Characterization of the dog Agouti gene and a nonagouti Agouti gene and a nonagouti

The interaction between two genes, Agouti and Melanocortin-1 receptor (Mc1r), produces diverse pigment patterns in mammals by regulating the type, amount, and distribution pattern of the two pigment types found in mammalian hair: eumelanin (brown/black) and pheomelanin (yellow/red). In domestic dogs (Canis familiaris), there is a tremendous variation in coat color patterns between and within breeds; however, previous studies suggest that the molecular genetics of pigment-type switching in dogs may differ from that of other mammals. Here we report the identification and characterization of the Agouti gene from domestic dogs, predicted to encode a 131-amino-acid secreted protein 98% identical to the fox homolog, and which maps to chromosome CFA24 in a region of conserved linkage. Comparative analysis of the Doberman Pinscher Agouti cDNA, the fox cDNA, and 180 kb of Doberman Pinscher genomic DNA suggests that, as with laboratory mice, different pigment-type-switching patterns in the canine family are controlled by alternative usage of different promoters and untranslated first exons. A small survey of Labrador Retrievers, Greyhounds, Australian Shepherds, and German Shepherd Dogs did not uncover any polymorphisms, but we identified a single nucleotide variant in black German Shepherd Dogs predicted to cause an Arg-to-Cys substitution at codon 96, which is likely to account for recessive inheritance of a uniform black coat. Diversity in mammalian skin and hair color is achieved by differential expression and regional distribution of two pigment types: brown/black eumelanin and red/yellow pheomelanin. Switching between the synthesis of these two pigment types is regulated by a paracrine signaling molecule, Agouti protein, which acts as an antagonistic ligand for the melanocortin-1 receptor (Mc1r), a seven-transmembrane protein expressed on the surface of melanocytes (Bultman et al. 1992; Miller et al. 1993; Robbins et al. 1993; reviewed in Jackson 1994). Mc1r activation due to constitutive activity or an exogenous agonist such as a-melanocyte stimulating hormone (a-MSH) promotes eumelanin synthesis; by contrast, Agouti protein inhibits Mc1r activation, causing a switch from eumelanin to pheomelanin synthesis (Lu et al. 1994; reviewed in Barsh 1996; Jordan and Jackson 1998; Ollmann et al. 1998). Biochemical and pharmacologic aspects of this pathway have become apparent over the last decade but were predicated by classical genetic studies in laboratory mice (Searle 1968; Silvers 1979). Gain-of-function Agouti alleles and loss-of-function Mc1r alleles each cause a pheomelanic phenotype, loss-of-function Agouti alleles and gain-of-function Mc1r alleles each cause a eumelanic phenotype, and animals carrying combinations of Agouti and Mc1r alleles with potentially opposite effects exhibit a coat color phenotype predicted by Mc1r rather than the Agouti genotype; in other words, Mc1r is epistatic to Agouti. Comparative genetic and zoological studies suggest that the antagonistic ligand–receptor relationGenbank accession numbers are AC092250 (bacterial artificial chromosome clone RP81-20712) and AY714374 (Doberman Pinscher Agouti cDNA). *Current address: Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 Correspondence to: Gregory S. Barsh, Beckman Center B271A, Stanford University School of Medicine, Stanford, CA 94305-5323, USA; E-mail: [email protected] 798 DOI: 10.1007/s00335-004-2377-1 Volume 15, 798–808 (2004) Springer Science+Business Media, Inc. 2004 ship between Agouti protein and the Mc1r applies to many vertebrate species (reviewed in Mundy et al. 2003). Mc1r missense mutations that promote eumelanin synthesis have been identified in sheep (Vage et al. 1999), cattle (Klungland et al. 1995), pigs (Kijas et al. 1998), jaguars (Eizirik et al. 2003), bananaquits (Theron et al. 2001), and chickens (Ling et al. 2003); in each of these cases, the mutations are thought to cause constitutive receptor activation, i.e., gain-of-function, and exhibit dominant transmission. Conversely, Mc1r mutations that promote pheomelanin synthesis have been identified in cattle (Klungland et al. 1995; Joerg et al. 1996), pigs (Kijas et al. 1998), chickens (Takeuchi et al. 1996), horses (Marklund et al. 1996), humans (Ha et al. 2003; Sturm et al. 2003), bears (Ritland et al. 2001), and dogs (Everts et al. 2000; Newton et al. 2000); in each of these cases, the mutations are thought to cause a loss-of-function and exhibit a recessive pattern of inheritance. The domestic dog, Canis familiaris, is especially well-suited to studies of coat color genetics. Its evolutionary ancestors in the canid family, whose representatives today include foxes, jackals, coyotes, and wolves, span a range of hair color types and patterns (Vila et al. 1999; reviewed in Wayne and Ostrander 1999; Savolainen et al. 2002). Domestication and selective breeding have established a large number of different breeds with a diversity of pigmentation phenotypes including uniform pheomelanin (the Irish Setter, Vizsla, or Golden Retriever), uniform eumelanin (the Newfoundland, Portuguese Water Dog), and a mixture of pheomelanin and eumelanin in a distinctive ‘‘black-and-tan’’ pattern (the Doberman Pinscher or Rottweiler). Twoand threegeneration kindreds with different coat color phenotypes are common and often readily available from an active community of dog breeders and owners with a deep interest in genetics. Most important, certain aspects of pigment-type switching in dogs are either not represented in other mammals or are thought to proceed by different mechanisms (Little 1957; Burns and Fraser 1966; Searle 1968). In particular, the uniform eumelanic appearance referred to above for breeds such as the Newfoundland or Portuguese Water Dog has been attributed to an allele of the Agouti locus, A, that is dominant to a, an Agouti allele thought to be responsible for the pheomelanic appearance characteristic of fawn-colored Boxers or Basenjis. However, dominance of a ‘‘black’’ Agouti allele to a ‘‘yellow’’ Agouti allele is not consistent with the biochemical action of Agouti protein and the Mc1r described above, and would represent a departure from genetic studies of pigment-type switching in other mammals. Furthermore, we have recently demonstrated that dominant transmission of a black coat color in a large Labrador · Greyhound cross established by Lust and colleagues at Cornell University is unlinked to Agouti and to Mc1r (Kerns et al. 2003). In most breeds, eumelanic coat color (sometimes described as ‘‘self’’-coloration) is thought to be dominantly inherited, as in the Labrador · Greyhound cross. In a few breeds, however, a uniform black appearance can be inherited as a recessive trait. This has been most clearly documented for German Shepherd Dogs, in which black progeny may be produced from two sable, saddle-colored, or blackand-tan parents, with a distribution ratio of 1:3 for black vs. nonblack offspring (Carver 1984). Some of the uncertainty about the biochemistry and genetics of different pigment types in dogs stems from the paucity of molecular studies. We have previously described the isolation and characterization of the dog Mc1r gene and demonstrated complete association between homozygosity for a nonsense mutation, R306ter, and a uniform pheomelanic coat color in the Irish Setter, Golden Retriever, yellow Labrador Retriever, and Samoyed (Newton et al. 2000). We have also described an association between an Mc1r missense variant, M264V, and the presence of a melanistic mask (Schmutz et al. 2003), although a causal relationship has not yet been confirmed by pedigree and/or functional studies. Here we report isolation and characterization of the dog Agouti gene. Linkage studies reveal that recessive inheritance of a uniform black coat in a German Shepherd pedigree cosegregates with Agouti, and molecular genetic studies identify the probable causative mutation. Materials and methods Molecular cloning. A Doberman Agouti cDNA clone was isolated in 3 stages by RT-PCR with internal degenerate oligonucleotide primers based on the human, mouse, and bovine sequences, followed by Rapid Amplification of cDNA Ends (RACE) to obtain the 5¢ and 3¢ ends of the cDNA. Doberman Pinscher puppy tail fragments were collected during routine tail-docking procedures by a community veterinarian. Total RNA was extracted from the yellow-haired skin on the ventral side of the tail, and a partial cDNA was reverse transcribed and amplified with a reverse primer, 5¢-A(A/G)NACNC(G/ T)(G/A)CANGT(G/A)CA-3¢, and a forward primer, 5¢-G(G/T)ITTC(C/T)TITG(C/T)TT(C/T)TT(C/T)AC IG-3¢. A second round of PCR was carried out with the same forward primer and a nested reverse primer, J.A. KERNS ET AL.: Agouti MUTATION IN GERMAN SHEPHERD DOGS 799