Identification and molecular characterization of a new ovarian cancer susceptibility locus at 17q21.31

Abstract

Epithelial ovarian cancer (EOC) has a heritable component that remains to be fully characterized. Most identified common susceptibility variants lie in non-protein-coding sequences. We hypothesized that variants in the 3' untranslated region at putative microRNA (miRNA)-binding sites represent functional targets that influence EOC susceptibility. Here, we evaluate the association between 767 miRNA-related single-nucleotide polymorphisms (miRSNPs) and EOC risk in 18,174 EOC cases and 26,134 controls from 43 studies genotyped through the Collaborative Oncological Gene-environment Study. We identify several miRSNPs associated with invasive serous EOC risk (odds ratio=1.12, P=10(-8)) mapping to an inversion polymorphism at 17q21.31. Additional genotyping of non-miRSNPs at 17q21.31 reveals stronger signals outside the inversion (P=10(-10)). Variation at 17q21.31 is associated with neurological diseases, and our collaboration is the first to report an association with EOC susceptibility. An integrated molecular analysis in this region provides evidence for ARHGAP27 and PLEKHM1 as candidate EOC susceptibility genes.

Figure 1. Regional association plot for genotyped and imputed SNPs at 17q21.31

The middle portion of the plot contains the region of the inversion polymorphism (ch 17: 43,624,578-44,525,051, hg build 37), with the four blue dots representing the candidate miRSNPs (rs4640231, rs1052587, and rs17574361) and the tagSNP, rs916793. rs1052587 in the 3′UTR of MAPT has the strongest signal (P=4.6×10⁻⁸) among the miRSNPs. The cluster on the left side of the plot (around 43.5 MB) contains highly correlated SNPs (r²=0.99), including three directly genotyped intronic SNPs, rs2077606 and rs17631303 in PLEKHM1 (P=3.9 × 10⁻¹⁰ and P=4.7 × 10⁻¹⁰, respectively), and rs12942666 in ARHGAP27 (P=1.0 × 10⁻⁹). The linkage disequilibrium between each plotted SNP and the top-ranked SNP in the region with the best clustering, rs12942666, is depicted by the color scheme; the deeper the color red, the stronger the correlation between the plotted SNP and rs12942666. The top miRSNP, rs1052587, is moderately correlated (r²=0.76) with rs2077606, rs17631303, and rs12942666 in our study population. (n=8,371 invasive serous cases and n= 23,491 controls, of European ancestry).

Figure 2. Expression and methylation analyses at the 17q21.31 ovarian cancer susceptibility locus

(a) Genomic map and LD structure. The location and approximate size of 17 known protein coding genes (grey) and one microRNA (blue) in the region are shown relative to the location of rs12942666. Orange indicates the location of the inversion polymorphism, and green indicates the region outside the inversion. (b) Gene expression (EOC and normal cell lines). Gene expression analysis in Epithelial Ovarian Cancer (EOC) cell lines (T; n=51) compared to normal ovarian surface epithelial cells (OSECs) and fallopian tube secretory epithelial cells (FTSEC) (N; n=73) (* p<0.05, **p<0.01, ***p<0.001). (c) Gene expression (Primary EOCs and Normal Tissue). Boxplots of The Cancer Genome Atlas (TCGA) Affymetrix U133A-array based gene expression in primary high-grade serous ovarian tumors (T; n=568) and normal fallopian tube tissues (N; n=8). Where data were not available in TCGA, gene expression data from the Gene Expression Omnibus series GSE18520 dataset containing 53 high-grade serous tumors and 10 normal ovarian tissues are shown (indicated by a red asterisk). (d) Methylation (Primary Tumors and Normal Tissue). Methylation analysis of 106 high-grade serous ovarian tumors compared to normal ovarian tissues (n=7). Methylation data were generated for CpG site(s) associated with each gene using the Illumina 450 methylation array. Pairwise analysis of methylation for an individual CpG for each gene is based on the CpG with most significant inverse relationship to gene expression (i.e. cis negative), for a subset of 43 tumors having available gene expression data. Statistically significant cis-negative probes are indicated by a red open circle. (e) Expression quantitative trait locus (eQTL) analysis (OSECs/FTSECs). eQTL analysis comparing expression for each gene to genotype for the most statistically significant SNP at 17q21.31 (rs12942666), for 73 normal OSEC/FTSEC lines. Data are presented as box plots comparing expression levels in cases carrying rare homozygotes/heterozygotes, with cases homozygous for the common allele. (f) Expression quantitative trait locus (eQTL) analysis (Primary EOCs). eQTL analysis comparing expression for each gene to genotype using level 3 gene expression profiling data from Agilent 244K custom arrays and level 2 genotype data from the Illumina 1M-Duo BeadChip for 568 high-grade serous ovarian cancer patients from TCGA. In all panels * p<0.05, **p<0.01, *** p<0.001. Grey X's indicate data not available. Here, genotype data for rs2077606 is used (rather than rs12942666) because rs12942666 was not genotyped in the TCGA dataset. (g) Methylation quantitative trait locus (mQTL) analysis (Primary EOCs). mQTL analysis showing methylation status in 227 high-grade serous EOCs relative to rs12942666 genotype.

Figure 3. eQTL associations between the rs2077606 susceptibility SNP at 17q21

(a) Analysis of the chromatin landscape at ARHGAP27 and PLEKHM1 in normal ovarian surface epithelial and fallopian tube secretory epithelial cells (OSECs/FTSECs) by formaldehyde assisted isolation of regulatory elements sequencing (FAIRE-seq). Alignment with ENCODE FAIRE-seq tracks (shown) and ChIP-seq tracks (not shown) from non-EOC related cell lines reveals open chromatin peaks corresponding to (a) promoters (b) CTCF insulator binding sites and (c) H3K4me3 signals, suggestive of a dynamic regulatory region. An H3K4me3 signal at a coding ARHGAP27 mRNA variant (c) located between the genes is highly pronounced in OSEC/FTSEC, suggesting tissue-specific expression and function. Several of the top-ranking SNPs fall within transcription factor binding sites (TFFS) (Supplementary Table S2). rs12942666 did not coincide with TFBS, but tightly linked SNPs, rs12946900 and rs2077606 fell within predicted binding sites for SPIB and ZEB1, respectively. (b) We analyzed the expression of SPIB and ZEB1 in primary high-grade serous tumors from TCGA and found (i) no significant change in SPIB expression but (ii) significant down-regulation of ZEB1 in tumors compared to normal tissues. (iii) QPCR analysis of ZEB1 expression in 73 OCPT and 50 EOC cell lines replicated the finding that ZEB1 expression is lower in cancer cell lines compared to normal precursor tissues. (c) eQTL analysis in OSECs/FTSECs for different alleles of rs2077606. There was a (i) significant eQTL for ARHGAP27, with the minor (A) allele being associated with increased ARHGAP27 expression (P=0.034), (ii) no evidence of an association between rs2077606 genotypes and ARHGAP27 expression in lymphoblastoid cell lines suggesting this association may be tissue-specific. (iii) We observed a borderline significant eQTL association between ZEB1 mRNA and rs2077606 in tumors from TCGA, with the minor risk allele also associated with lower expression.