To date, more than 950 cats have been tested using the CatScan panel of 85 known variants associated with diseases and traits in cats (www.mycatscan.com). Of these, four Maine coons, which are typically longhaired cats, were identified to be heterozygous for only one of the known variants in the FGF5 gene. The samples from two of these cats were used for sequencing of the FGF5 gene or genotyped for the novel FGF5 variant, with results as described below. An additional 275 cats of various breeds, including 144 Maine coons, were screened via restriction enzyme digestion on a 2% E-Gel and 68 cats were screened via in silico sequence alignment for a total of 345 different cats included in this study (Table 2). Samples represented 32 different breeds of cats of varying hair lengths: 3 hairless breeds, 15 shorthaired breeds, 7 longhaired breeds, and 8 breeds that can have either short or longhair.
We used samples from two Maine coon cats heterozygous for one known mutation and a control cat that is a compound heterozygote for M3 and M4 to search for additional variants within the FGF5 gene. Based on a recessive mode of inheritance, it was hypothesized that cats with longhair having only one copy of a known mutation were likely compound heterozygotes for a different variant. Through sequencing, the known variants were confirmed in each cat. Furthermore, a single, novel missense variant was identified c.G>A625 in the FGF5 sequencing (Fig. 1). The sequence data was submitted to NCBI GenBank with the accession number MZ311544. Figure 2 shows the location of this novel variant, M5, as compared to the four previously known variants. This variant causes an amino acid substitution of alanine to threonine at amino acid position 193 (p.A193T). PolyPhen predicts this mutation to be deleterious with a score of 1 (PolyPhen). The amino acid sequence was also compared against other species and showed that the region is highly conserved among various species (Fig. 3). No other variants were identified.
A PCR assay was developed to screen cats for this mutation to correlate its occurence with longhair and to determine if the variant was associated with any of the known longhair mutations (in cis), as known longhair mutations were already known in each of the cats. The screening was performed using a Sau96I restriction digest, with the wildtype sequence resulting in a 350bp and 400bp digested product and M5 sequence resulting in a 600bp (uncut) product. Cats with an M5 allele and one other known variant, showed the 350bp, 400bp and 600 bp products (Fig. 4). A total of 277 cats were screened using this assay. No shorthair or hairless cats were found to have this variant and no longhaired cats with two known mutations were found to have this variant. Two additional Maine Coons, who were not used for the sequencing, were found to be heterozygous for M5, for a total of four Maine Coons with one known mutation and the new M5 variant: M3/M5 (n=2), M4/M5 (n=2). Of the 68 additional cat sequences from various breeds analyzed in silico, none were found to have the M5 variant. No Maine coons were among those sequences analyzed.
After finding the variant in four unrelated Maine Coons, the owners were contacted and we requested buccal swabs from related cats, preferably parents or offspring. We received sample from three offspring of one Maine Coon who has the M5 variant (Fig. 5A) and received samples from the mother of a second Maine Coon with M5 (Fig. 5B). Each related Maine Coon was tested for M5 using the PCR screening assay and tested for the four known variants with the CatScan. All three offspring (Fig. 5A) and the mother in the second pedigree (Fig. 5B) were found to have the M5 variant, consistent with a recessive mode of inheritance.