A mutation in the FAM83G gene in dogs with hereditary footpad hyperkeratosis (HFH)

Abstract

Hereditary footpad hyperkeratosis (HFH) represents a palmoplantar hyperkeratosis, which is inherited as a monogenic autosomal recessive trait in several dog breeds, such as e.g. Kromfohrländer and Irish Terriers. We performed genome-wide association studies (GWAS) in both breeds. In Kromfohrländer we obtained a single strong association signal on chromosome 5 (p(raw) = 1.0×10(-13)) using 13 HFH cases and 29 controls. The association signal replicated in an independent cohort of Irish Terriers with 10 cases and 21 controls (p(raw) = 6.9×10(-10)). The analysis of shared haplotypes among the combined Kromfohrländer and Irish Terrier cases defined a critical interval of 611 kb with 13 predicted genes. We re-sequenced the genome of one affected Kromfohrländer at 23.5× coverage. The comparison of the sequence data with 46 genomes of non-affected dogs from other breeds revealed a single private non-synonymous variant in the critical interval with respect to the reference genome assembly. The variant is a missense variant (c.155G>C) in the FAM83G gene encoding a protein with largely unknown function. It is predicted to change an evolutionary conserved arginine into a proline residue (p.R52P). We genotyped this variant in a larger cohort of dogs and found perfect association with the HFH phenotype. We further studied the clinical and histopathological alterations in the epidermis in vivo. Affected dogs show a moderate to severe orthokeratotic hyperplasia of the palmoplantar epidermis. Thus, our data provide the first evidence that FAM83G has an essential role for maintaining the integrity of the palmoplantar epidermis.

Figure 1. Mapping of HFH by GWAS and haplotype analysis.

(A) A genome-wide association study in Kromfohrländer using 13 cases and 29 controls indicates a strong signal with multiple associated SNPs on CFA 5. (B) The association is replicated in a cohort of 10 Irish Terrier cases and 21 controls. (C) Homozygosity mapping. Each horizontal bar corresponds to one of the 23 analyzed cases. Homozygous regions with shared alleles are shown in color. A shared homozygous interval of 611 kb delineates the exact boundaries of the critical interval from 40,521,040–41,131,739 (CanFam 3.1 assembly). (D) NCBI gene annotation of the CanFam3.1 assembly in the critical interval.

Figure 2. Electropherograms of the FAM83G:c.155G>C variant.

A fragment harboring exon 2 and flanking sequences of the FAM83G gene was PCR-amplified and sequenced with the Sanger method. The figure shows representative traces from Kromfohrländer with the 3 different genotypes. The position of the variant is indicated by an arrow.

Figure 3. Evolutionary conservation of the arginine residue at position 52 in the FAM83G protein.

All mammals share identical amino acid sequences in the region of the variant. The sequences were derived from the following database accessions: C. lupus XP_003434684.1, H. sapiens NP_001035088.2, B. taurus NP_001192445.1, M. musculus NP_848733.2, R. norvegicus XP_002727764.1.

Figure 4. Clinical phenotype of digital hyperkeratosis.

(A) Paw of a 1 year old affected Kromfohrländer. Note the cracked surface and deep fissures of the foot pads. (B) Paw of a control Kromfohrländer. (C) Hair coat of the dogs shown in panels A and B. The affected dog (left) has a more irregular coat appearance in comparison to the unaffected control dog (right). Both dogs are representatives of the wire-haired (“rough-coated”) Kromfohrländer variety.

Figure 5. Histopathological findings in the palmoplantar epidermis.

(A) Haematoxylin and eosin (HE) stained paw pad section from a non-affected control dog. Note the relatively thin layer of orthokeratotic keratin on the epidermal surface. (B) Paw pad of a 1 year old affected Kromfohrländer. Note the moderate epidermal hyperplasia with papillated epidermal protrusions to the outside. The epidermis is covered by abundant compact orthokeratotic keratin. (C) Higher magnification of the same biopsy as shown in panel B. Note that the epidermis is regularly differentiated and no nuclei are seen in the stratum corneum.