Genome-wide association study identifies a new melanoma susceptibility locus at 1q21.3.

We performed a genome-wide association study of melanoma in a discovery cohort of 2,168 Australian individuals with melanoma and 4,387 control individuals. In this discovery phase, we confirm several previously characterized melanoma-associated loci at MC1R, ASIP and MTAP-CDKN2A. We selected variants at nine loci for replication in three independent case-control studies (Europe: 2,804 subjects with melanoma, 7,618 control subjects; United States 1: 1,804 subjects with melanoma, 1,026 control subjects; United States 2: 585 subjects with melanoma, 6,500 control subjects). The combined meta-analysis of all case-control studies identified a new susceptibility locus at 1q21.3 (rs7412746, P = 9.0 × 10(-11), OR in combined replication cohorts of 0.89 (95% CI 0.85-0.95)). We also show evidence suggesting that melanoma associates with 1q42.12 (rs3219090, P = 9.3 × 10(-8)). The associated variants at the 1q21.3 locus span a region with ten genes, and plausible candidate genes for melanoma susceptibility include ARNT and SETDB1. Variants at the 1q21.3 locus do not seem to be associated with human pigmentation or measures of nevus density.

Introductory paragraph

We performed a genome-wide association study of melanoma in a discovery cohort of 2,168 Australian melanoma cases and 4,387 controls, confirming several previously characterised melanoma-associated loci and identifying two novel susceptibility loci on chromosome 1. The most significant genotyped SNPs in the novel loci were at 1q21.3 nearby several genes including ARNT and SETDB1 (rs7412746, P=2.5 × 10−7, OR=0.82) and at 1q42.12 in the DNA repair gene PARP1 (rs3219090, P=9.5 × 10−7, OR=0.82). Both new findings were replicated in three independent case-control studies (Europe: 2,804 cases, 7,618 controls; United States 1: 1,804 cases, 1,026 controls; United States 2: 585 cases, 6,500 controls). Estimates of the ORs in the combined replication cohorts were 0.89 for rs7412746 (P=1.5×10−5) and 0.91 for rs3219090 (P=3.4 × 10−3). Meta-analysis of all case-control studies combined showed genome-wide significance (P=9.0 × 10−11) for rs7412746 and suggestive significance (P=9.3 × 10−8) for rs3219090.

Main Text

To date, genome-wide association studies (GWAS) for melanoma[1,2], pigmentation[3] and nevogenesis[4,5] have identified a small number of low-penetrance melanoma susceptibility variants. These variants appear to exert their effect on melanoma risk through their role in the known melanoma-associated risk phenotypes of pigmentation and nevus count. In contrast to other cancers, these variants have shown relatively large effects on disease risk (odds ratios (OR) >1.5), with previous melanoma GWASs underpowered to detect variants of small effect. Here we describe a large melanoma GWAS with sufficient power to detect the small effects typically observed for other cancers (1.1 < OR <1.5).

Melanoma cases of European descent (n=2,168) were selected from the Queensland study of Melanoma: Environment and Genetic Associations (Q-MEGA)[6] and the Australian Melanoma Family Study[7] (AMFS). Three Australian samples of European descent were used as controls (n=4,387)[6-8]. Samples were genotyped on Illumina SNP arrays (Cases: Omni1-Quad or HumanHap610; Controls: Omni1-Quad or HumanHap610 or HumanHap670, Table 1). Cases and controls were combined into a single data set for quality control (including principal component analysis for outlier removal) and imputation (Supplementary Note). Imputation based on 1000 Genomes Project[9] data allowed association testing for 5,480,804 well imputed SNPs, which helped recover the full sample size for SNPs only typed on a subset of the arrays. After cleaning, the genomic inflation factor (λ) for those SNPs directly genotyped in all individuals in these discovery samples was 1.04 (Supplementary Fig. 1).

Table 1

Study Samples

Sample	Array	Cases	Controls
Discovery sample: Australia	Omni1-Quad	1,242	431
	610/670	926	3,956
Replication Sample 1: Europe -GenoMEL	610	2,804	7,618
Replication Sample 2: United States 1 - MDAnderson Cancer Center	Omni1-Quad	1,804	1,026
Replication Sample 3: United States 2 - Harvard	NA*	585	6,500

United States 2 samples were only typed for rs7412746 and rs3219090 using the OpenArray™ SNP Genotyping System

Results of tests of association for SNPs directly genotyped in all discovery samples are displayed in Figure 1 (a similar pattern was seen for imputed SNPs, data not shown). Three of the previously reported melanoma susceptibility loci (MC1R, ASIP, MTAP/CDKN2A)[1-4] reached genome-wide significance. Two additional regions were noteworthy at chromosome 1q42.12 and 1q21.3; for both loci there was at least one SNP directly genotyped in all discovery samples with P < 1 × 10−6 (Table 2, Supplementary Table 1, Supplementary Fig. 2A and 2B) as well as at least one imputed SNP with P < 5 × 10−7 (Fig. 2A and 2B, Supplementary Fig. 3A and 3B).

Figure 1

Association results for SNPs directly genotyped in all Australian samples. SNPs with P-values exceeding genome-wide significance (P < 5 × 10−8) are shown in black, while SNPs with 5 × 10−8 < P < 1 × 10−6 are shown in blue. The y-axis is truncated at 1 × 10−9, however, some SNPs from previously identified loci exceed this threshold (specifically at ~88 Mb on chromosome 16 near MC1R and at the ASIP locus 33 Mb on chromosome 20. The genome-wide significant signal on chromosome 9 is in the vicinity of the MTAP/CDKN2A region.

Table 2

Results for nine loci selected from the discovery sample

			Australia		UnitedStates 1		Europe		CombinedReplicationsamples*		Discoveryplusreplication
SNP	Chr	Coordinate	OR	P	OR	P	OR	P	OR	P	OR	P
rs7412746	1	149,127,095	0.82	2.5 ×10−7	0.85	2.7×10−3	0.92	0.014	0.90	2.6×10−5	0.87	9.0×10−11
rs3219090	1	224,631,314	0.82	9.5 ×10−7	0.88	0.028	0.91	0.048	0.90	3.5×10−3	0.87	9.3×10−8
rs10170188	2	205,757,059	1.19	3.3 ×10−5	0.99	0.86	0.99	0.74	0.99	0.70
rs17065828	3	62,017,865	0.83	3.9 ×10−5	1.00	1.00	1.00	0.95	0.99	0.96
rs13177645	5	115,031,773	0.83	2.1 ×10−5	1.01	0.80	0.96	0.50	0.99	0.76
rs7811987	7	136,176,803	1.19	1.1 ×10−5	1.01	0.82	1.00	0.95	1.00	0.87
rs6478444	9	121,721,401	1.19	5.6 ×10−6	0.90	0.067	1.01	0.92	0.96	0.41
rs10766295	11	16,061,966	1.17	2.1 ×10−5	1.02	0.70	1.03	0.32	1.03	0.30
rs1584186	11	25,137,541	1.21	4.4 ×10−5	1.04	0.54	0.96	0.37	0.98	0.65

Results for Europe plus United States 1 samples only. The results for the two chromosome 1 SNPs in all three replication samples (Europe, United States 1, United States 2) are given in the text

Figure 2

Discovery sample association results at two novel melanoma susceptibility loci on chromosome 1 for both SNPs directly genotyped in all Australian samples and imputed SNPs. Genotyped SNPs are indicated by solid triangles and imputed SNPs by hollow circles. The top ranked SNP at each locus is shown as a solid purple diamond (this SNP is an imputed SNP at both loci). Imputation p-values for all SNPs are plotted. Note imputed and genotyped p-values for genotyped SNPs differ slightly because for the imputed result, analysis is based on dosage scores whereas with genotyped SNPs hard genotype calls are used. Association results shown are for (A) the chromosome 1 locus near 149 Mb, and (B) SNPs in the vicinity of the PARP1 association signal. The color scheme indicates linkage disequilibrium between the most strongly-associated SNPs for the 149 Mb and PARP1 region (shown in purple, rs267735 and rs2695238, respectively) and other genotyped SNPs in the two regions.

For replication, we selected nine novel genomic regions and evaluated them in silico from array data in two additional case-control studies from Europe[1] and the United States[10] (Table 1). In each of the nine regions, we selected the most strongly associated SNPs present on both the Omni1-Quad and HumanHap610 arrays (since such SNPs were directly genotyped in all our samples, as well as in both sets of replication samples). We further limited follow-up region choice to those with at least two SNPs with P < 10−4 (i.e. there must be a supporting SNP in addition to the primary SNP). Both chromosome 1 regions show significant associations in the replication samples whilst the other seven regions did not (Table 2, Supplementary Fig. 4 and 5). We sought further replication of the two chromosome 1 regions in an additional set of cases and controls from the United States (Table 1, Supplementary Table 2); rs7412746 clearly replicated (OR=0.86, P=0.0076, one-sided; meta-analysis of all three replication cohorts OR=0.89, P=1.5 × 10−5), with rs3219090 showing a trend toward significance in the expected direction (OR=0.95, P=0.20, one-sided; meta-analysis of all three replication cohorts OR=0.91, P = 3.4 × 10−3). Based on the ORs seen in the replication cohorts, rs7412746 and rs3219090 each explain 0.1% of the genetic variance in melanoma risk. The meta-analysis P-values for all case-control studies combined were P=9.0 × 10−11 (genome-wide significant) for rs7412746 and P=9.3 × 10−8 (suggestively significant) for rs3219090.

We tested for association of rs7412746 and rs3219090 with pigmentation and nevus phenotypes, available on a subset of our discovery sample (up to 1,146 cases and 1,080 controls, Supplementary Note). SNP rs7412746 showed nominally significant association with blue versus non-blue eye colour (P=0.02), fair versus non-fair hair colour (P=0.01) and dark brown versus non-dark brown hair colour (P=0.02), as well as borderline association with nevus count (P=0.06). The direction of effect of rs7412746 on blue eye colour, fair hair colour, dark hair colour and nevus count was the same in the case and control subsets of our discovery sample. No association was seen between rs7412746 and skin colour or freckling. SNP rs3219090 was not associated with any pigmentation or nevus traits. Adjusting for pigmentation or nevus traits did not appreciably change the association of either locus with melanoma (rs7412746 OR before correction 0.82, after correction 0.84, P=0.33 for difference; rs3219090 OR before correction 0.82, after correction 0.83, P=0.61 for difference).

We also tested for differences in the strength of the associations of rs7412746 and rs3219090 with melanoma in early versus late onset (=<40 compared with >40 years at age of onset) and in situ versus invasive (79% of cases were invasive) subsets of the Australian cases. We found no differences in the association OR for these subsets. For early onset versus controls rs7412746 yielded OR=0.83, 95% CI 0.75,0.91 (P=0.79 for difference in frequency between early and late) and rs3219090 yielded OR=0.81, 95% CI 0.73,0.90 (P=0.63 for difference in frequency between early and late). For invasive versus controls rs7412746 yielded OR=0.80, 95% CI 0.73,0.88 (P=0.60 for difference in frequency between invasive and in situ) and rs3219090 yielded OR=0.84, 95% CI 0.76,0.93 (P=0.38 for difference in frequency between invasive and in situ).

The ratio of males to females was similar in cases and two of the control samples but the third control sample was all-female (samples from an endometriosis study[8]). We repeated our analysis without the all-female sample set and the results were similar; rs7412746 OR=0.82 in full data set, OR=0.84 with the all-female control set removed (P=0.42 for difference in frequency between endometriosis control set and remaining controls); rs3219090 OR=0.82 in full data set, OR=0.82 with the all-female control set removed (P=0.96 for difference in frequency between endometriosis control set and remaining controls). In the full Australian sample, there were no differences in the strength of association in only male or only female cases and controls; rs7412746 OR=0.82 and rs3219090 OR=0.81 in male only samples; rs7412746 OR=0.84 and rs3219090 OR=0.81 in female only samples (P=0.83, P=0.90 for OR difference between sexes for rs7412746 and rs3219090, respectively).

The associated region at 149 Mb on chromosome 1 spans approximately 450 Kb and harbours ten genes. The peak imputed SNP at this locus in the Australian case control sample, rs267735 (P=5.5 × 10−8) maps 1 Kb upstream of the transcription start site (TSS) of LASS2 (genome build 36 position 149,215,120), although there is substantial linkage disequilibrium (LD) that spans several genes in the region. All but one (ANXA9) of these genes are expressed in normal cultured human melanocytes, and most are also expressed across the vast majority of melanoma cell lines examined[11]. Several of the genes in the region have been implicated in cancer or cancer-related processes, including MCL1 (anti-apoptotic protein), ARNT (hypoxia-inducible factor 1 beta), and LASS2 (ceramide synthase 2). The SNP rs7412746 significantly influences the expression (i.e. is an expression quantitative trait loci or eQTL) of several genes in the region including CTSK (Chicago EQTL browser). Perhaps the strongest candidate in the region is SETDB1; a recent study in zebrafish has shown a role for variation in this gene in melanoma development[12]. Further study will be required to determine which gene or genes at this locus mediate melanoma risk.

In contrast to the 149 Mb region, the associated region at 224 Mb spans only 70 Kb and encompasses a single gene in its entirety (45 Kb), poly (ADP-ribose) polymerase 1 (PARP1). The peak imputed SNP is rs2695238 (P=3.8 × 10−7 in the Australian case-control sample, genome build 36 position 224,671,142) and lies ~9 Kb upstream of the TSS of PARP1 with several highly correlated SNPs lying within the gene. PARP1 encodes a chromatin-associated enzyme which modifies various nuclear proteins by poly-ADP-ribosylation. PARP1 plays a key role in multiple cellular processes such as differentiation, proliferation, and tumor transformation and plays a key role in the repair of single-strand DNA breaks. Interestingly, a recent candidate gene study[13] reported a nominally significant association between the intronic PARP1 SNP rs3219125 and melanoma in a set of 585 melanoma cases and 585 controls (OR 1.89, 95% CI 1.34–2.68), with stronger effect in patients with melanoma of the head and neck. SNPs rs3219090 and rs3219125 are not highly correlated in 1000 Genomes CEU samples (r2=0.042). SNP rs3219125 was not genotyped in our Australian discovery cohort but was well imputed (imputation r2=0.70) and showed marginal evidence for association (P=0.053). While no strongly-associated imputed or genotyped SNPs within the PARP1 locus alter the protein-coding sequence of the gene, two SNPs directly adjacent to each other and located within a nuclear factor 1 (NF1) transcription factor binding site were strongly associated (rs3754376: imputed P=7.39 × 10−7, OR=1.22; rs3754375: imputed P=3.0 × 10−3, OR=1.16). Both SNPs are in complete LD with each other and rs2695238 (pairwise D’=1 for all 3 pairs, pairwise r2 in the range 0.39 to 0.83). Further study will be required to assess whether these or other variants within this region directly mediate melanoma risk.

In our Australian discovery cohort, there remains an excess of positive results in the Q-Q plot after the removal of SNPs located within previously identified melanoma susceptibility regions (Supplementary Fig. 1). A small proportion of this excess was explained by the two novel chromosome 1 regions described here. Work examining the distribution of effect sizes obtained from GWAS suggests that for a wide range of traits, there are many more loci that will be found by conducting GWAS on larger samples[14]. Our data are consistent with there being further common SNPs influencing melanoma risk and we expect that further studies of additional melanoma samples will allow us to identify and characterize further loci.

In summary, our GWAS of melanoma identified two novel melanoma risk loci on chromosome 1 and replicated findings from previous melanoma GWASs. The observed effect size for the two novel loci was smaller than that observed for previously reported loci. Neither appears to be strongly correlated with human pigmentation or measures of nevus density, suggesting they may influence melanoma risk through distinct mechanisms. Identification of the causal variants at these loci will help refine estimates of risk for this increasingly common cancer.