Genome-wide association studies (GWAS) have identified~20 melanoma susceptibility loci, most of which are not functionally characterized. Here we report an approach integrating massively-parallel reporter assays (MPRA) with cell-type-specific epigenome and expression quantitative trait loci (eQTL) to identify susceptibility genes/variants from multiple GWAS loci. From 832 high-LD variants, we identify 39 candidate functional variants from 14 loci displaying allelic transcriptional activity, a subset of which corroborates four colocalizing melanocyte cis-eQTL genes. Among these, we further characterize the locus encompassing the HIV-1 restriction gene, MX2 (Chr21q22.3), and validate a functional intronic variant, rs398206. rs398206 mediates the binding of the transcription factor, YY1, to increase MX2 levels, consistent with the cis-eQTL of MX2 in primary human melanocytes. Melanocytespecific expression of human MX2 in a zebrafish model demonstrates accelerated melanoma formation in a BRAF V600E background. Our integrative approach streamlines GWAS followup studies and highlights a pleiotropic function of MX2 in melanoma susceptibility.

Background: Population age structure may confound the comparison of age at cancer diagnosis across racial/ethnic groups. We compared age at cancer diagnosis for U.S. Hispanics, a population that is younger on average, and non-Hispanic whites (NHW), before and after adjustment for the age structure of the source population. Methods: We used Surveillance, Epidemiology, and End Results data from 18 U.S. regions in 2015 for 34 cancer sites to calculate crude and adjusted (using age- and sex-specific weights) mean ages at diagnosis. Differences in age at diagnosis comparing Hispanics to NHWs (δ) were assessed using independent sample t tests. Results: Crude mean ages at diagnosis were lower among Hispanic males and females for all sites combined and for most cancer sites. After age-adjustment, Hispanic (vs. NHW) males remained younger on average at diagnosis of chronic myeloid leukemia [δ = −6.1; 95% confidence interval (CI), −8.1 to −4.1 years], testicular cancer (δ =−4.7; 95% CI, −5.4 to −4.0), Kaposi sarcoma (δ =−3.6; 95% CI,−6.3 to −0.8), mesothelioma (δ =−3.0; 95% CI,−4.3 to −1.7), and anal cancer (δ =−2.4; 95% CI, −3.9 to −0.8), and older at diagnosis of gallbladder cancer (δ = +3.8; 95% CI, 1.8 to 5.7) and Hodgkin's lymphoma (δ = +7.5; 95% CI, 5.7 to 9.4), and Hispanic (vs. NHW) females remained younger at diagnosis of mesothelioma (δ = −3.7; 95% CI, −6.7 to −0.7) and gallbladder cancer (δ = −3.0; 95% CI, −4.3 to −1.7) and older at diagnosis of skin cancer (δ = +3.8; 95% CI, 3.1 to 4.5), cervical cancer (δ = +4.1; 95% CI, 3.3 to 4.8), and Hodgkin's lymphoma (δ = +7.0; 95% CI, 5.0 to 9.1). Conclusions: On average, Hispanics are diagnosed with cancer at younger ages than NHWs; however, for many cancers these differences reflect the younger age structure in Hispanics. Impact: Population age structure should be considered when comparing age at cancer diagnosis across racial/ethnic groups.

Genome-wide association studies (GWAS) have identified ∼20 melanoma susceptibility loci. To identify susceptibility genes and variants simultaneously from multiple GWAS loci, we integrated massively-parallel reporter assays (MPRA) with cell type-specific epigenomic data as well as melanocyte-specific expression quantitative trait loci (eQTL) profiling. Starting from 16 melanoma loci, we selected 832 variants overlapping active regions of chromatin in cells of melanocytic lineage and identified 39 candidate functional variants displaying allelic transcriptional activity by MPRA. For four of these loci, we further identified four colocalizing melanocyte cis-eQTL genes (CTSS, CASP8, MX2, and MAFF) matching the allelic activity of MPRA functional variants. Among these, we further characterized the locus encompassing the HIV-1 restriction gene, MX2, on chromosome band Chr21q22.3 and validated a functional variant, rs398206, among multiple high LD variants. rs398206 mediates allelic transcriptional activity via binding of the transcription factor, YY1. This allelic transcriptional regulation is consistent with a significant cis-eQTL of MX2 in primary human melanocytes, where the melanoma risk-associated A allele of rs398206 is correlated with higher MX2 levels. Melanocyte-specific transgenic expression of human MX2 in a zebrafish model demonstrated accelerated melanoma formation in a BRAFV600E background. Thus, using an efficient scalable approach to streamline GWAS follow-up functional studies, we identified multiple candidate melanoma susceptibility genes and variants, and uncovered a pleiotropic function of MX2 in melanoma susceptibility.

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microRNAs (miRNA) are important mediators of post-transcriptional gene regulation and play critical roles in tumorigenesis. In melanoma, miRNAs are involved in proliferation, migration, invasion and drug resistance modulating RAS/MAPK and MITF pathways and others. Melanoma arises in melanocytes, and understanding heritable regulation of miRNAs in primary melanocytes could enhance our knowledge of genes and pathways contributing to melanoma risk. While a handful of studies in limited tissue types have identified miRNA transcripts with expression levels that are correlated with common genotypes using an expression quantitative loci approach (miQTL), expression levels of miRNAs are highly tissue-specific, further correlating with tissue-specific mRNA expression. Given the lack of cell-type specific miQTL data, we established a miQTL dataset using primary cultures of human melanocytes from 106 newborn males mainly of European decent. To assess comprehensive miRNA transcript levels from these samples, we performed RNA sequencing of the small RNA population (single-end, stranded, 51bp). An average of 63M reads per sample were obtained, and subsequently processed following miRMaster pipeline (ccb-compute.cs.uni-saarland.de/mirmaster) to detect known and novel miRNAs. A total of 961 known and 1262 novel miRNA transcripts were expressed above the background noise (> 0.05 Read Per Million and >5 reads per transcript in >=10 samples). Genotypes of 106 individuals were also assessed by direct genotyping using Illumina OmniExpress arrays followed by imputation using Michigan Imputation Server to produce a total of 10,687,550 genotypes. Subsequent QTL analyses using FastQTL (fastqtl.sourceforge.net) and QTLtools (qtltools.github.io/qtltools) identified 33 known and 34 novel miRNA transcripts as cis-miQTLs (q-value < 0.05) and 2 known and 6 novel miRNAs as trans-miQTLs (FDR < 0.1), respectively. We also observed a subset of significant cis-miQTLs overlapping with marginally significant mRNA trans-eQTLs from the same RNA samples, suggesting miRNA-mediated trans regulation. Further identification of melanocyte-specific miQTLs and their target genes will provide better understanding of melanocyte-specific miRNA regulation network. To identify candidate melanoma susceptibility genes functioning through heritable miRNA expression, melanocyte miQTLs will also be overlaid with melanoma genome-wide association data. In addition to the results from these analyses, further analyses of differentially expressed miRNAs and target genes between our primary melanocytes and melanoma tumors (available through The Cancer Genome Atlas) will shed light on the altered gene networks in melanomagenesis that might have therapeutic relevance through miRNA-mediated intervention. Citation Format: Jiyeon Choi, Tongwu Zhang, Michael Kovacs, Mai Xu, Andrew Vu, NISC Comparative Sequencing Program, Stacie Loftus, William Pavan, Kevin Brown. Understanding the roles of miRNAs in melanoma susceptibility through miQTL study using miRNA transcriptomes from 106 individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 491.