SLC45A2: a novel malignant melanoma-associated gene

Human pigmentation appears to be one of the strongest risk factors for malignant melanoma (MM). In humans, there is a long list of genes known to be involved in rare pigmentary disorders such as albinism. These genes explain most of the variation in pigmentation phenotypes seen in human populations, and they do this by regulating the level of synthesis, chemical composition, packaging, and distribution of melanin. This Spanish case-control study included 131 consecutive melanoma patients and 245 control subjects frequency-matched for sex and age. A total of 23 SNPs in six candidate genes (ASP, OCA2, TYR, TYRP1, SILV, and SLC45A) belonging to the pigmentation pathway were genotyped. We found that the variant allele of c.1122C>G, p.Phe374Leu (NCBI dbSNP rs16891982) in SLC45A2 (membrane associated transporter previously known as MATP) was associated with protection from MM (OR, 0.41; 95% CI, 0.24-0.70; P=0.008 after adjustment for multiple testing). This association was validated by the consistent link observed with dark hair, dark skin, dark eye color, and the presence of solar lentigins and childhood sunburns. This is the first time SLC45A2 has been described as a melanoma susceptibility gene in a light-skinned population.     

Variants of the MATP/SLC45A2 gene are protective for melanoma in the French population

In this study, we investigated whether variants in three key pigmentation genes-MC1R, MATP/SLC45A2, and OCA2--were involved in melanoma predisposition. A cohort comprising 1,019 melanoma patients (MelanCohort) and 1,466 Caucasian controls without skin cancers were studied. A total of 10 polymorphisms, including five functional MC1R alleles (p.Asp84Glu, p.Arg142His, p.Arg151Cys, p.Arg160Trp, and p.Asp294His), two nonsynonymous SLC45A2 variants (p.Phe374Leu and p.Glu272Lys), and three intronic OCA2 variants previously shown to be strongly associated with eye color (rs7495174 T>C, rs4778241 G>T, and rs4778138 T>C) were genotyped. As expected, MC1R variants were closely associated with melanoma risk (P value <2.20.10(-16); odds ratio [OR]=2.29 [95% confidence interval, CI=1.85-2.82 and OR=3.3 [95% CI=2.00-5.45], for the presence of one or two variants, respectively). Interestingly, the SLC45A2 variant p.Phe374Leu was significantly and strongly protective for melanoma (P-value=2.12.10(-15); OR=0.35 [95% CI=0.26-0.46] and OR=0.32 [95% CI=0.24-0.43], considering the genotypes Phe/Leu and Leu/Leu, respectively). MC1R and SLC45A2 variants had additive effects on melanoma risk, and after adjusting for pigmentation characteristics, the risk was persistent, even though both genes had a strong impact on pigmentation. Future studies may show whether genetic information could provide a useful complement to physical examination in predicting melanoma risk.     

Interactive effects of MC1R and OCA2 on melanoma risk phenotypes

The relationships between MC1R gene variants and red hair, skin reflectance, degree of freckling and nevus count were investigated in 2331 adolescent twins, their sibs and parents in 645 twin families. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. Of nine MC1R variant alleles assayed, four common alleles were strongly associated with red hair and fair skin (Asp84Glu, Arg151Cys, Arg160Trp and Asp294His), with a further three alleles having low penetrance (Val60Leu, Val92Met and Arg163Gln). These variants were separately combined for the purposes of this analysis and designated as strong 'R' (OR=63.3; 95% CI 31.9-139.6) and weak 'r ' (OR=5.1; 95% CI 2.5-11.3) red hair alleles. Red-haired individuals are predominantly seen in the R/R and R/r groups with 67.1 and 10.8%, respectively. To assess the interaction of the brown eye color gene OCA2 on the phenotypic effects of variant MC1R alleles we included eye color as a covariate, and also genotyped two OCA2 SNPs (Arg305Trp and Arg419Gln), which were confirmed as modifying eye color. MC1R genotype effects on constitutive skin color, freckling and mole count were modified by eye color, but not genotype for these two OCA2 SNPs. This is probably due to the association of these OCA2 SNPs with brown/green not blue eye color. Amongst individuals with a R/R genotype (but not R/r), those who also had brown eyes had a mole count twice that of those with blue eyes. This suggests that other OCA2 polymorphisms influence mole count and remain to be described.     

Interactions Between HERC2, OCA2 and MC1R May Influence Human Pigmentation Phenotype

Human pigmentation is a polygenic trait which may be shaped by different kinds of gene–gene interactions. Recent studies have revealed that interactive effects between HERC2 and OCA2 may be responsible for blue eye colour determination in humans. Here we performed a population association study, examining important polymorphisms within the HERC2 and OCA2 genes. Furthermore, pooling these results with genotyping data for MC1R , ASIP and SLC45A2 obtained for the same population sample we also analysed potential genetic interactions affecting variation in eye, hair and skin colour. Our results confirmed the association of HERC2 rs12913832 with eye colour and showed that this SNP is also significantly associated with skin and hair colouration. It is also concluded that OCA2 rs1800407 is independently associated with eye colour. Finally, using various approaches we were able to show that there is an interaction between MC1R and HERC2 in determination of skin and hair colour in the studied population sample.     

Comprehensive evaluation of allele frequency differences of MC1R variants across populations

The melanocortin 1 receptor (MC1R), a member of the G protein-coupled receptors superfamily, mediates the response to melanocortins and is currently the best-described contributor to normal pigment variation in humans. A remarkably large number of natural polymorphisms, or variants, of the MC1R gene have been identified in different populations. Some of these variants have been associated with specific hair and skin color phenotypes, the presence of freckling, and melanoma and nonmelanoma skin cancer risk. Interestingly, some MC1R variants have been associated with skin cancer beyond their effects on pigmentation. Although the red hair color variants (RHC variants) have been associated with skin cancer risk in the Celtic population, studies in darkly-pigmented Caucasian populations have demonstrated the importance of non-RHC MC1R variants on skin cancer risk as well. We have reviewed and compared allele frequency differences of MC1R variants across geographic regions. We observed large differences in the distribution of variants across populations, with a prominent difference between lightly and darkly-pigmented individuals. Moreover, among Caucasian groups, there were seven variants (p.V60L, p.V92M, p.D84E, p.R151C, p.R160W, p.R163Q, and p.D294H) with significantly different allele frequencies. Exploring differences in allele frequencies of MC1R variants across populations with varying pigmentation and differing skin cancer risk may improve our understanding of the complex relationship between MC1R, pigmentation, and carcinogenesis.     

Genome-wide association study identifies novel loci predisposing to cutaneous melanoma

We performed a multistage genome-wide association study of melanoma. In a discovery cohort of 1804 melanoma cases and 1026 controls, we identified loci at chromosomes 15q13.1 (HERC2/OCA2 region) and 16q24.3 (MC1R) regions that reached genome-wide significance within this study and also found strong evidence for genetic effects on susceptibility to melanoma from markers on chromosome 9p21.3 in the p16/ARF region and on chromosome 1q21.3 (ARNT/LASS2/ANXA9 region). The most significant single-nucleotide polymorphisms (SNPs) in the 15q13.1 locus (rs1129038 and rs12913832) lie within a genomic region that has profound effects on eye and skin color; notably, 50% of variability in eye color is associated with variation in the SNP rs12913832. Because eye and skin colors vary across European populations, we further evaluated the associations of the significant SNPs after carefully adjusting for European substructure. We also evaluated the top 10 most significant SNPs by using data from three other genome-wide scans. Additional in silico data provided replication of the findings from the most significant region on chromosome 1q21.3 rs7412746 (P = 6 × 10(-10)). Together, these data identified several candidate genes for additional studies to identify causal variants predisposing to increased risk for developing melanoma.     

The melanocortin-1-receptor gene is the major freckle gene

Ephelides and solar lentigines are different types of pigmented skin lesions. Ephelides appear early in childhood and are associated with fair skin type and red hair. Solar lentigines appear with increasing age and are a sign of photodamage. Both lesions are strong risk indicators for melanoma and non-melanoma skin cancer. Melanocortin-1-receptor (MC1R) gene variants are also associated with fair skin, red hair and melanoma and non-melanoma skin cancer. The purpose of this study was to investigate the relationship between MC1R gene variants, ephelides and solar lentigines. In a large case-control study, patients with melanoma and non-melanoma skin cancer and subjects without a history of skin cancer were studied. In all participants, the presence of ephelides in childhood and solar lentigines by physical examination was assessed according to strict definitions. The entire coding sequence of the MC1R gene was analyzed by single-strand conformation polymorphism analysis followed by sequence analyses. Carriers of one or two MC1R gene variants had a 3- and 11-fold increased risk of developing ephelides, respectively (both P < 0.0001), whereas the risk of developing severe solar lentigines was increased 1.5- and 2-fold (P = 0.035 and P < 0.0001), respectively. These associations were independent of skin type and hair color, and were comparable in patients with and without a history of skin cancer. The population attributable risk for ephelides to MC1R gene variants was 60%, i.e. 60% of the ephelides in the population was caused by MC1R gene variants. A dosage effect was found between the degree of ephelides and the number of MC1R gene variants. As nearly all individuals with ephelides were carriers of at least one MC1R gene variant, our data suggest that MC1R gene variants are necessary to develop ephelides. The results of the study also suggest that MC1R gene variants play a role, although less important, in the development of solar lentigines.     

Genome-wide association study identifies novel alleles associated with risk of cutaneous basal cell carcinoma and squamous cell carcinoma

We conducted a genome-wide association study on cutaneous basal cell carcinoma (BCC) among 2045 cases and 6013 controls of European ancestry, with follow-up replication in 1426 cases and 4845 controls. A non-synonymous SNP in the MC1R gene (rs1805007 encoding Arg151Cys substitution), a previously well-documented pigmentation gene, showed the strongest association with BCC risk in the discovery set (rs1805007[T]: OR (95% CI) for combined discovery set and replication set [1.55 (1.45-1.66); P= 4.3 × 10(-17)]. We identified that an SNP rs12210050 at 6p25 near the EXOC2 gene was associated with an increased risk of BCC [rs12210050[T]: combined OR (95% CI), 1.24 (1.17-1.31); P= 9.9 × 10(-10)]. In the locus on 13q32 near the UBAC2 gene encoding ubiquitin-associated domain-containing protein 2, we also identified a variant conferring susceptibility to BCC [rs7335046 [G]; combined OR (95% CI), 1.26 (1.18-1.34); P= 2.9 × 10(-8)]. We further evaluated the associations of these two novel SNPs (rs12210050 and rs7335046) with squamous cell carcinoma (SCC) risk as well as melanoma risk. We found that both variants, rs12210050[T] [OR (95% CI), 1.35 (1.16-1.57); P= 7.6 × 10(-5)] and rs7335046 [G] [OR (95% CI), 1.21 (1.02-1.44); P= 0.03], were associated with an increased risk of SCC. These two variants were not associated with melanoma risk. We conclude that 6p25 and 13q32 are novel loci conferring susceptibility to non-melanoma skin cancer.     

Novel MC1R variants in Ligurian melanoma patients and controls

Several variant forms of the melanocortin-1 receptor gene (MC1R) have been associated with red hair, fair skin and an increased risk for melanoma. Their involvement in melanoma susceptibility is apparently linked both to skin sensitivity and to non-pigmentary pathways. We investigated the frequency of the MC1R variants in the Italian region of Liguria, where the occurrence and penetrance of melanoma are low and primary susceptibility is characterized by prevalence of the CDKN2A c.301G>T [p.G101W] founder mutation. Additionally, we attempted to establish the frequency of the red hair/fair skin phenotype in our region. As predicted by anecdotal evidence, the frequency of red hair/phototype I was very low (0.7%). Screening of 17 red-haired individuals and their red-haired relatives, 207 controls and 214 melanoma patients unselected for hair color but all of Ligurian descent, led to the detection of 8 novel substitutions (c.133T>C [p.F45L], c.248C>T [p.S83L], c.332C>T [p. A111V], c.479G>A [p.R160Q], c.637C>T [p.R213W], c.793G>A [p. V265I], c.923C>T [p. T308M], c.943T>C [p.C315R]), 1 novel deletion (c.520_523delGTC [p.V174del]) and 3 novel synonymous variants (c.366G>C [p. V122V], c.684G>A [p. Q228Q], c.726C>T [p.T241T]). Preliminary genotype/phenotype correlation seems to indicate that other genes involved in the regulation of human pigmentation may mask the recessive action of high-penetrance MC1R alleles, thus determining the low frequency of at-risk phototypes and of incidence and/or penetrance of melanoma in Liguria.     

Receptor function, dominant negative activity and phenotype correlations for MC1R variant alleles

The human melanocortin-1 receptor (MC1R) is a G-protein coupled receptor involved in the regulation of pigmentation. Several MC1R variant alleles are associated with red hair, fair skin and increased skin cancer risk. We have performed a systematic functional analysis of nine common MC1R variants and correlated these results with the strength of the genetic association of each variant allele with pigmentation phenotypes. In vitro expression studies revealed that variant receptors with reduced cell surface expression, including V60L, D84E, R151C, I155T, R160W and R163Q, showed a corresponding impairment in cAMP coupling. The R142H and D294H variants demonstrated normal cell surface expression, but had reduced functional responses, indicating that altered G-protein coupling may be responsible for this loss of function. The V92M variant cAMP activation was equal to or higher than that for wild-type MC1R. In co-expression studies, the D84E, R151C, I155T and R160W variants showed a dominant negative effect on wild-type receptor cell surface expression, which was reflected in a decreased ability to elevate intracellular cAMP levels. The D294H variant also demonstrated a dominant negative effect on wild-type MC1R cAMP signalling, but had no effect on wild-type surface expression. Importantly, comparison of the in vitro receptor characteristics with skin and hair colour data of individuals both homozygous and heterozygous for MC1R variant alleles revealed parallels between variant MC1R cell surface expression, functional ability, dominant negative activity and their effects on human pigmentation. These findings show the first direct correlations between variant MC1R biochemical properties and pigmentation phenotype.     

The Asp84Glu variant of the melanocortin 1 receptor (MC1R) is associated with melanoma

Melanocyte stimulating hormone (MSH) plays an important role in determining the cutaneous response to ultraviolet radiation and may also influence melanoma progression. We have previously shown that variants of the melanocortin receptor present on melanocytes, MC1R, are associated with sun sensitivity and red hair in a UK population and therefore now consider the gene as a candidate for melanoma susceptibility. We have compared the frequency of known MC1R variants in the second and seventh transmembrane domains in 43 melanoma cases and 44 controls. MC1R variants were more common in cases than controls (chi 2 = 6.75, 1 d.f.; P = 0.0094) with a relative risk to carriers of variant alleles compared with normal homozygotes of 3.91 (95% c.l.: 1.48-10.35), and a population risk attributable to carriers of 34.6% (95% c.i. 10.7-52.1%). The Asp84Glu variant was only present in melanoma cases and appears to be of particular significance. The contribution of variant MC1R alleles was largely independent of skin type. Variants of the MC1R gene are likely to be causally associated with the development of melanoma.      

Signatures of Positive Selection in Genes Associated with Human Skin Pigmentation as Revealed from Analyses of Single Nucleotide Polymorphisms

Phenotypic variation between human populations in skin pigmentation correlates with latitude at the continental level. A large number of hypotheses involving genetic adaptation have been proposed to explain human variation in skin colour, but only limited genetic evidence for positive selection has been presented. To shed light on the evolutionary genetic history of human variation in skin colour we inspected 118 genes associated with skin pigmentation in the Perlegen dataset, studying single nucleotide polymorphisms (SNPs), and analyzed 55 genes in detail. We identified eight genes that are associated with the melanin pathway ( SLC45A2, OCA2, TYRP1, DCT, KITLG, EGFR, DRD2 and PPARD ) and presented significant differences in genetic variation between Europeans, Africans and Asians. In six of these genes we detected, by means of the EHH test, variability patterns that are compatible with the hypothesis of local positive selection in Europeans ( OCA2, TYRP1 and KITLG ) and in Asians ( OCA2, DCT, KITLG, EGFR and DRD2 ), whereas signals were scarce in Africans ( DCT, EGFR and DRD2 ). Furthermore, a statistically significant correlation between genotypic variation in four pigmentation candidate genes and phenotypic variation of skin colour in 51 worldwide human populations was revealed. Overall, our data also suggest that light skin colour is the derived state and is of independent origin in Europeans and Asians, whereas dark skin color seems of unique origin, reflecting the ancestral state in humans.     

Mutations in the MATP gene in five German patients affected by oculocutaneous albinism type 4

Oculocutaneous albinism (OCA) is caused by a deficiency of melanin synthesis and characterized by generalized hypopigmentation of skin, hair, and eyes. Due to the hypopigmentation of the retinal pigment epithelium, OCA is usually associated with congenital visual impairment, in addition to an increased risk of skin cancer. OCA is a genetically heterogeneous disease with distinct types resulting from mutations in different genes involved in the pathway which results in pigmentation. OCA1 is associated with mutations in the TYR gene encoding tyrosinase. OCA2 results from mutations in the P gene encoding the P protein and is the most common form of OCA. OCA3, also known as rufous/red albinism, is caused by mutations in the TYRP1 gene, which encodes the tyrosinase-related protein 1. Recently, OCA4 was described as a new form of OCA in a single patient with a splice site mutation in the MATP gene (or AIM1), the human ortholog of the murine underwhite gene. The similarity of MATP to transporter proteins suggests its involvement in transport functions, although its actual substrate is still unclear. We screened 176 German patients with albinism for mutations within the MATP gene and identified five individuals with OCA4. In this first report on West European patients, we describe 10 so far unpublished mutations, as well as two intronic variations, in addition to two known polymorphisms.     

Four novel variants in MC1R in red-haired South African individuals of European descent: S83P, Y152X, A171D, P256S

Skin, hair and eye pigmentation is a polygenic multifactorial trait determined by the cumulative effects of multiple genetic variants and environmental factors. MC1R is one of the genes involved in pigmentation, and has been implicated in the red hair and pale skin trait in human Caucasoid individuals. This study was undertaken to investigate variants in the MC1R gene in Caucasoid individuals in South Africa, who are of European decent. Seven unrelated individuals were studied, all of whom were found to be either homozygous for a single mutation or compound heterozygous for two different mutations. We report four novel MC1R missense mutations: S83P, Y152X, A171D and P256S. This study supports the view that two mutations are necessary, but not necessarily sufficient, to give rise to the red hair and pale skin phenotype.     

MC1R, ASIP, TYR, and TYRP1 gene variants in a population-based series of multiple primary melanomas

Allelic variants of the low-penetrance melanoma gene MC1R increase the risk of both melanoma and non-melanoma skin cancer. Common variants of the genes ASIP, TYR, and TYRP1, which regulate the melanogenic pathway, have also been shown to associate with melanoma. In this population-based study, we investigated SNPs of MC1R, ASIP, TYR, and TYRP1 as risk factors for development of multiple primary melanomas (MPM) in 388 Norwegian cases. The MPM patients had a significantly higher likelihood of carrying any MC1R variant than the control group of 420 blood donors [86.8 vs. 78.3%, OR = 1.73, and confidence intervals (CI) 1.18-2.52]. When MC1R variants were analyzed individually, Asp84Glu and Arg151Cys were significantly more frequent among the MPM cases than among the controls (OR = 5.77, CI 1.97-16.90, and OR = 1.80, CI 1.36-2.37, respectively). In addition, there was an allele dose-dependent increase in MPM risk for carriers of red hair color (RHC) MC1R variants. The AH haplotype of ASIP was also a significant risk factor for MPM development (OR = 1.72 and CI 1.12-2.49), whereas no association was observed for previously reported risk variants of the TYR and TYRP1 genes. In summary, by using a population-based material of high-risk melanoma cases, we demonstrate a significant effect of both MC1R RHC variants and an ASIP haplotype, but could not replicate an association with postulated risk SNPs of TYR and TYRP1.     

Identification of four novel melanocortin 1 receptor (MC1R) gene variants in a Mediterranean population

The melanocortin 1 receptor (MC1R) gene is a major determinant of human pigmentation and specific allelic variants have been associated with red hair and sun sensitive skin types as well as increased skin cancer risk in Caucasian individuals. We screened for allelic variants the entire MC1R coding region of 100 unrelated individuals sampled from an Italian population who has darker pigmentary traits than populations analyzed to date. Twenty MC1R variants were identified, eighteen located at non-synonymous sites and two at synonymous sites. We report four novel MC1R allelic variants: C35Y (g.104G>A), V38M (g.112G>A), L44V (g.130C>G) and I120T (g.359T>C).     

Assessment of multiple displacement amplification for polymorphism discovery and haplotype determination at a highly polymorphic locus, MC1R

The identification of common genetic variants such as single nucleotide polymorphisms (SNPs) in the human genome has become central in human population genetics and evolution studies, as well as in the study of the genetic basis of complex traits and diseases. Crucial for the accurate identification of genetic variants is the availability of high quality genomic DNA (gDNA). Since popular sources of gDNA (buccal cells, lymphocytes, hair bulb) often do not yield sufficient quantities of DNA for molecular genetic applications, whole genome amplification methods have recently been introduced to generate a renewable source of double-stranded linear DNA. Here, we assess the fidelity of one method, multiple displacement amplification (MDA), which utilizes bacteriophage Phi29 DNA polymerase to generate amplified DNA from an original source of gDNA, in a representative SNP discovery and genetic association study at the melanocortin 1 receptor (MC1R) locus, a highly polymorphic gene in humans involved in skin and hair pigmentation. We observed that MDA has high fidelity for novel SNP discovery and can be a valuable tool in generating a potentially indefinite source of DNA. However, we observed an allele amplification bias that causes genotype miscalls at heterozygous sites. At loci with multiple polymorphic sites in linkage disequilibrium, such as at MC1R, this bias can create a significant number of heterozygote genotype errors that subsequently misrepresents haplotypes.     

Molecular genetic characterization of Congolese patients with oculocutaneous albinism

BackgroundOculocutaneous albinism (OCA) is an autosomal recessive genetic disorder associated with reduced or absent pigmentation in the skin, hair and eyes. OCA type 2 (OCA2) is the most common type in Sub-Saharan Africa, related to a recurrent 2.7 kb intragenic deletion. Genomic data from Congolese patients are lacking. We aimed to describe genetic causes of OCA2 in a cohort of Congolese persons with OCA, and explore possible genotype-phenotype correlations.MethodsA cross sectional study was conducted from January 2015 to December 2017 in Kinshasa, Democratic Republic of Congo (DRC). 165 Congolese unrelated families with non-syndromic OCA, identified through patients' associations, consented to participate to this study. All index cases were tested for the known 2.7 kb deletion involving the exon 7 of the OCA2 gene. Patients heterozygous for the deletion underwent Sanger sequencing of all exons and flanking sequences in the OCA2 gene. Family segregation was performed for candidate pathogenic variants.ResultsThe 2.7 kb deletion in the OCA2 gene was identified in 136/165 (82.4%) index cases, including 113 (68.5%) homozygotes and 23 (13.9%) heterozygotes. Sanger sequencing identified a pathogenic or likely pathogenic variant in the OCA2 gene in 12 out of 23 heterozygotes investigated (52.1%). Segregation analysis allowed us to locate the point mutation on the trans allele in the three patients from whom parental DNA was available.ConclusionThe OCA2 2.7 kb deletion is the major cause of non-syndromic OCA in Congolese patients recruited in this study, confirming results from other Sub-Saharan African populations. Several additional mutations were detected in OCA patient's heterozygote for the deletion, with to date no evidence for a second frequent founder mutation. The confirmation of a single mutation as the major cause will facilitate genetic counselling in this country.     

Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk

The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surface receptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.