In other species: Tasmanian devil , common brushtail possum , gray wolf , coyote , red fox , domestic cat , leopard , ass (donkey) , horse , pig , Arabian camel , llama , Western roe deer , impala , taurine cattle , goat , sheep , rabbit , tassel-eared squirrel , North American deer mouse , Mongolian gerbil , meadow voles , domestic guinea pig , fallow deer , alpaca , leopard cat , oldfield mouse , Kodkod , Colocolo , Asiatic golden cat , Northern mole vole , Eurasian water mole

Categories: Pigmentation phene

Links to possible relevant human trait(s) and/or gene(s) in OMIM: 611742 (trait) , 600201 (gene)

Mendelian trait/disorder: yes

Mode of inheritance: Autosomal

Disease-related: no

Key variant known: yes

Year key variant first reported: 2004

Cross-species summary: This locus, ASIP, encodes the agouti signalling protein, a peptide antagonist of the melanocyte-stimulating hormone receptor (MC1R), which is the product of the extension locus. As explained by Schneider et al. (PLoS Genet 10(2): e1004892; 2015), "The most common causes of melanism (black coat) mutations are gain-of-function alterations in MC1R, or loss-of function alterations in ASIP, which encodes Agouti signaling protein, a paracrine signaling molecule that inhibits MC1R signaling".

History: The A locus was initially described in 1957 by Clarence Little (Little, 1957 ). Several alleles were postulated by many researchers, but allele designations and breed-specific phenotype names remained inconsistent and controversial for decades. The following allelic series was mostly accepted prior to 2020: A^y (dominant yellow, fawn or sable) > a^w (wildtype, agouti or wolf sable) > a^s (saddle tan) > a^t (black-and-tan) > a (recessive black). A new nomenclature was proposed by Bannasch et al. (2021), see below, Molecular Basis. By cloning and sequencing a very likely comparative candidate gene (based on knowledge that the Agouti locus is actually the ASIP gene in many species), Kerns et al. (2004) were the first to characterise the ASIP gene in dogs. They reported "a missense alteration in exon 4, C427T [according to HGVS designated as c.286C>T], predicted to cause an Arg-to-Cys substitution at codon 96 (R96C)" and concluded tentatively that "the R96C mutation is the biochemical cause for recessive black", which corresponds to the a allele. Berryere et al. (2005) "identified an Agouti allele with two [adjacent] missense alterations, A82S and R83H, which was present (heterozygous or homozygous) in 41 dogs (22 breeds) with a fawn or sable coat, but was absent from 16 dogs (8 breeds) with a black-and-tan or tricolor phenotype". Bannasch et al. 2021 demonstrated that these missense variants were not causal, but in strong linkage disequilibrium with the causal variant. Dreger and Schmutz (2011) "characterize[d] a mutation consisting of a short interspersed nuclear element (SINE) insertion in intron 1 of ASIP that allows for the differentiation of the a(w) wolf sable and a(t) black-and-tan alleles. The SINE insertion is present in dogs with the a(t) and a alleles but absent from dogs with the a(w) and a(y) alleles. Dogs with the saddle tan phenotype were all a(t)/a(t). Schnauzers were all a(w)/a(w)." Bannasch et al. 2021 demonstrated that this SINE insertion was not causal, but in strong linkage disequilibrium with the causal variant. Dreger et al. (2013) identified a 16-bp duplication (g.1875_1890dupCCCCAGGTCAGAGTTT) in an intron of the RALY gene encoding hnRNP associated with lethal yellow that discriminated black-and-tan and saddle tan Basset Hounds and Pembroke Welsh Corgis. The RALY gene is located upstream of ASIP and the intronic duplication is in linkage disequilibrium with the causal variant in the ventral promoter of ASIP (Bannasch et al. 2021). [Compiled by Prof. Tosso Leeb 6/9/2021]

Mapping: Kerns et al. (2004) mapped the canine Agouti/ASIP gene to "the mid-distal region of CFA24".

Markers: The markers identified for A^y (Berryere et al. 2005), a^s (Dreger et al. 2013) and a^t (Dreger and Schmutz, 2011) were widely used in commercial genetic testing in 2020. The strong linkage disequilibrium with the true causal variant and the ease of genotyping were the main reasons that especially the two A^y associated missense variants represented valuable diagnostic markers. Dreger et al. (2019) reported an analysis of genetic testing results from 11,790 dogs, which revealed a small proportion of dogs that appeared to have three ASIP alleles and dogs with discordant genotype/phenotype combinations. These results highlight the challenges of using associated markers instead of the causal variants for genetic testing. [Compiled by Prof. Tosso Leeb 6/9/2021]

Molecular basis: Kerns et al. (2004) were the first to characterise the ASIP gene in dogs. They identified the c.286C>T missense variant, “predicted to cause an Arg-to-Cys substitution at codon 96 (R96C)" and concluded tentatively that "the R96C mutation is the biochemical cause for recessive black", which corresponds to the a allele. This allele represents a complete loss of ASIP function and is the most recessive allele in the allelic series. Bannasch et al. (2021) reinvestigated the molecular genetics of the remaining ASIP alleles, which all involve distinct distributions of yellow pheomelanin and black eumelanin according to body region (ventral/dorsal) or hair cycle. The authors proposed a new nomenclature of ASIP alleles involving one additional allele termed “shaded yellow”. The new allele designations were: DY (dominant yellow) > SY (shaded yellow) > AG (agouti) > BS (black saddle) > BB (black back) > a (recessive black) Bannasch et al. (2021) hypothesized that the causal variants for different ASIP-related patterns represent regulatory promoter variants. They characterized three different ASIP promoters in the canine ASIP gene and identified pattern-specific genetic variation in two of them. These promoters were termed “ventral promoter (VP)” and “hair cycle specific promoter (HCP)” and correspond to comparable VP and HCP in mice (Vrieling et al. 1994). Similar to the situation in mice, Bannasch et al. (2021) recognized that the VP and HCP act independently from each other in dogs and provide a modular system for the fine-tuned regulation of ASIP expression at the transcription level. The allelic series at the ASIP locus mentioned above corresponds to haplotypes consisting of one VP and one HCP allele each. Bannasch et al. identified a series of structural variants within 1.5 kb of the transcription start sites that are likely to affect promoter function. The CanFam3.1 genome reference assembly represents a DY allele derived from a dominant yellow boxer. Bannasch et al. termed the corresponding promoter haplotype VP1-HCP1, which is characterized by a gain-of-function or increased ASIP expression at both promoters. The wildtype AG haplotype corresponds to a VP2-HCP2 haplotype with “normal” promoter activity at both promoters that gives the pattern of a grey wolf with a light ventrum and banded hairs at the dorsum. The VP2-HCP1 haplotype that has “normal” activity at the VP but an increased activity at the HCP gives rise to the shaded yellow (SY) allele. The two remaining alleles, black saddle (BS) and black back (BB) are characterized by functionally inactive HCP alleles and consequently do not show any hair banding. The difference between BS and BB is entirely due to the activity of the VP. Bannasch et al. (2021) discovered three independent loss-of-function variants at the HCP and termed the corresponding promoter alleles HCP3, HCP4 and HCP5. All studied BS dogs carried a VP1-HCP4 haplotype. In BB dogs, three different but functionally equivalent haplotypes were observed: VP2-HCP3, VP2-HCP4 and VP2-HCP5 (named BB1, BB2 and BB3 in the likely causal variant table below). Bannasch et al. (2021) validated their proposed promoter haplotypes for association to the five patterns in a cohort of 366 dogs. They found perfect concordance of the promoter haplotypes with the four traditionally recognized patterns. However, 13 of the 366 dogs had diplotypes that did not correspond to the phenotypic difference between DY and SY. All 13 discordant dogs had a eumelanistic mask (see OMIA 001199-9615 : Coat colour, extension in Canis lupus familiaris), “which prevented reliable phenotype distinction between dominant yellow and shaded yellow” (Bannasch et al. 2021). An evolutionary analysis revealed that the gain-of-function variants at VP and HCP arose prior to the domestication of dogs. The completely white wolves of the American Arctic carry a virtual identical DY haplotype as dominant yellow dogs. Light colored wolves from the Himalaya or Inner Mongolia carry an SY haplotype. The mutation event that led to an overactive VP1 occurred more than 50,000 years ago in the grey wolf lineage. The HCP1 haplotype represents ghost DNA from a now extinct canid species that must have been introgressed into grey wolves more than 2 million years ago (Bannasch et al. 2021). [Compiled by Prof. Tosso Leeb 6/9/2021] The likely causal variant table for this phene has been updated to reflect the recent findings by Bannasch et al. (2021) [5/11/2021]: Three previously listed variants (published by Berryere et al., 2005; Dreger and Schmutz, 2011; Ciampolini et al., 2013) were removed from the table of likely causal variants, as these variants were shown to be in strong linkage disequilibrium with the causal variants. The variants and haplotypes reported by Bannasch et al. (2021) were added to the table. While Bannasch et al. (2021) did not claim causality, these promotor haplotypes were added as they were reported to be in perfect concordance with the four phenotypes. Belyakin et al. (2022) “investigate the genetics of the red sesame coat color in Shiba Inu dogs. [The] ... study revealed that red sesame dogs carry a specific heterozygous ASIP promoter diplotype, VP2-HCP1/VP2-HCP3, where VP2-HCP1 [Ays (or YS in OMIA) haplotype, OMIA variant ID: 1381] is responsible for the red coat with a dark overlay, and VP2-HCP3 [at (or BB1 in OMIA) haplotype, OMIA variant ID: 1383] for a tan point-like pattern. … The phenotypically darker sesame dogs were confirmed to have the aw/at genotype. In these cases, the aw allele was directly determined by the VP2-HCP2 [AG in OMIA; OMIA variant ID: 1380] promoter combination.” A single Shibu Inu dog was genotyped as Ays/Ays and had “significantly more red in their coat color, although with obvious dark shading, clearly distinguishing it from red Shiba Inu dogs.” Shiba Inu dogs with red coat colour were genotyped and presented with one of the following promoter haplotype combinations Ay/Ay, Ay/Ays, Ay/at or Ay/aw, where Ay corresponds to the ‘DY’ haplotype in OMIA with the variant ID 1382.

Genetic testing: Details to genotype the c.286C>T variant causing the a allele for recessive black were reported in Kerns et al. (2004). In their supplementary table 5, Bannasch et al. (2021) provided primer sequences for 5 different PCRs that enable the unequivocal characterization of the ASIP promoter alleles that control the ASIP-related patterns DY (dominant yellow), SY (shaded yellow), AG (agouti), BS (black saddle), and BB (black back). [Compiled by Prof. Tosso Leeb 6/9/2021]

Associated gene: