Affiliations of authors: Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Bethesda, MD (MTL, MT-M, AMG, RMP); University of Pennsylvania School of Medicine, Philadelphia (PAK, TR, JS); Laboratory of Molecular Technology (ST, DM) and Laboratory of Genomic Diversity (BG), National Cancer Institute, SAIC, NIH, DHHS, Frederick, MD; Department of Biochemistry, Johns Hopkins University, Baltimore, MD (MH, LG); Dermatology Unit, Maurizio Bufalini Hospital, Cesena, Italy (DC)
Correspondence to: Maria Teresa Landi, MD, PhD, Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, 6120 Executive Blvd., EPS 7114, Bethesda, MD 208927236 (e-mail: landim{at}mail.nih.gov).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Families at high risk of developing melanoma have provided evidence of additional genetic factors that may be involved in melanoma susceptibility. Studies of large melanoma-prone families have demonstrated linkage of melanoma susceptibility to a locus on chromosome 9p21 (MIM 600160) in the majority of kindreds and probable linkage to 1p22 (9) and 1p36 (10) in others. About one-third of families in which melanoma risk is linked to 9p21 carry mutations in the CDKN2A tumor suppressor gene (11), which encodes the p16 cell cycle inhibitor. Rare kindreds may also possess mutations in the coding sequences of CDK4 (MIM 12829) (12) or p14ARF, which is translated from exons 1 and 2 of the CDKN2A gene (13). Another potential candidate gene is CDKN2B, which is also part of the cell-cycle regulation pathway (51).
One low-penetrance gene that has been associated with melanoma risk is the melanocortin-1 receptor gene (MC1R, MIM *155555). Its protein product binds to the -melanocytestimulating hormone (
-MSH) and adrenocorticotropin (ACTH) (14), and regulates pigmentation. MC1R is highly polymorphic, and more than 65 human MC1R alleles with nonsynonymous changes have been identified to date (15). Studies of Northern European populations have identified several MC1R variants that are associated with fair pigmentation phenotypes. In particular, the presence of the Arg151Cys, Arg160Trp, or Asp294His variant of MC1R, any of which is known as a "red hair color" (RHC) variant, has been associated with red hair, low tanning ability, light skin color, and freckling (1618). Other MC1R variants have a weak or no association with red hair (18) and are referred to as "non-red hair color" (NRHC) variants. Several studies in fair-skinned populations of Northern European origin have reported that the risk of melanoma is higher among individuals who carry MC1R variant alleles than among individuals who are wild type for MC1R (17,19,20). MC1R can also act as a modifier of melanoma risk within CDKN2A-positive melanoma-prone families (20,21), whereas the risk associated with MC1R variant alleles in CDKN2A-negative families is unknown.
Another pigmentation gene that has the potential to be associated with melanoma risk and is highly conserved across species is the Agouti Signaling Protein gene (ASIP). In mice, its protein product acts as an inverse agonist of the MC1R gene, which may be able to decrease melanogenesis (21). In humans, an A-to-G substitution (g.8818A>G) in the 3' untranslated region (UTR) of the ASIP gene (MIM, *600201) has been associated with dark hair and eye color among Caucasians (22). However, it is not known whether ASIP is associated with melanoma risk.
We examined the associations of variant alleles of the MC1R and ASIP genes with pigmentation, nevi count, and melanoma risk in a Mediterranean population that is mostly negative for mutations in additional melanoma candidate genes. We also assessed whether these genes and/or DNA repair capacity were involved in disease progression.
![]() |
SUBJECTS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study included 267 melanoma patients and 382 control subjects identified from a casecontrol study and a family study in northeastern Italy. The casecontrol study, which has been described in detail by Landi et al. (1), included 183 melanoma patients (87 males and 96 females) and 179 control subjects (89 males and 90 females) aged 1777 years. Melanoma patients had no family history of melanoma. Of the 179 control subjects, 134 were spouses or close friends of the melanoma patients, 14 were outpatients referred to the hospital because of minor accidental trauma, and 31 were healthy volunteers from hospital personnel. Control subjects were frequency-matched to melanoma patients by decade of age and sex. All control subjects were from the same geographic area as the melanoma patients. None of the subjects in this study had mutations in any of the major candidate genes for melanoma, including CDKN2A, CDK4, or p14ARF.
The family study, which has been described in detail by Landi et al. (23), included 55 melanoma-prone families, which are defined as having two or more individuals affected with melanoma per family. As in many melanoma-prone kindreds, several individuals with melanoma had dysplastic nevi, multiple melanomas, or, more rarely, pancreatic cancer. Some of the melanoma case patients were deceased at the time of family recruitment; thus, DNA was obtained from 84 melanoma patients and 203 unaffected individuals (23 of whom were spouses, i.e., not blood relatives). All families have been tested for presence of mutations in CDKN2A, p14ARF, CDK4, and CDKN2B. Only four families (nine subjects) had mutations in CDKN2A, and no family had a mutation in any of the other genes.
All 649 subjects from both studies were recruited at the Dermatology Department of the Maurizio Bufalini Hospital of Cesena, Italy. During the study period, the Bufalini Hospital examined approximately 85% of all melanoma patients diagnosed in the area, as verified by comparison of patient lists with the Romagna region cancer registry (24) and with records of melanoma diagnoses from the main hospitals of the area. Both studies were approved by the Bufalini Hospital and National Cancer Institute's ethical committees, and written informed consent was obtained from all participants. A standardized in-person questionnaire was administered to all participants by trained interviewers, who asked questions on lifetime residential history, medical history, family history of cancer and other diseases, drug consumption (i.e., corticosteroids or photosensitizing antibiotics), skin reaction to the first half-hour of sun exposure, tanning ability after prolonged sun exposure according to the Fitzpatrick score (25), and sunscreen use. Participants in the casecontrol study were also asked questions about exposure to sun and artificial UV radiation. The entire skin of each participant's body, except for the genital area, was examined by a single dermatologist (DC) to assess pigmentation characteristics, freckling, presence of skin lesions, and presence of other skin cancers.
To assess all diagnoses of dysplastic nevi and verify the number of nevi, an expert oncologist, blinded to melanoma status, was provided with standardized photographs of the back of each subject. Nevus count was restricted to the back to decrease the amount of time spent examining the patient and to maximize subject participation rate. However, restricting nevus count to the back should not negatively impact classification based on this characteristic, since the number of nevi on the trunk has been shown to be highly correlated (r = .82 in female Caucasians and r = .90 in male Caucasians) with the number of nevi on the entire body (26). Moreover, recognition and classification of nevi and dysplastic nevi from photographs have been shown to be accurate and reproducible (27). To be defined as dysplastic, a nevus had to be 5 mm or larger, be predominantly flat, and have at least two of the following characteristics: variable pigmentation, indistinct borders, and an irregular outline (8,10,28). The study dermatologist assessed the skin, hair, and eye color of each study subject (1).
Standardized Measurements of Skin Color and UV Radiation Sensitivity
In addition to qualitatively examining skin color and sensitivity to the sun, we objectively assessed these traits. Detailed description of the two instrumental assessments has been reported by Brenner et al. (29). Briefly, skin color was measured using a Minolta 300 spectrophotometer/colorimeter (CR-300, Konica-Minolta, Osaka, Japan). The colorimeter measures skin color by measuring reflected light from the visible spectrum (range, 400700 nm) and records colors in a three-dimensional space of dimensions, L*, a*, and b* (30), where L* = the total quantity of light reflected or brightness; a* = color ranging from red (positive values) to green (negative values); and b* = color ranging from blue (negative values) to yellow (positive values). Cutaneous sensitivity to UV radiation, or minimal erythema dose, was determined by measuring the minimal UV radiation dose able to provoke perceptible erythema of the skin 2024 hours after exposure (31). Minimal erythema dose was measured in an area of the body that is unexposed to the sun.
Sequencing of MC1R
MC1R was sequenced by two different laboratories at NCI-Frederick and the University of Pennsylvania in Philadelphia, using similar protocols. The 951-bp coding region of MC1R, either in its entirety or in smaller overlapping segments, was amplified by polymerase chain reaction (PCR) followed by complete direct sequencing of the amplicon(s). Specific primer sequences and sequencing chemistries from the NCI laboratory are given below, and protocol details from the second laboratory (32) are available upon request.
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') and 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') and MC1R_IVR (5'-GGA AAC AGC TAT GAC CAT GCT CTG CCC AGC ACA CTT AAA-3'). The underlined regions of the primers are specific to the target DNA. Amplification PCRs included 1x PCR buffer (Invitrogen High Fidelity PCR buffer); 1.5 mM MgSO4; a 175 nM concentration of each pair of primers; a 50 nM concentration of each of the four dNTPs; and 1 unit of HiFi Platinum Taq polymerase (Invitrogen, Carlsbad, CA) and were carried out under the following conditions: 95 °C for 3 minutes, followed by 40 cycles of 95 °C for 1 minute, 58 °C for 1 minute, 72 °C for 1 minute, and finally an extension of 72 °C for 7 minutes. All PCR products were analyzed on a DNA 1000 labchip using "lab-on-a-chip" capillary electrophoresis (Agilent Technologies, Palo Alto, CA). All PCR products were treated with exonuclease I and shrimp alkaline phosphatase (USB Corporation, Cleveland, OH) as described (26), and the PCR product was purified on Sephadex G-50 (Sigma-Aldrich, St. Louis, MO) as (33) described prior to sequencing. All PCR products were sequenced with the ABI prism BigDye Terminator Cycle Sequencing Kit 1.0 (Applied Biosystems, Foster City, CA) on an ABI3100 sequence analyzer using the sequence primer pairs 1F (5'- GCT CCC TCA ACT CCA CC-3') and IR (5'- GAA GAC GAC ACT GGC CAC-3') and M13F (5'-GTA AAA CGA CGG CCA GT-3') and M13R (5'-GGA AAC AGC TAT GAC CAT G-3'). All sequences were analyzed and variants were detected using Mutation Surveyor (Soft Genetics LLC, State College, PA).
Genotyping of ASIP Polymorphism With 5'-Nucleotidase Assay
Sequences of the ASIP gene surrounding g.8818A>G (rs6058017) were submitted to an Applied Biosystems Assays-by-Design service. Primer sequences were 5'-CGTGCT CAGCCTCA-3' and 5'-AGCCGCCCTGTTAG-3', and probe sequences were 5'-VIC-CCGCGAGCGGGCAGGG-MGBNFQ-3' and 5'-FAM-CCGCGAGCAGGCAGGGC-MGBNFQ-3'; VIC, MGBNFQ, and FAM are reporter dyes (Applied Biosystems). ASIP genotypes were determined using the 5'-nucleotidase assay as previously described (34). For genotype sequence confirmation, 50 ng of DNA from samples known to be homozygous wild-type, heterozygous, or homozygous variant was selected from a panel of 88 anonymous, ethnically diverse DNAs (39% of European American origin, 34% of African American origin, and 27% of Hispanic origin) from a large AIDS cohort studied by the Laboratory of Genomic Diversity at the NCI. These samples were amplified using 2.5 units of AmpliTaq Gold enzyme (Applied Biosystems, Foster City, CA) in 4 mM MgCl2, a 250 mM concentration of each nucleoside triphosphate, and 200 nmol of each primer, ASIP SEQF: 5'-GTAAAACGACGGCCAGTAGAAGGAGGCTTCGATG-3' and ASIP SEQR: 5'-GGAAACAGCTATGACCATGACCTTCGAGCAGCCTAT-3', under the following cycling conditions: 95 °C for 10 minutes followed by 35 cycles of 95 °C for 30 seconds, 58 °C for 30 seconds, 68 °C for 1 minute, followed by a final extension of 72 °C for 7 minutes. If necessary, the products were held overnight at 4 °C. PCR products were then treated with exonuclease I and shrimp alkaline phosphatase (USB Corporation) and were subjected to BigDye Terminator cycle sequencing in the presence of an M13 forward or reverse primer tag (M13 forward: 5'-GTAAAACGACGGCCAGT-3' or M13 reverse: 5'-GGAAACAGCTATGACCATG-3') for 15 minutes at 37 °C. PCR products were purified on Sephadex G-50, dried on a thermocycler (95 °C uncapped for 20 minutes), resuspended in 100% formamide, and electrophoresed on an ABI 3700 sequencer (Applied Biosystems, Foster City, CA). Forward and reverse trace files were examined visually, and sequences were determined using Mutation Explorer software (Soft Genetics, State College, PA).
Assessment of DNA Repair Capacity
DNA repair capacity was measured in cryopreserved lymphocytes of the subjects in the casecontrol study by the hostcell reactivation assay, as described by Athas et al. (35). Briefly, plasmid DNA containing a chloramphenicol acetyltransferase (CAT) reporter gene was UV irradiated (254 nm at 0, 350, and 700 J/m2) and then transfected into peripheral blood lymphocytes. The cells were incubated at 37 °C for 40 hours. After incubation, DNA repair capacity was calculated as the percentage of residual CAT gene expression after the repair of UV radiationdamaged plasmid DNA divided by that in undamaged plasmid DNA (100%). In all of the analyses, DNA repair capacity values measured after irradiation with UV doses of 350 and 700 J/m2 were similar. Consequently, we report only results obtained after irradiation at 350 J/m2.
CAT activity in unirradiated cells must be more than 1000 cpm to accurately measure DNA repair capacity with a signal-to-noise ratio that is at least double the background level (200 cpm) in cells with irradiated plasmids. Therefore, subjects with baseline CAT activities of less than 1000 cpm (51 of 183 melanoma patients, 27.9%, and 34 of 179 control subjects, 19.0%) were excluded from the analysis. We analyzed data from 132 melanoma patients and 145 control subjects from the casecontrol study. Details on the assay and related quality control measures are described by Landi et al. (8).
Statistical Methods
Most MC1R variants were rare, and we were unable to independently assess the association between each MC1R variant and melanoma risk or pigmentation characteristics. Therefore, we analyzed three different risk categories, as follows (all compared with homozygous carries of the wild-type [WT] MC1R sequence): 1) carriers of any MC1R variant; 2) carriers of at least one RHC variant (RHC/RHC, RHC/NRHC, or RHC/WT) or of only non-RHC variants (NRHC/NRHC or NRHC/WT); and 3) carriers of two variants (RHC/RHC, RHC/NRHC or NRHC/NRHC). We also repeated the analyses excluding the RHC/NRHC genotypes from the RHC group to assess the association of "pure" red hair color variants with melanoma risk and pigmentation characteristics. Subjects were also characterized by the presence or absence of the ASIP polymorphism: SNP/WT or SNP/SNP vs. WT/WT.
We assessed the associations between pigmentation characteristics and genotypes in the control subjects of the casecontrol study using chi-square and Fisher's exact tests. In the family study we used the generalized estimating-equations approach to account for correlations among controls from the same family. For most analyses, hair color was categorized into four groups: black, dark brown, light or reddish brown, and blond or red. Eye color was categorized into three groups: 1) black or dark brown; 2) light brown, brown-green, green or blue-green; and 3) light blue, blue, or grey. Skin color was categorized into three groups: dark/olive, medium, and light. Tanning ability after prolonged sun exposure was categorized into three groups: high, medium, and low. Freckling was categorized in two groups: presence and absence of freckles.
Odds ratios (ORs), 95% confidence intervals (CIs), and tests for trends were computed from unconditional logistic regression models (PROC GENMOD, SAS 8.2) for the casecontrol study and from conditional logistic regression models for the family study, conditioning on family (PROC PHREG, SAS 8.2). We adjusted the association for age, sex, presence of dysplastic nevi, tanning ability after prolonged sun exposure, and hair color by including these variables in the regression models. In the analyses involving only "pure" RHC and "pure" NRHC, we adjusted the models for age, sex, skin color, freckling, and Minolta b* index.
Odds ratios were computed for both studies combined by maximum likelihood estimation based on a likelihood function that multiplied conditional logistic regression terms for the families and unconditional logistic regression for the subjects from the casecontrol study, with adjustment for "study type," as well as for the variables mentioned above. For the family study, we also verified the validity of results from the conditional logistic models by comparing one randomly selected melanoma case patient from each family and all controls from the casecontrol study, using unconditional logistic regression. We obtained model-based 95% confidence intervals for the odds ratio estimates using the model information matrix. Computations were performed in Gauss (Aptech Systems Inc., Maple Valley, WA).
To tightly control for pigmentation characteristics, many variables would need to be included in the logistic regression models; however, many of these characteristics are highly correlated. To validate the pigmentation variables we ultimately included in the models, we compared the model containing each of these variables to the models that included all of the pigmentation characteristics in the form of independent factors from a factor analysis (36) (PROC FACTORS, SAS 8.2). Using both separate and combined data from the casecontrol and family studies, we constructed two factors for analysis based on the number of eigenvalues from the correlation matrix that were greater than 1 (37). Factors were extracted using principal factor analysis and then rotated to derive factors that were orthogonal (i.e., independent). The statistically significant contributors to the first factor or "pigmentation" factor included hair color, eye color, skin color, and tanning ability. In addition, the Minolta b* skin color was a statistically significant contributor in the casecontrol study. The statistically significant contributors to the second factor or the "nevi" factor were number of nevi and presence of dysplastic nevi. For the casecontrol study, freckling was also a statistically significant contributor to the nevi factor. We then calculated factor scores for each individual and categorized the scores into equal-sized quartiles. This categorical variable was then used in logistic regression analysis that also controlled for age and gender. Models that included the original pigmentation and nevi variables were compared to models that used the derived factors using the likelihood ratio test. All P values are two-sided, where a P of <.05 was considered statistically significant.
Associations between categories of pigmentation characteristics and MC1R genotypes were assessed in logistic regression models to investigate effect modification as well as direct association with the genotypes. Poisson regression models were used when the number of nevi was the outcome. Results that suggested an interaction in stratified analyses were further examined for multiplicative interactions between MC1R variant alleles and the subjects' characteristic, e.g., pigmentation or ASIP polymorphism.
Analysis of MC1R could be performed on 165 case patients and 171 control subjects, and analysis of ASIP could be done on 166 case patients and 172 control subjects. Five control subjects and 13 case patients lacked both MC1R and ASIP data and were excluded from the analyses involving genotypes. DNA repair capacity was measured in 183 case patients and 179 control subjects. Different models may include different number of subjects because of missing values in some of the covariates.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Overall, 32 different MC1R variants were found: 29 in subjects from the casecontrol study and 15 in subjects from the family study (Table 1). None of the nonsynonymous variants identified deviated from HardyWeinberg equilibrium. Three of these variants have not been reported previously, to our knowledge. One new variant, insA29, introduces a stop codon that results in a shorter, potentially nonfunctional protein. Frequencies of most variants were low. The most common variant was V60L. RHC variant alleles were more frequent in melanoma patients than in control subjects in both studies. Overall, 41 patients (26%) and 14 control subjects (8%) in the casecontrol study and 32 patients (38%) and 36 control subjects (18%) in the family study carried two variants of MC1R. In the casecontrol study, 41 melanoma patients (25%) and 46 control subjects (27%) carried the ASIP g.8818A>G polymorphism (data not shown).
|
The number of control subjects who carried either the RHC or NRHC variant alleles by age, gender, pigmentation characteristics, or nevi is shown in Table 2. In the casecontrol study, individuals who carried one or two RHC variants but not those who carried an NRHC variant had lighter hair (Ptrend = .03) and lighter skin (Ptrend = .005), were more likely to have freckles (P = .02) and dysplastic nevi (P = .02), and had more nevi (P<.001, in a Poisson regression model) than did those with wild-type alleles. In the family study, RHC variant genotypes were associated with tanning ability (Ptrend = .02). All RHC carriers (16/16) had freckles. By contrast with MC1R, the ASIP polymorphism was not associated with pigmentation characteristics (data not shown). It was also not associated with MC1R variants categorized by RHC or NRHC (OR = 0.5; 95% CI = 0.2 to 1.7, and OR = 1.2; 95% CI= 0.6 to 2.6, for RHC and NRHC, respectively).
|
Association of MC1R and ASIP with Instrument-Based Measures of Pigmentation and UV Radiation Sensitivity
Overall, there was no statistically significant association between the MC1R variants or the ASIP polymorphism with the Minolta L* and a* skin colors. Similarly, there was no statistically significant association between variants in either gene with UV radiation sensitivity, as measured by minimum erythema dose. However, we found a strong association between RHC and NRHC MC1R variants and high Minolta b* (i.e., yellowish) color of the skin (P = .006). When we stratified the analysis by sex, the association of MC1R variants with Minolta b* was statistically significant in men (P = .007) but not in women (P>.44). This difference may stem from men's generally thicker epidermis (38).
Association of MC1R and ASIP Variants with Melanoma Risk
The association between MC1R variants and melanoma risk in the casecontrol and the family studies separately as well as in both studies combined is shown in Table 3. In all three sets of analyses, subjects carrying any variant, one or more NRHC variants, one or more RHC variants, "pure" RHC variants, or two of any MC1R variants had a two- to threefold increase in melanoma risk compared with subjects who carried the wild-type sequence, after adjustment for age, sex, hair color, tanning ability, and presence of dysplastic nevi. These findings were confirmed in models that adjusted for skin color, freckling, or "pigmentation" and "nevi" factors (data not shown). For the family study, we verified the validity of our results by comparing one randomly selected melanoma patient from each family and all control subjects from the casecontrol study. Although this analysis was based on a smaller number of subjects, the results were similar (data not shown).
|
Of 55 families, only four were positive for CDKN2A mutations. Family members of two of the four families with CDKN2A mutations had no MC1R variant alleles. The exclusion of these four families did not substantially change the association between MC1R and melanoma risk (data not shown). We also found no association between MC1R variants and presence of multiple melanomas (12 cases) or age at first melanoma diagnosis (data not shown).
Carrying the ASIP polymorphism was not associated with melanoma risk overall (OR = 1.1; 95% CI = 0.6 to 1.8). However, we did find some associations with the ASIP SNP in selected subgroups (based on small numbers), as described below.
Association between MC1R Variants and Melanoma Thickness or Body Site
We investigated the association between melanoma thickness, an indicator of melanoma progression, and MC1R variants in case patients from the casecontrol study. Case patients with any MC1R variant were at three- to fourfold risk of having thick melanoma lesions compared to melanoma patients who were homozygous wild type for MC1R, after controlling for age, sex, hair color, tanning ability, and dysplastic nevi (Table 4). The addition of DNA repair capacity to the model did not substantially change the results (data not shown). We also investigated the association between presence of MC1R variants and the physical location of the melanomalesions. In the casecontrol study, carrying multiple MC1R variants was statistically significantly associated with melanoma in areas of the body that are continuously exposed to the sun (e.g., OR of having a melanoma on face, neck, or back of hands = 8.5; 95% CI = 2.2 to 33.6), although this conclusion was based on small numbers. In contrast, carrying the ASIP polymorphism was inversely associated with melanoma at those same body sites (OR = 0.08; 95% CI = 0.01 to 0.9). There was no association between carrying MC1R variants and developing melanoma in areas of the body that are intermittently or rarely exposed to sun (data not shown). We were unable to confirm these results in the family study because so few subjects had melanomas in continuously sun-exposed areas of the body. However, there was a statistically significant inverse association between carrying any MC1R variant (OR = 0.01; 95% CI = 0.001 to 0.3) or multiple MC1R variants (OR = 0.05; 95% CI = 0.005 to 0.4) and melanoma in areas of the body that are rarely, if ever, exposed to the sun (e.g., soles of the feet, nail beds, and mucosa).
|
We explored potential effect modifications of the association between MC1R genotypes and melanoma risk (Table 5)by nevi- and pigmentation-related variables. Although the results are based on small numbers, the association between RHC variants and melanoma was stronger in subjects who had characteristics that are associated with being at lower risk of developing melanoma, (i.e., few nevi, no dysplastic nevi, no freckling, low sun exposure, dark hair, and low Minolta b* index). In particular, the association between RHC variants and melanoma was modified by freckling, and this effect modification was statistically significant in a test for multiplicative interaction (P = .02). In subjects with NRHC, melanoma risk was not statistically significantly modified by nevi- and pigmentation-related variables. We could not explore this association in the family study because of the low frequency of subjects with pure RHC genotype, even after inclusion of the D84E variant, as suggested by Duffy et al. (18).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We observed a two- to fourfold-increased risk of melanoma among individuals who carried MC1R variant alleles, similar to the increased risks reported in analyses of MC1R and melanoma risk in subjects of Celtic or Germanic origin (17,19), but we found no association between melanoma risk and the ASIP polymorphism. Furthermore, the magnitude of the odds ratios was similar in the casecontrol and family studies, suggesting a similar role of the gene in sporadic and familial melanoma in this population. This similarity may be related to the lack of germline CDKN2A mutations in most families in the family study. In fact, only two families had members who carried a germline CDKN2A mutation and also carried an MC1R variant allele. Interestingly, sporadic melanoma patients had more variant MC1R alleles, including RHC alleles, than did familial melanoma patients. This difference may be related to the smaller number of independent subjects in the family study. Alternatively, genes other than MC1R or CDKN2A may have a greater role in familial melanoma in this population. Larger family studies from Mediterranean populations will be required to further investigate these associations.
Individuals carrying MC1R variants were at increased risk of melanoma even after controlling for hair color and tanning ability or for a "pigmentation" factor that took into account the combined effect of hair color, tanning ability, skin color, eye color, and Minolta b* index. The association between MC1R variants and melanoma also remained statistically significant after adjustment for the presence of dysplastic nevi or for a "nevi" factor that took into account number of nevi, presence or absence of dysplastic nevi, and freckles. This suggests that MC1R variants increase the risk for melanoma beyond the risk due to fair pigmentation, as previously seen in other populations (17,39,40), but also beyond the risk associated with nevi. However, the association between MC1R and melanoma was modified by these known phenotypic factors (i.e., pigmentation, freckling, and nevi). In fact, the association between RHC variants and melanoma risk was stronger in subjects without dysplastic nevi or with few nevi, no freckles, or darker pigmentation, suggesting that carrying RHC variant alleles is an independent risk factor for melanoma among phenotypically low-risk individuals. This finding is consistent with the associations reported in individuals with darker skin (19) and high tanning ability (41) in Australian and English populations, respectively, but contrasts with the associations found in a small study of French subjects (40).
A number of possible hypotheses could explain the stronger association between MC1R variants and melanoma risk in subjects with fewer risk factors. One explanation may be that MC1R has an additional role that is unrelated to its function in regulating pigmentation (11). Indeed, -MSH influences growth and development of melanocytes and melanoma cells in vitro (42) through MC1R. MC1R variants could alter the effect of
-MSH on melanoma cell behavior independent of their effects on pigmentation. Second, nevi represent melanocyte cell clones that have undergone cellular expansion followed by growth arrest (43). MC1R may play a role in enhancing melanocyte activity and at the same time limiting clonal expansion of melanocytes (18). If MC1R variants alter the latter function, clones may escape growth arrest, and individuals may develop melanoma; if melanocytic clones do not escape growth arrest, individuals may develop nevi or, in the absence of melanocyte clones, freckles. These changes may be affected by other factors. In our study, intense sun exposure, coupled with low DNA repair capacity, appeared to increase the association of RHC alleles of MC1R with nevi and freckling. Once melanoma develops, DNA repair (or at least the DNA repair that is measured in lymphocytes) may no longer be relevant, as suggested by the lack of an impact by DNA repair capacity on the association between MC1R variants and melanoma risk or progression. Third, nonfunctional MC1R may alter glucocorticoid induction mediated by ACTH. Glucocorticoid use (either topical or systemic) has been associated with decreased melanoma risk in a previous study in this population (44). One might speculate that altered MC1R function may decrease the potentially protective effect of ACTH-induced glucocorticoids. Finally, it is possible that the different MC1R variants have unique roles relative to pigmentation or nevi/melanoma formation. In fact, both RHC and NRHC alleles were associated with melanoma risk, but only RHC alleles were associated with nevi or many pigmentation characteristics. This finding is especially important since carriers of NRHC alleles may erroneously consider themselves to be at lower risk for melanoma, especially if they have darker skin or no nevi.
We also found that melanoma patients with multiple MC1R variants had more melanomas in continuously sun-exposed areas of the body (e.g., face, neck, back of hands) than did individuals who had the consensus sequence, whereas the ASIP polymorphism was inversely associated with melanoma at these body sites. These findings need to be reproduced, given the small number of subjects with melanomas at these body sites. UV radiation is known to induce the release of -MSH and to increase the expression of MC1R in the epidermis (45). Nonfunctional MC1R may alter local responses to UV radiation (46,47). In fact, it has been shown that melanocytes with nonfunctional MC1R are more sensitive to the cytotoxic effect of UV radiation than are melanocytes expressing functional MC1R in vitro (48).
MC1R variants were statistically significantly associated with melanoma thickness, which is an indicator of disease progression. This intriguing association may reflect a delay in diagnosis or a more aggressive form of melanoma in subjects with MC1R variants, and it may be related to the inability of nonfunctional MC1R variants to reduce growth and progression of melanoma cells (49). In addition, nonfunctional MC1R may reduce the glucocorticoid-related immunosuppressive effect that follows binding of ACTH with MC1R. Individuals with red hair, who are more likely to carry RHC variants, might be expected to have thicker lesions. We are not aware of any reports on the association between red hair and melanoma thickness. However, a study reported an interaction among cytochrome p450 2D6, MC1R and pigmentation with melanoma thickness (50). Further study of melanoma progression is thus required, given the importance of identifying subjects who would benefit from targeted therapies. We did not examine the association with melanoma thickness in the family study because frequent screenings may bias melanoma thickness in younger generations. Indeed, melanoma lesions in the patients from the casecontrol study (median thickness = 1.06 mm) were thicker than those from melanoma patients from the family study (median thickness = 0.60 mm).
The current study has several limitations. One is the low statistical power in some subgroup analyses, which showed imprecise odds ratio estimates with wide confidence intervals. In addition, it was not possible to examine the association of each individual MC1R variant with melanoma risk or pigmentation. Finally, it is important to note the possibility of false-positive findings due to multiple analyses. Further confirmatory work is thus needed, particularly for the results based on small numbers of subjects.
In conclusion, carrying MC1R variants, particularly RHC, was associated with fair pigmentation, more nevi, and increased melanoma risk in this Italian population. In contrast, the g.8818A>G ASIP polymorphism was not associated with pigmentation or melanoma risk overall, even though it may interact with RHC variants in modifying melanoma risk. Assessment of the role of pigmentation genes in melanoma risk may identify high-risk subjects who could benefit from prevention strategies and targeted therapies.
![]() |
NOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(1) Landi MT, Baccarelli A, Calista D, Pesatori A, Fears T, Tucker MA, Landi G. Combined risk factors for melanoma in a Mediterranean population. Br J Cancer 2001;85:130410.[ISI][Medline]
(2) Goldstein AM, Tucker MA. Screening for CDKN2A mutations in hereditary melanoma. J Natl Cancer Inst 1997;89:6768.
(3) Rivers JK. Is there more than one road to melanoma? Lancet 2004;363:72830.[CrossRef][ISI][Medline]
(4) Pfahlberg A, Uter W, Kraus C, Wienecke WR, Reulbach U, Kolmel KF, Gefeller O. Monitoring of nevus density in children as a method to detect shifts in melanoma risk in the population. Prev Med 2004;38:3827.[CrossRef][ISI][Medline]
(5) Carli P, Naldi L, Lovati S, La Vecchia C. The density of melanocytic nevi correlates with constitutional variables and history of sunburns: a prevalence study among Italian schoolchildren. Int J Cancer 2002;101:3759.[CrossRef][ISI][Medline]
(6) Bevona C, Goggins W, Quinn T, Fullerton J, Tsao H. Cutaneous melanomas associated with nevi. Arch Dermatol 2003;139:16204.
(7) Wei Q, Lee JE, Gershenwald JE, Ross MI, Mansfield PF, Strom SS, Wang LE, Guo Z, Qiao Y, Amos CI, Spitz MR, Duvic M. Repair of UV light-induced DNA damage and risk of cutaneous malignant melanoma. J Natl Cancer Inst 2003;95:30815.
(8) Landi MT, Baccarelli A, Tarone RE, Pesatori A, Tucker MA, Hedayati M, Grossman L. DNA repair, dysplastic nevi, and sunlight sensitivity in the development of cutaneous malignant melanoma. J Natl Cancer Inst 2002;94:94101.
(9) Gillanders E, Juo SH, Holland EA, Jones M, Nancarrow D, Freas-Lutz D, Sood R, Park N, Faruque M, Markey C, Kefford RF, Palmer J, Bergman W, Bishop DT, Tucker MA, Bressac-de Paillerets B, Hansson J, Stark M, Gruis N, Bishop JN, Goldstein AM, Bailey-Wilson JE, Mann GJ, Hayward N, Trent J, et al. Localization of a novel melanoma susceptibility locus to 1p22. Am J Hum Genet 2003;73:30113.[CrossRef][Medline]
(10) Bale SJ, Dracopoli NC, Tucker MA, Clark WH Jr, Fraser MC, Stanger BZ, Green P, Donis-Keller H, Housman DE, Greene MH. Mapping the gene for hereditary cutaneous malignant melanoma-dysplastic nevus to chromosome 1p. N Engl J Med 1989;320:136772.[Abstract]
(11) Kamb A, Shattuck-Eidens D, Eeles R, Liu Q, Gruis NA, Ding W, Hussey C, Tran T, Miki Y, Weaver-Feldhaus J. Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus. Nat Genet 1994;8:236.[CrossRef][Medline]
(12) Wolfel T, Hauer M, Schneider J, Serrano M, Wolfel C, Klehmann-Hieb E, De Plaen E, Hankeln T, Meyer zum Buschenfelde KH, Beach D. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 1995;269:12814.[ISI][Medline]
(13) Rizos H, Puig S, Badenas C, Malvehy J, Darmanian AP, Jimenez L, Mila M, Kefford RF. A melanoma-associated germline mutation in exon 1beta inactivates p14ARF. Oncogene 2001;20:55437.[CrossRef][ISI][Medline]
(14) Busca R, Ballotti R. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigment Cell Res 2000;13:609.[CrossRef][ISI][Medline]
(15) Rees JL. The genetics of sun sensitivity in humans. Am J Hum Genet 2004;75:73951.[CrossRef][ISI][Medline]
(16) Flanagan N, Healy E, Ray A, Philips S, Todd C, Jackson IJ, Birch-Machin MA, Rees JL. Pleiotropic effects of the melanocortin 1 receptor (MC1R) gene on human pigmentation. Hum Mol Genet 2000;9:25317.
(17) Kennedy C, ter Huurne J, Berkhout M, Gruis N, Bastiaens M, Bergman W, Willemze R, Bavinck JN. Melanocortin 1 receptor (MC1R) gene variants are associated with an increased risk for cutaneous melanoma which is largely independent of skin type and hair color. J Invest Dermatol 2001;117:294300.[CrossRef][ISI][Medline]
(18) Duffy DL, Box NF, Chen W, Palmer JS, Montgomery GW, James MR, Hayward NK, Martin NG, Sturm RA. Interactive effects of MC1R and OCA2 on melanoma risk phenotypes. Hum Mol Genet 2004;13:44761.
(19) Palmer JS, Duffy DL, Box NF, Aitken JF, O'Gorman LE, Green AC, Hayward NK, Martin NG, Sturm RA. Melanocortin-1 receptor polymorphisms and risk of melanoma: is the association explained solely by pigmentation phenotype? Am J Hum Genet 2000;66:17686.[CrossRef][ISI][Medline]
(20) Valverde P, Healy E, Sikkink S, Haldane F, Thody AJ, Carothers A, Jackson IJ, Rees JL. The Asp 84Glu variant of the melanocortin 1 receptor (MC1R) is associated with melanoma. Hum Mol Genet 1996;5:16636.
(21) Suzuki I, Tada A, Ollmann MM, Barsh GS, Im S, Lamoreux ML, Hearing VJ, Nordlund JJ, Abdel-Malek ZA. Agouti signaling protein inhibits melanogenesis and the response of human melanocytes to alpha-melanotropin. J Invest Dermatol 1997;108:83842.[CrossRef][ISI][Medline]
(22) Kanetsky PA, Swoyer J, Panossian S, Holmes R, Guerry D, Rebbeck TR. A polymorphism in the agouti signaling protein gene is associated with human pigmentation. Am J Hum Genet 2002;70:7705.[CrossRef][ISI][Medline]
(23) Landi MT, Goldstein AM, Tsang S, Munroe D, Modi W, Ter Minassian M, Steighner R, Dean M, Metheny N, Staats B, Agatep R, Hogg D, Calista D. Genetic susceptibility in familial melanoma from northeastern Italy. J Med Genet 2004;41:55766.
(24) Parkin D, Whelan S, Ferlay J, Raymond L, Young J. Cancer incidence in five continents. Vol VII. Lyon (France): International Agency for Research on Cancer; 1997.
(25) Fitzpatrick TB. The validity and practicality of sun-reactive skin types I through VI. Arch Dermatol 1988;124:86971.[CrossRef][ISI][Medline]
(26) English JS, Swerdlow AJ, MacKie RM, O'Doherty CJ, Hunter JA, Clark J, Hole DJ. Site-specific melanocytic naevus counts as predictors of whole body naevi. Br J Dermatol 1988;118:6414.[ISI][Medline]
(27) Hartge P, Holly EA, Halpern A, Sagebiel R, Guerry D, Elder D, Clark W, Hanson L, Harrison C, Tarone R. Recognition and classification of clinically dysplastic nevi from photographs: a study of interobserver variation. Cancer Epidemiol Biomarkers Prev 1995;4:3740.[Abstract]
(28) Landi MT, Calista D, Landi G, Bernucci I, Bertazzi PA, Clark WH Jr, Goldstein AM, Tucker MA. Clinical characteristics of 20 Italian melanoma-prone families. Arch Dermatol 1999;135:15545.
(29) Brenner AV, Lubin JH, Calista D, Landi MT. Instrumental measurements of skin color and skin ultraviolet light sensitivity and risk of cutaneous malignant melanoma: a casecontrol study in an Italian population. Am J Epidemiol 2002;156:35362.
(30) Weatherall IL, Coombs BD. Skin color measurements in terms of CIELAB color space values. J Invest Dermatol 1992;99:46873.[CrossRef][ISI][Medline]
(31) Damian DL, Halliday GM, Barnetson RS. Prediction of minimal erythema dose with a reflectance melanin meter. Br J Dermatol 1997;136:7148.[CrossRef][ISI][Medline]
(32) Kanetsky PA, Ge F, Najarian D, Swoyer J, Panossian S, Schuchter L, Holmes R, Guerry D, Rebbeck TR. Assessment of polymorphic variants in the melanocortin-1 receptor gene with cutaneous pigmentation using an evolutionary approach. Cancer Epidemiol Biomarkers Prev 2004;13:80819.
(33) Baccarelli A, Calista D, Minghetti P, Marinelli B, Albetti B, Tseng T, Hedayati M, Grossman L, Landi G, Struewing JP, Landi MT. XPD gene polymorphism and host characteristics in the association with cutaneous malignant melanoma risk. Br J Cancer 2004;90:497502.[CrossRef][ISI][Medline]
(34) Gold B, Kalush F, Bergeron J, Scott K, Mitra N, Wilson K, Ellis N, Huang H, Chen M, Lippert R, Halldorsson BV, Woodworth B, White T, Clark AG, Parl FF, Broder S, Dean M, Offit K. Estrogen receptor genotypes and haplotypes associated with breast cancer risk. Cancer Res 2004;64:8891900.
(35) Athas WF, Hedayati MA, Matanoski GM, Farmer ER, Grossman L. Development and field-test validation of an assay for DNA repair in circulating human lymphocytes. Cancer Res 1991;51:578693.[Abstract]
(36) Bartholomew D. Latent variable models and factor analysis. London (UK): Griffith; 1987.
(37) Kaiser HF, Caffrey J. Alpha factor analysis. Psychometrika 1965;30:114.[ISI][Medline]
(38) Sandby-Moller J, Poulsen T, Wulf HC. Epidermal thickness at different body sites: relationship to age, gender, pigmentation, blood content, skin type and smoking habits. Acta Derm Venereol 2003;83:4103.[CrossRef][ISI][Medline]
(39) Bastiaens M, ter Huurne J, Gruis N, Bergman W, Westendorp R, Vermeer BJ, Bouwes Bavinck JN. The melanocortin-1-receptor gene is the major freckle gene. Hum Mol Genet 2001;10:17018.
(40) Matichard E, Verpillat P, Meziani R, Gerard B, Descamps V, Legroux E, Burnouf M, Bertrand G, Bouscarat F, Archimbaud A, Picard C, Ollivaud L, Basset-Seguin N, Kerob D, Lanternier G, Lebbe C, Crickx B, Grandchamp B, Soufir N. Melanocortin 1 receptor (MC1R) gene variants may increase the risk of melanoma in France independently of clinical risk factors and UV exposure. J Med Genet 2004;41:e13.
(41) Ichii-Jones F, Lear JT, Heagerty AH, Smith AG, Hutchinson PE, Osborne J, Bowers B, Jones PW, Davies E, Ollier WE, Thomson W, Yengi L, Bath J, Fryer AA, Strange RC. Susceptibility to melanoma: influence of skin type and polymorphism in the melanocyte stimulating hormone receptor gene. J Invest Dermatol 1998;111:21821.[CrossRef][ISI][Medline]
(42) Suzuki I, Cone RD, Im S, Nordlund J, Abdel-Malek ZA. Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. Endocrinology 1996;137:162733.[Abstract]
(43) Robinson WA, Lemon M, Elefanty A, Harrison-Smith M, Markham N, Norris D. Human acquired naevi are clonal. Melanoma Res 1998;8:499503.[ISI][Medline]
(44) Landi MT, Baccarelli A, Calista D, Fears TR, Landi G. Glucocorticoid use and melanoma risk. Int J Cancer 2001;94:3023.[CrossRef][ISI][Medline]
(45) Chakraborty AK, Funasaka Y, Slominski A, Ermak G, Hwang J, Pawelek JM, Ichihashi M. Production and release of proopiomelanocortin (POMC) derived peptides by human melanocytes and keratinocytes in culture: regulation by ultraviolet B. Biochim Biophys Acta 1996;1313:1308.[CrossRef][ISI][Medline]
(46) Wintzen M, Gilchrest BA. Proopiomelanocortin, its derived peptides, and the skin. J Invest Dermatol 1996;106:310.[CrossRef][ISI][Medline]
(47) Haycock JW, Rowe SJ, Cartledge S, Wyatt A, Ghanem G, Morandini R, Rennie IG, MacNeil S. Alpha-melanocyte-stimulating hormone reduces impact of proinflammatory cytokine and peroxide-generated oxidative stress on keratinocyte and melanoma cell lines. J Biol Chem 2000;275:1562936.
(48) Scott MC, Wakamatsu K, Ito S, Kadekaro AL, Kobayashi N, Groden J, Kavanagh R, Takakuwa T, Virador V, Hearing VJ, Abdel-Malek ZA. Human melanocortin 1 receptor variants, receptor function and melanocyte response to UV radiation. J Cell Sci 2002;115:234955.
(49) Robinson SJ, Healy E. Human melanocortin 1 receptor (MC1R) gene variants alter melanoma cell growth and adhesion to extracellular matrix. Oncogene 2002;21:803746.[CrossRef][ISI][Medline]
(50) Strange RC, Fryer AA. The glutathione S-transferases: influence of polymorphism on cancer susceptibility. IARC Sci Publ 1999;148:23149.[Medline]
(51) Soto JL, Cabrera CM, Serrano S, Lopez-Nevot MA. Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B. BMC Cancer 2005;5:36.[CrossRef][Medline]
Manuscript received January 3, 2005; revised April 22, 2005; accepted May 26, 2005.
This article has been cited by other articles in HighWire Press-hosted journals:
Correspondence about this Article
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |