Hair is a unique structure that is characteristic of mammals and the shape of the hair follicle governs its form (Lindelof et al. 1988). The coat of the normal cat consists of three hair types: long and straight guard hairs of uniform diameter, thinner awn hairs, and fine undulating down hairs of uniform thickness. A variety of rexoid and hairless breeds are documented to have alterations in their hair types. The autosomal recessive Devon Rex coat is generally missing the guard hairs and had reduced length and thickness in the undercoat hairs. Hairless in Sphynx cats is allelic to Devon and some Sphynx have tuffs of hair on the chest, ears, or tail. Complementation testing with the other major curly breed, Cornish Rex, has shown that these autosomal recessive traits are not allelic (Robinson 1973).
Previous studies to identify the gene causing hairlessness in other species implicated KRT71 as the cause for curly coats in mice, rats, cattle, and dogs (Cadieu et al. 2009; Dunn 1937; Kikkawa et al. 2003; Poirier et al. 2002; Robinson 1981; Runkel et al. 2006; Sundberg et al. 1997; Taylor et al. 2000; A. D. Markey, unpublished). The keratin protein consists of three domains: a head domain, a low-complexity coiled-coil tail domain, and the helix-forming α-helical rod domain. The α-helical rod domain of KRT71 plays an important role in forming heteropolymers of specific type I and type II cytokeratin through interactions of these domains (Hatzfeld and Weber 1990). In mice, mutations such as Ca
9J, and Ca
16J induce single amino acid deletions in the α-helical rod domain and result in curly body hair (Kikkawa et al. 2003). In rat, the Re allele is identified as a 7-bp deletion at the 3′ splice site of intron 1 in KRT71 (Kuramoto et al. 2010). A SNP, c.451A>T, causing a nonsynonymous alteration, p.Arg151Trp, has been recently identified in curly hair in dogs (Cadieu et al. 2009). In cattle, an 8-bp deletion occurring in exon 1 causes an early truncated KRT71 protein resulting in a curly-hair coat (A. D. Markey, unpublished).
In the current study, KRT71 was analyzed in ten cat breeds with rexoid and hairless phenotypes. Hairless cats were included in this study because the naked phenotype of Sphynx cats is suggested to be allelic and dominant to the Devon Rex mutation (Robinson 1973). In cat, a 1575-bp nucleotide mRNA is produced and translated into 524 amino acids. Two of 55 identified mutations are likely associated with the Devon and Sphynx phenotypes. The Devon Rex mutation is a complicated set of two insertions and a deletion that leads to a splicing error. An 81-bp deletion removes the last 4 bp of intron 6, extending into the first 77 bp of exon 7 (c.1108-4_1184del). This deletion is followed by an 8-bp insertion of novel sequence (c.1184_1185insAGTTGGAG). This small insertion is followed by normal exon 7 sequence, except for one addition of thymine that is inserted 12 bp downstream (c.1196insT). As confirmed by cDNA analysis, these mutations disrupt the 3′ splice site of intron 6, the highly conserved AG, causing an alternative downstream acceptor to be employed (Fig. 2). A new alternative acceptor site is found within the first 37 bp of exon 7 (r.1108_1221del), which is 16 bp after the single thymine insertion. The resulting protein has a 35-amino-acid deletion at the 3′ C-terminus end of the α-helical rod domain (Supplementary Fig. 1). Approximately 200 cats were genotyped for the identified Rex (re) polymorphism and all of the Devon Rex were homozygous for the mutation. Thus, the mutation is fixed in the breed, suggesting that this mutation controls the re allele at the rex (R) locus and it could be considered a breed-defining genetic characteristic. These data suggest that two copies of the deletion are required to cause a curly-coat phenotype that meets the standard for the Devon Rex breed.
A guanine-to-adenine base substitution was observed at position +1 in intron 4 in the Sphynx cats (c.816+1G>A). The guanine is a highly conserved position in the intron at the 5′ splice site (Hastings and Krainer 2001; Ladd and Cooper 2002). Comparison of cDNA with genomic sequences of KRT71 suggested that the GT dinucleotide sequence 44 bp downstream of the original splice site, r[816+1_816+43ins; 816+1g>u], serves as an alternative splicing donor site in the Sphynx mutant allele (Fig. 2). Thus, an alternative splice site is recognized at base pair 44 in the modified exon 4 (Fig. 2). This alteration causes a stop codon at position 27 in the insertion, leading to a truncated KRT71 (Supplementary Fig. 1). The truncated protein deletes the majority of the α-helical rod domain that plays an important role in forming heteropolymers of specific type I and type II cytokeratin through interactions of these domains (Hatzfeld and Weber 1990). Together, sequence alterations in exon 7 and a truncated protein affecting the α-helical rod domain could also affect the formation of heteropolymers of specific type I and type II cytokeratins, cellular targeting, receptor binding, or proper folding of the protein after translation.
In the early stages of the hairless Sphynx breed development, crosses with Devon Rex were performed (Robinson 1973). Analysis of 34 Sphynx showed that 26 were homozygous for the intronic SNP and 8 were heterozygous for the mutation. Thus, this SNP is one of the causes of the hr allele at the H (hairless) locus. Six heterozygous Sphynx are compound heterozygotes for the SNP and the Devon Rex-associated deletion. These data suggest that the Sphynx allele is dominant to the Devon Rex allele, both being recessive to the wild type, supporting the phenotypic and segregation analyses previously published (Robinson 1973). However, two Sphynx were not homozygous for the hr mutation nor compound heterozygous with the re mutation, suggesting that these cats have some other epistatic mutations or hairless is dominant with variable penetrance in expression.
This genetic study identified the causative mutations for two mutant alleles, re and hr, at two reported loci, Rex (R) and Hairless (H), which are actually allelic mutations in KRT71. The Sphynx mutation leads to a complete loss-of-function allele where the structure of the hair is seriously compromised, i.e., the hair is produced but is easily dislodged because of the lack of a well-formed bulb. In the Devon Rex, the mutation causes a remarkable alteration in the protein as well, but evidently residual activity of the protein still exists. The KRT71
re and KRT71
hr are the suggested allelic designations, forming the allelic series KRT71
+ > KRT71
hr > KRT71
Sequence analyses of KRT71 did not identify genetic alterations causative for the other six Rexoids and the other two hairless cat breeds. Since much of the intronic and regulatory regions of domestic cat KRT71 have not been analyzed, this gene has not been excluded as causative for the other phenotypic alterations. Other genes involved in the keratinization process are also possible candidates. In humans, keratin genes are clustered as families in two regions of the genome: the type I genes on chromosome 17 and the genes encoding the type II keratins on chromosome 12 (Lessin et al. 1988; Rogers et al. 1995, 2005; Rosenberg et al. 1988). Family-based or genome-wide association studies may implicate the keratin clusters for the other cat rexoid and hairless phenotypes, leaving complex studies of the specific genes to follow.
Keratins have gained increasing attention during the last couple of decades because of their causal involvement in the etiology of a large heterogeneous group of hereditary epithelial diseases (see reviews Irvine and McLean 1999; Smith 2003). Our current study demonstrates the importance of the α-helical rod domain of KRT71 in hair formation in cats. Comparison of KRT71 mutations in various species may allow us to find associations of pelage phenotype with types of mutations and lead to better understanding of KRT71 functions. Moreover, the identification of feline coat variants should enhance the understanding of mammalian skin physiology, body homeostasis, and cell and tissue integration, and the development of a molecular test that could be used as a powerful instrument for the screening of different coat mutations will benefit the breeders.