SHORT COMMUNICATION
doi: 10.1111/age.12207
A missense mutation in melanocortin 1 receptor is associated with the red coat colour in donkeys M. Abitbol*†, R. Legrand*† and L. Tiret*† *Universite Paris-Est Creteil, Ecole Nationale Veterinaire d’Alfort, Maisons-Alfort, France. †UMR955 INRA-ENVA de G en etique Fonctionnelle et Medicale, Institut National de la Recherche Agronomique, Maisons-Alfort, France.
Summary
The seven donkey breeds recognised by the French studbook are characterised by few coat colours: black, bay and grey. Normand bay donkeys seldom give birth to red foals, a colour more commonly seen and recognised in American miniature donkeys. Red resembles the equine chestnut colour, previously attributed to a mutation in the melanocortin 1 receptor gene (MC1R). We used a panel of 124 donkeys to identify a recessive missense c.629T>C variant in MC1R that showed a perfect association with the red coat colour. This variant leads to a methionine to threonine substitution at position 210 in the protein. We showed that methionine 210 is highly conserved among vertebrate melanocortin receptors. Previous in silico and in vitro analyses predicted this residue to lie within a functional site. Our in vivo results emphasised the pivotal role played by this residue, the alteration of which yielded a phenotype fully compatible with a loss of function of MC1R. We thus propose to name the c.629T>C allele in donkeys the e allele, which further enlarges the panel of recessive MC1R loss-of-function alleles described in animals and humans. Keywords chestnut, melanocortin 1 receptor, melanocyte, miniature donkey, Normand donkey
Loss-of-function mutations in MC1R (melanocortin 1 receptor) generate extreme red-to-yellow hair or coat colours in various species including humans (www.ncbi.nlm.nih.gov/ omim), mice (www.informatics.jax.org), dogs, cats, cattle, goats, pigs, rabbits and horses (omia.angis.org.au). Few coat colours have been described in domestic donkeys, namely black, bay, grey, red, all with or without white spotting, and white. In the American miniature donkey breed, all coat colours and patterns are admissible, and red foals are often produced from red, bay, grey or black breeding stock. This prompted breeders to suspect a recessive inheritance pattern for the red coat colour. In the seven French breeds of donkey (Pyrenean, Berry Black, Poitou, Cotentin, Provence, Bourbonnais and Normand), the red colour is not recognised. However, light-red foals are seldom born to bay Normand parents (Fig. 1a). Because whole-genome mapping tools are still missing in donkey, we decided to directly screen for variants affecting MC1R function in four red and two control bay donkeys Address for correspondence M. Abitbol, UMR955 INRA-ENVA de Genetique Fonctionnelle et M edicale, Ecole Nationale Veterinaire d’Alfort, 7 Avenue du G en eral de Gaulle, F-94700 Maisons-Alfort, France. E-mail: [email protected] Accepted for publication 8 July 2014
878
excerpted from a comprehensive panel of 124 donkeys from eight breeds (Appendix S1). The Ensembl MC1R equine genomic sequence was used to design two sets of primers (Appendix S1) allowing successful amplification of the donkey MC1R sequences. We then sequenced the unique MC1R exon and observed that the coding sequences, as well as the intron–exon boundaries, were highly conserved between these two close species (Appendix S1, Table S1). However, pairwise base-to-base comparisons of sequences revealed nine variants between donkeys and the bay horse reference sequence (Table S1). Only the c.629T>C variant produced a substitution p.Met210Thr and was consistent with a recessive mode of inheritance of the red colour. Indeed, the four red donkeys were homozygous for this variant, whereas the two control donkeys and the bay horse were homozygous for the reference allele (Table S1). The other eight variants were either incompatible with the recessive mode of inheritance, or yielded putative benign substitutions in non-conserved residues (Q109, I119 and S137; Fig. 1b; Table S1) or were synonymous variants (Table S1). PROVEAN, PolyPhen-2 and SNAP (Appendix S1) predicted the p.Met210Thr substitution to be a deleterious variant. Our total cohort of 124 donkeys was further genotyped for the c.629T>C variant. The 20 red donkeys, including three red Normand and 17 red miniature
© 2014 Stichting International Foundation for Animal Genetics, 45, 878–880
MC1R mutation in red donkeys (a)
(c)
(b)
Figure 1 Red coat colour in donkeys is governed by a melanocortin 1 receptor (MC1R) allele. (a) Red phenotype in donkeys. The red coat colour exhibits a variety of shades ranging from a light-bay coat with cream-white points and light ancestral marking (up, red Normand foals with a bay Normand jenny) to a brown-red (bottom left, adult miniature donkey) to light-red (bottom right, adult miniature donkey) coat with light points and dark ancestral markings. Miniature donkey pictures are from BBS Miniatures. (b) Protein sequences alignment between human melanocortin receptors (MC1-4R) and MC1R of several vertebrate species. MC1R sequences from various animal species are depicted with the name of the species. Human MC1R (reference sequence) is on the top of the alignment, and human MC2-4R are the last four sequences identified by their numbers. Non-conserved residues are shown in grey. Conserved residues are shown in black in the reference sequence and with dots in other sequences. Dashes represent deletions. The conserved M210 residue in donkey MC1R, corresponding to the M215 residue in human MC4R and the M247 residue in human MC3R, is surrounded. Functional MC1R residues known to be involved in ligand orthosteric binding are shown with open arrowheads. Non-conserved Q109, I119 and S137 residues distinguishing horse and donkey proteins are shown with stars. (c) Two-dimensional transmembrane structure of donkey MC1R and functional mutations in mammals. Missense mutations are depicted by circles and non-sense mutations by squares. Human recessive red-hair mutations are shown in green, and recessive e red mutations in mammals are shown in blue. The start of the mouse frameshift mutation is shown with a hexagon. The rabbit deletion is shown with diamonds. The D84 residue, shown in blue, is mutated in amber cats but also associated with red hair in humans. The recessive e red mutation that we identified in donkeys is shown in red. When a residue differs between donkey and depicted species, it is shown in brackets.
© 2014 Stichting International Foundation for Animal Genetics, 45, 878–880
879
880
Abitbol et al. Table 1 Genotypes for the c.629T>C variant in donkeys.
Black Berry Black donkeys Black Pyrenean donkeys Black Poitou donkeys Bay Bourbonnais donkeys Bay Normand donkeys Grey Provence donkeys Grey Cotentin donkeys Red Normand donkeys Red miniature donkeys Black, bay and grey miniature donkeys Total
T/T
T/C
C/C
Total
6 4 11 2 16 8 9 0 0 27 83
0 2 0 0 5 2 0 0 0 12 21
0 0 0 0 0 0 0 3 17 0 20
6 6 11 2 21 10 9 3 17 39 124
Altogether, these convergent results strongly suggest that the M210 residue is essential for MC1R function. The observation that its alteration in donkeys leads to a phenotype fully compatible with a loss-of-function mutation in MC1R strengthens the pivotal role of this residue and also emphasises that this mutation has a causative role in determining the red coat colour in donkeys. We thus propose that the c.629T>C allele, which is easily detectable with a genetic test, be named the e allele (Fig. 1c) in donkeys.
Acknowledgements
donkeys, were all homozygous for the C allele of this variant, whereas the 104 black, bay or grey donkeys were either homozygous for the T reference allele (n = 83) or heterozygotes (n = 21). This perfect concordance between the recessively inherited red coat colour and the c.629T>C variant (Table 1) corroborated our hypothesis that it was associated with the red coat colour trait. To evaluate the functional importance of the M210 residue, we aligned the donkey MC1R protein sequence with the MC1R to MC5R human sequences and with the MC1R sequences of 10 vertebrates (Fig. 1b; Appendix S1). We found there is full conservation of this residue between MCRs. This high level of conservation was previously reported for the essential residues of the orthosteric site (Yang 2011). In addition, the donkey M210 residue of MC1R best aligns with M247 of human MC3R, a residue assigned to the fifth transmembrane domain (Yang 2011) or the third intracellular loop (ICL3) (Yang & Tao 2012; Wang & Tao 2013). This apparent discrepancy has been partially explained by the fact that, in crystal structures, ICL3 has a propensity in some receptors for forming a-helical structures which elongate the helices 5 and 6, causing variations in transmembrane domain length (Park et al. 2008; Moukhametzianov et al. 2011; Katritch et al. 2012). These structural features may have important signalling consequences, as ICL3 is believed to control receptor selectivity to different G proteins (Katritch et al. 2012). Moreover, the functional activities of several residues conserved among MCRs have been investigated in various mutagenesis experiments. Using an in vitro assay, Wang & Tao (2013) reported that a M247A-mutated MC3R protein displayed a decreased ligand binding capacity and an increased half maximal effective concentration. These in vivo data confirmed previous in silico results obtained in MC4R, predicting that this conserved methionine residue lies within a functional site (Bromberg et al. 2009).
We wish to thank owners and breeders for providing samples and pictures and Diana Warwick for editing the manuscript. This project was funded by the French National Donkey Institute, INAM (Institut National Asin et Mulassier, France).
References Bromberg Y., Overton J., Vaisse C., Leibel R.L. & Rost B. (2009) In silico mutagenesis: a case study of the melanocortin 4 receptor. FASEB Jou 23, 3059–69. Katritch V., Cherezov V. & Stevens R.C. (2012) Diversity and modularity of G protein-coupled receptor structures. Trends in Pharmacological Sciences 33, 17–27. Moukhametzianov R., Warne T., Edwards P.C., Serrano-Vega M.J., Leslie A.G., Tate C.G. & Schertler G.F. (2011) Two distinct conformations of helix 6 observed in antagonist-bound structures of a beta1-adrenergic receptor. Proceedings of the National Academy of Sciences of the United States of America 108, 8228–32. Park J.H., Scheerer P., Hofmann K.P., Choe H.W. & Ernst O.P. (2008) Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–7. Wang Z.Q. & Tao Y.X. (2013) Functions of the third intracellular loop of the human melanocortin-3 receptor. Current Pharmaceutical Design 19, 4831–8. Yang Y. (2011) Structure, function and regulation of the melanocortin receptors. European Journal of Pharmacology 660, 125–30. Yang F. & Tao Y.X. (2012) Functional characterization of nine novel naturally occurring human melanocortin-3 receptor mutations. Biochimica et Biophysica Acta 1822, 1752–61.
Supporting information Additional supporting information may be found in the online version of this article. Appendix S1. Supplementary methods. Table S1. Genomic variants in MC1R identified between donkey cDNA sequences and the horse coding reference sequence.
© 2014 Stichting International Foundation for Animal Genetics, 45, 878–880
doi: 10.1111/age.12207
A missense mutation in melanocortin 1 receptor is associated with the red coat colour in donkeys M. Abitbol*†, R. Legrand*† and L. Tiret*† *Universite Paris-Est Creteil, Ecole Nationale Veterinaire d’Alfort, Maisons-Alfort, France. †UMR955 INRA-ENVA de G en etique Fonctionnelle et Medicale, Institut National de la Recherche Agronomique, Maisons-Alfort, France.
Summary
The seven donkey breeds recognised by the French studbook are characterised by few coat colours: black, bay and grey. Normand bay donkeys seldom give birth to red foals, a colour more commonly seen and recognised in American miniature donkeys. Red resembles the equine chestnut colour, previously attributed to a mutation in the melanocortin 1 receptor gene (MC1R). We used a panel of 124 donkeys to identify a recessive missense c.629T>C variant in MC1R that showed a perfect association with the red coat colour. This variant leads to a methionine to threonine substitution at position 210 in the protein. We showed that methionine 210 is highly conserved among vertebrate melanocortin receptors. Previous in silico and in vitro analyses predicted this residue to lie within a functional site. Our in vivo results emphasised the pivotal role played by this residue, the alteration of which yielded a phenotype fully compatible with a loss of function of MC1R. We thus propose to name the c.629T>C allele in donkeys the e allele, which further enlarges the panel of recessive MC1R loss-of-function alleles described in animals and humans. Keywords chestnut, melanocortin 1 receptor, melanocyte, miniature donkey, Normand donkey
Loss-of-function mutations in MC1R (melanocortin 1 receptor) generate extreme red-to-yellow hair or coat colours in various species including humans (www.ncbi.nlm.nih.gov/ omim), mice (www.informatics.jax.org), dogs, cats, cattle, goats, pigs, rabbits and horses (omia.angis.org.au). Few coat colours have been described in domestic donkeys, namely black, bay, grey, red, all with or without white spotting, and white. In the American miniature donkey breed, all coat colours and patterns are admissible, and red foals are often produced from red, bay, grey or black breeding stock. This prompted breeders to suspect a recessive inheritance pattern for the red coat colour. In the seven French breeds of donkey (Pyrenean, Berry Black, Poitou, Cotentin, Provence, Bourbonnais and Normand), the red colour is not recognised. However, light-red foals are seldom born to bay Normand parents (Fig. 1a). Because whole-genome mapping tools are still missing in donkey, we decided to directly screen for variants affecting MC1R function in four red and two control bay donkeys Address for correspondence M. Abitbol, UMR955 INRA-ENVA de Genetique Fonctionnelle et M edicale, Ecole Nationale Veterinaire d’Alfort, 7 Avenue du G en eral de Gaulle, F-94700 Maisons-Alfort, France. E-mail: [email protected] Accepted for publication 8 July 2014
878
excerpted from a comprehensive panel of 124 donkeys from eight breeds (Appendix S1). The Ensembl MC1R equine genomic sequence was used to design two sets of primers (Appendix S1) allowing successful amplification of the donkey MC1R sequences. We then sequenced the unique MC1R exon and observed that the coding sequences, as well as the intron–exon boundaries, were highly conserved between these two close species (Appendix S1, Table S1). However, pairwise base-to-base comparisons of sequences revealed nine variants between donkeys and the bay horse reference sequence (Table S1). Only the c.629T>C variant produced a substitution p.Met210Thr and was consistent with a recessive mode of inheritance of the red colour. Indeed, the four red donkeys were homozygous for this variant, whereas the two control donkeys and the bay horse were homozygous for the reference allele (Table S1). The other eight variants were either incompatible with the recessive mode of inheritance, or yielded putative benign substitutions in non-conserved residues (Q109, I119 and S137; Fig. 1b; Table S1) or were synonymous variants (Table S1). PROVEAN, PolyPhen-2 and SNAP (Appendix S1) predicted the p.Met210Thr substitution to be a deleterious variant. Our total cohort of 124 donkeys was further genotyped for the c.629T>C variant. The 20 red donkeys, including three red Normand and 17 red miniature
© 2014 Stichting International Foundation for Animal Genetics, 45, 878–880
MC1R mutation in red donkeys (a)
(c)
(b)
Figure 1 Red coat colour in donkeys is governed by a melanocortin 1 receptor (MC1R) allele. (a) Red phenotype in donkeys. The red coat colour exhibits a variety of shades ranging from a light-bay coat with cream-white points and light ancestral marking (up, red Normand foals with a bay Normand jenny) to a brown-red (bottom left, adult miniature donkey) to light-red (bottom right, adult miniature donkey) coat with light points and dark ancestral markings. Miniature donkey pictures are from BBS Miniatures. (b) Protein sequences alignment between human melanocortin receptors (MC1-4R) and MC1R of several vertebrate species. MC1R sequences from various animal species are depicted with the name of the species. Human MC1R (reference sequence) is on the top of the alignment, and human MC2-4R are the last four sequences identified by their numbers. Non-conserved residues are shown in grey. Conserved residues are shown in black in the reference sequence and with dots in other sequences. Dashes represent deletions. The conserved M210 residue in donkey MC1R, corresponding to the M215 residue in human MC4R and the M247 residue in human MC3R, is surrounded. Functional MC1R residues known to be involved in ligand orthosteric binding are shown with open arrowheads. Non-conserved Q109, I119 and S137 residues distinguishing horse and donkey proteins are shown with stars. (c) Two-dimensional transmembrane structure of donkey MC1R and functional mutations in mammals. Missense mutations are depicted by circles and non-sense mutations by squares. Human recessive red-hair mutations are shown in green, and recessive e red mutations in mammals are shown in blue. The start of the mouse frameshift mutation is shown with a hexagon. The rabbit deletion is shown with diamonds. The D84 residue, shown in blue, is mutated in amber cats but also associated with red hair in humans. The recessive e red mutation that we identified in donkeys is shown in red. When a residue differs between donkey and depicted species, it is shown in brackets.
© 2014 Stichting International Foundation for Animal Genetics, 45, 878–880
879
880
Abitbol et al. Table 1 Genotypes for the c.629T>C variant in donkeys.
Black Berry Black donkeys Black Pyrenean donkeys Black Poitou donkeys Bay Bourbonnais donkeys Bay Normand donkeys Grey Provence donkeys Grey Cotentin donkeys Red Normand donkeys Red miniature donkeys Black, bay and grey miniature donkeys Total
T/T
T/C
C/C
Total
6 4 11 2 16 8 9 0 0 27 83
0 2 0 0 5 2 0 0 0 12 21
0 0 0 0 0 0 0 3 17 0 20
6 6 11 2 21 10 9 3 17 39 124
Altogether, these convergent results strongly suggest that the M210 residue is essential for MC1R function. The observation that its alteration in donkeys leads to a phenotype fully compatible with a loss-of-function mutation in MC1R strengthens the pivotal role of this residue and also emphasises that this mutation has a causative role in determining the red coat colour in donkeys. We thus propose that the c.629T>C allele, which is easily detectable with a genetic test, be named the e allele (Fig. 1c) in donkeys.
Acknowledgements
donkeys, were all homozygous for the C allele of this variant, whereas the 104 black, bay or grey donkeys were either homozygous for the T reference allele (n = 83) or heterozygotes (n = 21). This perfect concordance between the recessively inherited red coat colour and the c.629T>C variant (Table 1) corroborated our hypothesis that it was associated with the red coat colour trait. To evaluate the functional importance of the M210 residue, we aligned the donkey MC1R protein sequence with the MC1R to MC5R human sequences and with the MC1R sequences of 10 vertebrates (Fig. 1b; Appendix S1). We found there is full conservation of this residue between MCRs. This high level of conservation was previously reported for the essential residues of the orthosteric site (Yang 2011). In addition, the donkey M210 residue of MC1R best aligns with M247 of human MC3R, a residue assigned to the fifth transmembrane domain (Yang 2011) or the third intracellular loop (ICL3) (Yang & Tao 2012; Wang & Tao 2013). This apparent discrepancy has been partially explained by the fact that, in crystal structures, ICL3 has a propensity in some receptors for forming a-helical structures which elongate the helices 5 and 6, causing variations in transmembrane domain length (Park et al. 2008; Moukhametzianov et al. 2011; Katritch et al. 2012). These structural features may have important signalling consequences, as ICL3 is believed to control receptor selectivity to different G proteins (Katritch et al. 2012). Moreover, the functional activities of several residues conserved among MCRs have been investigated in various mutagenesis experiments. Using an in vitro assay, Wang & Tao (2013) reported that a M247A-mutated MC3R protein displayed a decreased ligand binding capacity and an increased half maximal effective concentration. These in vivo data confirmed previous in silico results obtained in MC4R, predicting that this conserved methionine residue lies within a functional site (Bromberg et al. 2009).
We wish to thank owners and breeders for providing samples and pictures and Diana Warwick for editing the manuscript. This project was funded by the French National Donkey Institute, INAM (Institut National Asin et Mulassier, France).
References Bromberg Y., Overton J., Vaisse C., Leibel R.L. & Rost B. (2009) In silico mutagenesis: a case study of the melanocortin 4 receptor. FASEB Jou 23, 3059–69. Katritch V., Cherezov V. & Stevens R.C. (2012) Diversity and modularity of G protein-coupled receptor structures. Trends in Pharmacological Sciences 33, 17–27. Moukhametzianov R., Warne T., Edwards P.C., Serrano-Vega M.J., Leslie A.G., Tate C.G. & Schertler G.F. (2011) Two distinct conformations of helix 6 observed in antagonist-bound structures of a beta1-adrenergic receptor. Proceedings of the National Academy of Sciences of the United States of America 108, 8228–32. Park J.H., Scheerer P., Hofmann K.P., Choe H.W. & Ernst O.P. (2008) Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–7. Wang Z.Q. & Tao Y.X. (2013) Functions of the third intracellular loop of the human melanocortin-3 receptor. Current Pharmaceutical Design 19, 4831–8. Yang Y. (2011) Structure, function and regulation of the melanocortin receptors. European Journal of Pharmacology 660, 125–30. Yang F. & Tao Y.X. (2012) Functional characterization of nine novel naturally occurring human melanocortin-3 receptor mutations. Biochimica et Biophysica Acta 1822, 1752–61.
Supporting information Additional supporting information may be found in the online version of this article. Appendix S1. Supplementary methods. Table S1. Genomic variants in MC1R identified between donkey cDNA sequences and the horse coding reference sequence.
© 2014 Stichting International Foundation for Animal Genetics, 45, 878–880
