Human Melanocortin-4 Receptor Gene Research Articles

Prediction of the damage-associated non-synonymous single nucleotide polymorphisms in the human MC1R gene.

The melanocortin 1 receptor (MC1R) is involved in the control of melanogenesis. Polymorphisms in this gene have been associated with variation in skin and hair color and with elevated risk for the development of melanoma. Here we used 11 computational tools based on different approaches to predict the damage-associated non-synonymous single nucleotide polymorphisms (nsSNPs) in the coding region of the human MC1R gene. Among the 92 nsSNPs arranged according to the predictions 62% were classified as damaging in more than five tools. The classification was significantly correlated with the scores of two consensus programs. Alleles associated with the red hair color (RHC) phenotype and with the risk of melanoma were examined. The R variants D84E, R142H, R151C, I155T, R160W and D294H were classified as damaging by the majority of the tools while the r variants V60L, V92M and R163Q have been predicted as neutral in most of the programs The combination of the prediction tools results in 14 nsSNPs indicated as the most damaging mutations in MC1R (L48P, R67W, H70Y, P72L, S83P, R151H, S172I, L206P, T242I, G255R, P256S, C273Y, C289R and R306H); C273Y showed to be highly damaging in SIFT, Polyphen-2, MutPred, PANTHER and PROVEAN scores. The computational analysis proved capable of identifying the potentially damaging nsSNPs in MC1R, which are candidates for further laboratory studies of the functional and pharmacological significance of the alterations in the receptor and the phenotypic outcomes.

Rescue of defective MC4R cell-surface expression and signaling by a novel pharmacoperone Ipsen 17.

Melanocortin 4 receptor (MC4R) is a key factor in regulating energy homeostasis, and null mutations occurring in the gene encoding MC4R cause severe early-onset morbid obesity in humans. Many obesity-causing mutations affecting MC4R clinically identified so far lead to failure of mutant receptors to shuttle to the plasma membrane. In this study, we show that a novel human MC4R antagonist, Ipsen 17, acted as an pharmacological chaperone of human MCR4. As tested with 12 obesity-causing human MC4R variants including S58C, E61K, N62S, I69T, P78L, C84R, G98R, T162I, R165W, W174C, C271Y, and P299H, Ipsen 17 was found to be the most universal pharmacological chaperone of MC4R reported so far because it can completely rescue nearly all mutant receptors (except P299H) with the highest potency (an EC50 value of approximately 10(-8) M) and efficiency when compared with results for other tested pharmacological chaperones of MC4R including ML00253764, PBA, MTHP, PPPone, MPCI, DCPMP, and NBP described in the literature. Once restored to the plasma membrane, defective human MC4R variants responded to α-MSH stimulation with an EC50 value of approximately 10(-8) M and displayed dramatically enhanced signaling ability (except for G98R) in a mutant-specific efficacy and potency profile. Taken together, these results indicate that Ipsen 17 represents a candidate for the development of a targeted treatment of severe early-onset morbid obesity caused by a large subset of inherited mutations in the human MC4R gene.

Letter to the Editor Adrenal function and MC1R gene analysis in a prepubertal girl with generalized hyperpigmentation:

Letter to the Editor Adrenal function and MC1R gene analysis in a prepubertal girl with generalized hyperpigmentation:

Sequence polymorphisms of MC1R gene and their association with depression and antidepressant response

Melanocortin 1 receptor (MC1R) is involved in various functions, such as pigmentation, antipyretic and anti-inflammatory actions, development of melanoma, susceptibility to ultraviolet-induced sun damage, modification of oculocutaneous albinism, development of freckles, and mediation of female-specific mechanisms of analgesia. MC1R's natural agonists include α-melanocyte-stimulating hormone and corticotrophin (ACTH1-39), which are important components of hypothalamic pituitary adrenal axis and increase in response to stress. Given the multiple relevant roles of MC1R, we studied whether the MC1R gene would be associated with susceptibility to major depressive disorder or with response to antidepressant treatment. The human MC1R gene is highly polymorphic; therefore, we sequenced the entire MC1R coding region of 1122 bp in 181 depressed Mexican-American patients and 185 Mexican-American controls. A total of 23 single nucleotide polymorphisms (SNPs, 15 known and eight new) were found within the sequenced region. Among the common SNPs, the nonsynonymous SNP, rs885479 (R163Q) was associated with the diagnosis of depression (P=0.04). The nonsynonymous SNP, rs2228479 (V92M) and the synonymous SNP, rs2228478 were found to be associated with the remission with desipramine treatment. No associations were found for remission with fluoxetine treatment or for the combined sample treated with fluoxetine or desipramine. The frequency of one (H2) of the five haplotypes identified was higher in depressed patients when compared with controls (P=0.05). In-silico functional analysis indicates that SNPs rs885479 and rs2228479 have significant impact on the protein function. The MC1R gene might be associated with major depressive disorder and with treatment response to desipramine.

Genetic analysis of three important genes in pigmentation and melanoma susceptibility: CDKN2A, MC1R and HERC2/OCA2

The CDKN2A gene is regarded as the major familial malignant melanoma (MM) susceptibility gene. Human pigmentation is one of the main modulators of individual risk of developing MM. Therefore, the genes involved in the determination of skin colour and tanning response are potentially implicated in MM predisposition and may be useful predictors of MM risk in the general population. The human melanocortin-1 receptor gene (MC1R) plays a crucial role in pigmentation and also appears to be important in MM. The OCA2 gene has emerged as a new and significant determinant of human iris colour variation. We present a case-control study in Spanish population including 390 consecutive patients with melanoma and 254 control subjects. Sequence analysis of the entire coding region and genotyping of 5 tag-SNPs in the genomic region of MC1R was performed. We identified 27 variants, two reaching statistical significance [R160W (OR: 4.18, 95% CI: 1.24-14.04, P = 0.02) and D294H (OR: 3.10, 95% CI: 1.37-7.01, P = 0.01)] and we detected two novel non-synonymous changes: V92L and T308M. Odds ratio for carrying two functional variants was 4.25 (95% CI: 2.30-7.84, P = 3.63 x 10(-6)). Haplotypes of the entire MC1R region have been established, and we observed an enrichment of a rare European haplotype similar to African values carrying variants V92M and I155T. In addition, three potentially functional SNPs were selected in p16/CDKN2A and in the promoter region of OCA2/HERC2. Our data for CDKN2A gene did not reach statistically significant results for any of the two studied alleles. We found that the variant allele A > G of OCA2/HERC2 (rs12913832) was associated with pigmentation features: eye, hair and skin colour; P-values = 1.8 x 10(-29), 9.2 x 10(-16), 1.1 x 10(-3), respectively, validating previous results.

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

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

Expression of the human melanocortin-4 receptor gene is controlled by several members of the Sp transcription factor family

The melanocortin-4 receptor (MC4-R) plays a key role in the hypothalamic control of food intake, lending importance to the understanding of the mechanisms that regulate its expression. To identify factors controlling the expression of the human (h) MC4-R gene, a fragment containing 1253 bp of the 5'-flanking region of the hMC4-R gene was isolated. A series of hMC4-R luciferase constructs were developed and used to transiently transfect HEK293 and GT1-7 cell lines, both expressing endogenous MC4-R mRNA. Deletion analysis of the 1253 bp fragment showed that the basal promoter activity is mainly restricted to the 179 bp upstream of the transcription start site in both cell types. Mutation of a putative Sp1-binding site located at position -76 bp resulted in a dramatic reduction of the luciferase activity in HEK293 and GT1-7 cells by 87 and 80% respectively. Both in vitro and in vivo studies (gel shift and chromatin immunoprecipitation analyses) revealed binding of both Sp1 and Sp3 to this site in HEK293 cells. Cotransfection with an Sp1 expression vector in Drosophila cells that do not express Sp1, in conjunction with treatment of HEK293 cells with mithramycin A, a specific inhibitor of Sp1, confirmed the role of Sp1. For the first time, we have demonstrated that the constitutive activity of the hMC4-R promoter is dependent upon Sp transcription factors.

Mutations in the human melanocortin-4 receptor gene associated with severe familial obesity disrupts receptor function through multiple molecular mechanisms.

Mutations in the melanocortin-4 receptor gene (MC4R) represent the commonest monogenic cause of human obesity. However, information regarding the precise effects of such mutations on receptor function is very limited. We examined the functional properties of 12 different mutations in human MC4R that result in severe, familial, early-onset obesity. Of the nine missense mutants studied, four were completely unable to generate cAMP in response to ligand and five were partially impaired. Four showed evidence of impaired cell surface expression and six of reduced binding affinity for ligand. One mutation in the C-terminal tail, I316S, showed reduced affinity for alpha-MSH but retained normal affinity for the antagonist AgRP. None of the mutations inhibited signaling through co-transfected wild-type receptors. Thus, in the most comprehensive study to date of the functional properties of naturally occurring MC4R mutations we have (1) established that defective expression on the cell surface is a common mechanism impairing receptor function, (2) identified mutations which specifically affect ligand binding affinity thus aiding the definition of receptor structure-function relationships, (3) provided evidence against the notion that these receptor mutants act as dominant-negatives, and (4) identified a potentially novel molecular mechanism of receptor dysfunction whereby a mutation alters the relative affinities of a receptor for its natural agonist versus antagonist.

Mutations in the human melanocortin-4 receptor gene associated with severe familial obesity disrupts receptor function through multiple molecular mechanisms

Mutations in the human melanocortin-4 receptor gene associated with severe familial obesity disrupts receptor function through multiple molecular mechanisms

Involvement of microphthalmia-associated transcription factor (MITF) in expression of human melanocortin-1 receptor (MC1R)

Analysis of the nucleotide sequence of human melanocortin-1 receptor (MC1R) promoter indicated that an E-box (CANNTG) is present immediately upstream of the transcriptional initiation site. The presence of the CATGTG motif suggests that MC1R gene expression may be regulated by a basic helix-loop-helix-leucine-zipper (bHLH-LZ) type transcription factor. The microphthalmia-associated transcription factor (MITF), which belongs to the family of bHLH-LZ type transcription factors, regulates the transcription of melanogenesis-related enzyme genes such as the tyrosinase and TRP-1 genes. We investigated whether MITF regulates human MC1R gene expression through the same transcriptional mechanism as tyrosinase and TRP-1 genes in melanocytes. For this purpose, the effect of co-expression of cDNA encoding MITF on MC1R promoter activity in NIH/3T3 cells was studied. MC1R promoter activity was induced to the extent of approximately 5-fold in the presence of MITF. In addition, electrophoretic mobility shift assay indicated that nuclear extracts of human SK-Mel-2 cells contain a protein that binds specifically to the MC1R promoter region containing the CATGTG motif. These results suggested that MITF regulates not only the expression of enzymes involved in melanin synthesis, but also the expression of a receptor which plays an essential role in melanocyte functions.

Thr40 and Met122 are new partial loss-of-function natural mutations of the human melanocortin 1 receptor

Activation by melanocortins of the melanocortin 1 receptor (MC1R), expressed in epidermal melanocytes, stimulates melanogenesis. Human MC1R gene loss-of-function mutations are associated with fair skin, poor tanning and increased skin cancer risk. We identified two natural alleles: Ile40Thr, probably associated with skin types I–II, and Val122Met. Val122Met bound [ 125I][Nle 4, D-Phe 7]-α-melanocyte stimulating hormone with lower affinity than the wild-type. Dose–response curves of cAMP accumulation were right-shifted for both forms. The Val122Met form failed to achieve maximal cAMP responses comparable to the wild-type or Ile40Thr receptors. Thus, the Ile40Thr and Val122Met variants are partial loss-of-function natural mutations of MC1R.

A missense mutation in the human melanocortin-4 receptor gene in relation to abdominal obesity and salivary cortisol.

The melanocortin-4 receptor (MC4-R) regulates food intake and possibly energy expenditure, and the inactivation of the MC4-R by gene targeting results in obesity, a phenotype strongly associated with Type II (non-insulin-dependent) diabetes mellitus. In our study, we addressed the hypothesis that a G-->A substitution at codon 103 (Val-103Ile) of the MC4R gene influences abdominal obesity, insulin, glucose, and lipid metabolism as well as circulating hormones, including salivary cortisol. We genotyped the missense variant at codon 103 of the MC4R gene in 284 unrelated Swedish men born in 1944 by using polymerase chain reaction amplification followed by digestion with the restriction enzyme HincII. The frequency of allele G was 0.97 and 0.03 for allele A. The observed genotype frequencies were 95 % and 5 % for G/G and G/A, respectively. The heterozygotes had lower waist-to-hip ratio (p = 0.023) and trends for lower body mass index (p = 0.054) and abdominal sagittal diameter (p = 0.095) compared to G/G homozygotes. Moreover, G/A subjects had borderline lower serum leptin concentrations (p = 0.087) and total cholesterol (p = 0.082). The heterozygotes had also, in comparison to G/G subjects, significantly (p < 0.01) higher mean cortisol concentrations in the morning (21.4 vs 14.6 nmol/l), at ll:45 h (11.6 vs 7.0 nmol/l), 30 min after a standardized lunch (15.3 vs 8.0 nmol/l) and finally, 60 min after lunch (10.8 vs 6.7 nmol/l). These findings suggest that the missense mutation in the MC4R gene could contribute to the variability in body mass, abdominal fat distribution and leptin concentrations in the general population. Moreover, the G/A mutation exhibits evidence of associations with diurnal cortisol levels.

A polymorphism study of the human Agouti gene and its association with MC1R.

To determine whether the Agouti Signalling Protein (ASP) gene is associated with skin and hair coloration in humans, the complete coding region of ASP was screened for polymorphisms. Analysis of ASP in Caucasian, African-American, Spanish Basque, Hispanic, Apache and Australian Aboriginal populations revealed no amino acid substitutions. A single polymorphism in the 3' untranslated region occurred at a frequency of 0.2 in African-Americans. Variants of the Melanocortin 1 Receptor (MC1R) gene have been found to be associated with red hair and fair skin in humans. Red hair individuals are usually compound heterozygotes or homozygous for one of a number of MC1R polymorphisms associated with red hair. Some individuals however are heterozygous for only one of these polymorphisms and dizygotic twins can be concordant for MC1R variants but discordant for hair colour. A recent study has also identified rare redheads carrying no MC1R variants indicating that polymorphisms of the human MC1R gene are required but not sufficient for the red hair phenotype. To address the question of whether ASP also contributes to the red hair phenotype, individuals previously identified as having unexpected MC1R genotypes were screened for polymorphisms at the ASP locus. No polymorphisms were found in any of these individuals. Results indicate that the human ASP gene is unlikely to function in normal human pigmentation in the same way as MC1R.

Melanocortin-1 Receptor Gene Variants Determine the Risk of Nonmelanoma Skin Cancer Independently of Fair Skin and Red Hair

Melanocortin-1 receptor (MC1R) gene variants are associated with fair skin and red hair and, independently of these, with cutaneous malignant melanoma. The association of MC1R gene variants with nonmelanoma skin cancer is largely unknown. A total of 838 subjects were included in the present study: 453 patients with nonmelanoma skin cancer and 385 subjects with no skin cancer. The coding sequence of the human MC1R gene was tested using single-stranded conformation polymorphism analysis followed by sequencing of unknown variants. Risk of skin cancer dependent on the various MC1R gene variants was estimated using the exposure odds ratio. We investigated whether subjects with MC1R variant alleles were at increased risk of developing nonmelanoma skin cancer and, if so, whether this increased risk was mediated by fair skin and red hair. A total of 27 MC1R gene variants were found. The number of carriers of one, two, or three MC1R gene variants was 379 (45.2%), 208 (24.8%), and 7 (0.9%), respectively. A strong association between MC1R gene variants and fair skin and red hair was established, especially the variants Arg151Cys and Arg160Trp (P < .0001). Carriers of two variant alleles were at increased risk for developing cutaneous squamous cell carcinoma (odds ratio 3.77; 95% confidence interval [CI] 2.11-6.78), nodular basal cell carcinoma (odds ratio 2.26; 95% CI 1.45-3.52), and superficial multifocal basal cell carcinoma (odds ratio 3.43; 95% CI 1.92-6.15), compared with carriers of two wild-type alleles. Carriers of one variant allele had half the risk. The highest relative risks of nonmelanoma skin cancer were found in carriers of the Asp84Glu, His260Pro, and Asp294His variant alleles, and the risk was only slightly lower for carriers of the Val60Leu, Val92Met, Arg142His, Arg151Cys, and Arg160Trp variant alleles. When subjects were stratified by skin type and hair color, analysis showed that these factors did not materially change the relative risks. These findings indicate that MC1R gene variants are important independent risk factors for nonmelanoma skin cancer.