Fine-mapping of the HNF1B multicancer locus identifies candidate variants that mediate endometrial cancer risk

Abstract

Common variants in the hepatocyte nuclear factor 1 homeobox B (HNF1B) gene are associated with the risk of Type II diabetes and multiple cancers. Evidence to date indicates that cancer risk may be mediated via genetic or epigenetic effects on HNF1B gene expression. We previously found single-nucleotide polymorphisms (SNPs) at the HNF1B locus to be associated with endometrial cancer, and now report extensive fine-mapping and in silico and laboratory analyses of this locus. Analysis of 1184 genotyped and imputed SNPs in 6608 Caucasian cases and 37 925 controls, and 895 Asian cases and 1968 controls, revealed the best signal of association for SNP rs11263763 (P = 8.4 × 10(-14), odds ratio = 0.86, 95% confidence interval = 0.82-0.89), located within HNF1B intron 1. Haplotype analysis and conditional analyses provide no evidence of further independent endometrial cancer risk variants at this locus. SNP rs11263763 genotype was associated with HNF1B mRNA expression but not with HNF1B methylation in endometrial tumor samples from The Cancer Genome Atlas. Genetic analyses prioritized rs11263763 and four other SNPs in high-to-moderate linkage disequilibrium as the most likely causal SNPs. Three of these SNPs map to the extended HNF1B promoter based on chromatin marks extending from the minimal promoter region. Reporter assays demonstrated that this extended region reduces activity in combination with the minimal HNF1B promoter, and that the minor alleles of rs11263763 or rs8064454 are associated with decreased HNF1B promoter activity. Our findings provide evidence for a single signal associated with endometrial cancer risk at the HNF1B locus, and that risk is likely mediated via altered HNF1B gene expression.

Figure 1.

Regional association and forest plots for the HNF1B locus associated with endometrial cancer: (A) Locuszoom (14) plot of the log₁₀ P-values for association between each SNP and endometrial cancer for the meta-analysis of the iCOGS fine-mapping dataset, and ANECS, SEARCH and NSECG GWAS datasets. The color of each point indicates the extent of LD with the top SNP rs11263763 (purple). Gene positions are given under the graph, and estimated recombination rates in cM/Mb are indicated by the blue line (right-hand scale). Genotyped SNPs are plotted as circles, and imputed SNPs as squares (info score ≥ 0.7 for all plotted SNPs). The small peak of signal ∼13 kb to the right of rs11263763 does not survive conditional analysis. (B) Forest plot of ORs for the GWAS and iCOGS fine-mapping datasets stratified by study and country for top SNP rs11263763 (study acronyms detailed in Supplementary Material, Table S1).

Figure 2.

Association of genotypes with HNF1B expression as measured by RNA_Seq for rs11263763 (A) and rs11658063 (B), and with average HNF1B CpG island methylation for rs11263763 (C) and rs11658063 (D).

Figure 3.

Genetic associations and epigenetic landscape at the HNF1B locus. (A) Enlarged image of the HNF1B intron 1 region, showing the epigenetic landscape in ENCODE cell lines. The top five likely causal SNPs are indicated in relation to marks of regulatory potential; (B) Histones H3K4Me1 (indicative of regulatory regions) and H3K4Me3 (indicative of promoters); (C) DNaseI hypersensitivity (DHS: indicative of open chromatin, with darker shading indicating stronger experimental signal) in 125 (layered) ENCODE cell lines and endometrial cancer ECC1 (DMSO and estradiol 10 m) and Ishikawa (4-OHTAM and estradiol 10 m) cell lines; (D) Transcription factor (TF) binding in 72 ENCODE cell lines; (E) Chromatin state in nine ENCODE cell lines, with the following color coding: bright red-active promoter; light red-weak promoter; purple-inactive/poised promoter; orange-strong enhancer; yellow-weak enhancer; blue-insulator; dark green-transcriptional transition; light green-weak transcribed; dark gray-repressed/heterochromatin; (F) HNF1B CpG island. The solid red box represents the extended promoter region, and the hatched box the minimal promoter region.

Figure 4.

Luciferase reporter assays in endometrial cell lines demonstrate that SNPs rs11263763 and rs8064454 reduce the extended HNF1B promoter activity. The minimal HNF1B (Min prom) or extended HNF1B (Ext prom) promoters were cloned upstream of a luciferase reporter. An Ext prom construct containing either the wild-type haplotype or minor alleles of rs11263763, rs11651052 or rs8064454 were also generated. Cells were transiently transfected with each of these constructs and assayed for luciferase activity after 48 h. Error bars denote standard error of the mean (SEM) from three independent experiments. P-values were determined with a two-tailed t test (*P < 0.05, **P < 0.01, ***P < 0.001).