Five endometrial cancer risk loci identified through genome-wide association analysis

Abstract

We conducted a meta-analysis of three endometrial cancer genome-wide association studies (GWAS) and two follow-up phases totaling 7,737 endometrial cancer cases and 37,144 controls of European ancestry. Genome-wide imputation and meta-analysis identified five new risk loci of genome-wide significance at likely regulatory regions on chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). We also found a second independent 8q24.21 signal (rs17232730). Functional studies of the 13q22.1 locus showed that rs9600103 (pairwise r(2) = 0.98 with rs11841589) is located in a region of active chromatin that interacts with the KLF5 promoter region. The rs9600103[T] allele that is protective in endometrial cancer suppressed gene expression in vitro, suggesting that regulation of the expression of KLF5, a gene linked to uterine development, is implicated in tumorigenesis. These findings provide enhanced insight into the genetic and biological basis of endometrial cancer.

Figure 1. Endometrial cancer meta-analysis Manhattan plot

Manhattan plot of −log₁₀-transformed P-values from meta-analysis of 22 autosomes. There are seven loci surpassing genome-wide significance including two known loci: 15q21 (CYP19A1) and 17q12 (HNF1B) and five novel loci: 6q22 (NCOA7, HEY2), 8q24 (MYC), 13q22 (KLF5), 14q32 (AKT1, SIVA1), 15q15 (EIF2AK4, BMF).

Figure 2. Forest plots of novel endometrial cancer risk loci

The odds ratio and 95% confidence intervals of each study of the meta-analysis are listed and shown in the adjacent plot. The I² heterogeneity scores (all <0.4) suggest that there is no marked difference in effects between studies. The SNPs represented are: a) rs11841589 (13q22), b) rs13328298 (6q22), c) rs4733613 (8q24), d) rs17232730 (8q24, pairwise r² 0.02 with rs4733613), e) rs937213 (15q15) and f) rs2498796 (14q32).

Figure 3. Regional association plots for the five novel loci associated with endometrial cancer.

The −log₁₀ P-values from the meta-analysis and regional imputation for three GWAS and eight iCOGS groups are shown for SNPs at: a) 13q22.1, b) 6q22, c) & d) 8q24, e) 15q15 and f) 14q32.33. The SNP with the lowest P-value at each locus is labeled and marked as a purple diamond, and the dot color represents the LD with the top SNP. The blue line shows recombination rates in cM/Mb. All plotted SNPs are either genotyped or have an IMPUTE info score of more than 0.9 in all datasets. Although genome-wide significant results for the 14q32.33 locus rely on imputed data, it should be noted that there is strong support from nearby genotyped markers. Supplementary Figure 6 displays similar regional association plots with a larger number of SNPs using a less stringent info score cut-off.

Figure 4. The 13q22.1 endometrial cancer susceptibility locus

a) Diagram showing the 16kb region around rs11841589, rs9600103 and correlated SNPs rs7981863, rs7988505 and rs7989799 (black marks), DNaseI hypersensitivity site (DHS) density signal in estrogen- and tamoxifen-treated ENCODE Ishikawa cells (Supplementary Note), and 100 vertebrates conservation. Vertical dotted line represents the position of rs9600103. FAIRE and ChIP assays for H3K4Me2 and H4Ac in endometrial cancer cell lines ARK-2 (rs9600103-TT), Ishikawa (rs9600103-AA) and AN3CA (rs9600103-AA) show evidence for enrichment of histone modifications. b) 3C experiment for KLF5-expressing Ishikawa cells. Relative interaction frequencies between an NcoI restriction fragment containing risk SNPs rs9600103 and rs11841589 (bait fragment) and NcoI fragments across the KLF5 promoter region, plotted against fragment position on chromosome 13. NcoI restriction sites are displayed below the schematic of KLF5 transcripts. H3K4Me3 binding, indicative of promoters, from multiple ENCODE cell lines are also shown.. The graph represents three biological replicates. Error bars represent standard deviation. A significant interaction was seen with the fragment containing a KLF5 transcriptional start site (fragment shaded in grey). c) Luciferase reporter assays to analyze the activity of 3kb fragments containing either rs9600103 or rs11841589 using the pGL3-Promoter vector in Ishikawa cells. Green arrows represent the low-risk alleles, and red arrows the high-risk alleles. Error bars represent the standard error of the mean (n=3). Luciferase activity for the rs9600103-A risk allele was more than double that of the rs9600103-T protective allele (P=0.018). There was no significant difference in luciferase activity between the rs11841589 alleles (Supplementary Table 7).