SERPINB11 frameshift variant associated with novel hoof specific phenotype in Connemara ponies

Abstract

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 (ncases = 15, ncontrols = 24). Population stratification (λ = 1.48) was successfully improved by removing outliers (ncontrols = 7) identified on a multidimensional scaling plot. A genome-wide significant association was detected on chromosome 8 (praw = 1.37x10-10, pgenome = 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 (ncases = 23, ncontrols = 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 (ncases = 4, ncontrols = 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.

Fig 1. Hoof from a Connemara pony foal affected with hoof wall separation disease (HWSD).

(A) Note the dorsal hoof wall separation at the sole and (1B) proliferative horn on the solar aspect of the hoof (1B). (1C) A sagittal section of a post-mortem HWSD-affected hoof demonstrates that the dorsal hoof wall fissure occurs outside of the white line. (1D) Coronary band, periople (hematoxylin & eosine stain): normal transition from haired skin into periople is evident.

Fig 2. Genome-wide case-control allelic association study results of Connemara ponies with HWSD (15 cases, 24 controls).

(2A) Multi-dimensional scaling plot. Controls circled contributed most to genomic inflation and were removed from analysis (2B). Quantile-Quantile plot of genome-wide association results after removing 7 control ponies. Grey dots represent the observed versus expected p-values of all SNPs. Black dots represent the observed versus expected p-values after removal of all SNPs on chromosome 8. The solid grey line represents the null hypothesis: observed p-values equal expected p-values. (2C) Manhattan plot of—log₁₀ of raw p-values by chromosome. The lowest p-values are on chromosome 8. (2D) 52K Max (T) permutation results. The line that parallels the X axis denotes genome-wide significance (p<0.05;-log₁₀ >1.3).

Fig 3. Genes and segregating variants within the region of homozygosity shared by HWSD-affected ponies.

(3A) Scaled schematic representation of SERPINB gene family situated between 79.77Mb and 80.34Mb on equine chromosome 8. Arrows show the relative positions of the SERPINB genes in the cluster, and aligned numbers report the percent identity of the horse proteins compared to corresponding human proteins at the amino acid level. Five copies of equine SERPINB3/4 are variably closer to human B3 or B4. Carets depict locations of variants that segregate with HWSD: two insertions and one substitution. (3B) Chromatograms of SERPINB11 exon 5 sequences where a variant of C to C/C was found in affected Connemara ponies. From left to right: unaffected ponies have a genotype of “C/C”; affected Connemara ponies have a genotype of “CC/CC”; carrier Connemara ponies have a genotype of “C/CC”.

Fig 4. Expression analysis of SERPINB2, SERPINB8, SERPINB10 and SERPINB11.

(4A) Relative SERPINB11 gene expression ratio of coronary band cDNA from four HWSD-affected Connemara ponies compared to four unaffected horses. Relative expression levels were normalized to the housekeeping gene B2M. Statistical significance is reported as p<0.001(*). B. Relative SERPINB2, B8, B10, and B11 gene expression levels in four affected Connemara ponies, compared to four unaffected horses and normalized to the housekeeping gene B2M. P(H1) = Probability of alternate hypothesis that difference between sample and control groups is due only to chance. P(H1) is significant (p<0.05) for only SERPINB11. C. Gel image of PCR amplification of SERPINB2, B8, B10, and B11 from spleen, heart, lung, kidney, stomach, liver, skin, coronary band, and spinal cord cDNA of an unaffected horse. Beta-actin was used to control for cDNA concentrations.