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Association of MC1R Variants and Risk of Melanoma in Melanoma-Prone Families with CDKN2A Mutations

¹ Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda and ² Laboratory of Molecular Technology, National Cancer Institute at Frederick, Science Applications International Corporation-Frederick, Frederick, NIH, Department of Health and Human Services, Maryland

Requests for reprints: Alisa M. Goldstein, Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Department of Health and Human Services, Executive Plaza South, Room 7004, 6120 Executive Boulevard, MSC 7236 Bethesda, MD 20892-7236. Phone: 301-496-4375; Fax: 301-402-4489. E-mail: goldstea{at}exchange.nih.gov

	Abstract

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Major risk factors for melanoma include many nevi, especially dysplastic nevi, fair pigmentation, freckling, poor tanning ability, and germ line mutations in the CDKN2A, CDK4, or MC1R genes. We evaluated the relationship between MC1R and melanoma risk in CDKN2A melanoma-prone families with extensive clinical and epidemiologic data. We studied 395 subjects from 16 American CDKN2A families. Major melanoma risk factors were assessed by clinical examination or questionnaire; MC1R was sequenced. Odds ratios were estimated by unconditional and conditional logistic regression models. We examined the distribution of MC1R variants and median ages at melanoma diagnosis in multiple primary melanoma (MPM) and single primary melanoma (SPM) patients. Presence of multiple MC1R variants was significantly associated with melanoma, even after adjustment for major melanoma risk factors. All 40 MPM patients had at least one MC1R variant; 65% of MPM patients versus only 17% of SPM patients had at least two MC1R variants (P < 0.0001). For all 69 melanoma patients combined, as well as the 40 MPM patients, there was a statistically significant decrease in median age at diagnosis as numbers of MC1R variants increased (P = 0.010 and P = 0.008, respectively). In contrast, no significant reduction in age at melanoma diagnosis was observed for SPM patients (P = 0.91). The current study suggests that the presence of multiple MC1R variants is associated with the development of multiple melanoma tumors in patients with CDKN2A mutations. Additional studies are needed to confirm these findings and to explore the mechanisms that may contribute to this relationship.

	Introduction

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Cutaneous malignant melanoma (CMM) is a potentially fatal form of skin cancer whose etiology is heterogeneous and complex. Major host and environmental risk factors for melanoma include many melanocytic nevi, especially dysplastic nevi, fair pigmentation (skin, eye, and hair), freckling, poor tanning ability, and a tendency to burn after sun exposure (1, 2). Genetic risk factors include germ line mutations in the CDKN2A, CDK4, or MC1R genes. CDKN2A and CDK4 have been designated "high-risk" melanoma susceptibility genes. The CDKN2A gene is the major known melanoma susceptibility gene. Germ line mutations have been detected in 20% of melanoma-prone families with three or more melanoma patients. In contrast, few families with germ line mutations in CDK4 have been identified. MC1R has also been shown to influence melanoma risk, but it is described as a "low risk" melanoma susceptibility gene (3, 4).

MC1R is involved in pigmentation primarily through its binding with -melanocyte-stimulating hormone (5). MC1R is very polymorphic, with >65 nonsynonymous alleles identified to date (6, 7). Three variants (R151C, R160W, and D294H) designated red hair color or RHC variants have been repeatedly shown to be associated with red hair color, poor tanning ability, pale/fair skin color, and extensive freckling (8-10). Most other variants (designated non-RHC or NRHC) have a weaker or no association with red hair (10, 11). Several studies conducted in generally fair-skinned populations of Northern European origin have shown that risk of melanoma is higher among MC1R variant carriers than among noncarriers, with the strongest effects observed for carriers of multiple variants (9, 12, 13).

MC1R has also been shown to be a risk factor for melanoma in families segregating CDKN2A mutations. A study of 15 Australian CDKN2A mutation–carrying families with nine different mutations (14) and a study of 101 p16-Leiden–mutation carriers from six Dutch families (15) both showed that the presence of MC1R variants increased the frequency/penetrance of melanoma among CDKN2A mutation carriers. The MC1R-melanoma association was primarily related to the R151C variant in the Dutch families and to the three RHC variants in the Australian families. There was also an inconsistent reduction in age at melanoma diagnosis associated with the presence of at least one MC1R variant; this age reduction was observed in the Australian study sample but not in the Dutch families. Further studies are needed to confirm and refine the findings from the Australian and Dutch CDKN2A families. The objective of the current study was to evaluate the relationship between MC1R and melanoma risk in 16 CDKN2A melanoma-prone American families with extensive clinical and epidemiologic risk factor data.

	Materials and Methods

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Participants and Design
Families were recruited for this non–population-based family study if there was a history of invasive melanoma in at least two first-degree relatives. The families were referred by health-care professionals or through self-referrals. Written informed consent was obtained prior to participation under an Institutional Review Board–approved protocol. All family members willing to participate in the study were clinically evaluated. Variables recorded during the clinical examination included the type and total number of nevi, extent of freckling, skin complexion, evidence for solar injury, and hair and eye color. In addition, a self-administered questionnaire obtained information on sun-related variables such as the skin's reaction to acute and chronic sun exposure (i.e., tanning ability). The subjects for this study were drawn from 16 families in which a CDKN2A mutation had been previously identified (16). The families had the following 12 mutations: 1_8dup8, L16R, M53I, R58X, N71S, R87P, S56fs (c.167_197del31), c.240_253del14, P75fs (c.225_243del19), G101W (n = 3), V126D (n = 3), and c.IVS2+1 G>T. The families have been followed prospectively from 4 to 26 years starting in the 1970s. All melanoma diagnoses were confirmed by review of histologic materials, pathology reports, medical records, or death certificates. Total numbers of primary melanomas were recorded for each melanoma patient.

Sequencing of MC1R
MC1R genotyping was conducted at the National Cancer Institute, Frederick, MD employing PCR amplification of the 951 bp coding region of MC1R, either in its entirety or in smaller overlapping segments, followed by complete direct sequencing of the amplicon(s). The coding region of MC1R was amplified from genomic DNA extracted from patient blood samples using two sets of M13-tagged PCR primers MC1R_1F: 5'-GTA AAA CGA CGG CCA GTG AAG ACT TCT GGG CTC CCT C-3'; MC1R_IIIR: 5'-GGA AAC AGC TAT GAC CAT GGC GTG CTG AAG ACG ACA CT-3'; and MC1R_IVF: 5'-GTA AAA CGA CGG CCA GTG TGC TGT ACG TCC ACA TGC T-3'; MC1R_IVR: 5'-GGA AAC AGC TAT GAC CAT GCT CTG CCC AGC ACA CTT AAA-3'. The underlined region of the primer is specific to the target DNA. The reaction mix for PCR amplification included 1x PCR buffer (Invitrogen high-fidelity PCR buffer), 1.5 mmol/L MgSO₄, 175 nmol/L each pair of primers, 50 nmol/L each of the four deoxynucleotide triphosphates, and 1 unit of HiFi Platinum Taq polymerase (Invitrogen, Carlsbad, CA). All PCR products were processed prior to sequencing. All products from two regions of PCR were sequenced with ABI prism BigDye terminator cycle sequencing kit 1.0 (Applied Biosystems, Inc.) on ABI3700 sequence analyzer using sequence primers 1F: 5'-GCT CCC TCA ACT CCA CC-3'; IR: 5'-GAA GAC GAC ACT GGC CAC-3' and M13F: 5'-GTA AAA CGA CGG CCA GT-3'; M13R: 5'-GGA AAC AGC TAT GAC CAT G-3', respectively. All sequences were analyzed and variants were detected using Mutation Surveyor (SoftGenetics Inc., PA) and sequence analysis software package developed at the Laboratory of Molecular Technology, National Cancer Institute.

Statistical Analyses
Initially, we evaluated each MC1R variant individually comparing 1+ variant to the consensus MC1R sequence (i.e., wild-type MC1R). Because many MC1R variants were too rare to examine their individual associations with melanoma risk after adjustment for major melanoma risk factors (i.e., CDKN2A status, nevus/pigmentation factors—see below), we also used the following MC1R variables in the analyses: carriers of any MC1R variant compared with wild-type MC1R; carriers of multiple (1, 2+) variants compared with the consensus sequence; carriers of 1 NRHC variant, 2+ NRHC variants, 1 RHC variant, 2+ RHC variants, or carriers of both RHC and NRHC variants compared with wild-type MC1R.

For purposes of this study, the measure of association between melanoma risk and the clinical, genetic, and environmental variables was the odds ratio (OR). Point estimates and 95% confidence intervals (CI) of adjusted ORs were calculated using logistic regression analysis as implemented in the EPICURE package (17).

We assessed the association of pigmentation and nevus characteristics with all nonsynonymous MC1R variants combined using ² and Fisher exact tests in the unaffected relative and spouse controls separately (Stata 8.2; ref. 18). Dysplastic nevi, hair color, eye color, skin complexion, freckling, solar injury, and tanning ability were all associated with MC1R. We also evaluated the ORs between these same factors and melanoma risk. Because of the relatively small number of cases, we created summary factors that combined the covariates showing the strongest associations with both MC1R variants and melanoma risk. A three-category nevus factor was created by combining dysplastic nevi (absent, indeterminate, present) and total numbers of nevi. Similarly, a three-category pigmentation factor was developed by combining skin complexion (medium/dark, pale/fair) and extent of freckling (none/few, moderate, many).

We conducted two logistic regression analyses (17). The first analysis conditioned on family membership using the entire data set (72 melanoma cases, 245 unaffected relative, and 78 spouse controls). We also conducted an unconditional logistic regression analysis on the subset of confirmed CDKN2A mutation carriers (69 cases and 72 unaffected relative controls). All analyses were adjusted for age as a continuous variable. Sex had no effect on risk of melanoma and therefore was excluded from all analyses (data not shown). For the conditional logistic regression analysis, three models were examined: univariate (with age adjustment); adjustment for CDKN2A status and age; and adjustment for age, CDKN2A, and the pigmentation/nevus factors. For the unconditional analysis of CDKN2A mutation carriers, two models were evaluated: univariate (adjusted for age) and adjustment for age and pigmentation/nevus factors.

We examined the distribution of MC1R variants in multiple primary melanoma (MPM) compared with single primary melanoma (SPM) patients using Fisher exact test as implemented in StatXact 4 (19). We also estimated the median ages at diagnosis of initial melanomas in all melanoma patients and in MPM and SPM patients separately. The nonparametric Jonckheere-Terpstra test was used to investigate the hypothesis of no differences among the ages at diagnosis of melanoma according to numbers or numbers/types of MC1R variants against the alternative that the ages at diagnosis decreased as the numbers or numbers/types of MC1R variants increased.

	Results

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Ten nonsynonymous and five synonymous (i.e., silent) MC1R variants were detected in the 395 subjects sequenced for MC1R. Table 1 shows the number of cases, unaffected relative controls, and spouse controls with each of the nonsilent variants observed. The five silent variants found (R34R, A166A, A240A, I264I, and T314T) were excluded from all analyses. The most frequent variants observed were V60L, R160W, and R151C. As has been previously observed in other studies, there was a strong association between the RHC variants R151C, R160W, and D294H and red hair color. Seventy-seven percent of the subjects (20 of 26) with red hair had at least two RHC variants. Also, only 29% of the subjects (8 of 28) without red hair had two RHC variants. These eight subjects had primarily brown or light brown hair. In contrast, few subjects with V60L, V92M, I155T, or R163Q had red hair color.

Table 2 shows the associations between melanoma risk and selected MC1R covariates. Table 2A presents the ORs and 95% CIs for all three conditional analysis models evaluated. For the three analyses, the presence of at least two MC1R variants was significantly associated with melanoma [OR, 5.6 (2.1-14.7); OR, 20 (5-80); and OR, 6.1 (1.2-29.7), respectively]. Any MC1R variant, the number of variants, and types of variants also showed significant but imprecise associations with melanoma when we adjusted for age only or age and CDKN2A status. R151C and R160W also showed significant associations with CMM after adjustment for both age and CDKN2A status [OR, 11.3 (1.4-93.3) and OR, 9.1 (1.6-52.4), respectively]. Table 2B shows the number of cases and unaffected relative controls who were CDKN2A mutation carriers and results from the unconditional subset analysis of CDKN2A mutation carriers. There were significant associations between melanoma risk and all three summary MC1R variables examined after adjustment for age only. In addition, after adjustment for age and the pigmentation/nevus factors, there were significant associations between melanoma and multiple MC1R variants [OR, 7.3 (1.6-33.2)] as well as suggestive associations with the presence of at least two NRHC variants [OR, 7.1 (1.0-49.4)] or the presence of both RHC and NRHC variants [OR, 5.7 (1.0-32.2)]. These analyses were, however, based on relatively small numbers that resulted in wide confidence intervals. It was not possible to fully evaluate the number of RHC variants. Specifically, seven cases and no controls had two RHC variants.

	Discussion

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To the best of our knowledge, this is the first study of MC1R variants in CDKN2A mutation carriers that examined the relationship between MPM and SPM patients from the same study sample. The MPM findings observed here are further supported by a small Italian study of 14 MPM patients without a positive family history for melanoma; Peris et al. (21) detected MC1R variants in 11 of 12 patients with nonfamilial MPM, a much higher frequency relative to that previously reported in other populations (22). Two of the patients with MC1R variants also had CDKN2A mutations as well as red hair color. The authors suggested that the results might represent an example of the effects of gene-gene interaction on disease risk (21). The current study with thrice the number of MPM patients plus 29 SPM patients, all with CDKN2A mutations, suggests that the presence of multiple MC1R variants is associated with the development of multiple melanoma tumors in patients with CDKN2A mutations. Although the small sample size precludes full evaluation of this association, the dampening of the complex host risk with sun-related factors (i.e., freckling/multiple nevi/dysplastic nevi) hints at the possible importance of sun exposure. Additional studies are needed to confirm these findings and to explore the mechanisms that may contribute to this relationship.

The Australian and Dutch studies of MC1R variants in melanoma-prone families with CDKN2A mutations showed inconsistent differences in age at melanoma diagnosis. In the Australian study, mean age at melanoma diagnosis decreased significantly from 58.1 to 37.8 years with the presence of one or more MC1R variants (14). In contrast, the Dutch study showed no such reduction in age at diagnosis; in fact, the mean age at melanoma diagnosis was 40 years in melanoma patients with no MC1R variants and 42 to 45 years in patients with two or more MC1R variants (15). The current study revealed a significant decrease in median age at melanoma diagnosis as the overall number of MC1R variants increased and when looking at the number of RHC and NRHC variants separately. However, this association resulted from melanoma patients with >1 melanoma tumor (i.e., MPM patients). That is, among the 29 patients with only one melanoma tumor, there was no significant association between MC1R variants and age at melanoma diagnosis. It is possible that differences in the number of MPM versus SPM patients between the Dutch and Australian studies may have contributed to the inconsistent results observed in these two studies. Alternatively (or in addition), differences in the types or distribution of CDKN2A mutations across the two studies—nine CDKN2A mutations in the Australian study versus one founder mutation (p16-Leiden) in the Dutch study—might have influenced the ages at melanoma diagnosis and/or the development of MPM tumors. Finally, distribution of major melanoma risk factors including relative amounts of sun exposure and the skin's reaction to sun exposure may have differed between the two studies. Further studies are needed to evaluate the age association between MC1R and numbers of melanoma tumors (and sun exposure).

The current study was limited by the small number of confirmed mutation carriers. The small size precluded adjustment for family membership in the CDKN2A mutation carrier subset analysis. In addition, it was not possible to examine individual CDKN2A mutations or CDKN2A mutations classified according to their type, location, or effect on the p14ARF protein. Also, it was difficult to evaluate MC1R variants separately. In addition, even though significant associations between melanoma risk and multiple MC1R variants were observed after adjustment for major melanoma risk factors, the odds ratio estimates were imprecise with wide confidence intervals. Finally, although all family members were invited to participate in the study, differential inclusion of mutation carriers, deceased melanoma cases or relatives with certain exposures could influence the results. It is difficult, however, to predict whether the odds ratios would be decreased or increased by this potential participation bias. In conclusion, this study of 16 melanoma-prone American families with CDKN2A mutations adds to the growing literature of studies demonstrating a relationship between multiple MC1R variants and melanoma risk. The study also provides new directions for research to further explore the differences in the distribution of MC1R variants and ages at melanoma diagnosis observed in MPM versus SPM patients. Studies with much larger sample sizes and extensive epidemiologic, clinical, and genetic risk factor data will be required to investigate these relationships further.

	Acknowledgments

 
We are indebted to the participating families, whose generosity and cooperation have made this study possible. We also acknowledge the contributions to this work that were made by Virginia Pichler, Laura Fontaine, Mary Wells, and Deborah Zametkin. We thank Benjamin Hulley for analytic support and Rashida Williams for help with manuscript preparation. This research was supported in part by the Intramural Research program of the NIH, NCI, DCEG.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/ 3/05; revised 6/21/05; accepted 7/ 5/05.

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