The D84E variant of the a-MSH receptor 1 gene isassociated with cutaneous malignant melanomaearly onset

Ricardo F.-de-Misa a,b,*, Jose Gerardo Hernandez-Jimenez b,Gregorio Carretero Hernandez c, Lina Perez-Mendez d,Armando Aguirre-Jaime b, Carlos Flores b, Jose Suarez Hernandez a,b,Antonio Perera Molinero e, Felix Claverıe-Martın b

Journal of Dermatological Science (2008) 52, 186—192

www.intl.elsevierhealth.com/journals/jods

aDermatology Department, Nuestra Senora de Candelaria University Hospital,Santa Cruz de Tenerife, SpainbResearch Unit, Nuestra Senora de Candelaria University Hospital, Santa Cruz de Tenerife, SpaincDermatology Department, Dr. Negrin University Hospital, Las Palmas de Gran Canaria, SpaindDivision of Clinical and Genetic Epidemiology, Research Unit, Nuestra Senora de CandelariaUniversity Hospital and Gerencia de Atencion Primaria, Santa Cruz de Tenerife, Spaine Pathology Department, Nuestra Senora de Candelaria University Hospital, Santa Cruz de Tenerife, Spain

Received 23 February 2008; received in revised form 11 May 2008; accepted 7 June 2008

KEYWORDSMelanoma;Onset;MC1R;D84E;Polymorphism

* Corresponding author at: DermatoSanta Cruz de Tenerife E-38010, Spai

E-mail address: fdemisa@msn.com

0923-1811/$30.00 # 2008 Japanese Sdoi:10.1016/j.jdermsci.2008.06.001

Summary

Background: Alpha-melanocyte-stimulating hormone receptor 1 (MC1R) has animportant role in skin pigmentation and variants of the gene have been establishedas independent risk factors for susceptibility to cutaneous malignant melanoma.Objective: To explore whether variants of the gene also influence the onset of thedisease.Methods: We analyzed 285 melanoma patients of European ancestry for commonvariation in codon 84 (D84E) of the a-MSH receptor 1 gene, which is known to havefunctional consequences in MC1R protein activity.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, eyesand hair colour, the risk for cutaneous malignant melanoma at any age was 2.07 timeshigher (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

logy Department, Nuestra Senora de Candelaria University Hospital, Carretera del Rosario 145,n. Tel.: +34 922 602037; fax: +34 922 602100.(R. F.de-Misa).

ociety for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

MC1R gene and early-onset malignant melanoma 187

84E carriers and non-carriers. Further analyses based on the Clark level and Breslow’sindex, both indicative for cancer invasion, reasonably supported an unbiased selec-tion of patients during the study enrolment. Additional exon re-sequencing of thecyclin-dependent kinase inhibitor 2A (CDKN2A) gene in MC1R 84E carriers ruled outthe presence of high penetrance mutations that have previously been associated withearly onset of the disease.Conclusion: Although our findings need to be confirmed by independent and largerstudies we have described for the first time the association of D84E variant of the a-MSH receptor 1 gene as an independent risk factor for an earlier onset of cutaneousmalignant melanoma.# 2008 Japanese Society for Investigative Dermatology. Published by Elsevier IrelandLtd. All rights reserved.

1. Introduction

Alpha-melanocyte-stimulating hormone (a-MSH)receptor 1 gene (MC1R), located on chromosome16q24.3, plays a crucial role in skin pigmentation[1]. MC1R (MIM#155555) encodes a 317 amino acidseven-pass transmembrane G-protein coupledreceptor with high affinity for the a-MSH. Bindingof a-MSH to the receptor results in activation ofadenylate cyclase triggering a variety of intracel-lular signalling pathways which promote a switch inmelanin synthesis from phaeo- to eumelanin thatincreases eumelanin/phaeomelanin ratio [2].MC1Ris highly polymorphic [3] and more than 60 non-synonymous non-conservative (disruptive) muta-tions of this gene have been reported to date [4].Some variants are strongly associated with fair skinand red hair [5—7] and several studies suggest thatMC1R variants are independent risk factors forcutaneous malignant melanoma (CMM) and non-melanoma skin cancer (NMSC), with independenceof main phenotypic pigmentation features [8—14].It has been very recently suggested that this riskassociation involves only patients carrying BRAFmutations in their CMM [15]. The first report sup-porting the association of MC1R gene with CMMfocused on a 252C>A single nucleotide polymorph-ism (SNP) originating a non-synonymous conserva-tive change from Asp to Glu at codon 84 (D84E) [8].Functional effects of this variation are not fullyunderstood. However, evolutionary constrains atthe locus (the Asp residue is conserved across mam-mal species and in other four members of themelanocortin receptor family) [16] and the associa-tion of the 84E allele with fair skin/red hair indivi-duals [5,8,16—18], support its direct contributionto skin pigmentation. Although the underlyingmechanisms accounting for its association withCMM remain unknown, it has been suggested thatD84E, as well as other reported functional MC1Rvariants, would favour a decrease in eumelaninsynthesis and an impaired protection against carci-

nogenic ultraviolet radiation [19]. Despite theaforementioned evidences, there are no studiesspecifically reporting on relationship of the D84Epolymorphism and clinical parameters in CMMpatients although other mutations in other geneshave been associated with them [20,21]. MC1Rvariants have been described to decrease age atdiagnosis of second or further melanoma diagnosisonly in presence of cyclin-dependent kinase inhi-bitor 2A (CDKN2A) gene mutations, increasing thepenetrance of this high CMM risk locus [22]. Hereweaimed to study the association of MC1R D84E withclinical parameters in CMM patients and found it tobe a risk factor for disease early onset in theabsence of any CDKN2A disrupting mutation.

2. Materials and methods

2.1. Patients

We recruited 285 consecutive CMM patients whowere followed up in the Dermatology Departmentsof Hospital Universitario Ntra. Sra. de Candelaria(Tenerife, Spain) and Hospital Universitario Dr.Negrin (Gran Canaria, Spain) from year 1982 to2007. Familial history of CMM (CMM in one or morefirst degree relatives) was recorded in 9% ofpatients. They were studied for main epidemiolo-gical, pathological and pigmentation features(Table 1). Two of the authors (RFM and GCH) per-formed a standardized personal interview and phy-sical examination. Skin type was assessed followingFitzpatrick’s classification: phototype I (burnseasily, never tans), phototype II (burns easily, tansminimally with difficulty), phototype III (burnsmoderately, tans moderately and uniformly),phototype IV (burns minimally, tans moderatelyand easily), phototype V (rarely burns, tans profu-sely) and phototype VI (never burns, tans profu-sely). Eye colour was categorized as blue, green,light brown or dark brown.Hair colourwas recorded

188 R. F.-de-Misa et al.

Table 1 Main features of studied patients

All cases (n = 285) 84E (n = 18) 84DD (n = 267) p-valuea

Gender (% of males) 34 39 34 0.678b

Familial melanoma (%)c 9 18 8 0.221b

Photoype (%)I 0.4 0 0.4II 52.7 52.9 52.8III 38.7 41.2 38.5IV 8.2 5.9 8.3 0.976b

Eyes colour (%)Blue 22 35 21Green 15 6 15Light brown 18 29 17Dark brown 45 29 46 0.182b

Hair colour (%)Red 5 18 4Blond 22 24 22Light brown 33 41 32Dark brown 41 18 42 0.024b

Invasivemelanomas (%)

86 100 85 0.139b

Median Breslowindex (P25—P75)

1.10 (0.65—2.10) 1.05 (0.64—1.47) 1.10 (0.64—2.11) 0.745d

Age at diagnosis(mean years � S.D.)

51.6 � 15.4 42.8 � 12.7 52.2 � 15.4 0.012e

Statistically significant differences in bold. 84E: carriers of 84E allele; 84DD: 84DD homozygous patients.a For the comparison of 84E carriers and non-carriers.b Chi-square test.c Malignant melanoma.d Mann—Whitney U-test.e Student’s t-test.

as red, blond, light brown, brown or black. Thisstudy was approved by local Ethics Committees andinformed consentwas obtained from all individuals.

2.2. Genotyping and mutation detection

Genomic DNAwas extracted using the QIAamp1 DNABloodMini Kit (Qiagen) fromwholebloodaccording tomanufacturer’s instructions. The 252C>A SNP (D84E)forMC1R gene (rs18050006) was genotyped using theSNaPshot1 Multiplex Kit (Applied Biosystems). 3 PCRamplification was done in a 25 ml reaction consistingon concentrations and including 5 pmol of forward(50-CAACGACTCCTTCCTGCTTC-30) and reverse (50-GCTGTGGGAGTAGCTCTTGG-30) primers. Amplifica-tion program consisted on a 9 min denaturing cycleat 95 8C, followed by 40 cycles of 95 8C for 50 s, 65 8Cfor 30 s and 72 8C for 1 min, and a final elongationcycle at 72 8C for 10 min. PCR products were purifiedusing the QIAquick1 PCR Purification Kit (Qiagen),according tomanufacturer’s instructions. SNaPshot1

extension reactions were performed under manufac-turer’s conditions using the primer 50-CCTGGCC-TTGTCGGA-30, designed by means of the SBEprimer

software [23], and products were resolved using theABI PRISMTM 310 Genetic Analyzer automatic sequen-cer (Applied Biosystems).

Patients harbouring the 252A allele (84E) werefurther investigated for exonic mutations in theCDKN2A gene. The four CDKN2A exons were PCRamplified and sequenced using the following com-binations of primer pairs and annealing tempe-ratures: Exon 1a, 50-ACCGGAGGAAGAAAGAGGAG-30 and 50-GCGCTACCTGATTCCAATTCC-30 at 63.5 8C;Exon 1b, 50-CTCAGAGCCGTTCCGAGAT-30 and 50-CGAAATCACACCAAACAAAAC-30 at 60 8C; Exon 2,50-AGCTTCCTTTCCGTCATGC-30 and 50-CAAATTCTCA-GATCATCAGTCCTC-30 at 60.5 8C; Exon 3, 50-GTGAA-GCCATTGCGAGAACT-30 and 50-TTTACGGTAGTGGG-GGAAGG-30 at 60.5 8C. All amplifications consistedon a 95 8C cycle for 10 min, 35 cycles of 95 8C for30 s, the indicated annealing temperature for 1 minand 72 8C for 1 min, followed by a final cycle of 72 8Cfor 10 min. 3 PCR amplification was done in a 50 mlreaction consisting on concentrations and including10 pmol of each primer. Sequencing was done usingthe amplification primers by means of the BigDye1

Terminator v3.1 Cycle Sequencing Kit (Applied

MC1R gene and early-onset malignant melanoma 189

Fig. 1 Survival analysis showing the probability of devel-oping cutaneous malignant melanoma according to diag-nosis age and MC1R status: Dashed line, 84D homozygousallele carriers; solid line, 84E allele carriers. Differencesare statistically significant ( p = 0.004).

Biosystems), according to manufacturer’ instruc-tions. Products were resolved with the ABI PRISMTM

310 Genetic Analyzer automatic sequencer.

2.3. Statistical analysis

Hardy—Weinberg equilibrium (HWE) was examinedusing an exact test. Differences for demographicaland clinical variables between 84E carriers and non-carriers (84D homozygotes) were analyzed usingPearson chi-squared tests, Fisher exact, t-Student,Mann—WhitneyU or ANOVA tests, as appropriate. TheMC1RD84Eeffects onCMMonsetwas testedbymeansof a survival analysis, considering the diagnosticonset age as the time of the study, and the CMMdiagnosis as the end point. A Cox modelling analysiswas further performed to test the influence of theMC1RD84E variant in early onset of CMMadjusting forrecorded variables. Potential biases, due to an inten-sified malignant melanoma screening over time ordue to geographical frequency differences, wereadditionally explored as alternative explanationsfor the observed association. All comparisons weretested at 0.05 statistical significance level usingSPSS# 13.0 computer statistical package (SPSS Inc.).

Table 2 Cox regression analysis proportional hazardratio results for the influence of theMC1R status on theage at diagnosis of skin melanoma

Variables Adjusted odds ratio(95% CI)a

p-value

D84E variant 2.07 (1.21—3.52) 0.008Gender 1.22 (0.94—1.58) 0.130Phototype 0.83 (0.67—1.03) 0.093Eyes colour 1.10 (0.97—1.23) 0.122Hair colour 0.95 (0.81—1.11) 0.531Clark’s level 1.05 (0.74—1.50) 0.783a Reference level: 84D homozygous genotype of the a-MSH

receptor 1 gene.

3. Results

Eighteen (6%) patients showed the 84E variant (allpatients were heterozygous) in our CMM cases andthe distribution followed HWE ( p = 1). Table 1 showsmain features of the total sample and stratified bythe MC1R D84E status, with data summarized aspercentage, median (25th—75th percentiles) ormean � S.D., depending on the variable. No patientshowed a phototype higher than IV. Similar distribu-tions were observed between 84E carriers and non-carriers for all recorded variables, except for thehair colour and the age at diagnosis. Hair colour wasstatistically different, most probably due to red hairprevalence among 84E carriers. The mean age dif-ference at diagnosis between 84D homozygouspatients and 84E carriers was 9 years (95% confi-dence interval [CI]: 2—17; p = 0.012), the 84D homo-zygous patients being older. Survival analysisindicated that the probability for a CMM at anyage was 1.99 times higher (95% CI: 1.23—3.22;p = 0.004) among 84E carriers (Fig. 1). An adjust-ment for gender, Clark’s level, phototype, eyescolour and hair colour, using a Cox regression modeldid not change this result, demonstrating a risk 2.07times higher (95% CI: 1.21—3.52; p = 0.008) for 84Eallele carriers (Table 2).

To further analyze the consistency of the associa-tion between the MC1R D84E variant and CMM early

onset, we then tested whether it could be derivedfrom an intensified CMM screening over time withdifferential impact in 84E carriers and non-carriers.Comparison of the Breslow’s indexes and Clark’slevels of invasion between 84E carriers and 84Dhomozygous over time showed no difference neitherin Clark’s level (p = 0.823) nor the Breslow’s index(p = 0.972). Additionally, although the percentageof patients enrolled increased over time, which maybe indicative of an intensified CMM screening, amean trend analysis showed no difference in thepercentage of patients included by year of diagnosisbetween 84E carriers and non-carriers ( p = 0.778)(Fig. 2). Because the D84E alleles may show geo-graphic heterogeneity in the region which may havebiased the association, we also explored if patientscame preferentially from any of the regions coveredby the hospital services and if such enrolment wasdifferential due to the D84E status. Although cases

190 R. F.-de-Misa et al.

Fig. 2 Percentage of enrolled patients by year of diagnosis according to MC1R status: Dashed line, 84D homozygousallele carriers; solid line, 84E allele carriers. Differences were not statistically significant ( p = 0.778).

were differentially enrolled by origin, they contrib-uted with no bias to the total sample studied accord-ing to the D84E genotype (Pearson chi-squaredp = 0.767): the 84E carriers were 47% from SantaCruz de Tenerife, 35% from Gran Canaria and 18%from other islands, while the 84D homozygous were42% from Santa Cruz de Tenerife, 37% from GranCanaria and 21% from other islands. This supportsthat the geographical distribution of D84E alleles,although geographically heterogeneous, did notbiased the detected association.

In order to rule out the presence of high pene-trance CDKN2Amutations in patients with the MC1RD84E genotype, as have previously been describedfor early onset CMM [20,21], the MC1R 84E carrierswere additionally re-sequenced for CDKN2A exons.We did not find any single variant of the CDKN2Agene other than a heterozygous 13G>C change inthe 30-UTR of the gene in a single patient.

4. Discussion

The MC1R D84E is not one of the classical ‘‘red haircolour’’ variants of the gene (i.e. R151C, R160WandD294H), which have been shown to confer a signifi-cant risk for CMM even in dark skinned and provedtanning ability patients [8,10,11]. However, it hasbeen also associated with red hair (as it may be inthe current series) and with risk for CMM and NMSCwith independence of skin type and hair colour[11,12]. In fact, MC1R D84E was the first non-clas-sical ‘‘red hair colour’’ variant to be associated with

risk for CMM and NMSC [8] and Mossner et al. [14]recently reported on 84E allele as an independentrisk factor for CMM on a multivariate analysis. CMMrisk also increases with the number of simultaneousMC1R variants [14,24]. Although the influence ofMC1R gene variants in CMM susceptibility is wellsupported by current evidence, clinical and patho-logical variables in CMM have not been stronglyassociated with MC1R gene variants. Here we foundCMM MC1R 84E carrier patients to be significantlyyounger (9.4 years) at diagnosis than 84D homozy-gous CMM patients, with independence of pigmen-tation features, and this association was verified notto be due to the co-occurrence of CDKN2A genemutations, as have been previously described forearly onset CMM [20,21], or to biases in patientenrolment (by time or geographical clustering). Inthis sense, it has recently been suggested [13] arelationship between MC1R gene variants and mel-anoma thickness. However, no differences in CMMthickness between 84E carriers and 84D homozygouspatients (which otherwise supports the CMM screen-ing homogeneity) were detected in this and otherstudies [25].

As a rule in genetic association studies, an unsa-tisfactory power is one of the main limitations of ourstudy [26]. D84E variant shows a low prevalence inEuropean populations [14,27,28], even amongpatients with CMM [14,28]. Although several studieshave pointed out the genetic homogeneity of thispopulation [29], we did not empirically estimatedpopulation stratification in our series of patients toprevent false positive associations, therefore we

MC1R gene and early-onset malignant melanoma 191

can not completely rule out albeit we demonstratedthat geographical clustering of patients was not asource of bias. Likewise, we did not explore theassociation of other variants of the MC1R gene,which could better or similarly explain the decreasein age at CMM diagnosis (currently under evalua-tion). In any case, it is well known that the Aspvariant at codon 84 is highly conserved across mam-mal species, across the melanocortin receptorfamily, and in other G-protein-coupled receptorfamilies, suggesting that the risk Glu allele is, mostlikely of functional significance [8]. Additionally, ithas been shown that, compared to Asp, Glu alleletriggers a dramatically lower adenylate cyclaseresponse and demonstrate a reduced cell surfaceexpression and intracellular retention of MC1Rreceptors [4].

Although our findings need to be confirmed byindependent and larger studies we have describedfor the first time the association of MC1R D84Evariant, predicting an amino acid change with func-tional significance, as an independent risk factor foran earlier onset of CMM. However, a prematuretranslational application of these results shouldbe avoided as several reasons [30,31] currentlypreclude predictive DNA testing for CMM in routinemedical practice.

Acknowledgement

This study was funded by grants 57/03 and 47/05from the Fundacion Canaria de Investigacion y SaludFUNCIS.

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