Peptides 26 (2005) 1901–1908

Review Worldwide polymorphism at the MC1R locus and normal pigmentation variation in

Kateryna Makova a,∗, Heather Norton b

a Department of Biology, The Pennsylvania State University, 518 Mueller Lab, University Park, PA 16802, USA b Department of Anthropology, The Pennsylvania State University, 409 Carpenter Building, University Park, PA 16802, USA Received 20 August 2004; accepted 16 December 2004 Available online 23 June 2005

Abstract

While there have been many advances in our understanding of the of pathological skin pigmentation in humans, our knowledge about what determines variation in normal skin color is still incomplete. Variation in one gene, (MC1R), has been associated with and fair skin in Europeans. However, this gene might also play an important role in shaping pigmentation of other populations, where it experiences different selective pressures. Below we review what is currently known about polymorphism and selection at the MC1R coding and promoter regions in human populations, the pattern of MC1R evolution in nonhuman primates, and the interaction of MC1R with other genes. © 2005 Elsevier Inc. All rights reserved.

Keywords: MC1R; Pigmentation; Natural selection; Human evolution

Contents

1. Introduction ...... 1901 2. Polymorphism of MC1R in human populations: coding region ...... 1902 2.1. European populations...... 1902 2.2. Asian populations ...... 1903 2.3. African populations ...... 1904 3. Polymorphism of MC1R in human populations: promoter ...... 1904 4. MC1R evolution in primates ...... 1905 5. Interactions between MC1R and other genes in human populations ...... 1905 6. Future directions ...... 1906 Acknowledgements...... 1907 References ...... 1907

1. Introduction This variation has long intrigued geneticists and anthro- pologists, especially because it is believed to have been Human skin and hair pigmentation are complex traits shaped, at least in part, by the action of natural selection that show variation both within and between populations. (e.g. [7,22,24,33,55]). While genes responsible for pigmenta- tion disorders such as have been known for nearly a decade (for a review, see [20]), it is only recently that we have ∗ Corresponding author. Tel.: +1 814 863 1619; fax: +1 814 865 9131. developed a better understanding of the genetic basis under- E-mail address: [email protected] (K. Makova). lying normal variation in human skin and hair pigmentation.

0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.12.032 1902 K. Makova, H. Norton / Peptides 26 (2005) 1901–1908

The major breakthrough in this area was due to the discovery MC1R isoform is a 317-amino acid 7 pass transmembrane of mutations in the melanocortin 1 receptor (MC1R) gene that protein. Mutagenesis studies of other MCRs suggest that the are associated with normal variation in skin color [53]. The transmembrane domains TM1, TM2, TM3, TM6, and TM7 MC1R is a cell-surface receptor of melanocytes and a major are likely to contain the MC1R’s primary hormone-binding player in the production pathway. The MC1R gene sites [39–41]. Robbins et al. [38] identified two gain-of- has been observed to regulate the switch between the pro- function point mutations in this region in the mouse, and duction of eumelanin (brown/black pigment) and pheome- human MC1R polymorphisms associated with red hair and lanin. Eumelanin together with pheomelanin (red/yellow fair skin have been observed here as well (e.g. [35,53]). pigment) constitute the two main pigments of the skin Like other G protein-coupled receptors, MC1R signal trans- and hair. duction is mediated via the GTP-binding protein to acti- The melanocortin 1 receptor belongs to a family of vate adenylyl cyclase and increase levels of cellular cAMP. G protein-coupled receptors known as the melanocortin Intracellular loops of G protein-coupled receptors are nec- receptors (MCRs). In mammals, the MC1R responds to ␣- essary for protein interactions with GTP-binding proteins, melanocyte-stimulating hormone (␣-MSH) and adrenocor- suggesting that these may be key functional regions of the ticotropic hormone (ACTH), both products of proopiome- MC1R. Mutations having an effect on pigmentation pheno- lanocortin (POMC). When the MC1R is activated by ␣-MSH, type have in fact been found in the second intracellular loop intracellular cAMP levels are increased via activation of [13]. adenylyl cyclase, leading to the production of elevated levels In the mouse, loss of function mutations at exten- of tyrosinase and ultimately to the production of brown/black sion lead to a yellow coat phenotype due to the over- eumelanin (Fig. 1). However, the antagonistic agouti signal- production of pheomelanin relative to eumelanin, while ing protein (ASIP) can compete with ␣-MSH for binding gain of function mutations at extension are associated with to the MC1R. When ASIP is able to completely block or melanic morphs [38]. Extension is also responsible for eume- inhibit ␣-MSH, pheomelanin is produced instead of eume- lanin/pheomelanin production in other mammals, such as lanin (Fig. 1). horses, dogs, pigs and sheep, as well as in birds (e.g. The human MC1R gene, located at chromosome 16q24.3 [18,38,52]). [14], has an open reading frame of 951 bp and until recently The human MC1R gene has been shown to be highly was thought to be composed of a single exon. However, Tan polymorphic (e.g. [6,15,35,47,53]) with many variants being et al. [51] identified an alternative mRNA splice variant at associated with a red hair/fair skin phenotype. Mutations in the 3 end of the gene. This “long” MC1R isoform (MC1RB) MC1R have also been associated with poor tanning ability contains a second exon encoding 65 amino acids, although and an increased risk of skin cancers, possibly since pheome- the functional significance of this alternative form is currently lanin can produce free radicals in the presence of unknown. radiation (UVR) and thus have deleterious effects on the The MC1R gene maps to the murine extension locus melanocytes [23,32]. and is expressed in a number of cell types, most notably There are a number of excellent reviews that focus on the melanocytes. The protein encoded by the “short” human relationship between the MC1R genotype and the red hair phenotype (e.g. [36,37,50]). In contrast, this review will focus on MC1R polymorphism across the entire human species (rather than limiting our discussion to variation in European populations), the role of selection in shaping MC1R diver- sity, and MC1R polymorphism and evolution in non-human primates.

2. Polymorphism of MC1R in human populations: coding region

2.1. European populations

Early reports indicated an association between the MC1R variants and a red hair/ phenotype. By screening 30 red-haired individuals, Valverde et al. [53] identified nine different changes in the MC1R coding region, all leading to amino acid substitutions. Frequently, there were two amino Fig. 1. Diagram of the alternate effects of the binding of ␣-MSH or ASIP to the melanocortin 1 receptor. Binding of ␣-MSH leads to the production of acid mutations within the same allele. Eight of these substitu- brown/black eumelanin, while binding of the antagonistic ligand ASIP leads tions span the second transmembrane domain of the MC1R. to the production of red/yellow pheomelanin. Another study investigated with red hair and identified K. Makova, H. Norton / Peptides 26 (2005) 1901–1908 1903

11 additional amino acid variants at the MC1R [6]. Three at most other genomic regions investigated in non-Africans substitutions were significantly associated with red hair. [34]. Note that this variation is mostly nonsynonymous. Interestingly, in several cases, dizygotic twins possessing Two alternative theories were proposed to explain the identical MC1R alleles had discordant hair color, suggesting high level of MC1R polymorphism in Europeans. First, Rana that the MC1R gene cannot be solely responsible for the red et al. [35] suggested that this might support the hair phenotype [6]. A more detailed study was later conducted hypothesis [9,22]. Vitamin D is produced by photoconver- in Irish populations [47], again providing evidence of signif- sion of 7-dehydrocholesterol in skin capillaries. In regions icant association of MC1R variants with red hair. Currently of low sunlight, light skin can be advantageous, as it allows more than 30 variant alleles have been reported for European more ultraviolet radiation to penetrate through the skin pre- populations with nine variants being at high frequency (each venting vitamin D deficiency and possibly diminishing the >1%; see [50] for a nearly complete list of variant alleles). occurrence of . Thus, when human populations moved In addition to its association with red hair and fair skin the from areas of high sun exposure to areas of low sun expo- MC1R gene variants have been found to be linked to sure, mutations resulting in lighter skin pigmentation could formation [2,10]. have been advantageous and as such increased in frequency. In familial studies (e.g. [36]), the inheritance of red hair High genetic polymorphism in Europeans could have been appears to follow an approximately autosomal recessive pat- explained by local selection for specific alleles. However, tern. Additionally, recent studies provided evidence that dif- this reasoning was not supported by a rigorous statistical test ferent MC1R polymorphisms can have either “strong” or [35]. “weak” effects on the red hair phenotype [10]. Combina- Second, Harding et al. [15] argued that the high poly- tions of these alleles suggest a heterozygote effect on red morphism at the MC1R locus in Europeans could be due hair phenotype, skin pigmentation, and freckling [11,10]. to relaxation of functional constraints. Indeed, they applied Indeed, most “pure” redheads (as opposed to individuals with several neutrality tests and found no statistical evidence that strawberry or auburn red hair) are homozygous/compound MC1R diversity has been enhanced by selection. Thus, the heterozygous for amino acid changing variants of MC1R [11]. data on MC1R alleles in European populations are consistent Combinations of particular MC1R variant alleles result in a with a removal of functional constraint rather than selection variety of hair shades [36,11]. in favor of loss-of-function mutations. One of the possible The effects of MC1R polymorphisms on red hair, skin explanations for the different conclusions of the two stud- reflectance, degree of freckling and nevus count were exam- ies is the low power of neutrality tests. Additionally, the ined by Duffy et al. [10] in 2331 twins, their siblings, and MC1R coding region is relatively small (∼1 kb). While its parents. Nine MC1R alleles were genotyped, four of which small size makes MC1R an attractive gene to investigate by could be classified as having a strong association () direct sequencing, it also limits the number of informative with the red hair/fair skin phenotype (Asp84Glu, Arg151Cys, nucleotide sites. Thus, while we cannot reject the neutral Arg160Trp, and Asp294His), and three of which showed only explanation, diversifying selection operating on the MC1R a weak association (Val60Leu, Val92Met, and Arg163Gln). coding region in Europeans is still a possibility. The authors classified the former group as strong (“R”) and weak (“r”) alleles. The majority of red-haired individuals in 2.2. Asian populations the study had either R/R (67.1%) or R/r (10.8%) genotypes; no red-haired individual was homozygous for the consensus Rana et al. [35] sequenced the MC1R coding region in sequence. The authors conclude that MC1R polymorphisms 85 Asians. The intriguing finding was the association of the have a multiplicative effect on hair pigmentation, while the Arg163Gln variant with the East and Southeast Asian pop- effects on skin reflectance are more subtle. ulations (Chinese, Japanese, Mongolian, Cambodian, Viet- MC1R mutations are loss-of-function alleles that have namese, and Yakut) with an average frequency of this variant either altered ability to bind ␣-melanocyte-stimulating hor- of 70%. Interestingly, Arg163Gln was found in the homozy- mone (e.g., Val92Met [56]) or to activate adenylyl cyclase gous state in five Native Americans studied, supporting a via a G-protein-coupled pathway (e.g., Arg151Cys [13], hypothesis that Native Americans originated in Northeast Val60Leu, Arg142His, Arg160Trp, and Asp294His; [42]). Asia. This variant has been observed at much lower frequen- Interestingly, some variants may be slightly impaired in both cies outside of East Asia: 7% in South Asian Indians [26] and their hormone binding and activation of adenylyl cyclase 4.7% among a population of European ancestry from South [42]. Additionally, melanocytes with a non-functional MC1R East Queensland [49]. have an increased sensitivity to the cytotoxic effects of UV The investigation of populations along the Silk Road of radiation [43]. [57] confirmed the high frequency of the Arg163Gln As a result of a large number of nucleotide substitutions, variant and detected the Val92Met variant at a high frequency the MC1R coding region is unusually polymorphic among among Asians. However, the frequency of the Arg163Gln Europeans. The average pairwise sequence difference per variant gradually declined from South East to North West: nucleotide (π) in Europeans is 0.11% with some populations from 72% in the Dai to 40% in the Uighur. A sim- reaching π of ∼0.16% [15]. This is higher than observed ilar cline was observed for the Val92Met mutation. These 1904 K. Makova, H. Norton / Peptides 26 (2005) 1901–1908 data support extensive gene admixture between Asian and of melanin may be important in preventing the breakdown European populations in the Silk Road of China. of folic acid by UVR [7]. Folic acid photolysis can lead to Peng et al. [31] examined MC1R variation in four Chinese meiotic errors during sperm production [26] as well as an ethnic populations (Uygur, Tibetan, Wa, and Dai). Similar to increased risk of neural tube birth defects [5,12]. The folic the results of Yao et al. [57], an east-west cline (from high acid hypothesis predicts that dark skin would be favored in to low) in the frequencies of the Arg163Gln was detected. high UVR environments. This suggests that conservation of Unlike the findings of Yao et al. [57], the frequencies of the the MC1R coding sequence in Africans may have been main- Val92Met were similar among the four studied populations. tained by purifying selection for dark skin to protect against Three additional nonsynonymous and two synonymous vari- toxic effects of ultraviolet radiation. The statistical support of ants were also reported. purifying selection acting in Africans was provided by Hard- Thus, the Arg163Gln variant reached high frequency in ing et al. [15]: the ratio of synonymous to nonsynonymous East and Southeast Asian populations in a very short time, variants in African polymorphism was significantly different following the divergence of Asians, Europeans, and Africans. from this same ratio at sites divergent between humans and Rana et al. [35] hypothesized that this allele increased in fre- chimpanzees. quency in Asians due to positive Darwinian selection. Indeed, Recently, John et al. [16] reported considerably greater under neutral expectation, the estimated arrival time of the MC1R polymorphism among Africans. The MC1R coding Arg163Gln allele is older than the age of modern humans region was sequenced in two populations—the San Africans [35], calling for a selective explanation to be invoked. Peng et (17 unrelated individuals) and the sub-Saharan Africans (22 al. [31] suggested that , migration and admixture unrelated individuals). Eight synonymous and three nonsyn- might also have an impact on the Arg163Gln frequency. For onymous mutations were detected. This is the first report instance, the frequency of Arg163Gln is highest in Tibetans of nonsynonymous mutations in African individuals in the (85%), possibly because of genetic drift. On the other hand, MC1R gene: L99I was found in a single San individual, S47I its lowest frequency is in Uygurs (40%), potentially due to was found in a single sub-Saharan individual, and F196L was gene admixture with Europeans. found in five sub-Saharan individuals. None of these muta- It is presently unknown whether the Arg163Gln substi- tions were found in Europeans. The functional significance tution is functionally significant. This variant, located in of these mutations is not presently known. This demonstrates the transmembrane region four of the protein, has not been that some nonsynonymous MC1R mutations are tolerated in observed in mice or other animals studied [35]. Site-directed individuals with relatively dark skin without dramatic effects mutagenesis or/and functional studies may shed light on the on skin phenotype. What can explain these differences in the potential importance of this variant in skin pigmentation in results between the studies of Harding et al. [15], John et al. Asians. [16], and Rana et al. [35]? The individuals investigated by The nucleotide diversity at the MC1R coding region in John et al. [16] originated from southern Africa while those Asians was found by Rana et al. [35] to be 0.21%, higher from the latter two studies were from more western and cen- than for other genes investigated in non-Africans [34]. Hard- tral African populations. Thus, it is conceivable that MC1R ing et al. [15] found the MC1R coding region to be somewhat selection is less strong in southern Africa than in the more less polymorphic (π = 0.09) in Asians, although more poly- equatorial regions where the samples for [15] and [35] came morphic than most other studied genes [34]. from. It is also possible that the observed nonsynonymous mutations might be selectively neutral and appeared in the 2.3. African populations studied populations only by chance. Several tests of neutrality were significant in these African The studies of Rana et al. [35] and Harding et al. [15] found populations when all individuals from the three studies no nonsynonymous substitutions and only three synonymous [15,16,35] were considered together. This supports the action substitutions among 25 and 53 African individuals screened, of purifying selection (functional constraint) at the MC1R respectively. This is unexpected since (1) African populations coding region in Africans. are usually more polymorphic than other populations as they are most ancient [34]; and (2) some African individuals are at one extreme of skin color (thus, there could be variants 3. Polymorphism of MC1R in human populations: specific to Africans). The level of polymorphism at the MC1R promoter coding region is the lowest in African populations. Rana et al. [35] interpreted these results again as support Observations of dizygotic twins discordant for the red hair of the vitamin D hypothesis. Dark skin reduces the amount phenotype but homozygous by state for MC1R haplotypes of ultraviolet radiation reaching skin, which in turn reduces on both chromosomes indicate the possibility that polymor- the amount of vitamin D formed. Rana et al. [35] assumed phisms in MC1R may be necessary but not sufficient to yield that an excess of vitamin D would be toxic, making dark skin the red hair color phenotype [6]. These results suggest that advantageous in regions of Africa where ultraviolet radiation either polymorphisms outside the MC1R coding region or (UVR) is strong. Alternatively, the photoprotective effects mutations in other genes might influence the phenotype. K. Makova, H. Norton / Peptides 26 (2005) 1901–1908 1905

The identification of the MC1R minimal promoter [28] gorilla), common chimpanzee ( troglodytes), and pygmy allowed researchers to examine the former possibility. In a chimpanzee (Pan paniscus). While MC1R was shown to vary recent study, Makova et al. [25] sequenced a 6.6-kb region extensively among these higher primates, all nonsynonymous located upstream of the MC1R coding region (this region substitutions (with the exception of the Val92Met variant in includes the MC1R promoter) in 54 humans (18 Africans, 18 the baboon) observed in humans were conserved. The results Asians, and 18 Europeans) and in one chimpanzee, gorilla, of KA/KS (the ratio of nonsynonymous to synonymous sub- and . The average nucleotide diversity, which was stitution rates) tests suggested that MC1R was not subject to 0.141%, is one of the highest such values for any study of strong selective constraint compared with other genes that nuclear sequence variation in humans [34]. In contrast to the have been studied in humans and the great apes. pattern observed in the MC1R coding region, π of the MC1R Mundy and Kelly [29] undertook a large-scale survey upstream region was highest in Africans (0.136%) compared of MC1R sequence polymorphisms in 57 non-human pri- to Asians (0.116%) and Europeans (0.122%). The pattern mate species in an effort to understand MC1R evolution in of genetic variation was consistent with purifying selection humans’ closest relatives. The authors attempted to assess and/or of population expansion in Africans. Makova et al. the rate and pattern of evolution of MC1R across various [25] also suggested a possible phase of population size reduc- primate taxa, identify polymorphisms associated with coat tion in non-Africans. The authors hypothesized diversifying color variation, and identify key amino acid residues that selection acting on some sites in the promoter and/or in the may have been conserved across primates. Simple associ- MC1R coding region in Asians and Europeans, although they ations between MC1R genotype and coat color phenotype could not reject the possibility of relaxation of functional among closely related primate taxa were difficult to iden- constraints on the MC1R gene in these populations. Pop- tify. Very few loss-of-function or gain-of-function mutations ulation screening and phylogenetic footprinting suggested were observed, and the authors concluded that this paucity potentially important sites for the MC1R promoter function. of loss-of-function mutations could be due to the involve- In another study, Smith et al. [46] investigated poly- ment of MC1R in other physiological systems of non-human morphism in the immediate 5 promoter region and a vari- primates. One possibility suggested by the authors is that able number tandem repeat (VNTR) minisatellite located MC1R, which is also expressed in immune cells, may play a upstream of the MC1R coding region in European and African role in the immune response [8,48]. Mundy and Kelly [29] populations. This study identified four and three polymorphic did observe numerous substitutions at amino acid residues sites within the promoter region in European and African across primate taxa (particularly in New World monkeys and samples, as well as 18 and 5 VNTR alleles, respectively. lemurs), suggesting that the biochemical function of MC1R The authors also found coding region variants and promoter may not have been conserved across primate lineages. Fur- variants to be in strong linkage disequilibrium. This allowed ther, the extensive variation observed in MC1R gene length them to estimate the mean age of one of the coding variants, in these non-human primates contrasts sharply to that which Arg151Cys, to be 7500 years before the present day, sug- is observed in other animals (including humans, most other gesting that this variant might have arisen in the European mammals, and birds), which show few deviations from a population more recently than previously thought [46]. product of 317 amino acids. This length variation did not Makova et al. [25] and Smith et al. [46] both suggest that seem to have a functional effect. there is considerable variation upstream from the MC1R cod- Purifying selection appears to have been common in non- ing region. However, further investigations, preferably those human primates. However, the lion tamarins, notable for their quantifying the MC1R RNA expression level, will be required reddish yellow pelage, showed a number of substitutions at for any potential genotype-phenotype associations to be elu- conserved amino acid residues, a 24 bp deletion, and a KA/KS cidated. ratio of 0.91 which was significantly higher than that observed for other anthropoid primates [29]. Based on this work, it appears that MC1R does not play the same dominant role in 4. MC1R evolution in primates pigmentation regulation in non-human primates as it does in humans. Studies of intraspecific variation of MC1R in non- While MC1R has been well studied in humans, until human primates might provide additional information about recently little was understood about MC1R variation in non- potential association of the MC1R mutations with particular human primates. This is of particular interest since several primate phenotypes. closely related primate species show coat phenotype variation that is consistent with MC1R loss-of-function or gain-of- function mutations (black or red coats). Initial information 5. Interactions between MC1R and other genes in about nonhuman primate MC1R variation came from a study human populations addressing evidence for selection at the human MC1R [35].In this work the MC1R coding region was sequenced in a single As pigmentation is a complex trait, it is likely that representative of the following species: baboon (Papio cyno- multiple genes play a role in determining normal variation in cephalus), orangutan (Pongo pygmaeus), gorilla (Gorilla skin and hair pigmentation. Indeed, studies in mouse models 1906 K. Makova, H. Norton / Peptides 26 (2005) 1901–1908 have shown this to be true [3,27]. In addition to having decrease in nevus count, suggesting a recessive effect. Two single-gene effects on pigmentation phenotype, the genes OCA2 SNPs typed in this study showed no association with may also interact with each other in producing normal varia- skin color, freckling, or nevus count, although these poly- tion in pigmentation. Akey et al. [1] typed three MC1R SNPs morphisms are associated with brown/green (not (Arg67Gln, Gln163Arg, and Val92Met) and two P (OCA2) blue). An investigation of the interactive effects of some of gene SNPs (IVS13-15 and Gly780Gly) in 1874 Tibetan the other known OCA2 polymorphisms may yield stronger subjects for whom quantitative skin pigmentation mea- results. Despite the failure to find associations between the surements were available. While these authors were unable specific OCA2 polymorphisms and the major risk to detect any single-locus effects, a gene–gene interaction factors examined, this work represents a major contribution model showed the Val92Met in MC1R and IVS13-15 SNPs to the understanding of the complex polygenic interactions of the P gene have a significant effect on pigmentation. The between MC1R and other pigmentation candidate genes. Val92Met allele has been shown to have a reduced binding Voisey et al. [54] investigated whether polymorphisms affinity for ␣-MSH [56], although it does not affect ability to in the agouti signaling protein gene (ASIP) act to suppress activate adenylyl cyclase [21]. It is unclear, however, what MC1R phenotype. ASIP produces an antagonistic ligand to the significance of the IVS13-15 variant may be; it may be in MC1R and can block the binding of ␣-MSH. No poly- linkage disequilibrium with a true functional variant rather morphisms were observed in the coding regions of ASIP, than have a direct functional effect. The Akey et al. study [1] although a SNP in the 3 untranslated region was typed in an shows that it may be possible to reconcile discordant results African–American sample at a frequency of 0.2. The absence about the effect of Val92Met on pigmentation phenotype of coding region variants in ASIP prevented Voisey et al. [54] seen in previous studies [6,11,53]. In cases where Val92Met from testing for gene–gene interactions between ASIP and was not observed to have a functional effect on pigmentation, MC1R. it may be because a crucial alternate variant was not present. Further evidence that MC1R may have interaction effects with other pigmentation loci was found in a study by King 6. Future directions et al. [19] examining the effects of MC1R polymorphism on the phenotype of one type of oculocutaneous albinism, OCA As evident from this review, MC1R has been well stud- type 2. OCA2 is the result of a decrease in the synthesis of ied for its effects on normal variation in pigmentation, as melanin, resulting in light (yellow) hair, sun sensitive skin, well as for being a possible target of purifying and/or diver- and foveal hypoplasia caused by mutations in the human P sifying selection during human evolution. However, a trait gene, also known as OCA2. King et al. reported eight indi- as complex as pigmentation cannot be simply characterized viduals with OCA type 2 that had red, rather than blonde by a single gene. In fact there have been a number of genes hair. Six of these eight individuals also exhibited MC1R poly- implicated in pigmentation variation, although many of these morphisms associated with red hair (Arg151Cys, Arg160Trp, also have broader physiological effects and were first stud- Val60Leu, Val92Met, and Asp84Glu), suggesting that MC1R ied for their association with pathological pigmentation. To polymorphisms can modify the classic OCA2 phenotype. date, mutations in pigmentation genes that lead to the vari- The relationship between MC1R and OCA2 was also ous forms of oculocutaneous albinism (OCA) have been well explored by Duffy et al. [10] in a study of melanoma risk studied. These genes include TYR, P (OCA2), TYRP-1, and phenotypes using data on 2331 twins, their sibs, and par- most recently MATP (for details see the Albinism Database ents. While many of the phenotypes associated with MC1R, at http://albinismdb.med.umn.edu/). Albinism-associated such as red hair, fair skin, and freckling, are risk factors mutations in these genes affect pigmentation phenotype for melanoma, an additional phenotype, eye color is also a (largely due to an absence of or decrease in tyrosinase produc- risk factor. Thus far, no effects of MC1R on eye color have tion). However, despite the broad-ranging effects of polymor- been observed, and the fact that eye color is a risk factor for phisms in these genes (ocular defects, etc.), Shriver et al. [44] melanoma (independent of hair color [4]) suggests that other demonstrated that a nonsynonymous variant in TYR was asso- genes may be involved. To investigate this relationship fur- ciated with normal variation in skin pigmentation between ther, the authors examined the relationship between eye color populations of European and African descent. In that same phenotype (believed to be associated with OCA2) and other study, the authors also demonstrated that skin pigmentation risk factors. The authors observed a decrease in skin pigmen- was also linked with a variant in the P (OCA2) gene. Other tation in individuals homozygous for strong (R) MC1R alleles studies have produced suggestive results supporting the role and the recessive b eye color allele, and suggest an additive of other genes in normal pigmentation variation. For instance, effect of MC1R and OCA2 on skin pigmentation. Freckling the allele frequencies of variants in pigmentation candidate count was increased in b/b homozygotes and MC1R consen- genes such as ASIP [17] and MATP [30] vary among popu- sus and heterozygote genotypes in an additive manner. When lations known to differ in skin pigmentation phenotype. nevus count was examined, a b/b genotype was found to As studies begin to focus on genes that may affect normal slightly increase nevus count for all non-R/non-R and R/non- variation in pigmentation that do not involve red hair (a rela- R individuals. Interestingly, b/bR/R homozygotes showed a tively easy phenotype to observe), it will become necessary to K. Makova, H. Norton / Peptides 26 (2005) 1901–1908 1907

Table 1 Comparison of the MC1R polymorphism and selection in human populations: contrast between coding region and promoter. Population/region African Asian European Coding region Polymorphism Low High High Selection Purifying Possible diversifying selection or relaxation Possible diversifying selection or relaxation of functional constraints of functional constraints Other Lack of synonymous variants High frequency of Arg163Gln >30 nonsynonymous variants Promoter Polymorphism High High High Selection Purifying Possible diversifying selection in parts of the Possible diversifying selection in parts of the promotor sequence promotor sequence use more precise measures of pigmentation. It will be critical to inter-individual variation in skin pigmentation phenotypes in a to refine the assessment of pigmentation phenotype from the Tibetan population. 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