Assessment of Polymorphic Variants in the Melanocortin-1 Receptor Gene with Cutaneous Pigmentation Using an Evolutionary Approach

The melanocortin-1 receptor gene (MC1R [MIM 155555]) encodes the melanocyte stimulating hormone receptor (MSHR), a membrane-bound protein central to pathways that signal production of eumelanin and pheomelanin. MC1R consists of a single exon that encodes a 317-amino acid protein (1). This gene is highly polymorphic with nearly 40 nucleotide variants reported to date (2). Specific MC1R variants have been associated with pigmentation phenotypes (3–9), risk for melanoma (10, 11) and non-melanoma skin cancers (12, 13), and can modify age of onset of melanoma of within melanoma-prone families (14, 15). Furthermore, studies have demonstrated that sequence variants in MC1R can alter protein function in vitro, expression of pigmentation in the mouse and primary human melanocyte cell lines, and in vivo response to UV radiation (16–19).

The large number of MC1R variants has made laboratory and statistical analyses challenging. To fully identify all MC1R variants, complete DNA sequence analysis may be required, which is cost- and labor-intensive. Furthermore, the highly polymorphic nature of MC1R can complicate statistical analyses in association studies. Analyses of MC1R variants in well-designed studies have focused on traditional multivariate analysis methods, but these methods are limited by the lack of power to detect significant associations involving numerous variants occurring at low to moderate frequencies. It is important to determine which of the many MC1R variants are most likely to have a relevant phenotypic effect, and which are phenotypically unimportant.

The purpose of our investigation was 2-fold. First, we report the frequency and distribution of MC1R variants and their associations with pigmentation characteristics in a sample of 179 U.S. Caucasian control subjects, a population that has not been previously studied in this context. Second, we used an evolutionary-based approach to determine which MC1R variants are likely to have functional significance, and we compared association analyses using different combinations of MC1R variants.

Between September 1997 and November 2000, we recruited 243 control subjects into a case-control study of genetic susceptibility to melanoma at the Pigmented Lesion Clinic of the Hospital of the University of Pennsylvania in Philadelphia, PA, who self-identified as white and not of Hispanic origin. Details of this study, participant recruitment, and data collection and measurement have been previously described (20). Each participant gave informed consent and the study's protocol was approved by the University's Institutional Review Board. Healthy control subjects were referred from enrolled clinic patients who had melanoma and/or dysplastic moles. The control subjects did not have a history of melanoma at the time of study enrollment. Of the 243 control subjects, 84.3% were partners/spouses of enrolled clinic patients, 12.4% were “friends,” and 3.3% were in-laws or adoptive parents. We report here on 179 (73.6%) control subjects for whom we have completed MC1R genotype data.

A brief self-administered questionnaire and physical examination was used to collect information on pigmentation characteristics and skin reaction to sun exposure. We asked about natural hair color as a teenager, skin reaction after initial exposure to strong sunlight of summer (‘acute sun exposure’), and skin reaction after long and repeated sun exposure (‘chronic sun exposure’). One research nurse (R.H.) completed a full skin examination, excluding the scalp and genitalia, for all study participants during which degree of freckling and eye color were recorded. The skin examination was not completed for 12 (6.7%) of the 179 controls.

Genomic DNA was extracted from collected buccal swab samples as previously described (21). Molecular techniques for PCR amplification of MC1R were modified from Box et al. (4). One of two PCR protocols was used to amplify the entire 951 nucleotide MC1R coding region. Briefly, the 50 μl total reaction volume contained 10 μl DNA template, 5 μm each forward 5′-GCAGCACCATGAACTAAGCA-3′ and reverse 5′-CAGGGTCACACAGGAACCA-3′ primers, 200 μm each dATP, dCTP, dGTP, dTTP, 5 μl DMSO, 5 μl 10× Herculase Taq polymerase buffer (containing MgCl₂; Stratagene, La Jolla, CA), 1.0 μl (5 units) Herculase Taq polymerase (Stratagene), and ddH₂O. Thermocycling occurred in a Robocycler 96 (Stratagene). DNA template was denatured at 94°C for 3 min and cycled 35 times through steps of denaturing at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 3 min; final DNA extension was at 72°C for 7 min. The original protocol used three sets of overlapping oligonucleotide primer pairs to amplify MC1R and was replaced with the more efficient single primer protocol. Regardless of amplification protocol, all PCR products were directly sequenced on an ABI Prism 377 or 3100 (Applied Biosystems, Foster City, CA) using Big Dye Terminators (Applied Biosystems) according to the manufacturer's specifications.

Data collected through the questionnaire on hair color were coded as red (including red and reddish-brown), blond, or dark (including light brown, medium brown, dark brown, gray, and black); on skin reaction to acute sun exposure as burn and blister, burn without blister, mild burn followed by a tan, or no burning (including no sunburn and no tan, tan with no sunburn, and no change in skin color); and on skin reaction to chronic sun exposure as no tan, light tan, or medium to dark tan. Data collected through clinical examination on eye color were coded as blue or gray, green or hazel, or dark (including light brown, dark brown, and black); and on freckling as extensive, moderate, mild, or none. In this paper, we refer to the phenotypes of lighter hair and eye color, reduced tendency to tan, tendency to burn, and freckling, as ‘fair pigmentation phenotypes.’

We used two approaches to evaluate MC1R genotype. First, we dichotomized variant MC1R alleles into risk categories based on previous reports. The high risk category indicates carriage of at least one “red hair color” (RHC) variant—R151C, R160W, or D294H—which has been associated with fair pigmentation phenotypes including red hair color and fair skin (3, 4). The low risk category indicates carriage of any other non-RHC missense (V60L, D84E, V92M, R142H, I155T, R163Q) or frameshift allele (86insA) found in our control subjects, most of which have not been strongly or consistently associated with fair pigmentation phenotypes. The referent category indicates homozygous carriage of the MC1R consensus sequence.

Second, we created risk categories for MC1R variants using an evolutionary approach, as implemented in the software program SIFT (22, 23). The underlying assumption for this analysis is that amino acid positions that are important to the native biological functioning of the protein should be conserved across the protein family and/or across evolutionary history. The SIFT algorithm compares sequence identity across related amino acid sequences to obtain probabilities for predicted tolerant amino acid substitutions. SIFT assigns a substitution probability (P_i) for each of the 20 amino acids at each position in the protein. For MC1R, 20 P_is are determined at each of 317 amino acid positions.

Two comparisons were made. First, we compared degree of MC1R sequence identity across species. Second, we compared degree of MC1R sequence identity across members of the melanocortin receptor family (MC1-, MC2-, MC3-, MC4-, and MR5-R) in humans. National Center for Biotechnology Information (NCBI) accession numbers and species for the melanocortin proteins used in SIFT analysis can be found in the Appendix. For each comparison, a P_i threshold of 0.05 was used to discriminate among amino acid substitutions predicted to be tolerant (P_i ≥ 0.05) and intolerant (P_i < 0.05). We categorized observed MC1R variants among control subjects as high risk if the specific substitution was predicted intolerant in both the MC1R cross-species or in the human MC cross-family comparison. Hence, the high risk category represents those MC1R variants that are more likely to adversely affect protein function, and hence contribute to fair pigmentation phenotypes. The low risk category includes variants that were predicted tolerant and thus have a lower likelihood of altering native protein function. The referent group indicates homozygous carriage of the MC1R consensus sequence. For each protein position, amino acid conservation was recognized if all tolerant amino acid substitutions were limited to those amino acids considered either favored or neutral substitutions for the consensus amino acid (24, 25).

To test for differences in the distribution of pigmentation characteristics and measures of skin reaction to sun exposure between controls with and without MC1R genotypes, we used χ² contingency table analyses and reported global P values. Differences among pigmentation and sun exposure measures according to classification of MC1R variants were tested using χ² contingency table analyses; however, P values determined by Fisher's Exact test are reported for those tables in which expected cell counts fell below 5. We combined exposure categories to create dichotomous variables for pigmentation and sun exposure variables (hair color: red, reddish-brown, blond versus light brown, medium brown, dark brown, gray, black; eye color: blue, gray, green, hazel versus light brown, dark brown, black; freckling: any versus none; skin reaction to acute sun: burn without tanning, regardless of blistering versus tanning or no effect, regardless of burning; and skin reaction to chronic sun: no or light tan versus medium or dark tan; freckling: any versus none). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to determine associations between outcome measures and MC1R variants. Mantel-Haenszel (M-H)-χ² was used to test for presence of a linear trend; two-sided M-H P values (P_M-H) are presented.

Demographic and pigmentation characteristics for the 179 control subjects are given in Table 1. We compared these measures to those of control subjects for whom MC1R genotyping was incomplete or not available (n = 64) to determine whether systematic differences existed between the groups. The groups were similar with respect to gender, hair color, eye color, acute sun exposure, and freckling. We found a difference in skin response to chronic sun exposure between the groups (χ² = 7.5, df = 2; P = 0.02), although no trend was apparent (χ²_M-H = 0.36; P = .55). Genotyped control subjects tended to be older (mean age = 50.7 years; SD = 13.1 years) than those not genotyped (mean age = 43.4 years; SD = 11.5 years; P < 0.0001).

Table 2 lists the MC1R variant alleles and their frequencies that were observed in our final sample of 358 alleles. One hundred thirty-five (75.4%) control subjects carried at least one MC1R variant, that is, any MC1R variant, including synonymous changes; 37 (20.7%) control subjects carried two variants, 17 (9.5%) carried three, and 1 (0.6%) carried four. Fig. 1 depicts the 55 homozygous and compound heterozygous variant combinations observed here. Overall, only 46.9% of alleles were identical to the consensus MC1R sequence. Counting only missense and frameshift variants and disregarding carriage of variants leading to silent changes, 129 (72.1%) persons carried at least one variant; 39 (21.8%) carried two.

The results from the two SIFT comparisons are shown in Fig. 2. For the observed MC1R variants in our sample, predicted intolerant (high risk) amino acid changes included D84E, R142H, R151C, I155T, R160W, and D294H. We included 86insA in the predicted intolerant category because this frameshift mutation results in a premature stop codon. The observed variants V60L, V92M, and R163Q were predicted tolerant (low risk) missense changes in these comparisons. There were regions of MC1R that were highly conserved in both comparisons. Overall, we observed greater conservation in amino acid substitutions in the MC1R cross-species comparison (65.6%) than in the human cross melanocortin receptor family comparison (53.0%). Of note, the first 15 amino acids were highly conserved in the human melanocortin receptor family, but were not conserved in MC1R across species. This result implies that this region is characteristic of human melanocortin genes but not MC1R in other species.

Table 3 presents associations of the three RHC alleles. Carriage of at least one RHC variant was consistently and statistically associated with fair pigmentation phenotypes. Carriage of at least one non-RHC variant, in the absence of a RHC variant, was suggestive of small increased risk for most exposure pigmentation measures, although the only association to reach statistical significance was for freckling. There was a significant trend with increasing number of RHC variants for hair color, eye color, and chronic sun exposure; these analyses were limited due to the small number of individuals (n = 6) who carried two RHC alleles. Results from analyses that used SIFT to predict variants more and less likely (intolerant and tolerant, respectively) to affect MC1R function are given in Table 4. Results were quite similar to those obtained from analyses of the three RHC alleles. Results from logistic regression models that adjusted for age and gender were not different from crude results for any pigmentation characteristic and for either method used to categorize MC1R variants.

We created a third high risk category that excluded carriage of the three RHC variants and indicated sole carriage of at least one of the three SIFT-identified predicted intolerant variants or 86insA. The results suggest that these four variants are also associated with fair pigmentation phenotypes although associations were not statistically significant. Only six individuals carried at least one of these alleles (see Table 4).

In an attempt to evaluate the incremental contribution of additional SIFT-identified predicted intolerant variants (plus ins86A), we informally compared goodness of fit measures of the models (Table 5). For all pigmentation phenotypes, models that used risk categories defined by SIFT analysis fit marginally better than did models that incorporated risk parameters based on published literature. Because these comparisons did not involve nested models, it was not possible to undertake formal hypothesis testing (e.g., via χ² analysis) to determine the difference between the model fit statistics.

This is the first study to describe the frequency and distribution of MC1R variants and their association with pigmentation characteristics derived from a sample of melanoma-free control subjects from the United States. Regardless of the method used to define MC1R allele risk categories that predict fair pigmentation phenotypes—either guided by published literature (RHC alleles) or based on evolutionary amino acid conservation (SIFT analysis)—we found statistically significant trends with MC1R high risk alleles for all measures of pigmentation phenotypes as well as significant associations with carriage number of risk MC1R alleles. All but one trend with increasing number of alleles were statistically significant despite the small number of observations of individuals who carry high risk variants. Even after controlling for the overall false discovery rate (i.e., the expected proportion of falsely rejected null hypotheses) (26), all trends remained statistically significant. Although this study is limited in scope by the small sample size, the selection of the control population and its potential biases, and the focus on MC1R to the exclusion of other genetic loci that likely also contribute to human pigmentation, it does provides a concrete example demonstrating the utility of using an evolutionary approach to guide the analysis of a complex genetic locus.

The allele frequencies of MC1R variants observed in our control sample are consistent with reports of MC1R variant from other populations of European ancestry (summarized in Ref. 2). This finding was unexpected because the U.S. Caucasian population is more ethnically heterogeneous than that of other Caucasian populations in which natural MC1R variation has been studied (e.g., Australia, Ireland, the Netherlands, and Scotland). In part, this may reflect that our control subjects were drawn from a clinic-based sample and hence may reflect a more homogenous Northern European population than the general U.S. population.

Our observed consensus allele frequency (46.9%) is likely to be slightly underestimated because we did not formally calculate linkage disequilibrium between the V92M and T314T variants. In our data, these two variants occur together greater than by chance alone (χ² = 73.7; P < 0.0001), and others have observed complete cosegregation of these variants (12). A direct comparison of the consensus allele frequency across populations is difficult because several studies genotyped only specific MC1R variants, rather than determining complete MC1R genotyping by direct sequencing. The true MC1R genotype distribution in those populations remains unknown. Still, our estimate was consistent with previous reports of Northern European-derived populations for which the frequency of the MC1R consensus allele was estimable (6, 12, 27). As expected, the consensus allele was more common among two populations of Southern European ancestry than in our sample, although the total number of alleles genotyped in these populations was small (27). It was not possible for us to further investigate the effect of ancestry patterns in our data because information on grandparental birthplace and/or ancestry was not collected.

Our results reinforce the hypothesis that MC1R contributes significantly to fair pigmentation phenotypes. Red hair color has been demonstrated to be strongly associated with the R151C, R160W, and D294H variants (11, 28). Our point estimate for this association was more conservative because it was necessary to combine subjects with red and blond hair into one category due to the low prevalence of redheads (5.6%) in our study sample. Analysis of the RHC variants comparing only subjects with red hair to those with dark hair (excluding blondes) resulted in a stronger point estimate akin to those previously reported [OR = 8.1, exact 95% CI (0.89, 383)]. MC1R variants have also been strongly associated with freckling, especially among persons reporting childhood freckling [OR = 10.8, 95% CI (7.0, 16.9) for carriage of two variants], although MC1R variants were also associated with solar lentigines [OR = 2.2, 95% CI (1.5, 3.1) for two variants] (7). In our data, freckling was the phenotypic characteristic most strongly associated with high risk MC1R variants. We did not collect information distinctly on childhood freckling versus solar lentigines, and thus our point estimate is likely be a weighted average between the two above estimates. MC1R variants have also been associated with the number of body sites on which freckling occurred (6). For measurement of skin reaction to sun exposure, many investigations use the Fitzpatrick scale that combines elements from skin reaction to acute and chronic sun exposures to score skin quality. Individuals with fair skin carry a greater proportion of variants, including RHC variants, and carriage was independent of ethnicity or hair color (11, 12). In contrast to two previous investigations (11, 12), we did observe an association between MC1R variants and eye color. This discrepancy could stem from differing methods used to measure eye color. In our study, all eye color measurements were obtained from a single trained research nurse who conducted all physical examinations.

A conservative definition of predicted tolerant and intolerant amino acid changes obtained through SIFT analysis was used for all MC1R-phenotype associations. We also analyzed our data using a more relaxed interpretation of tolerance obtained through SIFT analysis. The relaxed approach focused on non-conserved amino acid changes determined in the MC1R cross-species comparison alone, rather than non-conservative changes that occurred in both the cross-species and human MCR cross-family comparison. The resulting MC1R predicted intolerant high risk group included the addition of the V60L and V92M variants; the predicted tolerant category consisted only of the R163Q variant. Using these criteria, most phenotype associations were nonsignificant and were attenuated toward the null with odds ratios ranging from 1.9 to 2.1 (data not shown). An exception was the association with chronic sun exposure which despite being attenuated remained statistically significant [P_M-H = 0.0057; OR = 3.6, 95% CI (1.3, 9.8)]. While the number of observations was small, there was no apparent association with R163Q alone (data not shown). These analyses support the hypothesis that the V60L and V92M variants are not major contributors to fair pigmentation phenotypes despite experiments that have demonstrated that these variants are associated with decreased receptor function (29, 30).

Our results are limited by the protein sequences used for analyses and the assumptions inherent to SIFT algorithms that predict tolerant and intolerant amino acid substitutions. Because the protein environment for cytosolic, membrane, and extracellular proteins are different, we opted to determine amino acid conservation in MC1R based on favored or neutral amino acid substitutions among membrane proteins only (24, 25). Still, if amino acid conservation for MC1R is somehow distinct from that of other membrane proteins, our profile of MC1R conserved regions offered in Fig. 2 could be misleading. Despite the potential for overall misclassification of conserved regions, assignment of risk categories for codons corresponding to the nine missense variants observed in our data was straightforward, with the possible exception of codon 92. SIFT analysis indicated that substitution by methionine of the valine consensus amino acid was predicted tolerant in the comparison across the human MCR family; the only amino acid at this position with a P_i < 0.05 was tryptophan (W). However, this substitution was predicted intolerant in the cross-species analysis of MC1R. Here, only other hydrophobic amino acids were predicted tolerant at position 92. Interestingly, although the substitution of the consensus arginine residue by cysteine at position 151 was predicted intolerant in both the MC1R cross-species and human MCR family, codon 151 was not considered a conserved amino acid in the human MCR family comparison. This perhaps provides further support for the importance of the R151C variant in the pigmentation process.

The results from our literature- and evolutionary-based approaches to analysis of MC1R variants indicated that for predicting fair pigmentation phenotypes, SIFT analysis did not provide substantially more information about potentially important MC1R variants than that available through the literature. This observation, however, may be specific to the population of healthy controls and their MC1R genotypes under investigation here. For example, in a sample population demonstrating a greater prevalence of rarer MC1R variants, we would expect a different SIFT result and potentially a better ability of SIFT analysis to identify risk variants predictive of pigmentation phenotypes or disease outcomes. Regardless, our results do support previous reports that MC1R variants are strongly associated with cutaneous pigmentation characteristics and help to identify which of the many MC1R variants reported to date are likely to be predictive of fair pigmentation phenotypes.

GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for Alces alces alces MC1R [accession number P56442], Bos taurus MC1R [accession number P47798], Canis familiaris MC1R [accession number O77616], Capra hircus MC1R [accession number P56444], Capreolus capreolus MC1R [accession number P56443], Cervus dama MC1R [accession number P56446], Cervus elaphus MC1R [accession number P56445], Coereba flaveola MC1R [accession numbers AAK50782 and AAK50811], Equus asinus MC1R [accession number AAD33875], Equus caballus MC1R [accession numbers AAK70924.1 and P79166], Equus grevyi MC1R [accession number AAD33874], Equus przewalskii MC1R [accession number AAB96911], Equus zebra MC1R [accession number AAC68702], Gallus gallus MC1R [accession number P55167], Homo sapiens MC1R [accession number AL035458], Homo sapiens MC2R [accession numbers NP_000520 and JN0764], Homo sapiens MC3R [accession numbers B46647 and NP_063941], Homo sapiens MC4R [accession numbers A57055 and NP_005903], Homo sapiens MC5R [accession numbers JC5592 and NP_005904], Mus musculus MC1R [accession number NP_032585], Ovis aries MC1R [accession number O19037], Pan troglodytes MC1R [accession number Q9TUK4], Rangifer tarandus MC1R [accession number P56448], Sus scrofa MC1R [accession numbers AAK58422, AAD53963, and Q9TU05], Takifugu poecilonotus MC1R [accession number BAB71734], Takifugu porphyreus MC1R [accession number BAB71733], Takifugu radiatus MC1R [accession number BAB71732], Takifugu rubripes MC1R [accession number BAB71730], Tangara cucullata MC1R [accession number AAK50813], Vulpes vulpes MC1R [accession number Q29154]).

Online Mendelian Inheritance in Man (OMIM), http:/www.ncbi.nlm.nih.gov/Omim/.

We are deeply indebted to past and present physicians of the Pigmented Lesion Clinic, including Drs. David Elder, Rosalie Elenitsas, Stuart Lessin, Michael Ming, and Jonathan Wolfe, without whom this study would not have been possible. We also graciously acknowledge the help of several past and present University of Pennsylvania undergraduate students, Lindsay Ackerman, Sharon Sandel, and Zoe Harris, for their help in participant recruitment.