Gene polymorphisms and their effects in the melanocortin system

Peptides 26 (2005) 1871–1885

Gene polymorphisms and their effects in the melanocortin system Levi Carroll, Joanne Voisey, Angela van Daal ∗ CRC for Diagnostics, Level 5, Q Block, Queensland University of Technology, 2 George St, Brisbane 4000, Australia Received 1 July 2004; accepted 8 December 2004 Available online 23 June 2005

Abstract In addition to its role in human pigmentation, components of the melanocortin system regulate appetite, energy homeostasis and hormone production. Recent studies have suggested possible roles of this system in immunity, transmission of pain signals, and reproductive potential. A number of polymorphisms have been identified in genes of the melanocortin system and are associated with pigmentation in humans, as well as being causative of disorders of adrenal hormone production and obesity. This review gives an outline of these polymorphisms, their functional significance and possible application to or impact on diagnosis and pharmacotherapy based on melanocortin pathways. © 2005 Elsevier Inc. All rights reserved. Keywords: Polymorphism; SNP; Agouti; Melanocortin

1. Introduction Polymorphisms are small, sequence variations at a genetic locus found at varying frequencies in a population. Some single nucleotide polymorphisms (SNPs) are found within the coding region of a gene and hence may result in changes to the primary amino acid sequence of the encoded protein. These changes can lead to altered function of the protein, such as different substrate binding affinities or enzymatic activities. The majority of SNPs lie within non-coding regions of genes, namely in introns, promoters, or intergenic regions, and often appear to have little functional impact on the activity of a gene. However, SNPs that lie within the proximal promoter or enhancer regions of a gene can potentially alter gene expression at the transcriptional level. SNPs within both coding and non-coding regions have been statistically associated with a wide range of physical characteristics, such as hair or eye color, as well as mental disorders including anorexia nervosa and schizophrenia. The sequencing of the human genome has led to the creation of many useful databases including several that archive SNP data, such as dbSNP (http://www.ncbi.nih.gov/SNP), the SNP consortium ∗

Corresponding author. Tel.: +61 7 3864 2502; fax: +61 7 3864 1534. E-mail address: [email protected] (A. van Daal).

0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.12.031

(http://snp.cshl.org), HGVbase (http://hgvbase.cgb.ki.se), and the human genome mutation database (http://archive. uwcm.ac.uk/uwcm/mg/hgmd0.html). This review will present findings of studies that examine SNPs in genes of the melanocortin system and their potential effects on human traits and physiology. The melanocortin system is composed of a group of ligands derived from the pro-opiomelanocortin (POMC) precursor protein, their receptors and two antagonists. Several accessory proteins have also been identified in this system, but their roles are yet to be clarified. ␣-Melanocyte stimulating hormone (␣-MSH), ␤-MSH, ␥-MSH, and adrenocorticotropic hormone (ACTH), are the four major ligands which activate melanocortin receptors. Tissue-specific expression of these paracrine hormones is determined by levels of expression of the POMC gene [69,97] along with expression of prohormone convertases 1 and 2 (PC1 and 2), which cleave the POMC protein into its corresponding active forms [172]. Five melanocortin receptors have been identified, with between 60 and 80% protein sequence homology. They show varying tissue distributions and are members of the seven-transmembrane domain rhodopsin-like G-protein coupled receptor family [18]. Binding of the melanocortin ligands to these receptors generally results in release of stimulatory G-proteins, which activate adenylate cyclase.

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The resulting increase in cellular cyclic AMP (cAMP) is the major intracellular signaling pathway utilized by these receptors, although a range of other pathways appears to respond to melanocortin receptor/ligand interactions [13,31,58,63,64]. The two currently known antagonists of these receptors, agouti signaling protein (ASIP) and agoutirelated protein (AGRP), seem to prevent agonist-induced G-protein release and the subsequent production of cAMP by competitively binding to the receptors [75]. However, some studies have indicated that these proteins may act as inverse agonists in that they have functional effects even in the absence of melanocortin ligands [15,42,122]. These include effects on cAMP signaling apart from G-protein activation of adenylate cyclase and receptor internalization. Besides the traditional members of the melanocortin system, i.e. the ligands and their receptors, the membrane-bound heparan sulfate proteoglycans syndecan-1 and -3 have been implicated in facilitation of ASIP and AGRP binding to melanocortin receptors, in addition to attractin (ATRN) and attractin-like protein (ALP). Functional studies have revealed little about the mechanisms of these interactions, but suggest they are required for full “antagonism” of the melanocortin receptors by ASIP or AGRP [6,40,110]. A number of functions have been ascribed to the melanocortin system, including roles in pigmentation, steroid hormone production, and weight regulation. Many of these are conserved among mammalian species. ␣-MSH and ACTH are major regulators of skin and hair pigmentation, due to their interaction with melanocortin-1 receptors (MC1R) present on epidermal and follicular melanocytes [2]. Activation of MC1 receptors causes synthesis of eumelanin, the dark brown or black pigment, and is the main mechanism of UVinduced tanning of skin. Antagonism of melanocyte MC1R by agouti signaling protein in the mouse leads to production of light yellow or orange pheomelanin, resulting in a yellow or orange coat [75]. ACTH released by the anterior pituitary is the main stimulating signal in the hypothalamic-pituitaryadrenal (HPA) axis, in that it directly regulates production of steroid hormones (cortisol, androgens and estrogens) from the adrenal cortex by activation of the MC2 receptor and subsequent release of intracellular stores of cholesterol and upregulation of steroidogenic hydroxylases [121]. ␣-MSH also plays a significant role in weight regulation pathways. In the paraventricular nuclei of the hypothalamus, MC3 and MC4 receptors are activated by ␣-MSH secreted by arcuatenucleus-derived neurons in response to the adipostatic hormone leptin. Activation of these receptors leads to inhibition of appetite and increases in basal metabolic rate. Concordantly, agouti-related protein, expressed in the absence of leptin, antagonizes ␣-MSH-induced activation of these receptors, leading to hyperphagia and decreased metabolic activity [103]. Recent work on rats has also demonstrated a potential role for MC4 receptors in spinal transmission of pain signals [126], whereby intraspinal ␣-MSH administration leads to hyperalgesia, whereas injection of AGRP attenuates algesic responses. MC1 receptors have additionally been identified

in cells of the immune system [3,116,130,134] and ␣-MSH interaction with these cells has been seen to down-regulate expression of the immune response-stimulating transcription factor NF-␬B [78,93]. Several studies have shown that in vivo administration of ␣-MSH has potent anti-inflammatory effects, suggesting that therapeutic agents based on this molecule may be useful in the treatment of inflammatory diseases [169].

2. Pigmentation Human pigmentation varies greatly between individuals and particularly racial groups, because of the size, shape, density and type of melanin. Melanin is synthesized by a pathway that results in either pheomelanin (red/yellow pigment) or eumelanin (black/brown pigment) depending on the presence or absence of thiol compounds. In mammals a ratio of eumelanin: pheomelanin of greater than one results in brown or black hair and a ratio less than one produces red or yellow hair. In the mouse, agouti, which encodes ASIP and extension, which encodes MC1R, regulate the type of melanin produced by signaling through ␣-MSH (reviewed in [53]). ASIP antagonizes the binding of ␣-MSH to MC1R, switching the pigment pathway from eumelanin to pheomelanin synthesis. ␣-MSH is known to increase pigment production in mice and humans but it is still not clear whether ␣-MSH is needed for receptor activity in humans, although human melanocytes treated with ␣-MSH increase in proliferation and melanogenesis [50,51]. The phenotype of human POMC mutations indicates that ␣-MSH or ACTH is required for MC1R signaling in humans [65]. 2.1. MC1R The human MC1R gene encodes a 317 amino acid Gcoupled receptor [19,92] and ASIP encodes the 132 amino acid agouti signal protein. ASIP consists of a signal peptide with a glycosylation site, a basic domain and a cysteine rich C terminus [68,161]. The ASIP gene has been mapped and isolated in a number of species. It has been demonstrated to play a role in coat color determination in mouse (reviewed in [149]), sheep [105,106], standard silver fox [139] and rat [67] and has also been isolated in pig [71,152]. A role for MC1R in coat-color determination has also been identified in a number of species including mice [111], cattle [61], horses [79], pigs [60], sheep [138] and dogs [98]. MC1R has been strongly associated with normal hair and skin color variation in humans. Variants of the MC1R gene are associated with lighter skin types and red hair [11,34,41,56,143]. Red haired individuals are usually compound heterozygotes or homozygous for one of a number of MC1R polymorphisms (R151C, R160W, D294H, R142H, 86insA, and 537insC) [11,34,143] and homozygous individuals are more likely to have auburn or strawberry blonde

L. Carroll et al. / Peptides 26 (2005) 1871–1885 Table 1 MC1R variants associated with red hair in Caucasians Variant

Allele frequency in Caucasians

Reference

I40T V122M R142H R151C R160W D294H

0.04 0.06 0.004; 0.006; 0.008 0.012; 0.048; 0.099; 0.11 0.071; 0.085; 0.087; 0.11 0.008; 0.028; 0.034; 0.036; 0.11

[55] [55] [7,27,34] [7,27,34,41] [7,27,34,41] [7,27,34,41,143]

hair [34]. Two further MC1R variants I40T and V122M have also been associated with fair skin and hair color, and bind to ␣-MSH with lower affinity and produce less cAMP compared to the wild-type [55]. Interestingly, there are numerous non-synonymous MC1R SNPs associated with the red hair phenotype and the population frequencies these polymorphisms in Caucasians are quite high, with frequencies of about 11% for each of the three common polymorphisms (see Table 1). The involvement of another locus in the inheritance of red hair is evidenced by the existence of redheads carrying no MC1R variants [34,143] and dizygotic twin pairs that are concordant for MC1R variation, but discordant for hair color [11]. It may be that human variation in hair and skin pigmentation results from a combination of ASIP and MC1R alleles, as is the case for the German shepherd dog [73], with some MC1R variants interacting differently with ASIP, resulting in different ratios of eumelanin and pheomelanin production. Not surprisingly, MC1R genotyping in humans has identified variants that are associated with susceptibility to basal and squamous cell carcinoma [56,104]. Since MC1R signaling is associated with darker melanin production, a loss of MC1R function would be predicted to result in lower photo-protection. Individuals carrying polymorphisms R151C, R160W, and D294H have more than a two-fold increase in melanoma risk [104] and melanoma cells transfected with variant MC1R sequences (R151C, R160W, and D294H) have altered melanoma growth, providing further evidence that individuals with MC1R variants have increased risk of melanoma [112]. In addition, a recent study has determined that melanocytes with altered MC1R have a significant increase in UV radiation sensitivity [119]. Individuals carrying functional MC1R variants are therefore likely to have an increased risk of skin cancer. No single MC1R allele has been identified that renders MC1R completely non-functional. It is likely that two alleles together result in diminished MC1R activity. A bacterial artificial chromosome (BAC) containing the human MC1R sequence, as well as three MC1R variant sequences (R151C, R160W and D294H) were used to rescue loss of function MC1R transgenic mice [43]. Human MC1R sequence completely rescued MC1R deficient mice. The variant MC1R sequences showed reduced, but not completely absent, function in transgenic mice, suggesting that these three alleles alter melanin synthesis in humans but are not complete

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loss-of-function mutations. Transfection and binding studies were also performed to look at the functional significance of the V60L, R142H, R151C, A160W and D294H variants [118]. All variant receptors showed reduction in cAMP production in response to ␣-MSH stimulation. None of the mutant receptors displayed complete loss of ␣-MSH binding, with the R142H and D294H mutants displaying only a slight reduction in binding affinity. To analyze the effects of MC1R variation in a black population the MC1R sequence was analyzed in four Jamaican redheads [83]. As well as polymorphisms that have been previously identified as associated with red hair and fair skin several novel SNPs were identified. Each individual was a MC1R compound heterozygote (G89R/R151C; 23X/R160W; D294H/frameshift195; 23X/R151C). From this study, it is evident that MC1R affects hair color even on a genetic background that results in otherwise very dark pigmentation. The effects of MC1R seem to be greater on hair than skin since these individuals have red hair but not far skin [109]. A small percentage of Australian Aboriginals also have the unusual red-hair and dark skin phenotype [1] and analysis of MC1R genotypes may also prove interesting in this population. MC1R polymorphisms identified to date have all been loss of function mutations. Analysis of darker-skinned populations may reveal gain of function MC1R polymorphisms. 2.2. ASIP Since an agouti banded pattern is not characteristic of human hair there is doubt as to whether ASIP functions in human pigmentation. A 3 UTR polymorphism 25 nucleotides downstream from the stop codon was found at a higher frequency in an African-American population than a Caucasian population [148]. This polymorphism was shown to occur at a frequency of 0.12 in a population of 800 Caucasians and was found to be significantly associated with dark hair and eye color [57]. A more recent study has confirmed that darker-skinned populations have a higher frequency of this polymorphism [171]. Not surprisingly the highest frequency was reported in a West African population (0.80) followed by African–Americans (0.62). The polymorphism was seen at a much lower frequency in an East Asian population sample (0.28). Functional studies are required to determine whether this polymorphism is a significant factor in the production of eumelanin in humans. Although the polymorphism identified is not coding and as such does not result in an amino acid substitution, the 3 UTR of transcripts have been shown to be important in stability of messenger RNA [22,37,165]. The A to G transition may destabilize the ASIP mRNA, leading to premature degradation of the transcript. Decreased levels of ASIP would result in decreased antagonism of ␣-MSH signaling through MC1R and therefore increased production of eumelanin. Association studies do suggest that ASIP plays a part in human pigmentation.

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3. Hypothalamic-pituitary-adrenal axis

Table 2 Mutations found in MC2R

ACTH and the melanocortin-2 receptor play a major role in the production of steroid hormones from the adrenal cortex. Corticotrophs of the pituitary secrete ACTH under stimulation of corticotropin-releasing hormone (CRH) from the hypothalamus. Binding of ACTH to its receptor in the zona fasciculata within the adrenal cortex leads to release of cortisol, though cells of the zona reticularis also respond to ACTH by secreting androstenedione [170]. Cortisol acts by negative feedback on regulatory neurons in the hypothalamus, decreasing release of CRH and preventing over-secretion of adrenal hormones. The role cortisol plays in stress responses has been studied extensively. This steroid is secreted during times of stress and stimulates gluconeogenesis, leading to higher blood glucose levels [59]. Continually high levels of cortisol secretion are associated with fat deposition and non-insulin dependent diabetes mellitus. High blood cortisol levels have also been associated with a subset of clinically depressed patients [146], perhaps an indicator of impaired stress response mechanisms. Cortisol has potent anti-inflammatory and anti-immune functions as well, and related glucocorticoids are currently used in treatment of inflammatory diseases, including some forms of asthma and acute respiratory distress syndrome [84].

Mutation

References

C21R I44M S74I D103N D107N F119fs S120R R128C R137W V142L R146H T159K L192fs R201X G217fs A233P S247G C251F Y254C P273H F278C

[48] [30,156] [20,29,30,35,120,155,156,163] [8,29,30,52,107] [94] [29] [107,136] [30,156] [35,52] [8,107] [30,125,156] [29,30,120] [156] [136] [94] [107] [48] [94] [35,137] [163] [128]

3.1. MC2R Familial glucocorticoid deficiency (FGD) is an inherited disorder characterized by neonatal hyperpigmentation of the skin, failure to thrive, weakness and fatigue. This disorder presents mainly with low to undetectable cortisol levels in serum combined with very high ACTH. Serum aldosterone levels are normal, indicating that the renin pathway is unaffected. Often tall stature is also seen in patients with this syndrome. Mutations in the MC2 receptor gene (MC2R) are responsible for about 50% of FGD cases, and all homozygotes of these mutations appear to have the disease in some form. Some mutations have been found in multiple unrelated families, such as S74I, R146H, T159K, V142L, and D103N, though the remainder have only been seen in single cases and as such are extremely rare (see Table 2). All of these mutations are caused by single nucleotide changes, suggesting that they arose as spontaneous mutations. Many are inherited from heterozygous parents, so often it is unclear how recently they arose. Cellular transfection studies have examined responsiveness of mutant MC2 receptors to ACTH in a variety of cell lines, and indicate that ACTH resistance is usually a result of reduced affinity for the ligand, reduced activation of intracellular signaling, or a combination of both [30,35,52,94,107,128,136,137,155,156,163]. F278C actually results in an ACTH-independent Cushing’s syndrome, in that MC2 receptors are not desensitized or internalized following ligand interactions, leading to apparent constitutive activity [128]. Another interesting proband, a Japanese compound heterozygote patient, presented ACTH

The majority is only found in FGD cases and is as such extremely rare.

hypersensitivity [48], with low to non-detectable serum ACTH but normal cortisol levels. The two novel mutations in this case were C21R and S247G. Functional work on these receptors indicates that they are not constitutive, but are extremely sensitive to ligand interactions. A more recent study on these mutations and this patient found that they are present as a haplotype. Transfection assays of MC2R constructs for each individual mutation found they were both unable to bind ligand or produce signal, but had high constitutive activity when both were present in the same molecule [129]. The relevance of polymorphisms in the MC2R gene to normal populations is questionable. Studies have not examined the frequency of non-synonymous SNPs in the MC2R gene, so the frequency of carriers of FGD is unknown. Furthermore, many cases of FGD have been identified where no mutations have been detected in the MC2R gene [95,131,153,154,163,166]. 3.2. POMC Mutations in the ACTH precursor gene proopiomelanocortin (POMC) can also lead to adrenal insufficiency syndromes. Two patients with symptoms of obesity, red hair, fair skin, and adrenal insufficiency have been identified, caused by mutations of the coding sequence of the POMC gene [65]. One of the probands was a compound heterozygote for a G → T substitution, resulting in a nonsense mutation at codon 97, and for a frameshift mutation in codon 83, which also results in truncation of the mature protein. Both of these mutations prevent translation of the ACTH/␣-MSH domain of the pro-opiomelanocortin peptide. The second, unrelated, patient was homozygous for

L. Carroll et al. / Peptides 26 (2005) 1871–1885

a C → A transition 11 base pairs upstream from the initiating ATG codon of the POMC gene. This produces a novel ATG codon, out of frame with the normal POMC sequence, and is an example of upstream translation initiation site utilization preventing normal translation of the protein. The phenotype of both probands was quite extreme, with close to zero detectable ACTH or adrenal steroids present in serum. More recently, three new European patients with null allele mutations of the POMC gene were identified by recognition of this set of symptoms [66]. The mutations found include the previously identified C → A transition upstream of the ATG, a novel K51X mutation, a frameshift in codon 92 and a frameshift mutation in codon 127. All of these mutations lead to truncation of the mature protein, with the ACTH/MSH ligand sequence remaining untranslated. Interestingly, attempted compensation by nasal administration of ACTH4-10, a potent agonist of melanocortin receptors, had no effect on weight regulation in two of the three patients. These mutations highlight the importance of POMC-derived peptides, in particular ACTH and ␣-MSH, in weight regulation, skin and hair pigmentation, and regulation of adrenal hormones. However, they are extremely rare and are unlikely to play significant roles in normal population variation.

4. Weight regulation Melanocortin-3 and -4 receptors (MC3R, MC4R) are highly expressed on neurons in specific areas of the brain, including the ventromedial, arcuate and paraventricular nuclei of the hypothalamus [74,113]. Neurons in these regions regulate appetite and basal metabolic activity, by mediating signaling of the adipostatic hormone leptin. Leptin stimulates expression of POMC and ␣-MSH interaction with MC3 and MC4 receptors on arcuate neurons leads to inhibition of appetite and a general increase in metabolic activity [88]. Conversely, antagonism of these receptors by agoutirelated protein, which is downregulated by leptin, results in hyperphagia and decreased energy expenditure [89]. As such, MC3 and MC4 receptors in the hypothalamus act as molecular switches of weight regulation pathways. Because of their apparent importance in weight regulation pathways, the genes for POMC, AGRP, MC3R, and MC4R have been screened for variation in the coding regions of obese patients and controls. 4.1. POMC Null mutations in the POMC gene have been associated with obesity, hypopigmentation, and adrenal insufficiency [65]. However, these deleterious mutations appear to be rare. More frequent polymorphisms have been identified, though their affects on function are not as extreme. Single, double and triple 9 bp insertions have been found in varying populations, resulting in single or multiple SSG insertions

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into the mature POMC protein at codon 73 [45]. An insertion of 6 bp at codon 176, encoding RA, has also been found, and is linked to an L180X mutation, which prevents translation of ␤-MSH and ␤-endorphin. E188G, D90N, R236Q, R236G and several silent polymorphisms have also been identified [17,28,87]. Some studies have not found any SNPs in the POMC gene, highlighting their rarity [26,102]. No significant association has been seen between any of the polymorphisms identified and body mass index (BMI), although a screen of adult Swedish obesity patients found association between a homozygous 8246C → T transition in the 3 UTR and leptin levels in lean individuals [127]. A second Swedish study found the relatively common 9 bp insertion to be associated with higher leptin concentrations in serum in lean adults. However, because polymorphisms in POMC are rare, they are unlikely to be linked to a significant proportion of obesity cases. 4.2. MC4R Many mutations have been identified in the human MC4R gene, with some studies reporting up to 4% of early-onset obesity cases being caused by missense or nonsense mutations in this gene. MC4R was first identified as a candidate gene for obesity based on its elucidated function in the hypothalamic appetite-regulating neurons. Initial linkage analysis of the genomic region containing the MC4R gene in the Quebec Family Study found an association with fat mass and percent body fat [14]. One of the first mutations reported in the MC4R coding region was a 4 bp insertion at codon 244, resulting in a frameshift and subsequent truncation of the protein [141]. This mutation was found in obese members of a single family in heterozygous form only, suggesting that MC4R haploinsufficiency has an effect on weight. A second frameshift mutation has been found in codon 211, caused by a deletion of four base pairs [167], and also appeared to be dominant. In vitro assays found both truncated variants are unable to activate intracellular signaling and did not bind the high affinity NDP-MSH ligand [49]. This study also found that the frameshift mutants were not transported correctly to the cell surface, and so could not interact with ligands nor activate intracellular pathways. Further transfection experiments found that specific deletion of the cytoplasmic tail of the MC4R protein was responsible for prevention of cellsurface expression. Co-expression of either frameshift mutant with the wildtype MC4R gene did not appear to affect the ability of the normal protein to respond to NDP-MSH ligand, so it is likely that obesity seen in heterozygotes of these mutations are due to haploinsufficiency, rather than deleterious dimerisation between normal and mutant proteins. A separate study examined subjects with a heterozygous chromosomal deletion of 18q covering the MC4R gene and found no correlation between deletion and obesity, suggesting that the MC4R gene is not haploinsufficient, and mutations in the heterozygous form have a dominant negative effect [21]. As such, it is hard to determine the role of haplosufficiency

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in MC4R-associated obesity. Furthermore, a recent study examined the possibility of functional dimerisation between normal and mutant MC4 receptor molecules [9]. In particular, co-transfection studies of the D90N mutant and normal MC4R found decreased receptor activity in comparison to transfection of normal MC4R. Further, sandwich ELISA and fluorescence resonance energy transfer studies revealed that normal and D90N receptors form homodimers and heterodimers, which could potentially affect receptor activation and signaling. This would explain the dominant negative affect seen with some MC4R mutations. However, it raises the question of whether normal MC4R molecules need to form dimers in order to function properly. This has yet to be examined. Over 50 additional missense polymorphisms have been found in the MC4R gene of obese patients and control individuals (Table 3). V103I appears to be the most common MC4R variant, and a mega-analysis of more than 7000 individuals found a negative association (p = 0.03) between the I103 variant and obesity. However, functional analyses of the receptor showed no difference in function between the V and I variants. It is possible that the negative dominant affect seen statistically is due to other mutations in linkage disequilibrium with the V103I polymorphism [38]. Another common variant, I251L, is also a conservative substitution and is found at a frequency of around 2%. Other missense polymorphisms not associated with obesity include T112M and V253I. Most of the mutations in MC4R have only been found in obese patients, and are presumed to be linked to the patients’ phenotypes (Table 3). Most variants of MC4R are found at very low frequencies, which are likely to be an overestimate because of the small sample sizes in most studies. It is tempting to use these data to determine a likely percentage of obesity cases caused by MC4R mutations, but the rarity of the SNPs and the possibility of overlap of each study’s sample groups make this not feasible. Furthermore, differing definitions of morbid obesity and BMI cut-offs for each study make it difficult to determine what proportion of obese people these studies represent. Missense mutations of MC4R can affect receptor function in several ways. Alteration of amino-acids in the ligand binding site can change the affinity of MC4R for the melanocortin ligands. N97D, L106P, I125K, and I316S all appear to have deleterious affects only on ligand–receptor interactions [168], suggesting that these residues form part of ligand-binding regions. Previous studies have identified D122 as the main ligand-binding residue, but F284 and F261 have also been found to interact with MSH ligands [99]. A high proportion of MC4R mutations lead to intracellular retention of receptor molecules [77,168], preventing expression of binding sites on the cell surface, and hence appear to be a reduction in binding affinity for melanocortins. Recent studies describing novel MC4R mutations have examined cell surface display of receptors using immunofluorescence microscopy or fluorescent antigen cell sorting [9,77,100,132,168]. The majority of mutations that cause retention of receptor molecules in the

endoplasmic reticulum are found in the C-terminal region of the receptor (see Fig. 1 and Table 3). Interestingly, a transmembrane protein with binding affinity for the intracellular tail (amino acids 303–332) of MC4R was recently identified by yeast-two hybrid screen [40]. This novel protein shares 63% amino acid similarity to the previously identified melanocortin accessory protein attractin and has been termed attractin-like protein. This study found that residues 303-313 in MC4R and 1280-1317 in ALP are required for interaction, and it is likely that mutation of any of these residues would lead to impaired MC4R function. The fact that deletion of the intracellular tail of MC4R has a high impact on the cell surface expression of the receptor suggests that perhaps ALP binding to this domain is required for proper intracellular transport to the cell membrane. However, no mutations or SNPs have been found in this region of MC4R. Finally, some mutations (D90N and I170V) appear to affect G-protein activation and signaling directly, but not binding affinity or cell surface expression [9,100]. The mechanisms of this have not been fully elucidated, but are likely to involve a reduction of binding between intracellular loops of the MC4R and G-proteins. From the data currently available it would seem reasonable to suggest that MC4R mutations are causative factors in a significant proportion of morbid obesity cases [54], but their role in mild obesity and normal weight regulation is likely to be negligible due to lack of mutations in these individuals. Cell transfection studies have revealed that MC4R variants can have a range of effects on MC4 receptor activity, from no effect through to varying levels of loss of binding affinity, signaling, and cell surface expression. It would be difficult to predict obesity outcomes based on MC4R mutations, unless they were clear null alleles. A further complication is the possibility of dimerisation between mutant MC4R proteins and the wildtype receptor, which may explain lack of haploinsufficiency seen in heterozygous chromosomal deletions of the MC4R gene, and the current unpredictability of the effect of such interactions on receptor signaling. 4.3. MC3R Several SNPs in the MC3 receptor (MC3R) gene coding region have been identified, but none have been associated strongly with obesity. An I81V missense polymorphism, in complete linkage disequilibrium with a K6T polymorphism, has been found in two studies, with a heterozygote frequency of 0.12–0.15 for both obese and normal weight Caucasian groups [10,39,72,117]. African–American populations have heterozygote frequencies of around 0.50 in both obese and normal weight samples. This missense variant is unlikely to affect MC3 receptor function, but does show significant frequency variation between racial groups. One study found a novel I183N mutation in an obese proband and her father that has not been found in any other individuals, and so appears to be a rare mutation that influences weight regulation [70]. Other polymorphisms found outside the coding region of

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Table 3 MC4R variants associated with obesity (A) and non-functional polymorphisms (B) Mutation

(A) Obesity T11A T11S W16X codon16ins(G) R18C S30F Y35X + D37V D37V P48S V50M F51L S58C N62S L64X P78L Codon88-92del D90N S94R V95I N97D G98R I102S I102T L106P Codon112ins(A) I121T I125K S127L I137T Codon148del (4 bp) T150I Y157S R165W R165Q I169S I170V A175T G181D M200V Codon211del(CTCT) I226T P230L A244E Codon244ins(GATT) L250Q Codon250del (2 bp) G252S G255S C271Y C271R N274S Codon279ins(GT) Y287X P299H I301T I316S I317T Codon320del(T)

Cellular retention

Binding affinity

Null Null Yes

Normal

No No Yes Yes Yes

Null

Yes No No

Null Null WT

No

Low

Yes No Yes

Low

Yes No Yes Yes

Low

Yes Yes Yes No No

WT

WT WT WT WT

Yes

No Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes No Yes

Low

Half WT Low Low Low

Signaling

Heterozygote frequency

References

Obese

Normal

0.0021 0.0048 0.0063 0.0048 0.0048 0.0027 0.0067

0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000

0.0048 0.0159 0.0021 0.0159 0.0060 0.0046 0.0018 0.0042 0.0050

0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000

0.0012 0.0040

0.0000 0.0000

0.0159

0.0000

0.0020 0.0012 0.0040 0.0027

0.0000 0.0000 0.0000 0.0000

0.0048

0.0000

0.0028 0.0027

0.0000 0.0000 0.0000 0.0000

Null

0.0074 0.0020 0.0012 0.0043 0.0025

0.0000 0.0000

High Partial Low High Null Normal

0.0012 0.0012 0.0012 0.0048 0.0012 0.0018

0.0000 0.0000 0.0000 0.0000 0.0000 0.0000

Null None Normal Null Null Null Partial Low Partial Partial

0.0027 0.0040 0.0250 0.0054 0.0040

0.0000 0.0000 0.0000 0.0000 0.0000

0.0048 0.0059 0.0018 0.0010

0.0000 0.0000 0.0000 0.0000

Partial Normal Null Null Normal Low Null Normal Normal Partial Partial Low Null Low Low Low Null Null None Null Null Null Partial Null Low Partial None Low Partial Low Low Normal Partial Partial Null

[12,32] [140] [81] [140] [140] [46,47,77,100] [44,46,47] [132] [86,132] [26,77,132] [12] [26,77,132] [32,33,77,132,168] [54] [46,47,77,100,132] [24] [9] [46] [46] [32,168] [62,132] [26,77] [54] [168] [32,168] [46] [32,168] [46,77,142] [100] [168] [140] [132] [12,46,47,77,100] [32,33,100,168] [49] [26,77,100,132,140] [32,168] [46] [12] [32,46,47,49,123] [142] [46] [46,77] [49,141] [100,140] [46] [46,47,100] [77] [32,33,77,132,168] [32,133] [85,132] [32,33] [32,168] [77] [100,140] [32,168] [46,47,77,100,145] [46]

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Table 3 ( Continued ) Mutation

Cellular retention

Binding affinity

(B) Non-functional polymorphism V103I No WT T112M No Half WT T178M F202L N240S I251L No V253I No WT

Signaling

Normal Normal Normal

Normal Partial

Heterozygote frequency Obese

Normal

0.0210 0.0043

0.0350 0.0046 0.0031 0.0038 0.0038 0.0212 0.0065

0.0000 0.0000 0.0146 0.0041

MC3R have shown no correlation with weight phenotypes [72,117]. These results indicate that variation in MC3R is an uncommon contribution to development of obesity, despite its apparent role in weight regulation. 4.4. AGRP Very few SNPs in the AGRP gene have been identified. The most common variant is A67T, which has been found with varying frequencies in different racial populations. This polymorphism is found at low frequency in Hispanics [80], not at all in African–American populations [4] and with heterozygote frequencies of up to 10% in Caucasians. A significant correlation between heterozygosity of the A67T

References

[12,26,33,38,44,46,47,49,54,81,86,101,115,140,142] [12,33,46,47,54,100,142] [46] [54] [54] [12,26,33,44,47,54,81,140] [33,77,81,100,168]

allele and anorexia nervosa suggests that this missense change may affect AGRP protein function and lead to hypophagia [147]. A study of families involving 183 parents and 299 offspring found a significant association between the common A67 allele in homozygous form and late-onset obesity. Differences in BMI and other adiposity measures were significantly different between A67T heterozygotes and A67 homozygotes in the parent group (average age of 51 years), but not in the offspring group (average age 25 years) [4]. This suggests that the T67 allele has a protective effect against development of obesity later in life. A very recent study looking at frequencies of this variant in Caucasian and Hispanic populations had similar findings [80]. No T67 homozygotes were found in the Hispanic population (1415 individuals),

Fig. 1. Protein sequence of the MC4 receptor. Roman numerals indicate number of transmembrane domain, and Arabic numbers are adjacent to the corresponding amino acid of the primary sequence. Shaded amino acids represent positions of missense mutations and polymorphisms found in human populations.

L. Carroll et al. / Peptides 26 (2005) 1871–1885

and no significant difference was observed between heterozygotes and A67 homozygotes in this group. However, T67 homozygotes in the Caucasian sample had significantly lower BMI, fat mass, lean mass, and serum leptin levels than heterozygotes or A67 homozygotes. This reinforces the hypothesis that the T67 allele is protective against obesity, but only in the homozygous form. Furthermore, food intake and metabolic activity in T67 homozygotes were normal, indicating that MC4R signaling was not adversely affected by variant AGRP. The T67 residue does not fall within the MC4R-binding region of AGRP, so it is unknown at this stage what the mechanism of T67-induced leanness could be. It is possible that this region interacts with one or more accessory receptors, such as attractin or attractin-like protein, both of which are implicated in ASIP or MC4R signaling, although the function of these accessory proteins is still unknown.

5. Other physiological pathways The melanocortin system plays a role in a variety of other physiological activities. Recently MC1R has been implicated in modulation of the immune system. MC1R is expressed on many cells of the immune system, especially macrophages and mast cells [3,116,130]. ␣-MSH treatment of such cells reduces expression of NF␬B, a transcription factor involved in stimulating immune responses, suggesting that ␣-MSH and the MC1 receptor down-regulate immune function. There has been speculation that MC1R may play a role in inflammatory conditions of the skin or neurological tissues [116], and it has been suggested that ␣-MSH may have application as an anti-inflammatory drug [169]. One question that has not been addressed is the effect of MC1R variants on this response. Patients with polymorphisms of MC1R affecting the protein would be expected to have decreased efficacy of ␣-MSH-based anti-inflammatory drugs. Considering that some MC1R variants with known effects on receptor function can have frequencies of up to 0.10 in Caucasian populations, a significant proportion of individuals could be resistant to such treatment. This would suggest that pharmacotherapy of such conditions should be tailored to patient genotype. MC4R has also been suggested as a target for pharmacotherapy for obesity and other weight-related illnesses by administration of selective melanocortin agonist or antagonist molecules [76]. Some mutations of the MC4R gene are the cause of a significant proportion of morbid obesity cases, so knowledge of MC4R genotype would also be necessary before prescription of these drugs. It is important to note that the expression and function of these receptors in other tissues is not fully understood. As well as being expressed in skin and cells of the immune system, the MC1R gene is expressed in a wide range of tissues including testis, ovary, liver, and mucosa of the duodenum [96,116,135]. While its role in these tissues is unclear, it is likely that altered function mutations of the MC1R gene would also affect its function in these other tissues. For

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example, if the immune tolerance effect of ␣-MSH is mediated via MC1 receptors expressed on immune cells, one would expect this activity to be diminished in persons with reduction of function polymorphisms, such as those that lead to red hair color and fair skin. In the future, it should be feasible to predict different immune functionality in persons with polymorphisms that affect MC1R function. A recent review has considered this hypothesis with regard to the immune-associated pathology of multiple sclerosis [36], since this disease occurs at higher rates in populations likely to have higher frequencies of MC1R variants. The role of MC1R in mast cells has also been examined in several studies [3,130], and it appears likely that activation of this receptor inhibits the release of cytokines involved in allergic responses. It follows that individuals with loss of function MC1R variants would be more prone to suffer allergies due to a lower inhibition of release of histamines and other pro-inflammatory cytokines from mast cells. Surveys of women suffering endometriosis have also found red hair to be a predisposing factor to this disease [162,164], and endometriosis patients have higher risk of having or developing allergies and other immune-associated diseases [124]. Job’s syndrome, characterized by mild-moderate immune deficiency leading to continual formation of staphylococcal abscesses and hyperimmunoglobulinemia E, has also been associated with red hair and fair skin [5]. All of these associations suggest that MC1R variation may play a role in immune function, though further investigation is required. Red hair has been anecdotally linked to altered responsiveness to anesthesia. A recent study on the effects of pentazocine on analgesia found that women, but not men, with loss of function MC1R variants were more responsive to this drug than people with normal MC1 receptors [90]. This is surprising since MC1R expression has not been reported in the central nervous system. The MC4 receptor has been linked to transmission of pain signals through the spinal cord in rats [150,151]. Intraspinal administration of ␣-MSH led to hyperalgesia, suggesting that spinal MC4 receptors are activated during transmission of pain. Conversely, administration of SHU9119, a selective MC4R antagonist, acted alone or in concert with morphine to increase analgesia. It was suggested that SHU9119-related compounds could be used in future anesthetics to increase the efficacy of current pain treatments, especially for neuropathic pain. Such treatments would require functional MC4 receptors, so knowledge of patient genotype would be advantageous in prescribing their use. Melanotan-II is a potent synthetic agonist of melanocortin-1 and -4 receptors. It has been tested in trials as a human tanning agent, with the hypothesis that hyperactivation of MC1 receptors in the skin would increase production of eumelanin [25]. In addition to increased coloration of the skin, subjects exhibited mild nausea, increased stretching and yawning responses and spontaneous penile erection. Several studies since have also revealed similar responses to administration of synthetic melanocortin

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ligands [23,91,114,157–160] and it is likely that they are mediated by melanocortin-4 receptors [82,144]. Due to the effects of melanocortin ligands on erectile function, they are a likely candidate for treatment for erectile dysfunction. It has been hypothesized that melanocortins may also be used for treatment of cachexia, a form of anorexia associated with advanced stages of cancer [108]. This syndrome is thought to be caused by abnormal hormone and neuroendocrine signaling in the hypothalamus associated with the high levels of inflammatory cytokines released during late-stage cancer pathogenesis. Considering the major role played by MC4R in weight control, one would expect individuals with loss-of-function mutations in this gene to be more susceptible to development of cachexia. A study in rodents has also demonstrated a stimulatory effect of MC4R activation on stress responses [16], suggesting that MC4 receptors in the hypothalamus may regulate HPA-associated hormone release. Futhermore, this indicates that pharmacological blockade of MC4R could be used to treat stress-related disorders such as anxiety. Presumably such treatment would also affect other physiological pathways regulated by MC4R, including sexual function and weight control. It also suggests that loss of function MC4R polymorphisms would reduce stress responses. The widespread expression patterns of melanocortin receptor genes in humans [18] suggest that this system is involved in other physiological pathways. MC receptors and POMC are expressed in testis, ovary, placenta, small intestine, heart, lung, and kidney, but as yet no function has been described in these tissues. It is likely that polymorphisms or mutations affecting the function of MC receptors would have a significant effect on these as yet to be determined pathways. Future studies may find association between, for example, MC1R-associated red hair phenotypes and possible MC1R-mediated functions in the ovary, similar to the association of red hair to endometriosis and allergies. MC4R mutations leading to obesity may also affect pathways of this receptor in the testis, perhaps affecting erectile function, which may explain why obesity is a risk factor for development of erectile dysfunction. Until these roles of melanocortin receptors are elucidated, the degree to which polymorphisms of their corresponding genes are clinically relevant will be difficult to ascertain. Furthermore, the effectiveness of drugs based on melanocortin ligands will be determined by the type of melanocortin receptors present in a patient, and variants leading to reduction in function are likely to inhibit full functionality of these drugs.

6. Conclusions Polymorphisms of genes of the melanocortin system have been observed to cause or be associated with a range of diseases and phenotypic states. Mutations of MC4R affecting the tertiary protein structure can prevent its normal appetite inhibitory function in the hypothalamus, leading

to dysregulation of energy homoeostasis and consequent obesity. Similarly, MC1R variants cause production of red/yellow pheomelanin instead of photo-protective eumelanin, resulting in red hair, fair skin, freckles and increased risk of melanoma and non-melanoma skin cancers. While mutations of the MC2R gene are quite rare, they also prevent its normal role in the stimulation of production and secretion of adrenal steroid hormones. As would be expected, null mutations of the melanocortin ligand-encoding gene POMC result in manifestation of all three syndromes due to disruption of signaling via these receptors. The existence of other physiological pathways affected by melanocortins, such as nociception, immune functionality and sexual function, suggest that other traits may also be affected by polymorphisms in melanocortin genes. Clarification of the role played by the melanocortin system in these pathways is likely to reveal the extent of these effects.

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