Explicit evidence for a missense mutation in exon 4 of SLC45A2 gene causing the pearl coat dilution in horses

Four loci seem responsible for the dilution of the basic coat colours in horse: Dun (D), Silver Dapple (Z), Champagne (CH) and Cream (C). Apart from the current phenotypes ascribed to these loci, pearl has been described as yet another diluted coat colour in this species. To date, this coat colour seems to segregate only in the Iberian breeds Purebred Spanish horse and Lusitano and has also been described in breeds of Iberian origin, such as Quarter Horses and Paint Horse, where it is referred to as the ‘Barlink Factor’. This phenotype segregates in an autosomal recessive manner and resembles some of the coat colours produced by the champagne CH^CH and cream C^Cr alleles, sometimes being difficult to distinguish among them. The interaction between compound heterozygous for the pearl C^prl and cream C^Cr alleles makes SLC45A2 the most plausible candidate gene for the pearl phenotype in horses. Our results provide documented evidence for the missense variation in exon 4 [SLC45A2:c.985G>A; SLC45A2:p.(Ala329Thr)] as the causative mutation for the pearl coat colour. In addition, it is most likely involved as well in the cremello, perlino and smoky cream like phenotypes associated with the compound C^Cr and C^prl heterozygous genotypes (known as cream pearl in the Purebred Spanish horse breed). The characterization of the pearl mutation allows breeders to identify carriers of the C^prl allele and to select this specific coat colour according to personal preferences, market demands or studbook requirements as well as to verify segregation within particular pedigrees.     

Melanocortin receptor 1 (MC1R) mutations and coat color in pigs.

The melanocortin receptor 1 (MC1R) plays a central role in regulation of eumelanin (black/brown) and phaeomelanin (red/yellow) synthesis within the mammalian melanocyte and is encoded by the classical Extension (E) coat color locus. Sequence analysis of MC1R from seven porcine breeds revealed a total of four allelic variants corresponding to five different E alleles. The European wild boar possessed a unique MC1R allele that we believe is required for the expression of a wild-type coat color. Two different MC1R alleles were associated with the dominant black color in pigs. MC1R*2 was found in European Large Black and Chinese Meishan pigs and exhibited two missense mutations compared with the wild-type sequence. Comparative data strongly suggest that one of these, L99P, may form a constitutively active receptor. MC1R*3 was associated with the black color in the Hampshire breed and involved a single missense mutation D121N. This same MC1R variant was also associated with EP, which results in black spots on a white or red background. Two different missense mutations were identified in recessive red (e/e) animals. One of these, A240T, occurs at a highly conserved position, making it a strong candidate for disruption of receptor function.     

Pigmentation in Black-boned sheep (Ovis aries): association with polymorphism of the MC1R gene

Variations in vertebrate skin and hair color are due to varied amounts of eumelanin (brown/black) and phaeomelanin (red/yellow) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eumelanin and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many vertebrates. We have sequenced the entire coding region of the MC1R gene in Black-boned, Nanping indigenous and Romney Marsh sheep populations and found two silent mutation sites of A12G and G144C, respectively. PCR-RFLP of G144C showed that frequency of allele G in Black-boned, Nanping indigenous and Romney Marsh sheep was 0.818, 0.894 and 0, respectively. Sheep with GG genotype had significantly higher (P < 0.05) tyrosinase activity than sheep with CC genotype in the all investigated samples. Moreover, there was significant effect of MC1R genotype on coat color, suggesting that MC1R gene could affect coat color but not black traits. There would be merit in further studies using molecular techniques to elucidate the cause of black traits in these Black-boned sheep.     

白色基因座(I)在中国地方猪种毛色遗传中的作用研究

通过利用PCR—RFLP和PCR—SSCP技术对中国地方猪种KIT基因内含子17、18的序列进行多态性分析。结果表明：内含子17上的替换突变(G→A)发生于毛色为白色的个体——白色五指山猪、大白猪、长白猪上,其基因型(AB型)频率分别为1、1和0．8;其他中国地方猪种的此基因型频率均为0。内含子18上的缺失突变(AGTT)也同样发生在上述3个猪种的白色个体中,其基因型(AA型)频率分别为1、1和0．93;而且同样在其他的地方品种中其基因型频率均为0。这充分证明KIT基因对于猪的白毛色有重要的调控作用,而且I基因座对于其他的经典遗传基因座有上位作用。另一方面,中国地方猪种荣昌猪虽然在表型上与引入猪种大白猪、长白猪相似(白毛色),但是在KIT基因上发生的突变完全不同,推测它们分别属于不同的毛色遗传体系。     

MC1R studies in dogs with melanistic mask or brindle patterns

Black mask is a characteristic pattern in which red, yellow, tan, fawn, or brindle dogs exhibit a melanistic muzzle which may extend up onto the ears. Melanistic mask is inherited in several breeds as an autosomal dominant trait, and appears to be a fixed trait in a few breeds of dogs. A MC1R nonsense mutation, R306ter, has been shown to cause a completely red or yellow coat color in certain breeds such as Irish setters, yellow Labrador retrievers, and golden retrievers. The amino acid sequence for the melanocortin receptor 1 gene (MC1R) was examined in 17 dogs with melanistic masks from seven breeds, 19 dogs without melanistic masks, and 7 dogs in which their coat color made the mask difficult to distinguish. We also examined nine brindle dogs of four breeds, including three dogs who also had a black mask. No consistent amino acid change was observed in the brindle dogs. All dogs with a melanistic mask had at least one copy of a valine substitution for methionine at amino acid 264 (M264V) and none were homozygous for the premature stop codon (R306ter). These results suggest that black mask, but not brindle, is caused by a specific MC1R allele.     

TYRP1 is associated with dun coat colour in Dexter cattle or how now brown cow?

Tyrosinase related protein 1 (TYRP1), which is involved in the coat colour pathway, was mapped to BTA8 between microsatellites BL1080 and BM4006, using a microsatellite in intron 5 of TYRP1. The complete coding sequence of bovine TYRP1 was determined from cDNA derived from skin biopsies of cattle with various colours. Sequence data from exons 2-8 from cattle with diluted phenotypes was compared with that from non-diluted phenotypes. In addition, full-sib families of beef cattle generated by embryo transfer and half-sib families from traditional matings in which coat colour was segregating were used to correlate TYRP1 sequence variants with dilute coat colours. Two non-conservative amino acid changes were detected in Simmental, Charolais and Galloway cattle but these polymorphisms were not associated with diluted shades of black or red, nor with the dun coat colour of Galloway cattle or the taupe brown colour of Braunvieh and Brown Swiss cattle. However, in Dexter cattle all 25 cattle with a dun brown coat colour were homozygous for a H424Y change. One Dexter that was also homozygous Y434 was red because of an "E+/E+" genotype at MC1R which lead to the production of only phaeomelanin. None of the 70 remaining black or red Dexter cattle were homozygous for Y434. This tyrosine mutation was not found in any of the 121 cattle of other breeds that were examined.     

A missense mutation in the gene for melanocyte-stimulating hormone receptor (MCIR) is associated with the chestnut coat color in horses

The melanocyte-stimulating hormone receptor gene (MC1R) is the major candidate gene for the chestnut coat color in horses since it is assumed to be controlled by an allele at the extension locus. MC1R sequences were PCR amplified from chestnut (e/e) and non-chestnut (E/−) horses. A single-strand conformation polymorphism was found that showed a complete association to the chestnut coat color among 144 horses representing 12 breeds. Sequence analysis revealed a single missense mutation (83Ser → Phe) in the MC1R allele associated with the chestnut color. The substitution occurs in the second transmembrane region, which apparently plays a key role in the molecule since substitutions associated with coat color variants in mice and cattle as well as red hair and fair skin in humans are found in this part of the molecule. We propose that the now reported mutation is likely to be the causative mutation for the chestnut coat color. The polymorphism can be detected with a simple PCR-RFLP test, since the mutation creates a TaqI restriction site in the chestnut allele. Received: 20 May 1996 / Accepted: 31 July 1996     

犬MC1R基因T105A基因座多态性及 与毛色性状相关性的研究The

为了检测犬MC1R基因T105A基因座的多态性,并分析该多态性与犬毛色表型的相关性,抽取111只外科手术学实验用杂种犬血液并提取DNA,记录毛色表型。采用PCR-RFLP技术,对MC1R基因T105A基因座进行基因多态性分析,并对该基因座DNA进行克隆测序;用二元变量相关分析的统计学方法分析基因座多态性与毛色性状之间的相关性。经PCR-RFLP分析结果表明,T105A基因座序列具有多态性,表现为A、B二个等位基因和AA、AB及BB 3种基因型。A、B等位基因频率分别为72.97%和27.03%,基因杂合度（H）为0.39。基因型AA频率为55.86%,BB为9.91%,AB为34.23%。对T105A多态性片段DNA克隆测序后发现,MC1R基因在编码第105位氨基酸的密码子第一个碱基存在由G到A的单碱基突变,该突变导致第105位氨基酸发生由丙氨酸向苏氨酸的改变。统计分析结果表明MC1R基因T105A基因座的多态性与毛色性状不存在显著的相关性,这可能是由于外科手术学实验用犬是杂种犬,其遗传背景不同所致,尚须在纯种犬群体中进一步研究MC1R基因对毛色的影响。 Abstract: In order to detect the polymorphism of T105A in MC1R gene in dogs and to analyze the relationship between the genetic polymorphisms and phenotypes of dog coat color, the blood samples of 111 cross-breed dogs were taken and their genomic DNAs were extracted. The phenotypes of dog coat color were recorded. The T105A locus of MC1R gene in the canine was detected through the technology of PCR-RFLP. Furthermore, the polymorphic fragments at T105A were sequenced. The relationships between the polymorphism of T105A and coat color trait were analyzed by the statistical methods of bivarate correlation analysis. By the method of PCR-RFLP, the T105A polymorphism was found with two alleles A and B and three genotypes AA, AB and BB. The frequencies of two alleles were 72.97% and 27.03%, respectively. The heterozygosity of T105A locus was 0.39. The frequencies of three genotypes were 55.86%, 34.23% and 9.91%, respectively. According to the results of sequencing, one base change from G to A at the position 105 was found at T105A locus and it altered amino acid at the position 105 from alanine to threonine. According to the statistical analysis, no significant association between the polymorphism of MC1R gene and the coat color was found and the result may be due to the differences of genetic background. Further research on MC1R gene should be done in pure breed dogs.     

Association of an Agouti allele with fawn or sable coat color in domestic dogs

The type of pigment synthesized in mammalian hair, yellow–red pheomelanin or black–brown eumelanin, depends on the interaction between Agouti protein and the Melanocortin 1 receptor. Although the genetics of pigmentation is broadly conserved across most mammalian species, pigment type-switching in domestic dogs is unusual because a yellow–tan coat with variable amounts of dark hair is thought to be caused by an allele of the Agouti locus referred to as fawn or sable (a^y). In a large survey covering thirty seven breeds, we identified an Agouti allele with two 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. In an additional 33 dogs (14 breeds) with a eumelanic coat, 8 (German Shepherd Dogs, Groenendaels, Schipperkes, or Shetland Sheepdogs) were homozygous for a previously reported mutation, non-agouti R96C; the remainder are likely to have carried dominant black, which is independent of and epistatic to Agouti. This work resolves some of the complexity in dog coat color genetics and provides diagnostic opportunities and practical guidelines for breeders.     

Differential expression of MC1R gene in Liaoning Cashmere goats with different coat colors

Melanocyte stimulating hormone receptor (MC1R) has been known as a regulator of eumelanin and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many vertebrates; however, there are no research reports about the differentially expression of MC1R gene and its coding protein in Cashmere goats with different coat color. We examined the presence of MC1R distribution and MC1R protein and gene expression in the white Cashmere goats and black Cashmere goats, respectively; q-PCR, Western blot and immunhistochemical analysis showed that the expression of the MC1R gene in the black Cashmere goats was 3.39 fold more than the white ones (p?<?0.01), and Cashmere goats with black genotype had significantly higher (2.03, p?<?0.01) MC1R protein expression than white genotype in the all investigated samples. Moreover, all Cashmere goats with different coat color available for immunhistochemical analysis showed either lower (white Cashmere goats) or higher (black Cashmere goats) expression of the MC1R protein; these findings suggested that it had a relationship between the MC1R and the coat color of Cashmere goats. That could lay the foundation for the further research of the MC1R and coat color controllability regulation of the Cashmere goats.     

Investigation of the role of the agouti signaling protein gene (ASIP) in coat color evolution in primates

We investigated variation in the gene encoding the agouti signaling protein (ASIP) in relation to coat color evolution in primates. We found little evidence that mutations in the coding region of ASIP have been involved in color changes among closely related primate species. Among many closely related species with differing coat color, the coding region of ASIP was identical. In two cases (Sulawesi macaque and black lion tamarin) where species with almost completely black coat color had derived point mutations in exon 4 of the ASIP coding sequence, the same mutations did not alter coloration in other mammals and so probably do not affect ASIP function. Evolutionary reconstructions of two key phenotypes that are typically related to ASIP function—transverse phaeomelanin bands on hairs and pale ventral coloration—showed that these usually evolved concurrently, suggesting that loci acting downstream of ASIP may be involved. Analysis of dN/dS ratios revealed a likely change in functional constraint on ASIP following loss of agouti-banded hairs + pale ventral coloration, particularly in catarrhine primates (humans, apes, and Old World monkeys). Together with previous results on a lack of association of coat color with MC1R variation, these results suggest that other loci probably have an important role in primate coat color evolution.     

Characterization of the dog <Emphasis Type="Italic">Agouti</Emphasis> gene and a <Emphasis Type="Italic">nonagouti</Emphasis>mutation in German Shepherd Dogs

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.Genbank accession numbers are AC092250 (bacterial artificial chromosome clone RP81-20712) and AY714374 (Doberman Pinscher Agouti cDNA).     

A customized pigmentation SNP array identifies a novel SNP associated with melanoma predisposition in the SLC45A2 gene

As the incidence of Malignant Melanoma (MM) reflects an interaction between skin colour and UV exposure, variations in genes implicated in pigmentation and tanning response to UV may be associated with susceptibility to MM. In this study, 363 SNPs in 65 gene regions belonging to the pigmentation pathway have been successfully genotyped using a SNP array. Five hundred and ninety MM cases and 507 controls were analyzed in a discovery phase I. Ten candidate SNPs based on a p-value threshold of 0.01 were identified. Two of them, rs35414 (SLC45A2) and rs2069398 (SILV/CKD2), were statistically significant after conservative Bonferroni correction. The best six SNPs were further tested in an independent Spanish series (624 MM cases and 789 controls). A novel SNP located on the SLC45A2 gene (rs35414) was found to be significantly associated with melanoma in both phase I and phase II (P<0.0001). None of the other five SNPs were replicated in this second phase of the study. However, three SNPs in TYR, SILV/CDK2 and ADAMTS20 genes (rs17793678, rs2069398 and rs1510521 respectively) had an overall p-value<0.05 when considering the whole DNA collection (1214 MM cases and 1296 controls). Both the SLC45A2 and the SILV/CDK2 variants behave as protective alleles, while the TYR and ADAMTS20 variants seem to function as risk alleles. Cumulative effects were detected when these four variants were considered together. Furthermore, individuals carrying two or more mutations in MC1R, a well-known low penetrance melanoma-predisposing gene, had a decreased MM risk if concurrently bearing the SLC45A2 protective variant. To our knowledge, this is the largest study on Spanish sporadic MM cases to date.     

Genome-Wide Association Analysis Identifies a Mutation in the Thiamine Transporter 2 (SLC19A3) Gene Associated with Alaskan Husky Encephalopathy

Alaskan Husky Encephalopathy (AHE) has been previously proposed as a mitochondrial encephalopathy based on neuropathological similarities with human Leigh Syndrome (LS). We studied 11 Alaskan Husky dogs with AHE, but found no abnormalities in respiratory chain enzyme activities in muscle and liver, or mutations in mitochondrial or nuclear genes that cause LS in people. A genome wide association study was performed using eight of the affected dogs and 20 related but unaffected control AHs using the Illumina canine HD array. SLC19A3 was identified as a positional candidate gene. This gene controls the uptake of thiamine in the CNS via expression of the thiamine transporter protein THTR2. Dogs have two copies of this gene located within the candidate interval (SLC19A3.2 – 43.36–43.38 Mb and SLC19A3.1 – 43.411–43.419 Mb) on chromosome 25. Expression analysis in a normal dog revealed that one of the paralogs, SLC19A3.1, was expressed in the brain and spinal cord while the other was not. Subsequent exon sequencing of SLC19A3.1 revealed a 4bp insertion and SNP in the second exon that is predicted to result in a functional protein truncation of 279 amino acids (c.624 insTTGC, c.625 C>A). All dogs with AHE were homozygous for this mutation, 15/41 healthy AH control dogs were heterozygous carriers while 26/41 normal healthy AH dogs were wild type. Furthermore, this mutation was not detected in another 187 dogs of different breeds. These results suggest that this mutation in SLC19A3.1, encoding a thiamine transporter protein, plays a critical role in the pathogenesis of AHE.     

Genetic mapping of canine multiple system degeneration and ectodermal dysplasia loci

We characterized a movement disorder of Chinese Crested dogs clinically and pathologically indistinguishable from canine multiple system degeneration (CMSD) previously recognized in Kerry Blue Terriers. This fatal disease segregated as an autosomal recessive in a 51-dog pedigree of both breeds and their crosses. The occurrence of affected dogs among first-generation crosses demonstrated that the mutations causing multiple system degeneration in these breeds are allelic. The CMSD locus maps to CFA1 (LOD > 18) and haplotype analysis narrowed the CFA1 target region to a 15-Mb segment that contains orthologs of genes on HSA6, including PARK2, the gene for the ubiquitin ligase parkin. Mutations in human PARK2 cause the most common form of familial Parkinson's disease, autosomal recessive juvenile parkinsonism, which has clinical and pathological similarities to canine multiple system degeneration. A second phenotype, canine ectodermal dysplasia (CED), segregated in the pedigree as an autosomal dominant with homozygous lethality. Dogs with ectodermal dysplasia have a sparse hair coat and abnormal dentition that is characteristic of the "hairless" variety of Chinese Cresteds. CED mapped to a region of CFA17 (LOD > 14) containing orthologs from HSA2. EDAR, the gene for the ectodysplasin A1 receptor, occurs on HSA2 but was excluded as the cause of canine ectodermal dysplasia.     

Factors affecting gestation duration in the bitch

A retrospective analysis was performed to determine the effects of age, breed, parity, and litter size on the duration of gestation in the bitch. Bitches at two locations were monitored from breeding to whelping. A total of 764 litters whelped from 308 bitches (36 large hounds, 34 Golden Retrievers, 23 German Shepherd Dogs (GSD), and 215 Labrador Retrievers). By breed, the number of whelpings was 152, 72, 58, and 482 for the hounds, Golden Retrievers, German Shepherd Dogs, and Labrador Retrievers, respectively. Whelping was predicted to be 57 d from the first day of cytologic diestrus in the hounds or 65 d from the initial progesterone rise in the other breeds. The average gestation duration (calculated as 8 d prior to Day 1 of cytologic diestrus in hounds or measured from the initial progesterone rise in other breeds) by breed (days +/- S.D.) was 66.0 +/- 2.8, 64.7 +/- 1.5, 63.6 +/- 2.1, and 62.9 +/- 1.3 for the hounds, Golden Retrievers, German Shepherd Dogs, and Labrador Retrievers, respectively. The relationship of age, breed, parity, and litter size with the difference in gestation duration was evaluated using log linear modeling. Age or parity had no effect on gestation duration. Compared to Labrador Retrievers, the German Shepherd Dogs, Golden Retrievers and hounds were more likely to have a longer gestation duration; three, four and nearly eight times as likely, respectively. Bitches whelping four or fewer pups were significantly more likely to have a longer gestation duration than those whelping five or more pups; the prolongation averaging 1 d.     

马毛色遗传的分子基础与应用

毛色不仅是马品种和个体识别的重要依据,而且还可以作为某些疾病筛查的有力工具和手段。马的毛色主要由黑色素细胞产生的真黑素和褐黑素两种黑色素的分布及比例所决定,许多基因对黑色素的产生和分布的调控起着重要的作用,各基因相互间共同作用最终形成各种单毛色和复毛色,这些基因主要包括MC1R、ASIP、KIT、TYRP和EDNRB。另外STX17、MATP和PMEL17也在马毛色形成过程具有重要的作用,同时还发现个别毛色基因与黑色素瘤疾病有关。文章对近年来马主要毛色候选基因的作用机理、DNA序列多态性与毛色性状及黑色素瘤疾病的关系等研究进行了详细的阐述,为今后马匹育种工作和疾病防治提供重要理论依据。