This paper attempts to address two topics. The first is to confirm and quantify the overall contribution of currently recognized pigmentation loci to variation in risk of cutaneous melanoma. Secondly, we expand the evidence of the recently recognized association between SNPs near ASIP
and CMM, and attempt to reconcile results from different studies (Gudbjartsson et al. 2008
; Brown et al., 2008
We have replicated association of SNPs at our chosen loci MC1R, OCA2, ASIP, SLC45A2, SLC24A4, TYR, TYRP1, and IRF4 with skin, hair and eye color, and shown that most of these SNPs are also significant predictors of CMM risk in our population. Because some SNPs are not genotyped in all subjects, the dataset suitable for multivariate analysis is reduced, so the confidence intervals around simultaneously adjusted effect sizes are broader than for (most of) the univariate analyses. The pattern is broadly consistent with multiplicative epistasis., with only weak evidence for non-multiplicative interactions The combination of SNPs in the five associated loci (MC1R, SLC45A2, OCA2, TYR, ASIP) explains roughly one-third to one-half of the variation in risk due to observed pigmentation phenotype. Pigmentation phenotype itself explains approximately one-third of the familial aggregation of melanoma. By contrast, the high penetrance CDKN2A mutations segregating in families with multiple cases of melanoma explain only 0.1% of recurrence risk in the total population. This is because they are extremely rare, involved in 0.2% of all Queensland melanoma cases (Aitken et al, 1999), with an allele frequency ~5×10−6. Attributable risk is another measure of the importance of a risk locus, but is not completely satisfactory in that genotypes that are rare in our population may offer the lowest absolute reduction in risk (for example, the rare protective allele in SLC45A2).
This is only the third study reporting an association between SLC45A2
and CMM. Fernandez and coworkers (Fernandez et al., 2008
) genotyped the coding polymorphism rs16891982 (F374L) in a collection of Spanish CMM cases and controls, while Guedj et al. (2008)
report results from a French case-control study. In keeping with our findings, the minor (L374
) allele was protective (7% frequency in Spanish cases and 16% frequency in Spanish controls; 4% and 10% in the French sample). This protective allele is associated with olive and dark skin, and so is least common in northern European populations (1.7% in the Utah-derived CEU HapMap sample), more common in southern Europeans, but is fixed in African and most Asian populations (Yuasa et al., 2006
). In our Australian sample, examination of the 3 SNP haplotypes, as well as logistic regression analysis showed that rs16891982 is the SNP most strongly associated with melanoma risk in our population.
The variant in the tyrosinase gene most strongly associated with CMM risk by Gudbjartsson and coworkers (2008)
was rs1126809 (R402Q
). We found significant association with rs1126809, and any association to the intronic SNP rs10765198 disappeared in multivariate models where rs1126809 was included as a covariate. The Q402
variant form of tyrosinase has 25% of the catalytic activity of the R402
form at 37°C (Tripathi et al., 1991
), and causes autosomal recessive ocular albinism when co-occurring with more penetrant OCA mutations in compound heterozygote form (Fukai et al., 1995
; Hutton et al., 2008
). It is therefore a strong candidate for the causative risk variant in this gene. Like Gudbjartsson and coworkers (2008)
, we did not detect any association between rs1042602 (TYR
*S192Y) with CMM in our Australian sample, even though those authors did detect an association with freckling in their Scandinavian sample.
In the case of OCA2
, we found that the SNP (rs12913832) most strongly associated with blue eye colour in our sample (Sturm et al., 2008
) was not associated with CMM, especially after adjusting for ancestry. Rather, the less common coding mutation R419Q
(rs1800407) was a significant risk factor, notably in the multivariate analysis adjusting for other pigmentation loci. This association has also been seen in a Spanish dataset (Fernandez et al., 2009
), with similar effect sizes. In the French case-control study of Jannot et al. (2005)
was not a significant risk factor for melanoma, but the nearby R305W
and neighbouring intronic SNPs were. The R305W
variants are in complete linkage disequilibrium in our population. The OCA2
variants screened by Gudbjartsson et al. (2008)
were not associated with CMM.
) variants we genotyped have been previously shown to be associated with eye and hair colour (Sulem et al. 2008
), and with CMM risk (Gudbjartsson et al., 2008
). We confirmed these associations and effect size estimates. These variants differ significantly in frequency between the main HapMap ethnic samples, and we could demonstrate a gradient with proportion Northern European ancestry paralleling hair and eye colour. We did not detect specific interactions between TYR
in their effects on melanoma risk or pigmentation phenotypes.
The two recent reports of association of SNPs in the region of ASIP
to CMM risk (Gudbjartsson et al., 2008
; Brown et al., 2008
) implicated widely separated SNPs. We observed that the association of CMM to the ASIP
region closely paralleled that with red hair color, and seems to be due largely to a single long (approximately 1.8 Mb) haplotype that straddles a number of haplotype block boundaries. This is likely to represent the effects of selection along with a relatively recent origin, as has been observed around other pigmentation loci (Barreiro et al., 2008
), given that this particular haplotype is also associated with lighter hair and skin color.
Given the role of Agouti signalling protein (ASIP
) as an antagonist of the melanocortin pathway, it seems biologically plausible that there would be an interaction between MC1R
genotype and ASIP
genotype. For red hair color, this was markedly so, with the rs4911442*C allele being roughly equivalent to an “r” MC1R haplotype. This is seen most easily seen comparing the penetrances of the joint “R/w; C/T” genotype to the “R/r; T/T” genotype, and in the linear dose-response relationship between number of rs4911442*C alleles on a “R/r” background (Table 8). In the same fashion, MC1R “R” haplotypes were dominant to rs4911442*C. Therefore ASIP
is a red hair color locus, in the same way as MC1R
. In keeping with this, rs4911442 genotype was a significant predictor of facial freckling (P=0.0001), as noted by others (Gudbjartsson et al., 2008
). A similar pattern was observed for CMM, but did not reach formal statistical significance. The association signal for red hair color and CMM across chromosome 20 did track quite strongly, so we expect that the epistatic pattern seen for these two loci in respect to hair color would also be demonstrable for CMM given a larger sample size.
An interesting negative finding in this study is the absence of an association between IRF4
genotype and melanoma risk, despite the very strong evidence for association with this locus and skin coloring in this and other studies (Han et al., 2008
; Sulem et al., 2008
). This was also noted by Gudbjartsson et al. (2008)
, and is in some ways the obverse of our previous observation (Palmer et al., 2000
) that some pigmentation loci such as MC1R seem to affect CMM risk by pathways other than that mediated by measurable changes in skin coloring or tanning ability. Here, we observe an effect on coloring which one would expect to significantly influence response of the organism to exposure to ultraviolet light, but appears neutral with respect to CMM risk. In the case of OCA2
, we and others have previously described how a single SNP rs12913832 strongly predicts eye color (Sturm et al., 2008
; Kayser et al., 2008
; Eiberg et al., 2008
), but has little or no effect on CMM risk. We do see an effect of another OCA2
polymorphism rs1800407 on CMM risk, but this is less impressive in terms of statistical significance.
A peculiarity of these analyses is that most of these pigmentation loci markedly differ in allele frequencies between different ethnic groups, in a way that parallels the differences in CMM rates between these groups. The simplest hypothesis, given that we have shown that all the selected variants are associated with pigmentation, is that these variants underlie ethnic variation in CMM risk. To definitively exclude ethnic confounding as a cause of the observed association between ancestry informative variants and disease risk, we would need either a cohort design, where we could show that the rate of CMM in carriers of all low risk variants is comparable to that seen in ethnic groups of known low “intrinsic” risk, or a family based design where we could also demonstrate linkage of these variants. Unfortunately, the age of onset of CMM in our sample and sampling design means that we cannot pursue the latter strategy, although we can demonstrate significant transmission-disequilibrium test results for pigmentation phenotypes such as skin colouring.
In conclusion, the variants in pigmentation genes we describe explain approximately 40% of the variation in familial risk of melanoma ascribable to phenotypes such as skin, hair and eye color. In the cases of TYR, SLC45A2, OCA2 and MC1R, it is likely that the major causative risk variants have been identified, but this is still open for ASIP, and other minor risk variants at all loci await characterization.