Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA.
Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea.
Am J Hum Genet. 2022 Dec 1;109(12):2210-2229. doi: 10.1016/j.ajhg.2022.11.006. Epub 2022 Nov 23.
The most recent genome-wide association study (GWAS) of cutaneous melanoma identified 54 risk-associated loci, but functional variants and their target genes for most have not been established. Here, we performed massively parallel reporter assays (MPRAs) by using malignant melanoma and normal melanocyte cells and further integrated multi-layer annotation to systematically prioritize functional variants and susceptibility genes from these GWAS loci. Of 1,992 risk-associated variants tested in MPRAs, we identified 285 from 42 loci (78% of the known loci) displaying significant allelic transcriptional activities in either cell type (FDR < 1%). We further characterized MPRA-significant variants by motif prediction, epigenomic annotation, and statistical/functional fine-mapping to create integrative variant scores, which prioritized one to six plausible candidate variants per locus for the 42 loci and nominated a single variant for 43% of these loci. Overlaying the MPRA-significant variants with genome-wide significant expression or methylation quantitative trait loci (eQTLs or meQTLs, respectively) from melanocytes or melanomas identified candidate susceptibility genes for 60% of variants (172 of 285 variants). CRISPRi of top-scoring variants validated their cis-regulatory effect on the eQTL target genes, MAFF (22q13.1) and GPRC5A (12p13.1). Finally, we identified 36 melanoma-specific and 45 melanocyte-specific MPRA-significant variants, a subset of which are linked to cell-type-specific target genes. Analyses of transcription factor availability in MPRA datasets and variant-transcription-factor interaction in eQTL datasets highlighted the roles of transcription factors in cell-type-specific variant functionality. In conclusion, MPRAs along with variant scoring effectively prioritized plausible candidates for most melanoma GWAS loci and highlighted cellular contexts where the susceptibility variants are functional.
最近的一项皮肤黑色素瘤全基因组关联研究（GWAS）确定了 54 个与风险相关的基因座，但大多数尚未确定其功能变体及其靶基因。在这里，我们使用恶性黑色素瘤和正常黑素细胞进行了大规模平行报告基因分析（MPRA），并进一步整合了多层注释，系统地优先考虑来自这些 GWAS 基因座的功能变体和易感基因。在 MPRA 中测试的 1992 个风险相关变体中，我们在两种细胞类型（FDR < 1%）中鉴定出来自 42 个基因座（已知基因座的 78%）的 285 个具有显著等位基因转录活性的变体。我们通过基序预测、表观基因组注释和统计/功能精细映射进一步对 MPRA 显著变体进行了特征描述，创建了综合变体评分，为 42 个基因座中的每个基因座确定了一个至六个合理的候选变体，并为其中 43%的基因座提名了一个单一变体。将 MPRA 显著变体与来自黑素细胞或黑色素瘤的全基因组显著表达或甲基化定量性状基因座（eQTL 或 meQTL）进行叠加，确定了 60%的变体（285 个变体中的 172 个）的候选易感基因。顶级评分变体的 CRISPRi 验证了它们对 eQTL 靶基因 MAFF（22q13.1）和 GPRC5A（12p13.1）的顺式调控作用。最后，我们鉴定了 36 个黑色素瘤特异性和 45 个黑素细胞特异性 MPRA 显著变体，其中一部分与细胞类型特异性靶基因相关。在 MPRA 数据集中转录因子可用性和 eQTL 数据集中变体-转录因子相互作用的分析强调了转录因子在细胞类型特异性变体功能中的作用。总之，MPRA 及其变体评分有效地优先考虑了大多数黑色素瘤 GWAS 基因座的合理候选者，并突出了易感变体在功能上的细胞环境。
