Background
Melanoma is the most aggressive skin tumor, and its incidence has been correlated with latitude of residence, occurring most frequently in fair-skinned individuals [
1]. In fact, the risk of developing melanoma diverges markedly according to skin pigmentation and geographical area, mainly due to the causative effect of ultraviolet radiation [
2,
3]. Currently, Australia and New Zealand have the highest incidence and mortality rates of melanoma in the world, with incidence reaching 33.6/100,000 and 33.3/100,000, respectively [
4]. In those two countries, the risk of developing melanoma before age 75 years is 1/24 and 1/34 for males and females, respectively [
5]. In Brazil, melanoma represents 4% of all skin cancers, and 6260 new diagnoses are estimated in 2018. The highest incidence rates per region are expected for southern Brazil, reaching 8.4/100.000 [
6]. These high rates are attributed to geographical location– the southernmost state of Brazil, Rio Grande do Sul, is within the same latitude as Australia (30.0346° South) [
7]. Social practices with intense and often unprotected sun exposure and a majority-European ancestry are associated with lighter pigmentation of the skin in these individuals [
7,
8].
Approximately 10% of melanomas are caused by germline mutations in cancer predisposition genes [
9]. These include genes predominantly associated with melanoma (such as
CDKN2A and
CDK4) but also genes related to multiple solid tumors including melanoma. Examples are, among others, the
BAP1 gene, the
PTEN gene related to Cowden’s syndrome and
XPD,
XPC and
XPA genes related to Xeroderma pigmentosum. Most identifiable heritable mutations associated with hereditary melanoma have variable penetrance [
10]. In addition to germline mutations, our understanding of the contribution of single nucleotide polymorphisms (SNP) proposed as risk modulators for melanoma is increasing [
11]. Several common SNPs, usually of low penetrance, are commonly investigated in polygenic risk models. These models can assess the joint effect of independent SNPs in genes with lower and intermediate penetrance such as
MC1R,
ARNT,
CDK10, identified by the GWAS, and can assist in the identification of individuals with a higher risk of susceptibility to melanoma [
12]. Some of these SNPs are located in genes of the melanogenic pathway, and some have been described in association with melanoma in different populations across the world [
13]. A complicating aspect of such studies is that, in multifactorial disorders such as melanoma, genetic and nongenetic factors, such as admixture, population substructure, and evolution patterns, can severely confound the results and result in false-positive associations [
14]. Thus, differences in allele frequency between cases and controls could be associated with differences in ancestry rather than reflect an association of genes with disease [
15].
The Consortium for Analysis of Diversity and Evolution in Latin America (CANDELA) is a multidisciplinary international study that involves researchers focused on studying the biological diversity of Latin Americans, analyzing samples from Mexico, Colombia, Peru, Chile, and Brazil for a wide range of issues relevant to anthropological, biological and medical research in these populations. In 2014, an analysis to evaluate a possible association between 18 SNPs in genes involved in the pigmentation pathway and Melanin Index (MI) was performed within the Brazilian cohort of Consortium for the Analysis of the Diversity and Evolution of Latin America (CANDELA) with participants born in Rio Grande do Sul (RS) and Bahia (BA), in the South and Northeast of Brazil, respectively. As a result of this analysis, four SNPs were associated with differences in MI in these populations: rs1126809 (p.Arg402Gln) on tyrosinase (
TYR), rs1129038 (3’UTR) in the hect domain and rcc1-like domain (
HERC2), rs1426654 (p.Thr111Ala) in solute support family 24, member 4 (
SLC24A5) and rs16891982 (p.Phe374Leu) in the family of solute, member 2 (
SLC45A2) carriers. Among these four SNPs, allele A of rs1426654 and allele G of rs16891982 were associated with less melanin content in the 352 participants of RS cohort (
P < .001) [
16].
In this study, we aimed to assess the association of these four SNPs with melanoma risk in southern Brazil, a region with important contribution of European ancestry and with the highest indices of melanoma in the country.
Discussion
Based on a study developed by CANDELA, we investigated 4 variants previously associated with skin pigmentation in southern Brazil in melanoma patients and unaffected controls from the same geographic region [
16]. A model of logistic regression including ancestry, melanoma risk factors and SNPs
HERC2 rs1129038 and
SLC24A5 rs1426654 in dominant models of inheritance showed significant associations with melanoma. The
SLC45A2 rs16891982CC SNP, in a log-additive model, was associated with a lower risk for developing the disease.
Pigmentation is a polygenic trait. and different variants have been associated with melanin levels in populations over the world [
20]. One of the four SNPs investigated rs1129038, occurs in the untranslated region of
HERC2, and three.
TYR rs1126809,
SLC24A5 rs1426654, and
SLC45A2 rs16891982, are present in genes involved in the synthesis of melanosomes, the vesicles where melanin production and deposition occurs. In fact, rs1426654 and rs16891982 polymorphisms are determinants of pigmentation in Europeans [
21], as well as in other populations [
20,
22]. Our findings corroborate the association of some genotypes with lighter pigmentation and predominant European ancestry. The association of European ancestry and fair skin, eyes and hair was previously demonstrated in a sample of 1594 individuals from the same geographic region of the present study [
23]. and the different ancestry profiles between darker and lighter individuals has also been previously reported [
24]. Although our sample is not representative of the tri-hybrid pattern seen in most Brazilians [
25], it reflects the massive colonization by Europeans in the specific region of the study [
16,
23]. Just as there are regional differences in the proportions of ancestral populations in Brazil, we also expect heterogeneity in the frequency of genetic variants of specific genes, especially those related to skin, eye and hair pigmentation. Although research with admixed populations can be useful for allele detection involved in susceptibility to common diseases, the population substructure is a potential bias and should be controlled [
26,
27]. In addition to a limitation in sampling (the sample was partially paired), the Hardy-Weinberg deviation found in our allelic frequencies for
SLC45A2 rs16891982 can also be explained by the occurrence of interethnic mix and population substructure. Previously identified in Europe and in other Brazilian populations [
28].
The nonsynonymous SNP
TYR rs1126809 (p.Arg402Gln) has been previously associated with light pigmentation of skin and is frequent in Caucasians. Its presence results in the reduction of activity of tyrosinase, a key enzyme in of the melanin production pathway, and some authors reported an increased risk of melanoma in carriers. Both in Europe and Australia [
29]. However, we did not observe a consistent association of this SNP with pigmentation nor with risk for melanoma in our series.
On other hand,
HERC2 rs1129038, which was previously associated with lighter eye pigmentation in European populations [
30,
31], showed a significantly association with fair skin. Eyes and hair in our sample. Forensic associations have described this SNP as a good predictor of blue eyes in Europeans [
30,
32] and Brazilians [
33]. and our findings reinforce these predictions.
Finally, SNPs
SLC24A5 rs1426654 and
SLC45A2 rs16891982 were associated with fair skin, eyes, and hair and with melanoma.
SLC24A5 rs1426654 (p.Thr111Ala) was first described in zebrafish as responsible for the golden phenotype due to a delay in melanin production during embryonic development [
34]. In melanocyte cultures, homozygous GG leads to an increase in
SLC24A5 gene transcripts and a consequent increase in tyrosinase activity and melanin production [
35]. The decrease of G allele frequency is gradual from Africa to Europe, indicating that a selection pressure in favor of the A allele acted on the determination of fair skin in places where the intensity of UV radiation is lower [
36,
37]. Evidence of natural selection makes this SNP a frequent component of ancestral and forensic informative panels [
38]. In our study, we confirmed the association of the AA genotype with fair skin and light eyes. and we identified allelic frequencies consistent with those observed in European populations [
3] and in previous studies of Brazilians from other regions [
39]. Likewise, SNP
SLC45A2 rs16891982 is also widely studied regarding its relationship with pigmentation in different populations.
SLC45A2 encodes the membrane-associated transporter protein carrier involved in melanin synthesis, and experimental studies in zebrafish. Mice and yeast have clearly demonstrated that the presence of the missense variant rs16891982 (p.Phe374Leu) results in decreased protein activity [
40]. This SNP is also considered an ancestry informative marker (AIM), since it is able to differentiate European populations due to G allele frequency. Which is similar to the rs1426654 A allele [
41]. These findings are aligned with the theory of vitamin D synthesis. Which proposes that light skin is a feature selected to compensate for the lower solar incidence in populations living far from the Equator [
42] and with increased ability of the skin to respond to ultra violet (UV) radiation [
43].
Alleles associated with lighter pigmentation were also associated with melanoma in our study, and this result remained significant after analysis with multivariate logistic regression adjusted for the risk factors ancestry, gender, age, eye, hair, and skin color, and number of Nevi. We considered this analysis essential to identify whether the variants studied could be considered independent risk factors for the occurrence of melanoma. Thus, SNPs
HERC2 rs1129038 and
SLC24A5 rs1426654 remained strongly associated with risk for the development of melanoma in a dominant model. The presence of homozygous genotypes of either SNP (AA for rs1426654 and AA for rs1129038) were associated with increased melanoma risk, while the
SLC45A2 rs16891982 C allele was associated with protection for melanoma as shown previously in a GWAS study in Greece composed of 284 patients and 284 controls (OR = 0.51. 95% CI 0.34–0.76;
P = 0.001) [
44]. On the other hand, an Australian sample with individuals of 100% Northern European ancestry (1.062 cases and 1.262 controls) showed the same allele associated with the risk to melanoma in logistic regression models including pigmentation features and ancestry, similar to the one presented here (OR = 2.04. 95% CI 1.27–3.40) [
45] The results remained unchanged after population substructure analysis. Furthermore, the independent effects of each of these SNP were also accessed by MDR analysis, and the analysis considering the entire sample showed a redundancy interaction between the same SNPs that displayed significance through logistic regression (
P = .031). The interaction illustrated in Fig.
1 shows that these three genes act redundantly to increase the risk of melanoma. The genotypic combinations
SLC24A5 rs1426654AA and
SLC45A2 rs16891982GG present a greater contribution in determining the risk for the disease, presenting a possible epistatic effect similar to that found between
SLC45A2 and
VDR [
46],
SLC45A2 and
OCA2. and
MC1R and
SLC24A5 [
45].
The two most important limitations of our study are sampling process (individuals showing mostly Euro descendant ancestry in the entire sample) and relatively limited sample size. However, despite these limitations, our results are in line with previous studies and demonstrate that SNPs in genes related to pigmentation confer an independent increase in the risk for developing melanoma. In determining complex human traits in general, common genetic variants tend to have small effect sizes individually, but together. They may reveal important information and contribute to the assessment of individual risk for complex diseases such as cancer [
47]. The development and evaluation of predictive models that combine environmental and genomic risk factors can help improve melanoma prevention and population screening by motivating risk reduction behaviors, especially in regions with high incidence rates. High UV radiation exposure and predominantly European ancestry [
48].
Additional studies should be performed to verify whether the same scenario occurs in other regions of Brazil and Latin America. Although an association between SLC24A5 rs1426654 and SLC45A2 rs16891982 and melanoma has been previously described in Europeans, to our knowledge, this is the first study that confirms this association in a South American high-risk population.
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