variant associated with a hoof wall specific phenotype and suggests a role of SERPINB11 in maintaining hoof wall structure. Author Summary Inherited diseases affecting only the nails in humans are rare; however, humans do not support themselves entirely on one appendage. Horses bear their entire weight on their third toe, resulting in a large amount of force on each hoof. An inherited disease characterized by a phenotype restricted to separation and breaking of the dorsal hoof wall was identified in a specific breed of pony, the Connemara. This disease has been termed hoof wall separation disease (HWSD). Parents of affected ponies appeared clinically normal, suggesting an autosomal recessive mode of inheritance. A genome-wide association analysis identified a region associated with HWSD which was further assessed through whole genome next-generation sequencing and genotyping of potential variants. Here, we present the discovery of a frameshift variant, leading to a premature stop codon in SERPINB11 of HWSD-affected ponies. Significantly decreased expression of the SERPINB11 transcript was identified in the hoof capsule of HWSD-affected ponies. This study describes the first genetic variant associated with a hoof wall specific phenotype and suggests a role of SERPINB11 in maintaining hoof wall structure.
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