# A Large French Case-Control Study Emphasizes the Role of RareMc1R Variants in Melanoma Risk

Authors: Hui-Han Hu; Mériem Benfodda; Nicolas Dumaz; Steven Gazal; Vincent Descamps; Agnès Bourillon; Nicole Basset-Seguin; Angélique Riffault; Khaled Ezzedine; Martine Bagot; Armand Bensussan; Philippe Saiag; Bernard Grandchamp; Nadem Soufir
Journal: BioMed Research International
 (2014)

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## Abstract

Background. The MC1R gene implicated in melanogenesis and skin pigmentation is highly polymorphic. Several alleles are associated with red hair and fair skin phenotypes and contribute to melanoma risk. Objective. This work aims to assess the effect of different classes of MC1R variants, notably rare variants, on melanoma risk. Methods. MC1R coding region was sequenced in 1131 melanoma patients and 869 healthy controls. MC1R variants were classified as RHC (R) and non-RHC (r). Rare variants (frequency < 1%) were subdivided into two subgroups, predicted to be damaging (D) or not (nD). Results. Both R and r alleles were associated with melanoma (OR = 2.66 [2.20–3.23] and 1.51 [1.32–1.73]) and had similar population attributable risks (15.8% and 16.6%). We also identified 69 rare variants, of which 25 were novel. D variants were strongly associated with melanoma (OR = 2.38 [1.38–4.15]) and clustered in the same MC1R domains as R alleles (intracellular 2, transmembrane 2 and 7). Conclusion. This work confirms the role of R and r alleles in melanoma risk in the French population and proposes a novel class of rare D variants as important melanoma risk factors. These findings may improve the definition of high-risk subjects that could be targeted for melanoma prevention and screening.


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## Body

## 1. Introduction

The incidence of cutaneous melanoma, the most lethal type of skin cancer, is increasing in western countries, doubling every ten years [1]. Melanoma is a complex disease that arises through multiple etiological pathways [2]. Ultraviolet radiation exposure is the main environmental cause, and pigmentation characteristics such as light skin, hair and eye colour, and high number of nevi have also been identified as melanoma risk factors [3].Germline mutations in high-penetrance melanoma predisposing genesCDKN2A andCDK4 have been found in 20% of familial melanoma cases [4]. Recently, a third major gene,BAP1, that predisposes to melanoma (mainly ocular), mesothelioma, and possibly additional cancers has been identified [5–7]. In addition, numerous low penetrant susceptibility variants, which modulate melanoma risk, have also been described. These genes are mainly involved in melanogenesis (MC1R, ASIP, TYR, TYRP1, andSLC45A2) and melanocyte differentiation (MITF, KIT, andEDNRB) [8–12].Recently, variants in other pathways, such as DNA repair, genome maintenance integrity, and immunological pathways (TERT-CLPTM1,CASP8,ATM, andMX2), have also been linked to melanoma predisposition [13–17].Among pigmentation genes,MC1R, which is the most studied, is associated with human skin pigmentation and melanoma susceptibility. MC1R, the receptor for α-melanocyte stimulating hormone (α-MSH), is a G protein coupled receptor with seven transmembrane domains that regulates the relative concentration of brown-black eumelanin and red-yellow pheomelanin [18]. Eumelanin has been shown to reduce the accumulation of DNA photodamage and to protect melanocytes from UV-induced apoptosis. Pheomelanin is, on the contrary phototoxic, generating oxidative stress by the production of reactive oxygen species [19–21].MC1R is highly polymorphic within Caucasian populations [21]. A recent review has documented 57 nonsynonymous and 25 synonymous polymorphisms in different populations [22]. RHC (red hair color) variants (also called “R” alleles) lead to nonfunctional or diminished functional receptors [23], preferentially induce pheomelanin production, and are therefore associated with red hair, light skin, poor tanning ability, and heavy freckling [24–27]. OtherMC1R variants that impact less strongly on MC1R function are called non-RHC variants and are labelled “r” alleles.Numerous association studies have demonstrated the important role ofMC1RR variants in melanoma predisposition [8, 11, 28, 29]. The influence ofMC1Rr variants on melanoma risk has also been reported but there are several discrepancies in the different published works [4, 29–31]. In addition, to date there is no conclusion on the role of rareMC1R variants in melanoma. In this large French case-control study, we investigated the role of different classes ofMC1R variants in melanoma risk, focussing particularly on the role of rareMC1Rvariants.


## 2. Patients and Methods

### 2.1. Studied Populations

A cohort of 1131 Caucasian melanoma patients was recruited between 2002 and 2008 from the dermatology departments of all university-affiliated hospitals in Paris (the MelanCohort). The main characteristics of the patients have been previously described [32]. Melanoma was sporadic in 784 patients (69%), including 81 patients (7%) who had multiple melanomas and 229 patients (20%) who had familial melanoma (at least 2 cases in first- or second-degree relatives, including the proband). Among the familial and multiple sporadic cases, 8.5% of patients carried mutations in theCDKN2A orCDK4 gene.The control group comprised 869 ethnically matched skin cancer-free blood donors recruited from the EFS (Etablissement Français du Sang) in Bichat and Saint-Louis hospitals over the same period. These subjects have previously been described and used as controls in case-control studies [32–34].Data from patients and controls regarding sex, age, ethnic origin, date of birth, anatomoclinical data, pigmentation characteristics (hair, eye, and skin colours), skin type (Fitzpatrick classification I to IV), nevus count (<10, 10–50, 51–100, and >100), and heavy freckles (yes/no) were collected in a standard document. Ancestry was investigated through birth location of parents and grandparents, and only those with a Caucasian ancestry were retained for the study. The pigmentation characteristics of patients and controls are summarized in Supplementary Table 1 (see Supplementary Materials available online athttp://dx.doi.org/10.1155/2014/925716). All subjects signed an informed consent form and provided a blood sample.


### 2.2. Sequencing and Mutational Analysis

The coding sequence ofMC1R deposited in GenBank (NM_002386.3) was amplified with 2 couples of primer selected by UCSC Genome Browser Gateway (data available upon request) in 1131 melanoma patients and 869 controls. The PCR mix contained 20 ng of genomic DNA, 2.5 mM MgCl2, 50 μM of each dNTP, 400 nM of each primer, and 0.5 U Ampli TaqGold polymerase (Applied Biosystems, Courtaboeuf, France). A 62°C annealing temperature was used for PCR amplification. PCR products were verified on a 2% agarose gel and purified by EXOSAP-IT (USB Corporation, OH, USA). Sequencing reaction was performed on 8900 Fast Thermal Cycler (Applied Biosystems), using 10 ng of purified PCR products and the Big-Dye Terminator Cycle Kit (Applied Biosystems). Sequence analysis was performed with an ABI-Prism 3130 automated sequencer (Applied Biosystems) and read with SeqScape software v2.5 (Applied Biosystems).


### 2.3. Classification ofMC1R Variants

The functional impact of numerousMC1R variants has been assessed in previous studies [47] (Supplementary Table 2). Some variants lead to poor MC1R expression, due to endoplasmic reticulum (ER) retention or aberrant trafficking from ER to Golgi [50]. Other variants result in a diminished functional receptor due to lower affinity for α-MSH, reduced coupling with cAMP, or decreased ability to stimulate cAMP production [47, 50–53].In order to predict the impact on protein function ofMC1R variants, we usedin silico prediction tools, SIFT (http://sift.jcvi.org/), SNPs3D (http://www.snps3d.org/), and PolyPhen (http://genetics.bwh.harvard.edu/pph/). Rare variants, which were defined by an allele frequency <1% and predicted to be damaging by at least 1 of 3 prediction tools, were predicted to be damaging (D) variants, while variants without any damaging effect were regarded as nondamaging (nD) variants. Variants that had a clear functional impact (i.e., nonsense or frameshift mutations) were classified as damaging (D) variants.


### 2.4. Statistical Analysis

Statistical analysis was performed by using PASW software version 18. The level of significance for all tests corresponded to an alpha error rate of 5%. All odds ratios (OR) were calculated with 95% confidence intervals.To assess the association ofR and r variants with melanoma, we used Fisher’s exact test, the number of haplotypes without any variants alleles (wild-type and synonymous variants) being considered as reference. Rare r variants were thereafter classified into two subgroups according to functional prediction (predicted to be damaging (D) or nondamaging (nD)), and their effects on melanoma risk were also tested.A multivariate analysis adjusted for hair and eye colours, skin type, and nevus count was carried out to investigate the independent effect ofR and r alleles on melanoma risk. P values and their corresponding OR were calculated with logistic regression. Due to the interdependency of all our tests and the magnitude of our results, no correction for multiple testing was performed.In order to quantify the impact ofMC1RR, r, and D variants on melanoma risk, their population attributable fractions (PAF) were calculated as follows: PAF=(p×(OR-1)/(p×(OR-1)+1), where p is the proportion of controls carrying the risk alleles [34].Finally, in order to investigate the respective role of MC1R protein domains in melanoma, the number ofD and nD variants in different domains was compared between patients and controls. Each protein domain was determined by ExPASy Bioinformatics Resource Portal (http://expasy.org/) using UniProtKB Q01726.2 as query.


## 2.1. Studied Populations

A cohort of 1131 Caucasian melanoma patients was recruited between 2002 and 2008 from the dermatology departments of all university-affiliated hospitals in Paris (the MelanCohort). The main characteristics of the patients have been previously described [32]. Melanoma was sporadic in 784 patients (69%), including 81 patients (7%) who had multiple melanomas and 229 patients (20%) who had familial melanoma (at least 2 cases in first- or second-degree relatives, including the proband). Among the familial and multiple sporadic cases, 8.5% of patients carried mutations in theCDKN2A orCDK4 gene.The control group comprised 869 ethnically matched skin cancer-free blood donors recruited from the EFS (Etablissement Français du Sang) in Bichat and Saint-Louis hospitals over the same period. These subjects have previously been described and used as controls in case-control studies [32–34].Data from patients and controls regarding sex, age, ethnic origin, date of birth, anatomoclinical data, pigmentation characteristics (hair, eye, and skin colours), skin type (Fitzpatrick classification I to IV), nevus count (<10, 10–50, 51–100, and >100), and heavy freckles (yes/no) were collected in a standard document. Ancestry was investigated through birth location of parents and grandparents, and only those with a Caucasian ancestry were retained for the study. The pigmentation characteristics of patients and controls are summarized in Supplementary Table 1 (see Supplementary Materials available online athttp://dx.doi.org/10.1155/2014/925716). All subjects signed an informed consent form and provided a blood sample.


## 2.2. Sequencing and Mutational Analysis

The coding sequence ofMC1R deposited in GenBank (NM_002386.3) was amplified with 2 couples of primer selected by UCSC Genome Browser Gateway (data available upon request) in 1131 melanoma patients and 869 controls. The PCR mix contained 20 ng of genomic DNA, 2.5 mM MgCl2, 50 μM of each dNTP, 400 nM of each primer, and 0.5 U Ampli TaqGold polymerase (Applied Biosystems, Courtaboeuf, France). A 62°C annealing temperature was used for PCR amplification. PCR products were verified on a 2% agarose gel and purified by EXOSAP-IT (USB Corporation, OH, USA). Sequencing reaction was performed on 8900 Fast Thermal Cycler (Applied Biosystems), using 10 ng of purified PCR products and the Big-Dye Terminator Cycle Kit (Applied Biosystems). Sequence analysis was performed with an ABI-Prism 3130 automated sequencer (Applied Biosystems) and read with SeqScape software v2.5 (Applied Biosystems).


## 2.3. Classification ofMC1R Variants

The functional impact of numerousMC1R variants has been assessed in previous studies [47] (Supplementary Table 2). Some variants lead to poor MC1R expression, due to endoplasmic reticulum (ER) retention or aberrant trafficking from ER to Golgi [50]. Other variants result in a diminished functional receptor due to lower affinity for α-MSH, reduced coupling with cAMP, or decreased ability to stimulate cAMP production [47, 50–53].In order to predict the impact on protein function ofMC1R variants, we usedin silico prediction tools, SIFT (http://sift.jcvi.org/), SNPs3D (http://www.snps3d.org/), and PolyPhen (http://genetics.bwh.harvard.edu/pph/). Rare variants, which were defined by an allele frequency <1% and predicted to be damaging by at least 1 of 3 prediction tools, were predicted to be damaging (D) variants, while variants without any damaging effect were regarded as nondamaging (nD) variants. Variants that had a clear functional impact (i.e., nonsense or frameshift mutations) were classified as damaging (D) variants.


## 2.4. Statistical Analysis

Statistical analysis was performed by using PASW software version 18. The level of significance for all tests corresponded to an alpha error rate of 5%. All odds ratios (OR) were calculated with 95% confidence intervals.To assess the association ofR and r variants with melanoma, we used Fisher’s exact test, the number of haplotypes without any variants alleles (wild-type and synonymous variants) being considered as reference. Rare r variants were thereafter classified into two subgroups according to functional prediction (predicted to be damaging (D) or nondamaging (nD)), and their effects on melanoma risk were also tested.A multivariate analysis adjusted for hair and eye colours, skin type, and nevus count was carried out to investigate the independent effect ofR and r alleles on melanoma risk. P values and their corresponding OR were calculated with logistic regression. Due to the interdependency of all our tests and the magnitude of our results, no correction for multiple testing was performed.In order to quantify the impact ofMC1RR, r, and D variants on melanoma risk, their population attributable fractions (PAF) were calculated as follows: PAF=(p×(OR-1)/(p×(OR-1)+1), where p is the proportion of controls carrying the risk alleles [34].Finally, in order to investigate the respective role of MC1R protein domains in melanoma, the number ofD and nD variants in different domains was compared between patients and controls. Each protein domain was determined by ExPASy Bioinformatics Resource Portal (http://expasy.org/) using UniProtKB Q01726.2 as query.


## 3. Results

### 3.1. Characterization ofMC1R Variants

By sequencing the entireMC1R coding sequence, we found 79MC1Rvariants: 2 nonsense, 3 frameshift, 53 missense, and 21 silent variants (Table 1), 9% of which were localized in the extracellular portion of the receptor, 65% in the transmembrane domains, and 25% in the cytoplasmic domains (Figure 1).Table 1
All variants found in the study.
Nucleotide change
Amino acid change
Number of chromosome
Predicted to be damaging (D) varianta
Rare variantb
Novel variantc
Reference
Patients (chr = 2262)
Controls (chr = 1738)
Nonsynonymous
c.100C > T
p.R34W
1
No
Yes
Yes
c.104G > A
p.C35Y
1
Yes
Yes
No
[35]
c.112G > A
p.V38M
2
3
No
Yes
No
[35]
c.122C > G
p.S41C
2
Yes
Yes
Yes
c.133T > C (rs61996344)
p.F45L
2
Yes
Yes
No
[36]
c.178G > T (rs1805005)
p.V60L
359
254
Yes
No
No
[25]
c.199C > T
p.R67W
2
Yes
Yes
No
[37]
c.200G > A (rs34090186)
p.R67Q
1
1
No
Yes
No
[38]
c.205C > G
p.L69V
1
Yes
Yes
Yes
c.241G > C
p.A81P
1
Yes
Yes
No
[24]
c.247T > C (rs34474212)
p.S83P
5
1
Yes
Yes
No
[39]
c.252C > A (rs1805006)
p.D84E
23
13
RHC
No
No
[27]
c.274G > A
p.V92M
188
113
Yes
No
No
[27]
c.284C > T (rs34158934)
p.T95M
6
2
Yes
Yes
No
[27]
c.296T > C
p.L99P
1
Yes
Yes
Yes
c.310G > A (rs2229617)
p.G104S
1
Yes
Yes
No
[24]
c.350A > T
p.D117V
1
Yes
Yes
Yes
c.359T > C (rs33932559)
p.I120T
1
Yes
Yes
No
[35]
c.364G > A
p.V122M
1
4
No
Yes
No
[40]
c.373T > C
p.C125R
1
Yes
Yes
No
[41]
c.389C > T
p.S130F
1
Yes
Yes
Yes
c.415G > A
p.A139T
1
Yes
Yes
No
[31]
c.417G > A
p.V140M
1
No
Yes
Yes
c.419T > G
p.V140G
1
Yes
Yes
Yes
c.424C > T
p.R142C
1
Yes
Yes
No
[42]
c.425G > A (rs11547464)
p.R142H
29
13
RHC
No
No
[25]
c.451C > T (rs1805007)
p.R151C
211
76
RHC
No
No
[25]
c.456C > A
p.Y152X
1
Yes
Yes
No
[43]
c.464T > C (rs1110400)
p.I155T
35
14
Yes
No
No
[25]
c.467T > C
p.V156A
1
Yes
Yes
No
[31]
c.478C > T (rs1805008)
p.R160W
152
59
RHC
No
No
[25]
c.479G > A
p.R160Q
2
Yes
Yes
No
[36]
c.488G > A (rs885479)
p.R163Q
75
57
Yes
No
No
[25]
c.512C > A
p.A171D
1
Yes
Yes
No
[43]
c.613G > C
p.V205L
1
Yes
Yes
Yes
c.637C > T  (rs144239448)
p.R213W
3
4
Yes
Yes
No
[36]
c.652G > A
p.A218T
1
Yes
Yes
No
[29]
c.664G > T
p.A222S
1
No
Yes
Yes
c.667C > T
p.R223W
1
Yes
Yes
Yes
c.707G < A
p.G236D
1
Yes
Yes
Yes
c.766C > T
p.P256S
1
1
Yes
Yes
No
[43]
c.801C > A
p.C267X
1
Yes
Yes
Yes
c.820G > A
p.G274S
1
No
Yes
No
[44]
c.832A > G
p.K278E
3
1
Yes
Yes
No
[24]
c.842A > G (rs141177570)
p.N281S
1
Yes
Yes
No
[45]
c.853G > A
p.A285T
1
No
Yes
Yes
c.854C > G
p.A285G
1
Yes
Yes
Yes
c.861C > G
p.I287M
2
Yes
Yes
No
[46]
c.865T > C
p.C289R
1
Yes
Yes
No
[47]
c.880G > C (rs1805009)
p.D294H
85
35
RHC
No
No
[27]
c.892T > C
p.Y298H
1
Yes
Yes
No
[42]
c.895G > A
p.A299T
1
Yes
Yes
No
[25]
c.917G > A
p.R306H
1
Yes
Yes
No
[45]
c.928A > C
p.K310Q
1
No
Yes
Yes
c.951G > T
p.W317C
1
Yes
Yes
Yes
Insertion/deletion
c.86_87 insA
4
Yes
Yes
No
[46]
c.481_482 insG
1
Yes
Yes
Yes
c.524_525 insT
1
Yes
Yes
Yes
Synonymous
c.366G > A
p.V122V
1
No
Yes
No
[29]
c.414C > T
p.I138I
1
No
Yes
No
[38]
c.426C > A
p.R142R
2
No
Yes
Yes
c.471C > T
p.T157T
1
No
Yes
Yes
c.477G > C
p.P159P
1
No
Yes
Yes
c.478C > A
p.R160R
1
No
Yes
No
[48]
c.483G > A
p.A161A
1
No
Yes
No
[29]
c.504C > T (rs34612847)
p.I168I
1
No
Yes
No
[38]
c.531G > A (rs145781072)
p.T177T
1
1
No
Yes
No
c.537C > T
p.F179F
1
No
Yes
Yes
c.621C > T
p.Y207Y
1
No
Yes
No
[31]
c.690G > C
p.P230P
3
No
Yes
No
[24]
c.699G > A (rs146544450)
p.Q233Q
6
10
No
Yes
No
[46]
c.792C > T
p.I264I
4
No
Yes
No
[49]
c.828C > T
p.I276I
1
No
Yes
No
[31]
c.873C > T
p.A291A
1
No
Yes
Yes
c.894C > T (rs143395134)
p.Y298Y
2
No
Yes
No
c.900C > T (rs3212367)
p.F300F
1
4
No
Yes
No
[38]
c.927C < G
p.L309L
1
No
Yes
Yes
c.942A > G (rs2228478)
p.T314T
249
170
No
No
No
[25]
c.948C > T (rs151318945)
p.S316S
5
5
No
Yes
No
dbSNP
aDamaging variants were those predicted as deleterious or intolerated by SIFT, SNPs3D, and PolyPhen in silico prediction tools.
bRare variants were defined as allele frequency less than 1%.
cVariants were absent in dbSNP by using NM_002386.3 as contig transcript and works of Gerstenblith et al. [22] and García-Borrón et al. [23].
RHC, red hair colour variant.
Frequent non-RHC variants are shown in bold.Figure 1
Structure of human melanocortin-1 receptor. Putative structure is drawn according to the two-dimensional model of Ringholm et al., 2004 [54]. The amino acid sequence corresponds to the wildtype consensus (UniProtKB Q01726.2). Each protein domain is framed by residues marked in blue. Residues with R, frequent r, and predicted-to-be-damaging (D) alleles are shown in red, pink, and yellow, respectively. Ec: extracellular domain; Ic: intracellular domain; TM: transmembrane domain; R: RHC alleles; r: frequent non-RHC alleles; D: predicted damaging variants.FiveR alleles (D84E, R142H, R151C, R160W, and D294H) and 5 frequent r alleles (V60L, V92M, I155T, R163Q, and T314T) were found in our cohorts. In addition, we found 69 rare r alleles, consisting of 44 missense, 2 nonsense, 3 frameshift, and 20 silent variants. Interestingly, 25 were novelMC1R variants, 15 of which (52%) were predicted to have a functional impact (D variants). Amongst the 69 rare r alleles, 40 (58%) were predicted to be D variants, including 35 missense, 2 nonsense, and 3 frameshift variants (Table 1).


### 3.2. Association of DifferentMC1R Variant Subgroups with Melanoma Risk

The association of different subgroups ofMC1R variants with melanoma risk was assessed as described in Section 2. Collectively, R alleles were strongly associated with melanoma (OR = 2.66 [2.20–3.23]; P=2.69E-25) (Table 2). In addition, association of individual R alleles with melanoma was significant (ORs = from 2.33 to 2.9) for all R variants except for D84E (P=0.092). We calculated an estimated PAF due to R variants of 15.8%. By decreasing order, the PAFs were, respectively, 7.4 (R151C), 5.7 (R160W), 2.9 (D294H), 1 (R142H), and 0.8 (D84E) (Table 2).Table 2
Association of different variant subgroups with melanoma risk.
Patientsc (chr = 2262)
Controlsc (chr = 1738)
OR [95% CI]
P value
PAF (%)
Reference sequencea
936
976
Ref.
Ref.
AllR variantsb
500/0.22
196/0.11
2.66 [2.20–3.23]
2.69
E
-
25
15.8
Individual RHC variant
D84E
23/0.01
13/0.007
1.85 [0.89–3.87]
0.092
0.8
R142H
29/0.01
13/0.007
2.33 [1.16–4.75]
0.012
1
R151C
211/0.09
76/0.04
2.90 [2.18–3.86]
7.95
E
-
15
7.4
R160W
152/0.07
59/0.03
2.69 [1.94–3.72]
1.60
E
-
10
5.7
D294H
85/0.04
35/0.02
2.53 [1.66–3.87]
3.10
E
-
06
2.9
Allr variants
980/0.43
678/0.39
1.51 [1.32–1.73]
1.40
E
-
09
16.6
Frequentr variants
V60L
359/0.16
254/0.15
1.47 [1.22–1.78]
3.50
E
-
05
6.5
V92M
188/0.08
113/0.07
1.73 [1.34–2.25]
1.30
E
-
05
4.5
I155T
35/0.02
14/0.008
2.61 [1.35–5.12]
0.002
1.3
R163Q
75/0.03
57/0.03
1.37 [0.95–1.98]
0.087
1.2
Rarer variants
57/0.025
31/0.017
1.92 [1.20–3.07]
0.004
1.6
D variants
48/0.02
21/0.01
2.38 [1.38–4.15]
0.001
1.6
nD variants
9/0.004
10/0.006
0.94 [0.35–2.51]
1
aNumber of alleles containing wild-type sequence or synonymous variants and used as reference.
bAll R variants including D84E, R142H, R151C, R160W, and D294H.
cNumber of alleles/MAF (minor allelic frequency).
R: red hair colour variant; r: nonred hair colour variant; D: predicted to be damaging; nD: predicted to be nondamaging; OR: odds ratio; CI: confidence interval; PAF: population attributable fraction.
Statistically significant results are shown in bold. Ref, used as reference.Interestingly,r alleles were also associated, although less strongly, with melanoma (OR = 1.51 [1.32–1.73]; P=1.40E-09) (Table 2). In addition, three frequent r alleles (V60L, V92M, and I155T) were associated individually with melanoma, but the association for the R163Q variant did not reach significance (P=0.087). Of note, the I155T variant associated almost as strongly as R alleles with melanoma (OR = 2.61 [1.35–5.12]) and the other frequent r alleles conferred risks equal to roughly half the risk of R alleles (ORs = from 1.47 to 1.73). The PAF of r variants was very similar to that of R variants (16.6%). By decreasing order, the PAFs were, respectively, 6.5 (V60L), 4.5 (V92M), 1.3 (I155T), and 1.2 (R163Q) (Table 2).Furthermore, rarer variants were also strongly associated with melanoma (OR = 1.92 [1.20–3.07]; P=0.004) (Table 2). We divided rare r alleles into D and nD subgroups according toin silico functional predictions. Interestingly, D variants were associated with melanoma susceptibility as strongly as R alleles (OR = 2.38 [1.38–4.15]; P=0.001). Of note, the estimated PAF of D variants was 1.6% and contributed almost completely to the PAF of rare r variants (Table 2). On the contrary, nD variants had no impact on melanoma susceptibility (P=1). Moreover, the average age at melanoma diagnosis of patients carrying D variants was younger than that of patients without D variants (45 and 54, resp.; P=0.04). Finally, D variants, like R variants, were also associated with familial melanoma (OR = 4.78).To investigate whether the effect of the two mainMC1R variant categories on melanoma susceptibility was independent of pigmentation traits, we conducted a multivariate analysis including the main clinical melanoma risk factors using logistic regression (Tables 3(a) and 3(b)). This showed a persistent role of R and r alleles on melanoma risk (respective ORs = 2.22 [1.66–2.97] and 1.26 [1.04–1.52]).Table 3
Multivariate analysis.
(a)
R variants
OR [95% CI]
P valuea
Hair colour
0.88 [0.69–1.12]
0.292
Eye colour
2.30 [1.63–3.26]
2.64
E
-
06
Skin type
1.64 [1.17–2.3]
0.004
Nevus count
1.39 [0.97–1.98]
0.07
R variants
2.22 [1.66–2.97]
6.00
E
-
08
(b)
r variants
OR [95% CI]
P valuea
Hair colour
0.98 [0.74–1.29]
0.88
Eye colour
1.93 [1.45–2.57]
1.20
E
-
06
Skin type
1.96 [1.49–2.57]
7.33
E
-
06
Nevus count
1.6 [1.18–2.15]
0.002
r variants
1.26 [1.04–1.52]
0.018
a
P value was calculated by logistic regression.
R: red hair colour variant;r: nonred hair colour variant.
Statistically significant results are shown in bold.


### 3.3. Impact of the Different MC1R Protein Domains

In order to study the impact of MC1R protein domains on melanoma predisposition, the different classes ofMC1R variants (R, r, D, and nD) were positioned on the different protein domains (Figure 1).MostMC1Rvariants were localized in six receptor domains: transmembrane 1, 2, 3, and 7, intracellular 2, and C terminal domains. Very few variants were located in the extracellular portion. Among these domains, variants located in the intracellular domain 2 and in the transmembrane domain 7 had the highest impact on melanoma risk (OR = 2.75 [2.22–3.40] and OR = 2.48 [1.67–3.69]).The repartition ofR, r, D, and nD variants in each protein domain indicated in Table 4 showed that 63% of D variants were located in four domains (intracellular 2 and transmembrane 2, 5, and 7) whereas only 18% of nD variants were located in these domains (P<0.0001). Importantly, three of these four domains also contain at least one R variant, suggesting an important role of these domains in MC1R function and pathogenicity.Table 4
Localisation ofMC1R variants according to their classification and to their demonstrated or predicted functional impact.
Protein region
RHC variant (R)
Non-RHC variant (r)
Frequent non-RHC (r)
Rarer
Predicted to be damaging (D)a
Predicted to be nondamaging (nD)a
N-terminus
M1-E37
1
1
Transmembrane 1
V38-I63
x
4
5
Intracellular 1
A64-P72
3
2
Transmembrane 2
M73-L100
x
x
16
2
Extracellular 1
L101-Q115
0
1
Transmembrane 3
L116-Y143
x
7
6
Intracellular 2
I144-R162
x
x
4
0
Transmembrane 4
R163-Y183
x
1
0
Extracellular 2
D184-L192
0
0
Transmembrane 5
V193-A218
9
0
Intracellular 3
Q219-G236
2
1
Transmembrane 6
L237-V265
2
0
Extracellular 3
L266-I276
1
1
Transmembrane 7
F277-A299
x
11
2
C-terminus
F300-W317
2
1
aNumber of alleles found in each protein domain.
The crosses indicate localisation of RHC variants or frequent non-RHC variants.


## 3.1. Characterization ofMC1R Variants

By sequencing the entireMC1R coding sequence, we found 79MC1Rvariants: 2 nonsense, 3 frameshift, 53 missense, and 21 silent variants (Table 1), 9% of which were localized in the extracellular portion of the receptor, 65% in the transmembrane domains, and 25% in the cytoplasmic domains (Figure 1).Table 1
All variants found in the study.
Nucleotide change
Amino acid change
Number of chromosome
Predicted to be damaging (D) varianta
Rare variantb
Novel variantc
Reference
Patients (chr = 2262)
Controls (chr = 1738)
Nonsynonymous
c.100C > T
p.R34W
1
No
Yes
Yes
c.104G > A
p.C35Y
1
Yes
Yes
No
[35]
c.112G > A
p.V38M
2
3
No
Yes
No
[35]
c.122C > G
p.S41C
2
Yes
Yes
Yes
c.133T > C (rs61996344)
p.F45L
2
Yes
Yes
No
[36]
c.178G > T (rs1805005)
p.V60L
359
254
Yes
No
No
[25]
c.199C > T
p.R67W
2
Yes
Yes
No
[37]
c.200G > A (rs34090186)
p.R67Q
1
1
No
Yes
No
[38]
c.205C > G
p.L69V
1
Yes
Yes
Yes
c.241G > C
p.A81P
1
Yes
Yes
No
[24]
c.247T > C (rs34474212)
p.S83P
5
1
Yes
Yes
No
[39]
c.252C > A (rs1805006)
p.D84E
23
13
RHC
No
No
[27]
c.274G > A
p.V92M
188
113
Yes
No
No
[27]
c.284C > T (rs34158934)
p.T95M
6
2
Yes
Yes
No
[27]
c.296T > C
p.L99P
1
Yes
Yes
Yes
c.310G > A (rs2229617)
p.G104S
1
Yes
Yes
No
[24]
c.350A > T
p.D117V
1
Yes
Yes
Yes
c.359T > C (rs33932559)
p.I120T
1
Yes
Yes
No
[35]
c.364G > A
p.V122M
1
4
No
Yes
No
[40]
c.373T > C
p.C125R
1
Yes
Yes
No
[41]
c.389C > T
p.S130F
1
Yes
Yes
Yes
c.415G > A
p.A139T
1
Yes
Yes
No
[31]
c.417G > A
p.V140M
1
No
Yes
Yes
c.419T > G
p.V140G
1
Yes
Yes
Yes
c.424C > T
p.R142C
1
Yes
Yes
No
[42]
c.425G > A (rs11547464)
p.R142H
29
13
RHC
No
No
[25]
c.451C > T (rs1805007)
p.R151C
211
76
RHC
No
No
[25]
c.456C > A
p.Y152X
1
Yes
Yes
No
[43]
c.464T > C (rs1110400)
p.I155T
35
14
Yes
No
No
[25]
c.467T > C
p.V156A
1
Yes
Yes
No
[31]
c.478C > T (rs1805008)
p.R160W
152
59
RHC
No
No
[25]
c.479G > A
p.R160Q
2
Yes
Yes
No
[36]
c.488G > A (rs885479)
p.R163Q
75
57
Yes
No
No
[25]
c.512C > A
p.A171D
1
Yes
Yes
No
[43]
c.613G > C
p.V205L
1
Yes
Yes
Yes
c.637C > T  (rs144239448)
p.R213W
3
4
Yes
Yes
No
[36]
c.652G > A
p.A218T
1
Yes
Yes
No
[29]
c.664G > T
p.A222S
1
No
Yes
Yes
c.667C > T
p.R223W
1
Yes
Yes
Yes
c.707G < A
p.G236D
1
Yes
Yes
Yes
c.766C > T
p.P256S
1
1
Yes
Yes
No
[43]
c.801C > A
p.C267X
1
Yes
Yes
Yes
c.820G > A
p.G274S
1
No
Yes
No
[44]
c.832A > G
p.K278E
3
1
Yes
Yes
No
[24]
c.842A > G (rs141177570)
p.N281S
1
Yes
Yes
No
[45]
c.853G > A
p.A285T
1
No
Yes
Yes
c.854C > G
p.A285G
1
Yes
Yes
Yes
c.861C > G
p.I287M
2
Yes
Yes
No
[46]
c.865T > C
p.C289R
1
Yes
Yes
No
[47]
c.880G > C (rs1805009)
p.D294H
85
35
RHC
No
No
[27]
c.892T > C
p.Y298H
1
Yes
Yes
No
[42]
c.895G > A
p.A299T
1
Yes
Yes
No
[25]
c.917G > A
p.R306H
1
Yes
Yes
No
[45]
c.928A > C
p.K310Q
1
No
Yes
Yes
c.951G > T
p.W317C
1
Yes
Yes
Yes
Insertion/deletion
c.86_87 insA
4
Yes
Yes
No
[46]
c.481_482 insG
1
Yes
Yes
Yes
c.524_525 insT
1
Yes
Yes
Yes
Synonymous
c.366G > A
p.V122V
1
No
Yes
No
[29]
c.414C > T
p.I138I
1
No
Yes
No
[38]
c.426C > A
p.R142R
2
No
Yes
Yes
c.471C > T
p.T157T
1
No
Yes
Yes
c.477G > C
p.P159P
1
No
Yes
Yes
c.478C > A
p.R160R
1
No
Yes
No
[48]
c.483G > A
p.A161A
1
No
Yes
No
[29]
c.504C > T (rs34612847)
p.I168I
1
No
Yes
No
[38]
c.531G > A (rs145781072)
p.T177T
1
1
No
Yes
No
c.537C > T
p.F179F
1
No
Yes
Yes
c.621C > T
p.Y207Y
1
No
Yes
No
[31]
c.690G > C
p.P230P
3
No
Yes
No
[24]
c.699G > A (rs146544450)
p.Q233Q
6
10
No
Yes
No
[46]
c.792C > T
p.I264I
4
No
Yes
No
[49]
c.828C > T
p.I276I
1
No
Yes
No
[31]
c.873C > T
p.A291A
1
No
Yes
Yes
c.894C > T (rs143395134)
p.Y298Y
2
No
Yes
No
c.900C > T (rs3212367)
p.F300F
1
4
No
Yes
No
[38]
c.927C < G
p.L309L
1
No
Yes
Yes
c.942A > G (rs2228478)
p.T314T
249
170
No
No
No
[25]
c.948C > T (rs151318945)
p.S316S
5
5
No
Yes
No
dbSNP
aDamaging variants were those predicted as deleterious or intolerated by SIFT, SNPs3D, and PolyPhen in silico prediction tools.
bRare variants were defined as allele frequency less than 1%.
cVariants were absent in dbSNP by using NM_002386.3 as contig transcript and works of Gerstenblith et al. [22] and García-Borrón et al. [23].
RHC, red hair colour variant.
Frequent non-RHC variants are shown in bold.Figure 1
Structure of human melanocortin-1 receptor. Putative structure is drawn according to the two-dimensional model of Ringholm et al., 2004 [54]. The amino acid sequence corresponds to the wildtype consensus (UniProtKB Q01726.2). Each protein domain is framed by residues marked in blue. Residues with R, frequent r, and predicted-to-be-damaging (D) alleles are shown in red, pink, and yellow, respectively. Ec: extracellular domain; Ic: intracellular domain; TM: transmembrane domain; R: RHC alleles; r: frequent non-RHC alleles; D: predicted damaging variants.FiveR alleles (D84E, R142H, R151C, R160W, and D294H) and 5 frequent r alleles (V60L, V92M, I155T, R163Q, and T314T) were found in our cohorts. In addition, we found 69 rare r alleles, consisting of 44 missense, 2 nonsense, 3 frameshift, and 20 silent variants. Interestingly, 25 were novelMC1R variants, 15 of which (52%) were predicted to have a functional impact (D variants). Amongst the 69 rare r alleles, 40 (58%) were predicted to be D variants, including 35 missense, 2 nonsense, and 3 frameshift variants (Table 1).


## 3.2. Association of DifferentMC1R Variant Subgroups with Melanoma Risk

The association of different subgroups ofMC1R variants with melanoma risk was assessed as described in Section 2. Collectively, R alleles were strongly associated with melanoma (OR = 2.66 [2.20–3.23]; P=2.69E-25) (Table 2). In addition, association of individual R alleles with melanoma was significant (ORs = from 2.33 to 2.9) for all R variants except for D84E (P=0.092). We calculated an estimated PAF due to R variants of 15.8%. By decreasing order, the PAFs were, respectively, 7.4 (R151C), 5.7 (R160W), 2.9 (D294H), 1 (R142H), and 0.8 (D84E) (Table 2).Table 2
Association of different variant subgroups with melanoma risk.
Patientsc (chr = 2262)
Controlsc (chr = 1738)
OR [95% CI]
P value
PAF (%)
Reference sequencea
936
976
Ref.
Ref.
AllR variantsb
500/0.22
196/0.11
2.66 [2.20–3.23]
2.69
E
-
25
15.8
Individual RHC variant
D84E
23/0.01
13/0.007
1.85 [0.89–3.87]
0.092
0.8
R142H
29/0.01
13/0.007
2.33 [1.16–4.75]
0.012
1
R151C
211/0.09
76/0.04
2.90 [2.18–3.86]
7.95
E
-
15
7.4
R160W
152/0.07
59/0.03
2.69 [1.94–3.72]
1.60
E
-
10
5.7
D294H
85/0.04
35/0.02
2.53 [1.66–3.87]
3.10
E
-
06
2.9
Allr variants
980/0.43
678/0.39
1.51 [1.32–1.73]
1.40
E
-
09
16.6
Frequentr variants
V60L
359/0.16
254/0.15
1.47 [1.22–1.78]
3.50
E
-
05
6.5
V92M
188/0.08
113/0.07
1.73 [1.34–2.25]
1.30
E
-
05
4.5
I155T
35/0.02
14/0.008
2.61 [1.35–5.12]
0.002
1.3
R163Q
75/0.03
57/0.03
1.37 [0.95–1.98]
0.087
1.2
Rarer variants
57/0.025
31/0.017
1.92 [1.20–3.07]
0.004
1.6
D variants
48/0.02
21/0.01
2.38 [1.38–4.15]
0.001
1.6
nD variants
9/0.004
10/0.006
0.94 [0.35–2.51]
1
aNumber of alleles containing wild-type sequence or synonymous variants and used as reference.
bAll R variants including D84E, R142H, R151C, R160W, and D294H.
cNumber of alleles/MAF (minor allelic frequency).
R: red hair colour variant; r: nonred hair colour variant; D: predicted to be damaging; nD: predicted to be nondamaging; OR: odds ratio; CI: confidence interval; PAF: population attributable fraction.
Statistically significant results are shown in bold. Ref, used as reference.Interestingly,r alleles were also associated, although less strongly, with melanoma (OR = 1.51 [1.32–1.73]; P=1.40E-09) (Table 2). In addition, three frequent r alleles (V60L, V92M, and I155T) were associated individually with melanoma, but the association for the R163Q variant did not reach significance (P=0.087). Of note, the I155T variant associated almost as strongly as R alleles with melanoma (OR = 2.61 [1.35–5.12]) and the other frequent r alleles conferred risks equal to roughly half the risk of R alleles (ORs = from 1.47 to 1.73). The PAF of r variants was very similar to that of R variants (16.6%). By decreasing order, the PAFs were, respectively, 6.5 (V60L), 4.5 (V92M), 1.3 (I155T), and 1.2 (R163Q) (Table 2).Furthermore, rarer variants were also strongly associated with melanoma (OR = 1.92 [1.20–3.07]; P=0.004) (Table 2). We divided rare r alleles into D and nD subgroups according toin silico functional predictions. Interestingly, D variants were associated with melanoma susceptibility as strongly as R alleles (OR = 2.38 [1.38–4.15]; P=0.001). Of note, the estimated PAF of D variants was 1.6% and contributed almost completely to the PAF of rare r variants (Table 2). On the contrary, nD variants had no impact on melanoma susceptibility (P=1). Moreover, the average age at melanoma diagnosis of patients carrying D variants was younger than that of patients without D variants (45 and 54, resp.; P=0.04). Finally, D variants, like R variants, were also associated with familial melanoma (OR = 4.78).To investigate whether the effect of the two mainMC1R variant categories on melanoma susceptibility was independent of pigmentation traits, we conducted a multivariate analysis including the main clinical melanoma risk factors using logistic regression (Tables 3(a) and 3(b)). This showed a persistent role of R and r alleles on melanoma risk (respective ORs = 2.22 [1.66–2.97] and 1.26 [1.04–1.52]).Table 3
Multivariate analysis.
(a)
R variants
OR [95% CI]
P valuea
Hair colour
0.88 [0.69–1.12]
0.292
Eye colour
2.30 [1.63–3.26]
2.64
E
-
06
Skin type
1.64 [1.17–2.3]
0.004
Nevus count
1.39 [0.97–1.98]
0.07
R variants
2.22 [1.66–2.97]
6.00
E
-
08
(b)
r variants
OR [95% CI]
P valuea
Hair colour
0.98 [0.74–1.29]
0.88
Eye colour
1.93 [1.45–2.57]
1.20
E
-
06
Skin type
1.96 [1.49–2.57]
7.33
E
-
06
Nevus count
1.6 [1.18–2.15]
0.002
r variants
1.26 [1.04–1.52]
0.018
a
P value was calculated by logistic regression.
R: red hair colour variant;r: nonred hair colour variant.
Statistically significant results are shown in bold.


## 3.3. Impact of the Different MC1R Protein Domains

In order to study the impact of MC1R protein domains on melanoma predisposition, the different classes ofMC1R variants (R, r, D, and nD) were positioned on the different protein domains (Figure 1).MostMC1Rvariants were localized in six receptor domains: transmembrane 1, 2, 3, and 7, intracellular 2, and C terminal domains. Very few variants were located in the extracellular portion. Among these domains, variants located in the intracellular domain 2 and in the transmembrane domain 7 had the highest impact on melanoma risk (OR = 2.75 [2.22–3.40] and OR = 2.48 [1.67–3.69]).The repartition ofR, r, D, and nD variants in each protein domain indicated in Table 4 showed that 63% of D variants were located in four domains (intracellular 2 and transmembrane 2, 5, and 7) whereas only 18% of nD variants were located in these domains (P<0.0001). Importantly, three of these four domains also contain at least one R variant, suggesting an important role of these domains in MC1R function and pathogenicity.Table 4
Localisation ofMC1R variants according to their classification and to their demonstrated or predicted functional impact.
Protein region
RHC variant (R)
Non-RHC variant (r)
Frequent non-RHC (r)
Rarer
Predicted to be damaging (D)a
Predicted to be nondamaging (nD)a
N-terminus
M1-E37
1
1
Transmembrane 1
V38-I63
x
4
5
Intracellular 1
A64-P72
3
2
Transmembrane 2
M73-L100
x
x
16
2
Extracellular 1
L101-Q115
0
1
Transmembrane 3
L116-Y143
x
7
6
Intracellular 2
I144-R162
x
x
4
0
Transmembrane 4
R163-Y183
x
1
0
Extracellular 2
D184-L192
0
0
Transmembrane 5
V193-A218
9
0
Intracellular 3
Q219-G236
2
1
Transmembrane 6
L237-V265
2
0
Extracellular 3
L266-I276
1
1
Transmembrane 7
F277-A299
x
11
2
C-terminus
F300-W317
2
1
aNumber of alleles found in each protein domain.
The crosses indicate localisation of RHC variants or frequent non-RHC variants.


## 4. Discussion

MC1R variants are usually classified into two main categories, RHC (R) and non-RHC (r), according to their association or not with the red hair colour phenotype [21, 24, 27, 55]. For the past ten years several association studies have demonstrated the importance of R alleles on melanoma predisposition [28, 29, 42, 56, 57]. However, the influence of rareMC1Rvariants on melanoma predisposition has been poorly investigated which prompted us to study in detail the role of these variants in the French population.In this study, we confirmed the association of mostR alleles and melanoma risk with strengths that were close to those observed in previous studies [28, 29, 42, 56, 57].We also showed a clear association of frequentMC1Rr alleles, especially V60L, V92M, and I155T, with melanoma. In an early meta-analysis neither of them was found to be associated with melanoma [55], whereas, in a more recent and larger meta-analysis, both were associated with melanoma [57]. V60L is a loss of function variant with reduced coupling to the cAMP signalling [50, 53] and V92M has a lower affinity for α-MSH than wild-type MC1R and a decreased ability to stimulate the production of cAMP [23]. These functional data argue for an association of theses variants with melanoma risk.Even though the OR ofR alleles was much higher than that of r alleles, their PAFs were very close (15.8% for R and 16.6% for r) suggesting that the impact of r alleles on melanoma seems to be important in the French population. Notably, in our work, the individual PAFs of V60L and V92M (6.5% and 4.5%) were very close to that of R alleles R151C and R160W (7.4% and 5.7%). Our results are different from that published by Williams et al., in which the PAF of r variants is only 7.4. This may be due at least, in part, to the high allelic frequency of r variants observed in French melanoma patients (0.43) (versus 0.33 in Williams meta-analysis) [57]. This further emphasises the role r alleles in melanoma risk according to the ethnical background of the population studied. In addition, both R and r alleles remained associated with melanoma risk in multivariate analyses, suggesting that they exert a role in melanoma risk independently of their effect on pigmentation, as previously suggested.In this work we identified 25 variants that have not been reported before [22, 23, 31, 47], further underscoring the highly polymorphic character ofMC1R. According to Gerstenblith’s work, the proportion of rareMC1R variants varies across populations and within Caucasian populations [23]. In our study, the proportion of rare variants (87%) was close to that described in Scherer’s work (74% in Germany and 78% in Spain) [31]. Interestingly, rare variants predicted to be damaging (D variants) were associated with melanoma as strongly as R alleles (OR = 2.38 [1.38–4.15]) whereas variants not predicted to be damaging (nD) had no effect on melanoma. In addition, PAF of D variants (1.6%) was higher than two R variants (R142H and D84E), which seems to indicate that the contribution of this subgroup in melanoma predisposition should be taken into consideration, at least in the French population.Yet, there is a limitation in our work: the absence of functional studies concerning the potential effects of these novelMC1R variants, notably on α-MSH binding, receptor cellular localisation, and cAMP signalling.Finally, the majority ofD variants were located in the same domains as the R alleles (intracellular 2 and transmembrane 2, 5, and 7, Table 4 and Figure 1). It had been shown before that there was a similar localization of D variants in the German, Spanish, and Italian populations [29, 31] suggesting an important role of these domains in the receptor’s function.


## 5. Conclusion

In this large study we confirmed the role ofMC1RR alleles in melanoma susceptibility and clearly showed thatMC1Rr alleles also significantly increase the risk of melanoma. In addition, we defined the role of rareMC1R variants and proposed a novel class of D variants that are strong melanoma risk factors. These findings might help in the definition of high-risk melanoma subgroups that could be targeted for melanoma prevention.



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*Source: 925716-2014-04-10.xml*