BRIEF COMMUNICATION

Association Between Endothelin Receptor B Nonsynonymous Variants and Melanoma Risk

Nadem Soufir, Roubila Meziani, Jean-Jacques Lacapère, Guylene Bertrand, Frederic Fumeron, Agnes Bourillon, Bénédicte Gérard, Vincent Descamps, Béatrice Crickx, Laurence Ollivaud, Alain Archimbaud, Céleste Lebbe, Nicole Basset-Seguin, Philippe Saiag, Bernard Grandchamp
for the Investigators of the Melan-Cohort

Affiliations of authors: Laboratoire de Biochimie Hormonale et Génétique, Hôpital Bichat-Claude Bernard, AP-HP, Faculté de Médecine Paris VII, Paris, France (NS, RM, GB, AB, BG); Inserm U 410 Faculté de Médecine Paris VII Bichat-Claude Bernard, Paris, France (J-JL); EA 3516, Université Paris 7, Faculté de Médecine Paris VII Bichat-Claude Bernard, Paris, France (FF); Service de Dermatologie, Hôpital Bichat-Claude Bernard, Paris, AP-HP, Faculté de Médecine ParisVII, Paris, France (VD, BC); Service de Dermatologie, Hôpital Saint-Louis, AP-HP, Faculté de Médecine Paris VII, Paris, France (LO, AA, CL, NB-S); Service de Dermatologie, Hôpital Ambroise Paré, AP-HP, Faculté de Médecine Paris-Ile de France Ouest, Boulogne Billancourt, France (PS)

Correspondence to: Nadem Soufir, MD, PhD, Laboratoire de Biochimie Hormonale et Génétique, IFR02; Hopital Bichat-Claude Bernard, 46 rue Henri Huchard, 75018 Paris, France (e-mail: nadem.soufir{at}bch.ap-hop-paris.fr).


    ABSTRACT
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The endothelin signaling pathway plays a crucial role in melanocyte differentiation and migration. In this study, we investigated whether germline mutations of endothelin receptor B (EDNRB), a gene involved in Hirschsprung disease (HSCR), could also predispose for malignant melanoma (MM). The coding region of EDNRB was sequenced in 137 MM patients and in 130 ethnically matched Caucasian control subjects. Six nonsynonymous EDNRB variants were found in 15 patients (11%), but only two were found in four control subjects (3%, odds ratio [OR] = 3.87, 95% confidence interval [CI] = 1.25 to 12; P = .012). Overall, 14 out of 15 MM patients carried EDNRB mutations reported in HSCR, some of which had previously been shown to lead to loss of function. In multivariable logistic regression analysis including skin type, eye and hair color, number of nevi, and dorsal lentigines (freckles), the association between EDNRB mutations and MM risk remained statistically significant (OR = 19.9, 95% CI = 1.34 to 296.2; P = .03). Our data strongly suggest that EDNRB is involved in predisposition for two different multigenic disorders, HSCR and melanoma.


The incidence of malignant melanoma (MM) has doubled every 10 years in most Caucasian populations (1). Multiple phenotypic traits, including the number of melanocytic nevi, dysplastic nevi, freckling and sunburn tendencies, and severe sunburn episodes during childhood, have been shown to be MM risk factors (2). In addition to phenotypic traits, two highly penetrating melanoma-predisposing genes, CDKN2A and CDK4, are involved in predisposition to familial cutaneous melanoma, which accounts for 5–10% of all melanoma cases (36). Furthermore, the inheritance of melanoma can also be polygenic, involving variant alleles in several different genes (7,8), including the melanocortin 1 receptor MC1R (911).

The endothelin receptor B (EDNRB) is a G-protein-coupled, seven-transmembrane receptor that interacts with endothelins (EDN1, EDN2, and EDN3), multifunctional peptides involved in many cellular and physiologic processes (12). EDNRB signaling is required during embryogenesis for the migration of melanoblasts from the neural crest to the interfollicular epidermidis, retinal epithelium, and follicular hair (13) and plays a crucial role in regulating the number of progenitor melanocytes and melanocyte differentiation (14). Loss of EDNRB function is associated with several genetic diseases: Hirschsprung disease (HSCR, 600155) (15,16), Waardenburg syndrome 4 (WS4; OMIM 277580) (17), and Waardenburg-Shah syndrome (18). These diseases are characterized by major defects in the neural crest cell lineages. In addition to its role in melanocyte development, activation of the endothelin system also plays a crucial role in the skin's response to ultraviolet irradiation. EDN1, a paracrine peptide that is secreted by keratinocytes in response to UVB radiation (1921), is a ligand for EDNRB and induces proliferation of mature melanocytes and stimulates melanogenesis, producing a photoprotective effect (22).

We therefore tested the hypothesis that a germline defect of the EDNRB pathway could be associated with a genetic predisposition to melanoma. The coding sequence and intronic flanking regions of the EDNRB gene were entirely sequenced in this case control study.

Melanoma patients (n = 137) and control subjects (n = 130) were prospectively recruited between 1999 and 2004 by the Dermatology Departments of the Bichat Claude-Bernard, the Percy, the Ambroise Paré, and the Saint-Louis Hospitals in Paris. The study population consisted of patients aged 20–80 years with histologically confirmed MM. Twenty-nine of the patients had at least one relative with melanoma, but none harbored a CDKN2A or a CDK4 germline mutation. The control group was extended from the one previously described (23) and was composed of individuals who had no personal or family history of skin cancer, were in the same age range, and were referred by the same departments and hospitals as the MM patients. The birthplaces of the parents and grandparents were recorded to ensure that all patients and control subjects were of Caucasian origin. The Hospital Medical Ethics Committee (CCPPRB) approved the study protocol. Informed consent was obtained from all patients and control subjects enrolled in the study.

The clinical characteristics of the MM patients and control subjects are summarized in Table 1. The strongest risk factors identified for MM were a mole count of >50 (P<.001), the presence of an atypical mole syndrome (P<.001), fair skin color (P<.001), and having dorsal lentigines (P<.001). Other pigmentation characteristics (light-colored eyes and hair, as well as skin type I or II) were not associated with MM risk. In addition, the presence of MC1R functional variants was strongly associated with MM risk (for the presence of one variant, odds ratio [OR] = 2.94, 95% confidence interval [CI] = 1.73 to 5.02, and for two variants, OR = 8.88, 95% CI = 3.19 to 24.67).


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Table 1.  Clinical characteristics and MC1R status of melanoma patients and control subjects*

 
A higher frequency of nonsynonymous EDNRB variants was observed in the MM patients than in the control subjects. Six nonsynonymous EDNRB variants were characterized in 15 of the 137 patients (11%, Table 2), two of which were also present in 4 of the 130 control subjects (3%, OR = 3.87, 95% CI = 1.25 to 12.0; P = .012,). A single variant, S305N, had a higher allelic frequency in patients than in control subjects (.044 versus .011, difference = .033, 95% CI on the difference = .0041 to .059; P = .025). In the National Center for Biotechnology Information (NCBI) databank, this variant is reported to have an average estimated allelic frequency of only .002 (single-nucleotide polymorphisms [SNP] identification number rs5352; http://www.ncbi.nlm.nih.gov/SNP/index.html). To confirm the value of the allelic frequency of this SNP in a larger group of control subjects residing in the Paris region, we genotyped an additional 305 Caucasian control subjects and calculated an overall S305N allelic frequency of 0.0115 in the control group.


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Table 2.  EDNRB variants in melanoma patients and control subjects*

 
Interestingly, three of the variants identified in 14 out of 15 patients (S305N, G57S, and W276C) had previously been reported in HSCR disease, and impaired protein function had been documented for two of them (16,2427) (Table 3). In addition, two of the three newly identified variants (R434C and D246F) (Table 3) were predicted to be damaging by the Polyphen software program (28) (http://tux.embl-heidelberg.de/ramensky/). The position of each variant is indicated on a three-dimensional model of the protein (Supplementary Fig. 1, available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue17).


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Table 3.  Characterization of EDNRB nonsynonymous variants*

 
Fourteen patients were heterozygous for a single variant, but one patient harbored three variants (S305N, V260F, and R434C). DNA analysis of the patient's daughter revealed that V260F and R434C were located on the same allele, suggesting that the patient was compound heterozygous for two functional EDNRB variants. Interestingly, she had an explosive melanoma of the scalp.

None of the patients or control subjects carrying EDNRB variants displayed any symptoms of HSCR or gross hypopigmentation disorders. However, the presence of nonsynonymous EDNRB variants was statistically associated with skin type I/II in melanoma patients (P = .007), although no effect on any other pigmentation characteristics could be detected. Multiple logistic regression analysis was performed on each skin type group separately. In the skin type I/II group, the association between EDNRB variants and melanoma risk remained statistically significant (OR = 19.9, 95% CI = 1.34 to 296.21; P = .030) (Table 4), suggesting that EDNRB variants may modulate MM risk independently of pigmentation characteristics and MC1R variants.


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Table 4.  Logistic regression analysis of all clinical and genetic risk factors for melanoma after stratification for skin type*

 
In addition to the observed differences in allele frequencies, the pattern of genetic diversity in the coding region of the EDNRB gene was characterized by a higher number of nonsynonymous variants in patients than in control subjects. After sequencing the EDNRB gene in three unrelated chimpanzees, we observed that the EDNRB coding sequence from chimpanzee differed from the consensus human sequence by only two synonymous substitutions, thus suggesting that EDNRB is being subjected to strong purifying selection. Population surveys have shown that nonsynonymous polymorphisms were skewed to lower frequencies than were synonymous polymorphisms (2931). This difference is considered to reflect the presence of a number of moderately deleterious alleles that are maintained at a low frequency by negative selection. If such variants predispose to a disease, one can expect that they will be found not only at a higher frequency but also in a higher number amongst patients expressing the disease than in a control group of similar size. This is precisely the situation observed here for EDNRB variants in MM patients.

One possible hypothesis for the association between loss of EDNRB function and risk of MM is that a defect in EDNRB signaling could prevent melanoblasts from achieving differentiation, thus increasing the risk of their transformation into a malignant melanoma. Second, individuals with EDNRB mutations may have had fewer melanocytes—even though this was not clinically apparent—which may make them constitutively less well protected against UV radiation. Similarly, mutant EDNRB may be less effective than the wild-type receptor in inducing an efficient response by mature melanocytes to UV radiation (via EDN1). However, the association of EDNRB variants with MM risk persisted after stratification for skin type, suggesting that the role of EDNRB variants may, at least in part, involve a pigmentation-independent mechanism.

Finally, EDNRB itself may have an important role in the pathogenesis of MM. EDNRB is widely expressed in melanocytes but downregulated in melanoma cells, possibly being related to malignancy or apoptosis (3234), suggesting that inactivating mutations of EDNRB could contribute to tumor progression.

The overall EDNRB mutational rate is reported to be 4.15% in familial and sporadic cases of HSCR disease (16,3548). However, the presence of the predisposing variant is neither necessary nor sufficient for the disease to be expressed, and genetic interactions between several loci have been demonstrated (4952).

A similar model may also apply to the genetic predisposition for melanoma, a disease with both familial and sporadic forms. Various combinations of hypomorphic alleles at different loci, including CDKN2A, EDNRB, and MC1R, may contribute to susceptibility to the disease. It will be interesting to find out whether interactions between EDNRB and other loci contribute to melanoma predisposition.


    NOTES
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Melan-Cohort is a prospective cohort of melanoma patients constituted by all the university-affiliated Dermatology Departments of in the Paris area.

VD, BC, LO, AA, CL, NB-S, and PS contributed equally to this work.

This work was supported by grants from contract grant sponsor L'Assistance Publique des Hôpitaux de Paris (AP-HP, number CRC00128) and from La Société Française de Dermatologie (SFD).


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Manuscript received January 25, 2005; revised May 24, 2005; accepted July 6, 2005.



             
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