Human Determinants and the Role of Melanocortin-1 Receptor Variants in Melanoma Risk Independent of UV Radiation Exposure

Importance  Despite the unquestioned relationship of UV radiation (UVR) exposure and melanoma development, UVR-independent development of melanoma has only recently been described in mice. These findings in mice highlight the importance of the genetic background of the host and could be relevant for preventive measures in humans.

Objective  To study the role of the melanocortin-1 receptor (MC1R) and melanoma risk independently from UVR in a clinical setting.

Design, Setting, and Participants  Hospital-based case-control study, including genetic testing, questionnaires, and physical data (Molecular Markers of Melanoma Study data set) including 991 melanoma patients (cases) and 800 controls.

Main Outcomes and Measures  Association of MC1R variants and melanoma risk independent from sun exposure variables.

Results  The 1791 participants included 991 with a diagnosis of melanoma and 800 control patients (mean [SD] age, 59.2 [15.6] years; 50.5% male). Compared with wild-type carriers, carriers of MC1R variants were at higher melanoma risk after statistically adjusting for previous UVR exposure (represented by prior sunburns and signs of actinic skin damage identified by dermatologists), age, and sex compared with wild-type carriers (≥2 variants, OR, 2.13 [95% CI, 1.66-2.75], P < .001; P for trend <.001). After adjustment for sex, age, sunburns in the past, and signs of actinic skin damage, the associations remained significant (OR, 1.65 [95% CI, 1.02-2.67] for R/R, OR, 2.63 [95% CI, 1.82-3.81] for R/r; OR, 1.83 [95% CI, 1.36-2.48] for R/0; and OR, 1.50 [95% CI, 1.01-2.21] for r/r, with P values ranging from <.001 to .04 when adjusted for facial actinic skin damage; OR, 2.36 [95% CI, 1.62-3.43] for R/r; and OR, 1.47 [95% CI, 1.08-1.99] for R/0 with P values ranging from <.001 to .01 when adjusted for dorsal actinic skin damage; and OR, 2.54 [95% CI, 1.76-3.67] for R/r, OR, 1.75 [95% CI, 1.30-2.36] for R/0; and OR, 1.50 [95% CI, 1.02-2.20] for r/r with P values ranging from <.001 to .04 when adjusted for actinic skin damage on the hands).

Conclusions and Relevance  Carriers of MC1R variants were at increased melanoma risk independent of their sun exposure. Further studies are required to elucidate the causes of melanoma development in these individuals.

In recent decades, numerous case-control studies have improved the understanding of risk factors in melanoma development. Since the first description of pigmentation genes and melanoma risk,¹ these endogenous risk factors have been accepted as contributing to the risk of developing melanoma in collaboration with exogenous factors such as intermittent sun exposure leading to sunburns in childhood and adolescence.^2-5 However, as the UV radiation (UVR) dependency of melanoma is not as clear or linear as in squamous cell carcinoma, the effect of pigmentation variants on melanoma development has become more important in the evaluation of melanoma risk factors.

The most important gene affecting pigmentation, which determines each individual’s phenotype (and melanoma risk), is the melanocortin-1 receptor (MC1R), which was described in 1996.¹ Some variants in this highly polymorphic gene lead to a change of receptor function and subsequently to altered receptor signaling, thereby influencing the ratio of eumelanin to pheomelanin. The former is brown to black, stable and photoprotective, whereas the latter is yellow to red in color, less photoprotective, and known to generate reactive oxygen species (ROS) and subsequent DNA damage.^6,7 Carriers of specific MC1R variants that lead to a shift from eumelanin to pheomelanin have red hair, freckles, and fair skin (red hair phenotype). Dependent on their association with red hair, the most common MC1R variants are classified as “R” (high risk) and “r” (low risk) variants.^8,9

The identification of MC1R variants and their association with phenotype and melanoma has helped to elucidate the importance of a genetic background pathway in melanoma development, which is probably different from a sun-induced pathway. Recently, a UVR-independent pathway to melanoma development was identified in mice¹⁰: Induction of oncogenic BRAF V600E in mice with an inactivating mutation of MC1R led to a high incidence of invasive melanomas without UVR exposure. These mice phenotypically correspond to the red hair phenotype in humans, exhibiting a high pheomelanin/eumelanin ratio. In contrast, the albino mice (no pheomelanin) and the brown or black mice (low levels of pheomelanin) developed melanomas to a much lesser extent. Additionally, higher levels of oxidative DNA damage and lipid damage generated by ROS were observed in red mice compared with albino and brown or black mice. These findings in mice strengthen the evidence for a pathway to melanoma development that is related to the genetic background of an individual rather than to sun exposure alone.

We therefore aimed to test this hypothesis in a human case-control setting. By performing multivariate analyses including variables that represent either genetic (MC1R variants) or exogenous risk factors (sunburns and sun exposure), the effect of these particular risk factors in our study population was tested.

Box Section Ref ID

Participants were recruited within the ongoing M3 Study program (Molecular Markers of Melanoma), which was established in 2008 at the Department of Dermatology at the Medical University of Vienna (MUV). The purpose of the study is the collection of survey data, genomic DNA, and melanoma tissue samples to study molecular risk factors for melanoma. Patients with melanoma were recruited at the oncology clinic of the MUV Department of Dermatology. Patients who visit the outpatient clinic of the MUV Department of Dermatology with diseases other than melanoma were recruited as control patients. In the present study, participants with available MC1R status were included (991 patients with melanoma and 800 controls). More detailed information about the study population has been reported previously.¹¹ Written consent was obtained from all participants. This study was approved by the ethics committee of the MUV.

Risk factors that represent past sun exposure were documented as follows: number of sunburns per decade, and clinically visible signs of actinic skin damage (solar lentigines, freckling, wrinkling) on the participants’ face, back/neck, and hands as determined independently by 2 dermatologists (I.O., J.W.): Wrinkling on the face was defined as deep skin lines occurring on the forehead, periorbital, and perioral region, whereas wrinkling on the neck was defined as deep skin lines, commonly appearing in a characteristic rhomboid pattern.¹¹ Freckling was defined as persistent areas of hyperpigmentation, resulting in a mottled, irregular skin appearance;^12,13 and solar lentigines were defined as brown, macular lesions with sharp margins. Dependent on their severity, these signs of actinic skin damage were graded, as described elsewhere,¹⁴ as absent, mild, moderate, or severe. Photographs were taken from each participant under standardized conditions for reassessment procedures. For an interobserver variability analysis, 50 photographs were chosen randomly and independently assessed by 2 observers (I.O., J.W.). Consistency among the 2 independent observers was calculated to be either substantial (κ = 0.61-0.80) or almost perfect (κ = 0.81-1.00), as described previously.¹⁵

Sequencing of MC1R was performed for 1791 participants. For determination of MC1R variants, the 951-bp coding region of MC1R was amplified from genomic DNA by polymerase chain reaction (PCR) followed by complete direct sequencing of the amplicons as described elsewhere.¹⁶ Briefly, 2 sets of M13-tagged PCR primers were used for PCR amplification: 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 PCR solutions included 1 × PCR buffer (Invitrogen High Fidelity PCR buffer, Invitrogen); 1.5mM MgSO₄; a 175nM concentration of each pair of primers; a 50nM concentration of each of the 4 deoxyribonucleotide triphosphates; and 1 unit of HiFi Platinum Taq polymerase (Invitrogen). Amplification was 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). All PCR products were treated with exonuclease I and shrimp alkaline phosphatase (USB Corporation) as described elsewhere¹⁶ and the PCR product was purified on Sephadex G-50 (Sigma-Aldrich) prior to sequencing. All PCR products were sequenced with the ABI prism BigDye Terminator Cycle Sequencing Kit 1.0 (Applied Biosystems) 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 using Mutation Surveyor (Mutation Surveyor, Soft Genetics LLC).

Signs of actinic skin damage were classified into 3 categories as previously described.¹¹ The number of sunburns was not normally distributed (Kolmogorov-Smirnov test, P < .001) and therefore categorized into tertiles based on the distribution of all participants as follows: 0 or 1, 2 to 19, and 20 or more sunburns. MC1R variants were grouped into “R” (complete loss of function) and “r” (partial loss of function) variants as described elsewhere.^17-21 In our study population, Ins86_87A, R142H, R151C, R160W, and D294H were classified as “R” and V60L, D84E, V92M, I155T, and R163Q as “r.” Participants who did not carry any of these 10 most common variants were pooled in the reference group (0/0).

First, the association of sunburns and melanoma risk, actinic skin damage and melanoma risk, and MC1R variants and melanoma risk was calculated separately by using logistic regression procedures to obtain odds ratios (ORs) and their 95% confidence intervals. Second, these risk factors were included in a multivariate analysis thereby adjusting MC1R variants for exogenous melanoma risk factors such as sunburns in childhood and adolescence (0-20 years) and signs of actinic skin damage. Additionally, we adjusted for age at recruitment and sex in all analyses (for details see eTables 7-9 in the Supplement). A 2-sided P < .05 was considered statistically significant. All statistical analyses were performed using SPSS Statistics, version 19.0 (SPSS, IBM Inc).

In total, 1791 participants were included in the analyses, including 991 participants with a diagnosis of melanoma and 800 control patients. The mean (SD) age of the study population was 59.2 (15.6) years. Slightly more males than females were included (50.5% vs 49.5%, respectively) (see eTable 1 in the Supplement). Distribution of melanoma subtype, site of tumor occurrence, and phenotypical characteristics of the study population are summarized in eTables 2, 3, and 4 in the Supplement.

Information about sunburn history was available for 1773 participants (Table 1). When the association of past sunburns and melanoma risk was investigated, there was a significantly increased risk for melanoma in participants who reported more than 12 sunburns (AOR, 2.20 [95 % CI, 1.63-2.96]; P < .001) in the past compared with participants with no sunburn history. As the number of sunburns in childhood and adolescence has been acknowledged as an important contributor to melanoma risk, we also tested the association of sunburns at age 0 to 20 years: Again, participants with 10 or more sunburns during this period in life were at increased melanoma risk (AOR, 2.19 [95% CI, 1.73-2.78]; P < .001).

Clinically visible signs of actinic skin damage were another important factor that reflects prior sun exposure. We found a significantly increased melanoma risk in participants with different types and grades of severity of actinic skin damage on the face, the back/neck, and the hands.

The highest ORs were calculated for severe signs of actinic skin damage on the back and neck (AOR, 3.83 [95% CI, 2.74-5.35] for wrinkling, AOR, 3.62 [95% CI, 2.64-4.95] for lentigines, and AOR, 3.02 [95% CI, 2.19-4.15] for freckling; P < .001) in a multivariate analysis (eTable 6 in the Supplement), but also actinic skin damage on the face (AOR, 1.79 [95% CI, 1.23-2.59] for wrinkling and AOR, 1.81 [95% CI, 1.31-2.52] for solar lentigines; P < .001) and the hands (AOR, 2.64 [95% CI, 1.88-3.71] for solar lentigines and AOR, 1.90 [95% CI, 1.35-2.59] for freckling; P < .001) could be shown to be significantly associated with melanoma risk (eTable 6 in the Supplement). A detailed description of actinic skin damage and melanoma risk in our study population and the particular role of actinic skin damage on the back has been reported elsewhere.¹⁵

Melanoma risk was significantly increased with the number of MC1R variants (P for trend <.001) (Table 2). After adjustment for age and sex, carriers of 2 or more MC1R variants were at 2-fold higher melanoma risk compared with wild-type carriers (OR, 2.13 [95% CI, 1.66-2.75]; P < .001). With regard to “R” and “r” variants, an association was found for carriers of R in either combination (R/R, R/r, or R/0) with melanoma risk (OR, 1.77 [95% CI, 1.12-2.79], 2.93 [95% CI, 2.07-4.16], and 1.94 [95% CI, 1.45-2.60], respectively), as for carriers of “r” variants (OR, 1.64 [95% CI, 1.13-2.38] for r/r and OR, 1.35 [95% CI, 1.05-1.73] for r/0, respectively).

When we studied single MC1R variants and melanoma risk, we identified carriers of 86_87insA, D294H, R160W, and R151C to be at significantly higher melanoma risk, with OR ranging from 1.53 (95% CI, 1.17-2.01) (R151C) to 3.69 (95% CI, 1.24-10.99) (86_87insA) after adjusting for age and sex. For 4 of the remaining variants, ORs were calculated from 1.20 (95% CI, 0.94-1.52) to 1.48 (95% CI, 0.70-3.13) (R142H, I155T, R163Q, and V92M); V60L was equally distributed in cases and controls (OR, 1.00 [95% CI, 0.80-1.25]) and D84E was negatively associated with melanoma risk. However, these results were not statistically significant (Table 3).

Actinic skin damage was available for 3 different parts of the body; therefore 3 different analyses were performed (for details see eTables 7-9 in the Supplement). When considering MC1R and melanoma risk independently from facial actinic skin damage, and sunburns in childhood and adolescence, we found a significantly increased risk for carriers of R variants (OR, 1.65 [95% CI, 1.02-2.67] for R/R, OR, 2.63 [95% CI, 1.82-3.81] for R/r, OR, 1.83 [95% CI, 1.36-2.48] for R/0) and r/r carriers (OR, 1.50 [95% CI, 1.01-2.21]). Similarly, when we adjusted MC1R variants for dorsal actinic skin damage and sunburns in childhood and adolescence in a separate analysis, several combinations of these variants remained significantly associated with melanoma risk (OR, 2.36 [95% CI, 1.62-3.43] for R/r and OR, 1.47 [95% CI, 1.08-1.99] for R/0). When we repeated our analysis for MC1R, skin damage on the hands, and sunburns in childhood and adolescence, we again found a significant association for carriers of R/r, R/0, and r/r (OR, 2.54 [95% CI, 1.76-3.67], OR, 1.75 [95% CI, 1.30-2.36], and OR, 1.50 [95% CI, 1.02-2.20], respectively) and melanoma risk (Table 4).

In this case-control study, melanoma risk was significantly increased with the number of MC1R variants and independent of UVR exposure. Carriers of 2 or more MC1R variants were at 2-fold higher melanoma risk compared with wild-type carriers (AOR, 2.13 [95% CI, 1.66-2.75]; P < .001). Additionally, this study confirmed the findings that participants who experienced 10 or more sunburns during the first 20 years of life were at increased melanoma risk, as were participants with severe signs of actinic skin damage on the back/neck, the face, and the hands, which was previously reported.¹⁵

Although UVR exposure is the most important exogenous risk factor for melanoma, the relationship between UVR exposure and melanoma is not as linear as for squamous cell carcinoma. The importance of the genetic background of an individual has become more evident with the identification of numerous different genetic variants as melanoma risk factors, indicating divergent roads to melanoma. Whiteman et al²² proposed a dual-pathway model in melanoma development, dividing melanomas into those that occur on chronically sun-exposed skin and others that occur on intermittently exposed sites. These 2 groups vary not only in terms of sun exposure but also reflect different properties of the host: The former were associated with increasing age, a low number of nevi, and signs of chronically sun-damaged skin, whereas the latter were found to be associated with younger age at diagnosis, an increased number of nevi, and only intermittent and/or low levels of sun exposure in the past.

Extrapolating this dual-pathway classification, one could hypothesize that for the first group of melanomas, sun exposure could be seen as the main driver, whereas the other group of melanomas could be assumed to be more genetically determined with only modest levels of sun exposure required—corresponding to younger age at diagnosis and high number of nevi. Accordingly, Mitra et al¹⁰ demonstrated a UVR-independent melanomagenesis by generating different models of mice, corresponding to a particular phenotype. These findings indicate that melanoma could develop independently of UVR exposure—highlighting the specific significance of pheomelanin, ROS, and subsequent DNA damage in a red hair phenotype.

In our study, carrying 2 or more MC1R variants increased melanoma risk 2-fold, compared with wild-type carriers even after correcting for signs of prior sun exposure. As described by others, particularly high-risk variants (R/R, R/r/, R/0) were associated with a characteristic pigmentation phenotype such as fair skin type, red hair color, and the presence of freckles in childhood (see eTables 4 and 5 in the Supplement). Additionally, the number of MC1R variants was associated with an increase in melanoma risk as were several single established MC1R variants: D294H, R160W, R151C, and an insertion at position 86_87 were strongly and significantly associated with melanoma risk (Table 3)—corresponding to previously described associations of MC1R variants and melanoma risk in populations of similar ethnic origin.^23,24

Most studies related to MC1R variants and melanoma risk have been performed in countries of or populations originating from northern Europe. Whereas there is no doubt about the role of these genomic variants in pigmentation genes in melanoma risk, these studies might be biased toward light skin complexion. As MC1R is not the only determinant of pigmentation phenotype, a population with a more balanced pigmentation phenotype might be helpful to obtain information with regard to the role of pigmentation genes as risk factors of melanoma. To the best of our knowledge, this is the largest study ever conducted in a central European population. In our study population, 81.6% of the patients with melanoma carried at least 1 MC1R variant, which corresponds to a previously reported frequency for Austrian patients with melanoma.²⁵ As MC1R is known to be highly polymorphic with diverse distribution of genomic variants across Europe, our study therefore fills the gap between northern and southern European countries.

Although the association of several MC1R variants with light pigmentation phenotype and melanoma risk could be confirmed in numerous case-control studies,^{1,18,20,26-29} the pathway beyond this mechanism seems to be complex as several studies suggest a phenotype-independent melanoma risk and increasing risk for dark-skinned individuals with specific MC1R variants, respectively.^19,20,30,31 This nonlinear association could be explained by a MC1R role other than in pigmentation. Only recently, MC1R variants have been shown to modulate immune functions and inflammation, which has led to the hypothesis that the MC1R/αMSH signaling axis contributes to an immunosuppressive setting with subsequent UV-induced tumorigenesis; this has been summarized by Nasti and Timares.³² Moreover, MC1R has been reported to be involved in pain of inflammatory origin.³³ It is possible that the reduced immunologic response and sensation of pain in MC1R variant carriers might contribute to melanoma risk. Other melanoma risk factors such as a history of sunburns in childhood and adolescence or the presence of actinic skin damage were associated with increased melanoma risk in our study population. These exogenous factors represented prior sun exposure; thus, they were particularly helpful when examining the role of MC1R and melanoma beyond sun exposure.

One limitation of the study is selection bias because the control population was also hospital based and therefore not a random sample of the healthy population. Another issue that needs to be addressed is the effect of recall bias in patients with melanoma, which might play a role in self-reported past sun exposure, tanning ability, and burning tendency.^34,35 Additionally, other factors that represent past sun exposure such as occupational and recreational behavior should be taken into account in further analyses.

Our study revealed that people with MC1R mutations have a high risk of melanoma, even after adjustment for sunburns and actinic damage; thus, the MC1R variants conferred additional melanoma risk beyond sun exposure. These data supported the recent mouse model research that showed a UV-independent pathway of melanoma development in mice.¹⁰ Due to the frequency of these variants among the high-risk population, this finding is of global relevance. Recent reports have revealed a survival benefit in patients with inherited MC1R variants, suggesting that pigmentation genes might not only affect melanoma risk or onset of melanoma but also prognosis.^36-38 These findings stress the biological and clinical relevance of genomic MC1R variants in melanoma and give reason to reevaluate the extent of sun exposure as a risk factor for melanoma, which might directly affect public health recommendations in the future.³⁸ Further studies are required to better elucidate the molecular mechanisms underlying melanoma development under altered MC1R function.

Accepted for Publication: February 13, 2016.

Corresponding Author: Judith Wendt, MD, PhD, Division of General Dermatology, Department of Dermatology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria (judith.wendt@meduniwien.ac.at).

Published Online: April 6, 2016. doi:10.1001/jamadermatol.2016.0050.

Author Contributions: Drs Wendt and Okamoto had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Wendt, Fae, Pehamberger, Okamoto.

Acquisition, analysis, or interpretation of data: Wendt, Rauscher, Burgstaller-Muehlbacher, Fischer, Okamoto.

Drafting of the manuscript: Wendt.

Critical revision of the manuscript for important intellectual content: Rauscher, Burgstaller-Muehlbacher, Fae, Fischer, Pehamberger, Okamoto.

Statistical analysis: Wendt.

Obtained funding: Wendt, Okamoto.

Administrative, technical, or material support: Rauscher, Burgstaller-Muehlbacher, Fae.

Study supervision: Pehamberger, Okamoto.

Conflict of Interest Disclosures: None reported.

Funding/Support: This study was supported in part by the Oesterreichische Nationalbank (Anniversary fund, project Nos. 13036 and 13470), the Medical Scientific Fund of the Mayor of the City of Vienna (project No. 10077), and the Hans und Blanca Moser Stiftung.

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.