Missense and nonsense mutations in melanocortin 1 receptor (MC1R) gene of different goat breeds: association with red and black coat colour phenotypes but with unexpected evidences

Abstract

BackgroundAgouti and Extension loci control the relative amount of eumelanin and pheomelanin production in melanocytes that, in turn, affects pigmentation of skin and hair. The Extension locus encodes the melanocortin 1 receptor (MC1R) whose permanent activation, caused by functional mutations, results in black coat colour, whereas other inactivating mutations cause red coat colour in different mammals.ResultsThe whole coding region of the MC1R gene was sequenced in goats of six different breeds showing different coat colours (Girgentana, white cream with usually small red spots in the face; Maltese, white with black cheeks and ears; Derivata di Siria, solid red; Murciano-Granadina, solid black or solid brown; Camosciata delle Alpi, brown with black stripes; Saanen, white; F1 goats and the parental animals). Five single nucleotide polymorphisms (SNPs) were identified: one nonsense mutation (p.Q225X), three missense mutations (p.A81V, p.F250V, and p.C267W), and one silent mutation. The stop codon at position 225 should cause the production of a shorter MC1R protein whose functionality may be altered. These SNPs were investigated in a larger sample of animals belonging to the six breeds. The Girgentana breed was almost fixed for the p.225X allele. However, there was not complete association between the presence of red spots in the face and the presence of this allele in homozygous condition. The same allele was identified in the Derivata di Siria breed. However, its frequency was only 33%, despite the fact that these animals are completely red. The p.267W allele was present in all Murciano-Granadina black goats, whereas it was never identified in the brown ones. Moreover, the same substitution was present in almost all Maltese goats providing evidence of association between this mutation and black coat colour.ConclusionAccording to the results obtained in the investigated goat breeds, MC1R mutations may determine eumelanic and pheomelanic phenotypes. However, they are probably not the only factors. In particular, the surprising not complete association of the nonsense mutation (p.Q225X) with red coat colour raises a few hypotheses on the determination of pheomelanic phenotypes in goats that should be further investigated.

Sequence characterization of the melanocortin 1 receptor (MC1R) gene in sheep with different coat colours and identification of the putative e allele at the ovine Extension locus

“…DNA was extracted using a standard phenol-chloroform protocol for blood, the Wizard ® Genomic DNA Purification kit for blood and milk (Promega Corporation, Madison, WI, USA) or rapid extraction methods for milk and hair roots (Russo et al, 2007). Primers for MC1R amplification and sequencing (Table 1) were designed with Primer 3 (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) from the published bovine, sheep and goat MC1R gene sequences (GenBank accession numbers: AF445641, Y13965, and FM212940, respectively; Rouzaud et al, 2000;Våge et al, 1999;Fontanesi et al, 2009a). PCR was performed using a TGradient thermal cycler (Biometra, Göttingen, Germany) or a PT-100 thermal cycler (MJ Research, Watertown, MA, USA) in a volume of 20 L containing 10-100 ng DNA template, 1 U DNA EuroTaq DNA polymerase (EuroClone Ltd., Paington, Devon, UK), 1× PCR Buffer, 2.5 mM dNTPs, 10 pmol of each primer and optimised MgCl2 concentrations (from 1.0 to 2.0 mM).…”

“…Gain of function and loss of function mutations of the MC1R gene determining dominant or partially dominant black/dark and recessive or partially recessive red/yellow/pale coat colour phenotypes, respectively, have been described in several mammals, such as mice (Robbins et al, 1993), humans (Valverde et al, 1995), cattle (Klungland et al, 1995;Joerg et al, 1996;Rouzaud et al, 2000), pigs (Kijas et al, , 2001, horses (Marklund et al, 1996), foxes (Våge et al, 1997;Våge et al, 2005), beach and pocket mice (Hoekstra et al, 2006;Nachman et al, 2003), rabbits (Fontanesi et al, 2006), and goats (Fontanesi et al, 2009a).…”

Coat colours in the Massese sheep breed are associated with mutations in the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes

“…Gain-of-function and loss-of-function mutations of the MC1R gene determining dominant or partially dominant black/dark and recessive or partially recessive red/yellow/pale coat colour phenotypes, respectively, have been described in a large number of mammals including, for example, mice (Robbins et al, 1993), humans (Valverde et al, 1995) and several farm animals like cattle (Klungland et al, 1995;Joerg et al, 1996;Rouzaud et al, 2000), pigs (Kijas et al, 1998 and, horses (Marklund et al, 1996), dogs (Everts et al, 2000;Newton et al, 2000), cats (Eizirik et al, 2003;Peterschmitt et al, 2009), rabbits (Fontanesi et al, 2006) and goats (Fontanesi et al, 2009a). In sheep, classical genetic studies have identified two alleles at the Extension locus: the dominant black (E D ) allele caused by two missense mutations in the MC1R gene (p.M73K and p.D121N) and present in the Norwegian Dala, Corriedale, Damara, Black Merino, Black Castellana and Karakul breeds (Vå ge et al, 1999 andRoyo et al, 2008); and the wild type (E 1 ) allele widely distributed in most breeds (Searle, 1968;Sponenberg, 1997).…”

Identification of c.483C>T polymorphism in the caprine tyrosinase-related protein 1 (TYRP1) gene

“…Fontanesi et al (2009) characterized the variability of the caprine melanocortin receptor 1 (MC1R) gene identifying one nonsense, three missense and one silent single nucleotide polymorphisms (SNP). Unfortunately, none of these mutations showed a clear association with coat colour when analysing their segregation in a pool of 317 goats belonging to six breeds with different pigmentation patterns (Fontanesi et al, 2009).…”

“…Fontanesi et al (2009) characterized the variability of the caprine melanocortin receptor 1 (MC1R) gene identifying one nonsense, three missense and one silent single nucleotide polymorphisms (SNP). Unfortunately, none of these mutations showed a clear association with coat colour when analysing their segregation in a pool of 317 goats belonging to six breeds with different pigmentation patterns (Fontanesi et al, 2009). Moreover, sequence analysis of the caprine agouti signaling protein (ASIP) gene has evidenced the existence of a duplicated copy, as previously demonstrated in sheep (Norris and Whan 2008), and three missense SNP.…”