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Review articles Human pigmentation genetics: the difference is only skin deep Richard A. Sturm,1* Neil F. Box,1 and Michele Ramsay2 Summary There is no doubt that visual impressions of body form and color are important in the interactions within and between human communities. Remarkably, it is the levels of just one chemically inert and stable visual pigment known as melanin that is responsible for producing all shades of humankind. Major human genes involved in its formation have been identified largely using a comparative genomics approach and through the molecular analysis of the pigmentary process that occurs within the melanocyte. Three classes of genes have been examined for their contribution to normal human color variation through the production of hypopigmented phenotypes or by genetic association with skin type and hair color. The MSH cell surface receptor and the melanosomal P-protein are the two most obvious candidate genes influencing variation in pigmentation phenotype, and may do so by regulating the levels and activities of the melanogenic enzymes tyrosinase, TRP-1 and TRP-2. BioEssays 20:712–721, 1998. r 1998 John Wiley & Sons, Inc. Introduction Color in our genes—what role does environment play? Being on the surface of the body and so readily observable, eye, hair, and skin pigmentation can offer some of the simplest means for studying and analyzing the action of genes.(1) Differences in these human color traits can be graded between individuals of darkest to those of lightest pigmentation along a continuum. Rather than recognition of such subtlety, however, distinct groupings are commonly spoken of as black, white, red, or yellow with a predominant black/white dualism in popular categorization. Many genes contribute toward producing these different color shades by 1Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Queensland, Australia. 2Department of Human Genetics, School of Pathology, South African Institute for Medical Research and University of the Witwatersrand, Johannesburg, South Africa. *Correspondence to: Richard A. Sturm, Centre for Molecular and Cellular Biology are at the University of Queensland, Brisbane, Qld 4072 Australia; E-mail: [email protected] Abbreviations used: BOCA, brown oculocutaneous albinism; DHICA, 5,6-dihydroxyindole-2-carboxylic acid; DOPA, 3,4-dihydroxyphenylalanine; EGF, epidermal growth factor; MC1R, melanocortin receptor-1 gene; MSHR, melanocyte-stimulating hormone receptor; OA, ocular albinism; OCA, oculocutaneous albinism; ROCA, rufous oculocutaneous albinism; TRP, tyrosinase-related protein; TYR, tyrosinase gene; TYRP, tyrosinase-related protein gene. 712 BioEssays 20.9 taking part in the synthesis of different amounts or kinds of substances that give rise to the visible color differences. In the case of skin and hair color, such differences are produced by virtually one pigment known as melanin,(2) which has a variety of functions,(3) including photoprotection, routing of the optic nerve tracts, and possibly the scavenging of free radicals. By incorporating different chemical subunits, the melanin polymer can vary from black/brown to red/yellow, which can account for some of the color qualities, with melanin particle size, shape, density, and distribution contributing to the degree of opacity. The degree of pigmentation of the small group of ancestral humans originating in Africa,(4) and who radiated through the rest of the world, was presumably uniform, of unknown pigmentation state, but probably within the range of darker skin tones. The first depiction of variable pigmentation in man(5) dates back to about 1300 BC and was found on the walls of the tomb of Sethos I. It shows a relatively fair-skinned Libyan and a very dark Nubian, together with an Asian and Egyptian who had skin tones between these two. Problems arise in defining an individuals true pigmentation however, because skin color can be influenced by environmental factors or may change with age, and hair can be bleached or simply dyed. Even brief exposure to sunlight may result in persistent changes in the amount of cutaneous melanin; hence evaluation of these traits can become quite subjective if not properly analyzed and will introduce a confounding variable when attempting to link genotype with phenotype. Attempts to study the genetic BioEssays 20:712–721, r 1998 John Wiley & Sons, Inc. Review articles basis of human pigmentation began with the work of Gertrude and Charles Davenport early in the twentieth century, when they examined the inheritance of eye,(6), hair,(7) and skin colors.(8) Sewall Wright(9) recognized that each of these traits are physiologically connected, and must be considered together when discussing their inheritance. As an extension of their work on these traits in Caucasians, the Davenports also began an analysis of skin color inheritance in the children of marriages between people of black African and white Caucasian descent, suggesting that as little as two gene pairs would be sufficient to explain the phenotypes of the offspring of these unions which was always of intermediate pigmentation. Further work by Stern,(10) in 1953, by Harrison and Owen,(11) in 1964, and by Kalla,(12) in 1969, raised the number to 3–6 gene pairs. These classical studies, however, were based on the hypothesis that the genes involved worked in simple and equally additive ways, which is clearly an inadequate model for how genes act and interact.(13) Despite these numerous formal genetic studies, there is no definitive understanding of the ways in which genes determine human pigmentation. Insight into the process is now coming from the new genomic strategies and genotyping technologies that use comparative genomics to identify mammalian pigmentation genes, the study of hypopigmented human phenotypes to characterize dysfunctional genes, and polymorphism studies on variation within these genes in diverse human populations. The goal of understanding the differences of human pigmentation, so often reduced to the single index of color, becomes one of much higher complexity in identifying and understanding the nature of the genes expressed in the melanocyte cell and how they interact. Only upon this understanding will the heritable basis of the physical trait of color be revealed. This review focuses on three classes of melanocyte genes that may well underlie and expand our knowledge of this most obvious of physical traits (Table 1). First, some of the enzymes involved in the synthesis of melanin are considered; second, the role of a protein mutated in several hypopigmented conditions is examined; and third, a cell surface receptor that may regulate the pigmentation process is discussed. Ultimately, studies to identify functionally significant polymorphisms and to determine their level of variation in human populations will provide an explanation for how pigmentation differences have evolved and allow a true molecular understanding of the dynamics of color genes in different cultures and societies. The three gene systems discussed here are the strongest candidates yet identified but will not be the only genes determining human pigmentation. Melanin and the melanosome—the basis of the human paint palette Melanin is synthesized in a multistep biochemical pathway that operates within a specialized intracellular organelle known as the melanosome (Figs. 1, 2). This particle is secreted through the dendritic processes of the melanocyte cell to the surrounding keratinocytes and hair follicles in cutaneous tissue or, alternatively, is retained by the melanocytes of the eye.(14) There are easily recognized differences in melanosome qualities of ethnic groups, as shown in ultrastructural studies of the skin.(15) Although the number of melanocytes is essentially constant, the number, size, and the manner in which the melanosomes are distributed within the keratinocytes vary. In general, more deeply pigmented skin contains numerous single large melanosomal particles that are ellipsoidal and intensely melanotic. Lighter pigmentation is associated with smaller and less dense melanosomes that are clustered in membrane bound groups. Melanosomes in black African skin are .0.8 µm, with Asian and Caucasian melanosomes averaging ,0.8 µm,(16) but there is variation in melanosome size within these groups. These distinct patterns of melanosome type and distribution are present at birth and are not determined by sun exposure.(17) It is possible that the formation of either single or aggregated melanosomes depends more on melanosome size, which may be influenced by nongenetic factors,(16) as well as genetic factors. Usually, the relative size and density of the melanosomes in the keratinocyte correlates with skin tone and dispersed larger granules give rise to darker complexions. Differences in the degree of melanization, as well as chemical differences in the melanin pigments contained within the melanosome are determining factors in the visual gradation of skin and hair color. Ultimately, all melanins are derived by oxidation of the amino acid tyrosine, with two distinct types of melanin compounds of variable molecular weight produced in a bifurcated biosynthetic pathway. The underlying structure of the melanin polymer, however, remains uncertain.(2) The black/brown pigments are produced by the synthesis of eumelanin, with the red/yellow colors produced through an alternative sulfur-containing compound commonly known as pheomelanin but which is of an indeterminate nature. Each melanocyte has the capacity to synthesize both types of pigment and when they do the outcome is mixed melanin.(18) After a century of speculation, theory and difficult degradative and synthetic chemical investigation (2) major advances in the understanding of the enzymology of the melanin biosynthetic pathway have only occurred during the past decade through a molecular genetic dissection of mouse coat colors.(1,19) Originally the production of the melanin biopolymer was thought to involve only one copper-containing enzyme, known as tyrosinase. Tyrosinase certainly catalyzes the first critical step of the melanin pathway, the hydroxylation of tyrosine to dopa; it is also involved in two subsequent steps. The first two tyrosinase-catalyzed reactions are common to eumelanogenesis and pheomelanogenesis. It is known that they diverge after the formation of dopaquinone (Fig. 2), but the mecha- BioEssays 20.9 713 Review articles TABLE 1. Human Pigmentation Genes Gene symbol TYR TYRP1 TYRP2 P MC1R Mouse homologue Albino (c) Brown (b) Slaty (slt) Pink-eyed dilute (p) Extension (e) Chromosome Phenotype Protein Function/ activity 11q14–21 9p23 13q31–32 15q11.2–12 16q24.3 OCA1 OCA3/ROCA Unknown OCA2, BOCA Red hair Tyrosinase TRP-1 TRP-2 P-protein MSHR Tyrosine hydroxylation; DOPA oxidase DHICA oxidase Dopachrome tautomerase Melanosomal transmembrane protein G-protein-coupled receptor The synthesis and polymerization of the melanin precursors take place in the specialized melanosomal organelle where tyrosinase has a characteristic pattern of posttranslational glycosylation. The melanosomal structure correlates with the type of melanin within,(14) as illustrated in Figure 1. Stage I of the developing eumelanosome is a spherical vacuole, derived from the endoplasmic reticulum, that elongates into an ellipsoidal organelle. Tyrosinase and other enzymes are transported from the Golgi complex to the developing melanosome (stage II) by vesiculoglobular bodies, which then begin to synthesize melanin (stage III). Melanin eventually fills the eumelanosome (stage IV). The early spherical vacuole of the developing pheomelanosome is similar (stages I–IV), but the pheomelanosome remains round throughout its maturation. Figure 1. Variation in melanosome structure and distribution in different groups. A single skin melanocyte cell interdigitating with keratinocyte cells is partitioned into three sections. Shown within the melanocyte are the four stages of melanosome formation from budding from the Golgi apparatus, to the fully pigmented stage IV melanosomes migrating up the dendritic processes of the cell and secreted into the keratinocytes. In African populations, the melanosomes remain as singular heavily pigmented particles while in Asians and Europeans the melanosomes cluster in membrane bound organelles giving different skin complexions. nisms responsible for this divergence are poorly understood. The eumelanins are derived from the metabolites of dopachrome, whereas the pheomelanins derive from metabolites of cysteinyldopa. The eumelanin pathway is favored in the absence of thiol compounds; the switch from pheomelanogenesis to eumelanogenesis might be regulated by the availability of cysteine.(20) In addition to tyrosinase, however, two other enzymes are now known also to take part in the eumelanic pathway: the isomerization of dopachrome to DHICA catalyzed by dopachrome tautomerase(21) and the oxidation of DHICA performed by the enzyme DHICA oxidase.(22,23) 714 BioEssays 20.9 Identification, function, and variation of representative human pigmentation genes The tyrosinase-related protein gene family The most dramatic example of gene action in pigmentation is seen in the complete loss of color resulting from the inability to form melanin. Albinism has been recorded in almost every species and the way in which the genes responsible for hypopigmented states have been identified demonstrates the power that a comparative molecular genetic approach has given to the study of pigmentation in humans. Hypopigmentation takes many forms and albinism in humans was first attributed to a single locus because two albinos produced similarly albino offspring.(8) This is not always the case; a single exception(24) provides evidence that more than one locus is involved. Cloning of the first albinism gene (TYR) was achieved in 1987, using antibodies to the tyrosinase enzyme(25) to screen a cDNA expression library. Cross-species comparison subsequently showed this cDNA to map to the mouse albino c-locus and the functionality of the TYR gene in albinism has been demonstrated beyond doubt through its ability to rescue the albino phenotype in transgenic animals.(26,27) In humans, loss-of-function mutations in the TYR gene are known as tyrosinase-negative or OCA1 albinism. Review articles Figure 2. Schematic of the pheomelanosome and eumelanosome with the biosynthetic pathway of the two types of melanin outlined. The globular structure of TYR (white), TRP-1 (brown), and TRP-2 (slate) proteins is illustrated showing the presence of an epidermal growth factor (EGF)like repeat and the metal-binding sites for copper (blue) or zinc (green) that make up the binuclear active sites. The P-protein is an integral membrane protein that may mediate the formation of a melanogenic complex formed in the eumelanosome. TYR was not the first gene identified using anti-tyrosinase antibodies, as a gene that was later found to map to the mouse brown b-locus on chromosome 4 had already been cloned in 1986 using a similar strategy.(28) The corresponding human gene became known as tyrosinase-related protein (TYRP) because of its apparent similarity to the tyrosinase enzyme.(29) This TRP-1 protein is thought to encode the enzymatic activity in melanin biogenesis known as DHICAoxidase.(22,23) In 1988, a second TYRP gene was identified by sequence homology, and the encoded protein became known as TRP-2; this gene encodes dopachrome tautomerase.(30,31) The three tyrosinase related proteins have an overall identity of approximately 40%, are around 500 amino acids in size, and are postulated to have a common globular structure (Fig. 2). The bulk of the protein is located inside the melanosome and a short C-terminal fragment in the cytosol is connected by a transmembrane domain.(32) An epidermal growth factor (EGF)-like repeat present toward the C-terminus is thought to mediate protein–protein interactions possibly in a high molecular weight melanogenic complex,(33,34) which includes the P-locus protein(35) (Fig. 2); two metal-binding domains serve as the active sites of each enzyme. The chromosomal locations of the loci for the three human TYRP genes have been determined, and searches have been conducted for functional polymorphisms that could explain natural variation in pigmentation phenotypes as well as several hypopigmented states. The TYR gene on chromosome 11q14–21 is encoded in five exons spanning more than 50–65 kb.(36,37) Many alleles responsible for OCA1 albinism have been identified,(38) but ethnic differences in the tyrosinase protein are rare, with only two apparently nonpatho- genic amino acid substitutions reported. The Y192S(39) and R402Q variant substitutions are found in all populations except in Asian.(40,41) Thus, the expectation that a polymorphism in the tyrosinase protein sequence would be a principal determinant of normal variation in human pigmentation would appear to be unlikely. Results obtained from melanocytes cultured from different skin types, however, apparently correlate melanin content with in situ tyrosinase activity,(42) there being 10 times more tyrosinase activity in black skin as compared with white skin. This difference was apparently not attributable to different levels of tyrosinase protein in skin (however, see ref. 43), and the molecular basis for this catalytic difference is unknown. Post-translational control of tyrosinase protein has been suggested as a possible explanation,(42) with alterations in formation of the melanogenic complex a valid possibility. The TYRP1 genomic locus encoding the TRP-1 protein is located on chromosome 9p23. The 24.6-kb region encompassing this locus has been completely sequenced with the coding region contained in seven exons.(44,45) Two polymorphic AC dinucleotide microsatellites have been identified that can be used for linkage studies.(44–47) The mouse Tyrp1 gene is known to be associated with lightening of coat color as a result of several b-locus mutations,(1) and for this reason the gene was studied as a candidate for the cause of two hypopigmented conditions known as brown and rufous oculocutaneous albinism (BOCA and ROCA) (Fig. 3). Linkage studies in families with BOCA have excluded TYRP1 from playing a role in this form of hypopigmentation,(47) but scanning of the TYRP1 exons has shown the molecular basis for ROCA. A form of albinism(48) in an African-American neonate BioEssays 20.9 715 Review articles The TRP-2 protein is associated with dopachrome tautomerase activity and is encoded by 8 exons at the TYRP2 locus on chromosome 13q31–32.(44,50) Amino acid substitutions are present in the mouse Tyrp2 gene, which maps genetically to the slaty coat color mutant.(1) It is to be expected that some form of human albinism would result from TYRP2 loss of function but, as yet, none has been reported. Analysis of the TYRP2 coding region from the same Australian Caucasian samples from individuals with different hair colors also exhibited a similar lack of variation (Box and Sturm, unpublished data). The collective absence or low level of polymorphism in the TYRP gene family in the human populations studied argues that differences in normal patterns of melanization are not produced by differences in the encoded catalytic activity of these enzymes. This does not rule out the possibility that different TRP protein levels or enzymatic activity within the melanosomal complex are responsible for variation in pigmentation. Indeed, such variation is apparent when melanocytes have been cultured from individuals of different skin types(43) and assays performed for each of the three melanogenic enzymes. It is the control of these proteins in the melanosome that is really the chief determinant of pigmentation phenotype and it is this regulation that must be understood. Figure 3. Individuals representing four different types of oculocutaneous albinism. Top left, defect in the TYR gene in a Caucasian child classified as OCA1 or tyrosinase-negative OCA. Hypopigmented phenotypes found in the South African black population include defects in the P-gene, classified as OCA2 or tyrosinase-positive OCA(top right), the TYRP1 gene, classified as OCA3 or ROCA(bottom left), and a BOCA phenotype heterozygous for a known P-gene ‘‘OCA2’’ causing mutation and possibly another P gene mutation (bottom right). was shown to be caused by the loss of TYRP1 gene expression via a homozygous single A-base deletion within exon 6. This same protein-truncating mutation and an additional loss-of-function mutation were also identified in adults with ROCA.(49) As TYRP1 is the third albinism locus to be identified, phenotypes caused by mutations in this gene are referred to as OCA3. Although the OCA3 newborn expressed normal amounts of tyrosinase that was catalytically active in cell lysates, tyrosinase activity was reduced by 70% when assayed in melanocytes cultured from the patient.(48) This may suggest that a loss of TRP-1 protein disrupts the correct formation of the melanosomal complex and thereby inhibits melanin synthesis, resulting in the hypopigmented phenotype. No other coding region mutations were detected in the analysis of South African(49) and Australian Caucasian samples from individuals with different hair colors,(45) indicating that TRP-1 protein sequence variation is unlikely to play a major role in normal color variation. 716 BioEssays 20.9 The P-locus and hypopigmentation Not all genes that cause clinically significant forms of hypopigmentation are members of the TYRP family. The most common form of albinism worldwide, tyrosinase-positive oculocutaneous albinism, is most often caused by mutations in a gene encoding a structural protein whose function remains poorly understood. As this was the second albinism gene to be identified, the locus was designated OCA2. The OCA2 locus maps to chromosome 15q11.2–12,(51) and the gene is the human homologue, P, of the mouse pink-eyed dilution locus, p.(52,53) Mutations at the p locus on mouse chromosome 7 are associated with a reduction in eumelanin synthesis, with apparently little effect on pheomelanin production. The P gene spans 250–600 kb, has 25 exons of which the first is noncoding, and codes for an 838-amino acid protein with 12 transmembrane domains.(53) Most functional studies on the P gene have been done in the mouse and, since the human P gene cDNA was shown to restore pigment production in mouse p null melanocyte cell lines, whereas mutant P gene cDNA did not,(54) the conclusions are probably applicable to both. The protein encoded by the P gene has been shown to be an integral component of the melanosomal membrane(55) and was originally thought to be a transporter of small molecules, possibly tyrosine, the precursor for melanin biosynthesis. A study by Gahl et al.,(56) however, suggested that it is unlikely to be a significant tyrosine transporter. The P protein has been shown to be part of a high-molecular-weight melanogenic complex, including TYR, TRP-1, and TRP-2, Review articles that is associated with the melanosomal membrane. The high-molecular-weight forms of TYR, TRP-1, and TRP-2 are absent in mice that do not express p, suggesting that the P protein may play a role in stabilizing the complex and in the regulation of the levels of expression of the TYRP gene family.(35,57) Mutations in the P gene are responsible for at least three different types of albinism—tyrosinase-positive oculocutaneous albinism (OCA2),(51–53) ocular albinism (OA),(58) and BOCA(59)—suggesting that different mutations alter the level of functioning of the P gene and the stability of the melanogenic complex (Fig. 3). A study on a large OCA2 pedigree of triracial origin demonstrated a common 2.7-kb deletion in the P gene,(60) and this mutation was found to be widespread throughout sub-Saharan Africa(61) and in African-Americans(62). Numerous other P gene mutations have been identified in individuals with OCA2, although none was present at high frequency.(58,62,63) During the search for mutations in the P gene, several apparently nonpathogenic variants were identified, and some of these had markedly different frequencies in different population groups. For example, R305W occurred at a frequency of 0.83 in caucasoids but at a frequency of only 0.10 in blacks.(63). If this is a functionally significant mutation, it may indicate that the P gene plays a role in normal pigment variation. The role of the P gene in normal pigment variation remains uncertain. A locus for brown eye color and brown hair color was linked to markers on 15q11–12, with the P gene the most likely candidate gene.(64) Skin reflectance tests on obligate OCA2 carriers, presumably with a mutation in one copy of the P gene, showed that they had significantly lighter skin pigmentation than individuals without a family history of albinism, with a more marked difference in males than in females.(65) Furthermore, individuals with Prader-Willi syndrome (PWS) and Angelman syndrome (AS) with deletions of 15q11–13, particularly deletions including part of the P gene, are often hypopigmented relative to other family members.(66,67) Since hemizygosity for the P gene in PWS patients is significantly correlated with hypopigmentation, the presence of functionally significant variants in the intact gene may account for the hypopigmented phenotype. A recent P gene haplotype study by Spritz et al.(68) in PWS patients showed many diverse haplotypes associated with hypopigmentation and suggested that the intact P allele may have reduced expression because of complex gene regulation in this chromosomal region or that it was due to the deletion of a second pigment locus close to the P gene. In BOCA, where one chromosome appears to have an ‘‘OCA2’’ mutation and the other chromosome an apparently ‘‘mild’’ mutation, only about 70% of patients have eye involvement.(47) Perhaps individuals with two mild mutations would fall within the normal range of pigmentation, but veer toward the lighter end of the spectrum. Only through the identification of functionally significant variants and analysis of their distribution in different populations would the role of the P gene in normal skin color determination become clearer. The melanocyte-stimulating hormone receptor Because of its striking character, red hair has featured as a primary focus of investigation among geneticists interested in human pigmentation traits. The Davenports(7) began a description of the inheritance of red hair color, and subsequent studies have investigated large numbers of family groups in an attempt to discover its mode of transmission. The description of red hair is recognized to include the hair colors that grade from very light strawberry blonde through carrot red to dark auburn(69,70); and it has also been noted that red hair may be present as beard and axillary hair in combination with scalp hair of another color. Furthermore, many red-haired children become brunettes as they grow older, or at the very least, the color darkens.(69,71) The red hair trait in most cases presents with fair skin and freckles or ephilides (72); freckles are not restricted to the red phenotype, as they may also be present in combination with other hair and skin types. Two investigators(69,73) have observed that a relatively high number of distinctly non-red-haired individuals have a small proportion of red scalp hairs when examined microscopically, although these investigators did not mention whether these cases were associated with freckled skin or with any red body hair. The inheritance pattern of red hair in a six-generation family was consistent with an autosomal recessive mode of inheritance,(74) although initial studies had suggested a more complex pattern.(9) More than a half-century ago, Neel(75) attempted to assess whether the mode of transmission of obviously visible red pigment within the scalp hair was recessive or dominant to other hair colors. Of 114 offspring from 26 couples in which both partners had red hair, 101 were redheads and 13 had hair of some other color. It is important to note that if red hair were transmitted as a simple recessive trait, all the offspring would exhibit the trait. The ratio of non-red-haired to red-haired offspring was still low, however, prompting the conclusion that red hair is dependent on a single incompletely recessive factor that is hypostatic to the factors determining black or dark brown hair color. Data obtained by Rife(71) supported this conclusion and provided evidence that red hair is inherited dominant to blonde. Mice with recessive mutations at the extension locus (e) show a yellow (pheomelanic) coat color, suggesting that loss of function at this locus was important in the switching between eumelanin and pheomelanin synthesis in the melanocyte. An understanding of how this gene was involved in directing melanin synthesis was established with the cloning of the extension gene and the finding that it encoded the mouse melanocyte-stimulating hormone receptor (Mc1r).(76,77) This finding led to the realization that human melanocyte- BioEssays 20.9 717 Review articles Switching of Melanin synthesis G T A R C L E V S I L L S NH A A L G L Q P I A T P T S N L S G L L R R Q S G Q V A M Q N S D F L G L L V N A L V A T ....L...... . .. .... ...... .N.. A L V A R A A G V A .. . ....L...... Q L E . ... ....M L . .... Q L L L I V D N V G A T E L D V I S V N S G S C S S V V L L L S S E D S L A C F L V L C C I A I A I F . .. A C .....E..... K Y N M R P N L H S . .. .....H..... 2 537insC Y D H V A I T M L G V D R Y I S Y A I T S V S W I V A F L F V A V A A R R A R P L . .... ...Q . .... ..... ..... . .. W .... ...... .T.. T V H P T C G C E I P C L V F I L T L N H L F F A P G W C C L I F F I G L L I I T V L L C L V V F F L A M L V L M A V L Y V H M L A R I S H Y I F Y A L R . .... ...Q . .... . .. ....C...... A C Q H A Q G I L T V K N F L F L L I I N A I D P L Y A F A G . .. H ....H...... S T C S .. .... Q K ...T.... E W L L L V G R E F R T L K G Q H COOH V A R L H K R Q R P G-protein activation EGF cAMP Pheomelanin TYR TRP1 EGF P, TRP1, TRP2 EGF EGF P-protein TYR TRP2 Eumelanin Figure 4. Diagrammatic structure of the consensus human MSHR protein is shown (blue) with the position of the variants(81) reported to be associated with red hair (red: R151C, R160W, D294H) or found in individuals but yet to be statistically associated with red hair (green), blonde or brown hair (yellow: V60L), with the two alleles common in Chinese individuals (black: R67Q, R163Q) and the base insertion reported in Fig. 5 (white: 537insC) indicated. Switching from the pheomelanogenic to the eumelanogenic pathway by receptor stimulated G-protein activation or the cAMP pathway is illustrated by the induction of the P, TRP-1, and TRP-2 proteins to form eumelanin. stimulating hormone receptor (MSHR) was a potential candidate for producing the red hair phenotype. The human MC1R gene encoding the MSHR protein has been localized to chromosome 16q24.3(78) and codes for a seven-transmembrane domain G-protein-coupled receptor belonging to the melanocortin receptor subfamily (Fig. 4). MSHR is highly expressed in melanocytes and interacts with a-MSH to stimulate pigment production. The first screen for MSHR variation in humans was performed in English and Irish individuals, identifying nine different amino acid substitutions that showed some correlation with red hair and fair skin.(79) Eleven of 38 redheads had 718 BioEssays 20.9 two variant MSHR alleles, 20 had only a single variant allele, and the remaining 7 failed to present any MSHR coding region variation, suggesting a relatively weak involvement of MSHR. This result indicates that secondary loci may produce red hair in the absence of MSHR changes in a significant proportion of cases,(80) a conclusion unforeseen by the historic model of inheritance that favored a single incompletely recessive genetic locus as the primary determinant of the distinctly red trait. A recent investigation of hair color in Caucasian twins(81) builds on the initial study of MSHR variation.(79) Twelve amino acid substitutions were identified (Fig. 4), with significant Review articles associations observed between the three variants R151C, R160W, D294H, and red hair, while a fourth variant, V60L, had a strong association with fair/blonde hair, perhaps indicating that the MSHR plays a role in both blonde and red hair. Twenty of the 25 red heads analyzed had two variant MSHR alleles, and the remaining 5 had only a single variant allele, a result appearing, in the absence of functional data for each variant, to be more consistent with the historic hypothesis. Apart from the V92M allele, the MSHR variant haplotypes were also significantly associated with lighter skin color in Caucasians; and two variants, R67Q and R163Q, were predominant in the Chinese population. As yet, no systematic population based study has been performed to assess MSHR variation between ethnic groups and its potential contribution to skin pigmentation differences. The observation that the MSHR gene is associated with different skin tones in Caucasians is reason to believe that it may have a major role in influencing pigmentation within other populations. However, it has been shown that gene variation within a population can in some circumstances be greater than that between populations,(82) and MSHR variation may be one such example. Another result that sheds light on the extent of the genetic interactions that underlie red hair is the observation that 5 of 13 sets of dizygotic red-haired/non-red-haired twins share identical MSHR genotypes. All five are most likely to be identical by descent for this gene, suggesting that MSHR variation is necessary but not sufficient to generate red hair. As stated above, the red hair trait is not a simple biological phenomenon, appearing in the fully developed form with red scalp hair or an incomplete form exhibiting freckles and red axillary hair or perhaps freckles alone.(72) The interplay between different strength alleles of the MSHR locus and those of a masking genetic background is a possible mechanism controlling the expressivity of the red hair phenotype. The genetic background is potentially responsible for determining the shade of red hair that is inherited, although possible association between MSHR alleles and subtype of red hair is yet to be addressed. Differential expression of any MSHR promoter variants may also explain the occurrence of nonred scalp hair in combination with red body hair and freckles. Of further interest is the report by Neel(75) that two red-haired parents may occasionally produce non-red-haired offspring, a situation now explainable by inheritance of a newly identified MSHR null allele. Figure 5 shows the MSHR genotypes of a family where two red haired parents have produced a red and a blonde daughter. The father is heterozygous for a unique 537insC variant that results in a frameshift and premature stop 58 amino acids later. It is significant that this insertion is very close to the original recessive extension deletion, which produces a truncated and inactive MSHR and therefore the classic mouse extension phenotype.(83) It is probable that 537insC also produces a null allele and that red 1000710 V60L R151C R151C 537insC V60L V60L R151C 537insC Figure 5. Inheritance of MSHR haplotyes in red- and blondehaired children of two red-haired parents. hair in the father is directed by the common R151C variant. The mother also has an R151C as well as a V60L allele. It has been suggested that red may be dominant to blonde; indeed, in the mother, R151C directs the appearance of red hair in a dominant fashion over the V60L allele, which has previously been associated with blonde hair. This situation is duplicated in the first girl, who has red hair, but her younger sibling, who has received V60L from her mother and the null allele from her father exhibits blonde hair, reinforcing the association of V60L to blonde hair and providing a possible molecular explanation for this curious situation. However, as already described in the analysis of the dizygotic red-haired/non-redhaired twins, other genetic factors besides MSHR could determine hair color in this family; further inheritance studies of the 537insC allele are needed to confirm this interpretation. All MSHR variants that have been identified to date produce amino acid substitutions, this being the first report of a null allele. It will be interesting to observe the phenotype of an individual carrying two null MSHR alleles. BioEssays 20.9 719 Review articles Conclusions: what makes people different? The wide variety of pigment phenotypes seen in human populations prompts the question of whether there is likely to have been selection for skin color. Most of the Earth is populated with more darkly pigmented peoples, with a striking northern European localization of more lightly pigmented peoples.(84) One might argue in favor of selection for darkerskinned individuals who are better protected from the harmful effects of ultraviolet (UV) irradiation, but perhaps this was the ancestral state. A more likely scenario is that mutations that arose for lighter skin color have been selected for in individuals with poor dietary vitamin D intake and little exposure to the sun. Natural selection, although a possible driving force through latitudinal variation in sunlight, may not readily apply to humankind, which can so easily alter its environment and behavior, and where other factors are more important in choosing partners. Advances in the study of human pigmentation have only now come of age as a consequence of using a comparative genomic approach to understand this complex biological system. Three well-conserved gene systems—TYRP, P, and MSHR— have been used to illustrate how this has applied to the study of pigmentation. However, when considering mammalian pigmentation in general, humans are without an outer coat of body hair and are somewhat unique, in that the melanocyte sits at the dermal–epidermal junction secreting melanin particles into fully exposed cutaneous keratinocytes. This arrangement does not generally occur in other animals; in the mouse, for instance, the melanocytes are located predominantly in the dermal compartment. Although major advances have been made in identifying pigmentation genes it may be expected that not all the genes influencing pigmentation in humans will be found through the use of a comparative genomics approach because of this fundamental biological difference. Genetic studies of human populations and family groups are still required to identify and confirm the role of pigmentation genes. Variation in human skin color is clearly a multifactorial trait with a number of major gene determinants, several modifier genes, and environmental influences such as exposure to UV irradiation and gender effects. Our current understanding suggests that protein sequence variation in the catalytic enzymes tyrosinase, TRP-1, and TRP-2 that are active in the melanin biosynthetic pathway is not a major determinant of pigmentary differences, with very few polymorphisms showing marked differences between population groups. MSHR appears to play a role in the level of expression of these enzymes and the P gene seems to be essential in stabilising the melanogenic complex within the melanosome. Since the major histological difference between heavily and lightly pigmented individuals seems to be the packaging and size of the melanosomes in the keratinocytes, one would perhaps expect genes that are involved with organelle membrane 720 BioEssays 20.9 structure and integrity to be important determinants in skin color variation. Thus, the variation in MSHR and the P-gene coding regions are the two most obvious determinants of skin type and hair color. A consequence of this variation is the regulation of the levels and activities of the tyrosinase, TRP-1, and TRP-2 proteins. In essence, the difference is only skin deep. Acknowledgment The authors thank Professor Jennifer Kromberg for providing photographs of albino patients. We apologize to those authors whose papers we could not cite due to space restrictions. 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