The D84E variant of the α-MSH receptor 1 gene is associated with cutaneous malignant melanoma early onset
Journal of Dermatological Science(2008)
摘要
Results The mean age difference at diagnosis between MC1R 84E carriers and non-carriers was 9 years (95% confidence interval [CI]: 2–17; p = 0.012), with 84E non-carrier patients being older. After adjusting for gender, Clark's level, phototype, eyes and hair colour, the risk for cutaneous malignant melanoma at any age was 2.07 times higher (95% CI: 1.21–3.52; p = 0.008) among MC1R 84E carriers. Enrolment criteria, geographical origin, Clark's levels and Breslow's indexes were similar between MC1R 84E carriers and non-carriers. Further analyses based on the Clark level and Breslow's index, both indicative for cancer invasion, reasonably supported an unbiased selection of patients during the study enrolment. Additional exon re-sequencing of the cyclin-dependent kinase inhibitor 2A ( CDKN2A ) gene in MC1R 84E carriers ruled out the presence of high penetrance mutations that have previously been associated with early onset of the disease. Conclusion Although our findings need to be confirmed by independent and larger studies we have described for the first time the association of D84E variant of the α-MSH receptor 1 gene as an independent risk factor for an earlier onset of cutaneous malignant melanoma. Keywords Melanoma Onset MC1R D84E Polymorphism 1 Introduction Alpha-melanocyte-stimulating hormone (α-MSH) receptor 1 gene ( MC1R ), located on chromosome 16q24.3, plays a crucial role in skin pigmentation [1] . MC1R (MIM#155555) encodes a 317 amino acid seven-pass transmembrane G-protein coupled receptor with high affinity for the α-MSH. Binding of α-MSH to the receptor results in activation of adenylate cyclase triggering a variety of intracellular signalling pathways which promote a switch in melanin synthesis from phaeo- to eumelanin that increases eumelanin/phaeomelanin ratio [2] . MC1R is highly polymorphic [3] and more than 60 non-synonymous non-conservative (disruptive) mutations of this gene have been reported to date [4] . Some variants are strongly associated with fair skin and red hair [5–7] and several studies suggest that MC1R variants are independent risk factors for cutaneous malignant melanoma (CMM) and non-melanoma skin cancer (NMSC), with independence of main phenotypic pigmentation features [8–14] . It has been very recently suggested that this risk association involves only patients carrying BRAF mutations in their CMM [15] . The first report supporting the association of MC1R gene with CMM focused on a 252C>A single nucleotide polymorphism (SNP) originating a non-synonymous conservative change from Asp to Glu at codon 84 (D84E) [8] . Functional effects of this variation are not fully understood. However, evolutionary constrains at the locus (the Asp residue is conserved across mammal species and in other four members of the melanocortin receptor family) [16] and the association of the 84E allele with fair skin/red hair individuals [5,8,16–18] , support its direct contribution to skin pigmentation. Although the underlying mechanisms accounting for its association with CMM remain unknown, it has been suggested that D84E, as well as other reported functional MC1R variants, would favour a decrease in eumelanin synthesis and an impaired protection against carcinogenic ultraviolet radiation [19] . Despite the aforementioned evidences, there are no studies specifically reporting on relationship of the D84E polymorphism and clinical parameters in CMM patients although other mutations in other genes have been associated with them [20,21] . MC1R variants have been described to decrease age at diagnosis of second or further melanoma diagnosis only in presence of cyclin-dependent kinase inhibitor 2A ( CDKN2A ) gene mutations, increasing the penetrance of this high CMM risk locus [22] . Here we aimed to study the association of MC1R D84E with clinical parameters in CMM patients and found it to be a risk factor for disease early onset in the absence of any CDKN2A disrupting mutation. 2 Materials and methods 2.1 Patients We recruited 285 consecutive CMM patients who were followed up in the Dermatology Departments of Hospital Universitario Ntra. Sra. de Candelaria (Tenerife, Spain) and Hospital Universitario Dr. Negrin (Gran Canaria, Spain) from year 1982 to 2007. Familial history of CMM (CMM in one or more first degree relatives) was recorded in 9% of patients. They were studied for main epidemiological, pathological and pigmentation features ( Table 1 ). Two of the authors (RFM and GCH) performed a standardized personal interview and physical examination. Skin type was assessed following Fitzpatrick's classification: phototype I (burns easily, never tans), phototype II (burns easily, tans minimally with difficulty), phototype III (burns moderately, tans moderately and uniformly), phototype IV (burns minimally, tans moderately and easily), phototype V (rarely burns, tans profusely) and phototype VI (never burns, tans profusely). Eye colour was categorized as blue, green, light brown or dark brown. Hair colour was recorded as red, blond, light brown, brown or black. This study was approved by local Ethics Committees and informed consent was obtained from all individuals. 2.2 Genotyping and mutation detection Genomic DNA was extracted using the QIAamp ® DNA Blood Mini Kit (Qiagen) from whole blood according to manufacturer's instructions. The 252C>A SNP (D84E) for MC1R gene (rs18050006) was genotyped using the SNaPshot ® Multiplex Kit (Applied Biosystems). 3 PCR amplification was done in a 25 μl reaction consisting on concentrations and including 5 pmol of forward (5′-CAACGACTCCTTCCTGCTTC-3′) and reverse (5′-GCTGTGGGAGTAGCTCTTGG-3′) primers. Amplification program consisted on a 9 min denaturing cycle at 95 °C, followed by 40 cycles of 95 °C for 50 s, 65 °C for 30 s and 72 °C for 1 min, and a final elongation cycle at 72 °C for 10 min. PCR products were purified using the QIAquick ® PCR Purification Kit (Qiagen), according to manufacturer's instructions. SNaPshot ® extension reactions were performed under manufacturer's conditions using the primer 5′-CCTGGCCTTGTCGGA-3′, designed by means of the SBEprimer software [23] , and products were resolved using the ABI PRISM™ 310 Genetic Analyzer automatic sequencer (Applied Biosystems). Patients harbouring the 252A allele (84E) were further investigated for exonic mutations in the CDKN2A gene. The four CDKN2A exons were PCR amplified and sequenced using the following combinations of primer pairs and annealing temperatures: Exon 1α, 5′-ACCGGAGGAAGAAAGAGGAG-3′ and 5′-GCGCTACCTGATTCCAATTCC-3′ at 63.5 °C; Exon 1β, 5′-CTCAGAGCCGTTCCGAGAT-3′ and 5′-CGAAATCACACCAAACAAAAC-3′ at 60 °C; Exon 2, 5′-AGCTTCCTTTCCGTCATGC-3′ and 5′-CAAATTCTCAGATCATCAGTCCTC-3′ at 60.5 °C; Exon 3, 5′-GTGAAGCCATTGCGAGAACT-3′ and 5′-TTTACGGTAGTGGGGGAAGG-3′ at 60.5 °C. All amplifications consisted on a 95 °C cycle for 10 min, 35 cycles of 95 °C for 30 s, the indicated annealing temperature for 1 min and 72 °C for 1 min, followed by a final cycle of 72 °C for 10 min. 3 PCR amplification was done in a 50 μl reaction consisting on concentrations and including 10 pmol of each primer. Sequencing was done using the amplification primers by means of the BigDye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), according to manufacturer’ instructions. Products were resolved with the ABI PRISM™ 310 Genetic Analyzer automatic sequencer. 2.3 Statistical analysis Hardy–Weinberg equilibrium (HWE) was examined using an exact test. Differences for demographical and clinical variables between 84E carriers and non-carriers (84D homozygotes) were analyzed using Pearson chi-squared tests, Fisher exact, t -Student, Mann–Whitney U or ANOVA tests, as appropriate. The MC1R D84E effects on CMM onset was tested by means of a survival analysis, considering the diagnostic onset age as the time of the study, and the CMM diagnosis as the end point. A Cox modelling analysis was further performed to test the influence of the MC1R D84E variant in early onset of CMM adjusting for recorded variables. Potential biases, due to an intensified malignant melanoma screening over time or due to geographical frequency differences, were additionally explored as alternative explanations for the observed association. All comparisons were tested at 0.05 statistical significance level using SPSS © 13.0 computer statistical package (SPSS Inc.). 3 Results Eighteen (6%) patients showed the 84E variant (all patients were heterozygous) in our CMM cases and the distribution followed HWE ( p = 1). Table 1 shows main features of the total sample and stratified by the MC1R D84E status, with data summarized as percentage, median (25th–75th percentiles) or mean ± S.D., depending on the variable. No patient showed a phototype higher than IV. Similar distributions were observed between 84E carriers and non-carriers for all recorded variables, except for the hair colour and the age at diagnosis. Hair colour was statistically different, most probably due to red hair prevalence among 84E carriers. The mean age difference at diagnosis between 84D homozygous patients and 84E carriers was 9 years (95% confidence interval [CI]: 2–17; p = 0.012), the 84D homozygous patients being older. Survival analysis indicated that the probability for a CMM at any age was 1.99 times higher (95% CI: 1.23–3.22; p = 0.004) among 84E carriers ( Fig. 1 ). An adjustment for gender, Clark's level, phototype, eyes colour and hair colour, using a Cox regression model did not change this result, demonstrating a risk 2.07 times higher (95% CI: 1.21–3.52; p = 0.008) for 84E allele carriers ( Table 2 ). To further analyze the consistency of the association between the MC1R D84E variant and CMM early onset, we then tested whether it could be derived from an intensified CMM screening over time with differential impact in 84E carriers and non-carriers. Comparison of the Breslow's indexes and Clark's levels of invasion between 84E carriers and 84D homozygous over time showed no difference neither in Clark's level ( p = 0.823) nor the Breslow's index ( p = 0.972). Additionally, although the percentage of patients enrolled increased over time, which may be indicative of an intensified CMM screening, a mean trend analysis showed no difference in the percentage of patients included by year of diagnosis between 84E carriers and non-carriers ( p = 0.778) ( Fig. 2 ). Because the D84E alleles may show geographic heterogeneity in the region which may have biased the association, we also explored if patients came preferentially from any of the regions covered by the hospital services and if such enrolment was differential due to the D84E status. Although cases were differentially enrolled by origin, they contributed with no bias to the total sample studied according to the D84E genotype (Pearson chi-squared p = 0.767): the 84E carriers were 47% from Santa Cruz de Tenerife, 35% from Gran Canaria and 18% from other islands, while the 84D homozygous were 42% from Santa Cruz de Tenerife, 37% from Gran Canaria and 21% from other islands. This supports that the geographical distribution of D84E alleles, although geographically heterogeneous, did not biased the detected association. In order to rule out the presence of high penetrance CDKN2A mutations in patients with the MC1R D84E genotype, as have previously been described for early onset CMM [20,21] , the MC1R 84E carriers were additionally re-sequenced for CDKN2A exons. We did not find any single variant of the CDKN2A gene other than a heterozygous 13G>C change in the 3′-UTR of the gene in a single patient. 4 Discussion The MC1R D84E is not one of the classical “red hair colour” variants of the gene (i.e. R151C, R160W and D294H), which have been shown to confer a significant risk for CMM even in dark skinned and proved tanning ability patients [8,10,11] . However, it has been also associated with red hair (as it may be in the current series) and with risk for CMM and NMSC with independence of skin type and hair colour [11,12] . In fact, MC1R D84E was the first non-classical “red hair colour” variant to be associated with risk for CMM and NMSC [8] and Mössner et al. [14] recently reported on 84E allele as an independent risk factor for CMM on a multivariate analysis. CMM risk also increases with the number of simultaneous MC1R variants [14,24] . Although the influence of MC1R gene variants in CMM susceptibility is well supported by current evidence, clinical and pathological variables in CMM have not been strongly associated with MC1R gene variants. Here we found CMM MC1R 84E carrier patients to be significantly younger (9.4 years) at diagnosis than 84D homozygous CMM patients, with independence of pigmentation features, and this association was verified not to be due to the co-occurrence of CDKN2A gene mutations, as have been previously described for early onset CMM [20,21] , or to biases in patient enrolment (by time or geographical clustering). In this sense, it has recently been suggested [13] a relationship between MC1R gene variants and melanoma thickness. However, no differences in CMM thickness between 84E carriers and 84D homozygous patients (which otherwise supports the CMM screening homogeneity) were detected in this and other studies [25] . As a rule in genetic association studies, an unsatisfactory power is one of the main limitations of our study [26] . D84E variant shows a low prevalence in European populations [14,27,28] , even among patients with CMM [14,28] . Although several studies have pointed out the genetic homogeneity of this population [29] , we did not empirically estimated population stratification in our series of patients to prevent false positive associations, therefore we can not completely rule out albeit we demonstrated that geographical clustering of patients was not a source of bias. Likewise, we did not explore the association of other variants of the MC1R gene, which could better or similarly explain the decrease in age at CMM diagnosis (currently under evaluation). In any case, it is well known that the Asp variant at codon 84 is highly conserved across mammal species, across the melanocortin receptor family, and in other G-protein-coupled receptor families, suggesting that the risk Glu allele is, most likely of functional significance [8] . Additionally, it has been shown that, compared to Asp, Glu allele triggers a dramatically lower adenylate cyclase response and demonstrate a reduced cell surface expression and intracellular retention of MC1R receptors [4] . Although our findings need to be confirmed by independent and larger studies we have described for the first time the association of MC1R D84E variant, predicting an amino acid change with functional significance, as an independent risk factor for an earlier onset of CMM. However, a premature translational application of these results should be avoided as several reasons [30,31] currently preclude predictive DNA testing for CMM in routine medical practice. Acknowledgement This study was funded by grants 57/03 and 47/05 from the Fundación Canaria de Investigación y Salud FUNCIS. References [1] G. Mountjoy S. Robbins T. Mortrud D. Cone The cloning of a family of genes that encode the melanocortin receptors Science 257 1992 1248 1251 [2] A. Sturm F. Box M. Ramsay Human pigmentation genetics: the difference is only skin deep Bioessays 20 1998 712 721 [3] M. Harding E. Healy J. Ray S. Ellis N. Flanagan C. Todd Evidence for variable selective pressures at MC1R Am J Hum Genet 66 2000 1351 1361 [4] C. Garcia-Borron L. Sanchez-Laorden C. Jimenez-Cervantes Melanocortin-1 receptor structure and functional regulation Pigment Cell Res 18 2005 393 410 [5] P. Valverde E. Healy I. Jackson L. Rees J. Thody Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans Nat Genet 11 1995 328 330 [6] F. Box R. Wyeth E. O’Gorman G. Martin A. 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Melanoma,Onset,MC1R,D84E,Polymorphism
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