Ancient R301C variant of the MC1R gene is present in various breeds of today
To screen for the presence and frequency of the ancient R301C variant of MC1R in today’s canine population, 11,750 dog samples were genotyped as a part of a custom-designed microarray panel test commercially available as MyDogDNATM/Optimal SelectionTM Canine Genetic Breeding Analysis. The R301C variant was present in a total of 265 tested dogs representing 35 different breeds and breed varieties as well as mixed breed dogs. The allele frequency for R301C in all dogs representing 304 different breeds and mixed breeds was 1.5% (N = 11,750; Figure 1 and Table S1). The R301C frequency in the tested Alaskan Malamute population was 100%. The additional 34 breeds in which the R301C variant was found could be classified into old Nordic Spitzes (East-Siberian Laika, Finnish Lapphund, Finnish Spitz, Karelian Bear Dog, Lapponian Herder, Nordic Spitz, Siberian Husky, West-Siberian Laika), other Primitive Spitz Type dogs (Basenji, Cirneco Dell’Etna, Kritikos Lagonikos, Peruvian Hairless Dog – Large, Medium and Miniature), Scent Hounds (Basset Fauve de Bretagne, Beagle, Drever, English Foxhound, Finnish Hound, Hungarian Hound, Plott, Serbian Hound), one gundog breed (Chesapeake Bay Retriever), one guardian dog breed (Pyrenean Mastiff), three Companion and Toy Dog breeds (Chihuahua, Chinese Crested Dog, Phalene),some recently created breeds (Alaskan Husky, Alaskan Klee Kai, Chinook, Northern Inuit, Tamaskan Dog, Saarlooswolfdog), and a nearly extinct sheepdog of the Auvergne region in France (Berger d’Auvergne) . In this study sample the R301C variant was not found in dog breeds with Eastern Asian origin (Akita, Chow Chow, Hokkaido, Kai, Kishu, Shar Pei, Shiba, Shikoku, Korean Jindo Dog) or Middle Eastern/Central Asian origin (Afghan Hound, Saluki, Tibetan Mastiff, Tibetan Spaniel, Tibetan Terrier, Lhasa Apso, Shih-Tzu, Central Asian Ovcharka).
R301C is a novel alternative allele of the E locus
To elucidate the relationship of R301C and other known E locus variants, genotypes were obtained for EM (melanistic mask), EG (grizzle/domino) and e1 (recessive red) alleles of the MC1R gene. Two rare additional recently characterized e allelic variants(1); e2 discovered in Australian Cattle Dog and e3 discovered in Siberian Husky were not genotyped as a part of this study. The R301C variant and the tested E locus variants showed no linkage disequilibrium in the 262 dogs with diverse breed background in which it was found present. The R301C variant was not present in dogs with two copies of the tested E locus variants; EM, EG or e1, while in dogs with two copies of the R301C variant no EM, EG or e1 variants were present. Also, no more than one copy of EM or e1 variants was present when one copy of R301C was found. The rarest MC1R coat color variant, the EG allele, is only found in one of the dog breeds, Kritikos Lagonikos, in which R301C was identified. However, in this study sample no individuals carrying both EG and R301C variants were identified.
Notably, using current conventional practices for calling of E locus genotypes at commercial genotyping laboratories, dogs carrying R301C would have been interpreted as carrying E. As our findings suggested that R301C rather represents an independent alternative allele at the E locus, we refer to it as eA (for ancient red e) for clarity hereafter.
eA allele of MC1R explains presence of domino patterning in Tamaskan Dog
To interpret the phenotypic impact of the R301C variant, eA, on the dog’s coat color, also genotypes for Canine Beta-Defensin 103 (CBD103) and Agouti Signaling Protein (ASIP) were obtained for the analysis. Color phenotypes were available for 125 (47%) dogs of the 265 dogs identified with one or two copies of the eA allele in this study. Phenotyping using dog owner-provided photos initially focused on the Tamaskan Dog breed, which represented 35% (N=43) of the dogs with phenotype information available. These dogs were carefully examined to elucidate the potential phenotypic effect of eA within a single breed. The expected coat colors for these dogs were aw wolf sable, at tan point or a recessive black determined by their A locus (due to only wild type variant ky being present on the K locus of these dogs). A maximum copy number of two allelic variants at the A locus was found in any of the Tamaskan Dogs and no ay variant, indicating that no A locus anomalies were present as recently observed in a small number of other dog breeds (12). The observed coat color phenotypes were in concordance with expected phenotypes for 26 of the Tamaskan Dogs, while 17 of the Tamaskan Dogs manifested more abundantly pheomelanic hairs in areas of head, legs and body on which the coat color pattern known as “domino” or “grizzle” is formed. This patterning, which is commonly observed in the two arctic breeds Alaskan Malamute and Siberian Husky, bears high phenotypic resemblance to previously characterized EG domino in Afghan Hound and EG grizzle in Saluki. The aforementioned have been suggested to be dependent on the A locus at/at genotype for their manifestation (3). Here, the domino pattern is observed independently from EG on divergent breed backgrounds (Figure 2). Domino phenotype encompasses pale facial markings with receded eumelanin line forming a widow’s peak in the forehead, and often also white markings expressed up the centerline of the face including reduced pigment in the centerline of the nose referred to as a dudley nose. The latter phenotypic feature (white markings and a dudley nose) is also common in recessive red dogs. Notably, two dogs expected to manifest solid black coat as a result of a/a genotype on their A locus also showed lightened body coat color with tan point like markings that were very profound in the newborn puppy, while the coat phenotype resembled wild sable or tan point in the adult recessive black dog (Figure 2 and Figure 3; U- X).
The 26 non-domino Tamaskan Dogs had one copy of the R301C variant in combination with either one copy of the E or EM variant. Presence of two copies of the R301C variant (N=4), or compound heterozygosity of eA with e recessive red (N=13), showed statistically significant association with domino phenotype (P=2.37-12) (Table 1). The phenotypic impact of the eA allele - recessivity to wild type E and dominance to e - is further demonstrated in a litter of Tamaskan Dogs (Figure 2). Thirty-nine additional Tamaskan Dogs without any copies of the eA allele manifested a non-domino phenotype, suggesting that the eA allele explains the presence of all domino phenotypes observed in Tamaskan Dog.
eA allele of MC1R is associated with partial recessive red phenotypes in multiple breeds
After associating the eA allele with a coat color phenotype within a single breed, we pursued characterization of the phenotypic impact of the eA allele across different breeds and coat color genotypes. Examination of additional dog owner-provided photos revealed that the eA allele is associated with apparent partial recessive red coat color patterning. The coat color phenotype was altered in all 70 dogs (including the 17 Tamaskan Dog study sample) with the eA allele present in homozygous form (N=35) or in heterozygous form paired with the recessive red e1 allele (N=35). These phenotypes manifested in dogs with eA/eA and eA/e1 genotypes as follows. All seven dogs with eA/eA or eA/e1 and KB (or alternatively the intermediate kbr) on the K locus express a non-solid and non-striped eumelanin shade phenotype (Table 2, Table S2 and Figure 3; A-G). In five out of seven dogs, three Cirneco dell’Etna’s and two Drevers (a breed in which the striped kbr brindle pattern is observed), the phenotype is clear fawn and virtually indistinguishable from recessive red e1/e1 (Table 2, Table S2 and Figure 3; A-B and Y). Of the remaining two KB dogs, one Siberian Husky is wolf sable and one mixed breed dog is tan point (modified into saddle tan) (Table 2, Table S2, Figure 3; E-F). Given that the five clear fawn dogs have ay/ay genotype, the wolf sable dog has aw/at genotype and the mixed breed has at/a genotype on the A locus, we conclude that these dogs express the coat color pattern of their A locus despite the presence of one copy of dominant variant on the K locus.
Altogether all 63 dogs with eA/e1 or eA/eA genotype expressing A locus manifested altered phenotype (Table 2 and Table S2). Of 49 out of 56 dogs with eA/e1 or eA/eA genotype expressing A locus ay fawn, aw wolf sable, at tan point produced domino color patterning, but in all of the 56 dogs with eA genotype resulted in increase of pheomelanin expression. One of the four ay fawn dogs manifested domino patterning on eumelanin shaded fawn phenotype, whereas three dogs with eA/e1 or eA/eA genotype combined with ay fawn were phenotypically similar to recessive red e/e dogs..All of the 40 aw wolf sample dogs had domino patterning. Typical domino patterning was also manifested in eight of the 12 at tan point dogs, while in four out of 12 at tan point dogs variation in the level of pheomelanin expression was observed. . One Drever homozygous for the eA allele had no visible increase in its coat color pheomelanin expression; the dog expresses normal tan points, but also the white markings on the centerline of the face and a dudley nose. In contrast, almost no eumelanin pigment is present in two Hungarian Hounds with eA/e1 genotype manifesting rich red coat color with white markings on the centerline of the face and a dudley nose (Table 2, Table S2). Moreover, in one at tan point Beagle in which tan point coat color modifier Saddle Tan (20) is present, the ‘saddle’ consists of only a few eumelanic hairs and the dog manifests a dudley nose(Figure 3; R). This resulting coat color phenotype in the Beagle breed is called as “pied”, and we now demonstrate it to be caused by eA ancient red. In addition, all seven out of seven a recessive black dogs had pheomelanic markings despite of the loss-of-function a variantresulting in coat phenotypes resembling tan point or wolf sable (Table 2, Table S2, Figure 3; H-I and U-W).
We observed no phenotype change in 52 dogs genotyped EM/eA (N=16) or E/eA (N=37) strongly suggesting an allelic hierarchy in which eA is recessive to EM and E and dominant to e, while further information on phenotypes produced by EG/eA genotype remains to be collected (Table 2 and Table S2). In two Siberian Huskies with one copy of eA and no other tested E alleles present the phenotype was altered to domino as if no wild type E was present. We did not have DNA availability to test for the presence of a rare e3 variant discovered in Huskies (1), but we hypothesize that the actual genotype of these dogs is eA/e3 based on the observed phenotype.
Taken together, phenotype data available in 15 different breeds consistently shows that eA results in various increased pheomelanin pigment-containing phenotypes that we interpret to be partial recessive red coat colors. In dogs with KB dominant black or kbr genotype, the K locus is masked and A locus is expressed instead, while in dogs expressing the A locus (in the absence of KB variant) the ability to produce eumelanin is reduced resulting in coat color patterns known by the names “domino”, “grizzle” and “pied” depending on the breed background, but may also result in phenotypes indistinguishable from recessive red (cream), tan point or wolf sable.