Iverson, Edwin S.

Abstract Background: Genome-wide association studies (GWAS) have identified multiple loci associated with epithelial ovarian cancer (EOC) susceptibility, but further progress requires integration of epidemiology and biology to illuminate true risk loci below genome-wide significance levels (P < 5 × 10−8). Most risk SNPs lie within non–protein-encoding regions, and we hypothesize that long noncoding RNA (lncRNA) genes are enriched at EOC risk regions and represent biologically relevant functional targets. Methods: Using imputed GWAS data from about 18,000 invasive EOC cases and 34,000 controls of European ancestry, the GENCODE (v19) lncRNA database was used to annotate SNPs from 13,442 lncRNAs for permutation-based enrichment analysis. Tumor expression quantitative trait locus (eQTL) analysis was performed for sub-genome-wide regions (1 × 10−5 > P > 5 × 10−8) overlapping lncRNAs. Results: Of 5,294 EOC-associated SNPs (P < 1.0 × 10−5), 1,464 (28%) mapped within 53 unique lncRNAs and an additional 3,484 (66%) SNPs were correlated (r2 > 0.2) with SNPs within 115 lncRNAs. EOC-associated SNPs comprised 130 independent regions, of which 72 (55%) overlapped with lncRNAs, representing a significant enrichment (P = 5.0 × 10−4) that was more pronounced among a subset of 5,401 lncRNAs with active epigenetic regulation in normal ovarian tissue. EOC-associated lncRNAs and their putative promoters and transcription factors were enriched for biologically relevant pathways and eQTL analysis identified five novel putative risk regions with allele-specific effects on lncRNA gene expression. Conclusions: lncRNAs are significantly enriched at EOC risk regions, suggesting a mechanistic role for lncRNAs in driving predisposition to EOC. Impact: lncRNAs represent key candidates for integrative epidemiologic and functional studies. Further research on their biologic role in ovarian cancer is indicated. Cancer Epidemiol Biomarkers Prev; 26(1); 116–25. ©2016 AACR.

Abstract Background: Genome wide association studies (GWAS) have identified 20 loci associated with epithelial ovarian cancer (EOC) risk. Additional EOC risk loci await identification, and biological approaches may represent a useful strategy for overcoming sample size limitations. The ENCODE project recently concluded that only ∼1.2% of the genome encodes proteins, but at least 20% exhibits biological function and over 80% exhibits biochemical indices of function. Post-GWAS follow-up studies have firmly established that some associations are due to inter-individual differences in non-coding RNAs (ncRNAs). To estimate the magnitude of impact of variants in long non-coding RNAs (lncRNAs), we assessed enrichment of EOC-associated SNPs in lncRNA regions by comparing the density of EOC-associated loci between lncRNA regions, non-lncRNA regions, and the whole genome. Methods: The study population included ∼18,000 invasive EOC cases and 34,000 healthy controls of European ancestry from the Ovarian Cancer Association Consortium with GWAS data imputed to 1000 Genomes Project (1KGP) density. Density was compared with two measures: average chromosome length required for one EOC-associated SNP (kb/locus) and average number of tested SNPs containing one EOC-associated SNP (SNPs/locus). Coordinates and annotation for 13,870 lncRNA genes were downloaded from the publicly available GENCODE (v19) database and were used to annotate SNPs from the 1KGP imputed GWAS data, yielding 13,192 lncRNA genes with biallelic variants imputed at rsquare ≥0.25. A SNP was considered EOC-associated if it reached a significance level of P<10−5. Results: Genome-wide, 11.6% of the 15,123,646 tested SNPs fell within lncRNA regions. Of 5,294 EOC-associated SNPs that were identified, 27.7% were located within lncRNAs, a higher frequency than expected (<v:shape style = “WIDTH: 16.5pt; HEIGHT: 18pt” id = _x0000_i1026 equationxml = ‘14pχ2′ type = “#_x0000_t75”> = 9.35×10−22). Additionally, EOC-associated SNPs were located on average every 237.6 kb (or 1204 tested SNPs) in lncRNA regions versus every 657.7 kb (or 3489 SNPS) in non-lncRNA regions and every 541.5 kb (or 2857 SNPs) in the whole genome, clearly demonstrating an enrichment of EOC-associated loci in lncRNA regions. A total of 1,464 lncRNA SNPs from 53 unique lncRNAs associated with EOC risk (P<10−5); 70 of the identified lncRNA SNPs had P-values between 10−20 and 10−32, and 221 had P-values between 10−10 and 10−15, representing 21 unique regions > 500kb apart. Conclusions: These results justify efforts to integrate functional and population level genetic data on lncRNA regions to explore a new paradigm that addresses the problem of the missing heritability for cancer where restrictions in sample size and statistical power limit the ability to discover additional risk loci. Citation Format: Thomas A. Sellers, Brett M. Reid, Y. Ann Chen, Hui-Yi Lin, Edward Richards, Jamie Teer, Alvaro Monteiro, Zhihua Chen, Andrew Berchuck, Georgia Chenevix-Trench, Jennifer Doherty, Ellen Goode, Edwin Iverson, Leigh Pearce, Paul Pharoah, Catherine Phelan, Susan Ramus, Mary Anne Rossing, Joellen Schildkraut, Jin Cheng, Simon Gayther, Jennifer Permuth-Wey, on behalf of the Ovarian Cancer Association Consortium. Evidence that long non-coding RNA variants associate with epithelial ovarian cancer risk. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4633. doi:10.1158/1538-7445.AM2015-4633

Abstract Mitochondria have been implicated in carcinogenesis because of their central role in apoptosis, free radical production, and cellular energy metabolism. Although studies have identified somatic mitochondrial DNA mutations in ovarian cancers, little attention has been directed to the possible role of germline mitochondrial variants in mitochondrial dysfunction and subsequent ovarian cancer development. Through an ongoing epithelial ovarian cancer genome-wide association study of 1,856 invasive cases (59% with serous carcinomas) and 1,912 controls (frequency-matched on age, race, and study site), we genotyped 138 SNPs tagging variation in the maternally-inherited mitochondrial genome (mtDNA) to evaluate the hypothesis that single nucleotide polymorphisms (SNPs) in mtDNA are associated with ovarian cancer risk. All subjects were non-Hispanic, non-Jewish Caucasians, and were genotyped with the Illumina 610-quad Beadchip. Samples and SNPs with call rates less than 95% were excluded. Subjects with ambiguous gender, unresolved identical genotypes, and less than 80% European ancestry were excluded. For each SNP, logistic regression was used to estimate odds ratios (OR) and corresponding 95% confidence intervals (CI) between carriers of the minor versus major maternally-inherited allele and case status. Subgroup analysis was conducted to estimate allele-specific risks between serous-only cases and controls. Haplotype analysis was performed for regions containing statistically significant SNPs (p<0.05). Two rare SNPs, C16329T in the displacement loop gene (MT-D-loop) (OR: 6.72, 95%CI: 1.5-29.8, p=0.004; minor allele frequency (MAF)=0.001) and C15905T in the tRNA threonine gene (MT-TT) (OR: 1.60, 95%CI: 1.1-2.3, p=0.009; MAF=0.027), were associated with increased ovarian cancer risk; whereas T6777C in the cytochrome c oxidase subunit 1 gene (MT-CO1) (OR: 0.68, 95%CI: 0.51-0.91, p=0.009; MAF=0.063) was associated with decreased risk. All three SNPs remained statistically significant after adjustment for multiple comparisons within the mitochondrial SNPs using permutation testing. C16329T and T6777C were also significantly associated among cases with serous histology, with ORs (95% CIs) of 6.13 (1.2-29.6) and 0.61 (0.43-0.87), respectively. Haplotype analysis for each of the three regions investigated revealed significant haplotype-disease associations; however, findings suggest that the respective associations were driven by the most significant SNP(s) listed above. This is the first large-scale epidemiologic study to provide evidence that SNPs in mitochondrial genes may be novel risk factors for ovarian cancer. Future investigation of additional mitochondrial variants (i.e. nuclear-encoded mitochondrial proteins) is necessary to more comprehensively investigate this association. Furthermore, replication of the most promising SNPs in a larger population is warranted to validate these findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2835.