Discordant Haplotype Sequencing Identifies Functional Variants at the 2q33 Breast Cancer Risk Locus

Abstract

The findings from genome-wide association studies hold enormous potential for novel insight into disease mechanisms. A major challenge in the field is to map these low-risk association signals to their underlying functional sequence variants (FSV). Simple sequence study designs are insufficient, as the vast numbers of statistically comparable variants and a limited knowledge of noncoding regulatory elements complicate prioritization. Furthermore, large sample sizes are typically required for adequate power to identify the initial association signals. One important question is whether similar sample sizes need to be sequenced to identify the FSVs. Here, we present a proof-of-principle example of an extreme discordant design to map FSVs within the 2q33 low-risk breast cancer locus. Our approach employed DNA sequencing of a small number of discordant haplotypes to efficiently identify candidate FSVs. Our results were consistent with those from a 2,000-fold larger, traditional imputation-based fine-mapping study. To prioritize further, we used expression-quantitative trait locus analysis of RNA sequencing from breast tissues, gene regulation annotations from the ENCODE consortium, and functional assays for differential enhancer activities. Notably, we implicate three regulatory variants at 2q33 that target CASP8 (rs3769823, rs3769821 in CASP8, and rs10197246 in ALS2CR12) as functionally relevant. We conclude that nested discordant haplotype sequencing is a promising approach to aid mapping of low-risk association loci. The ability to include more efficient sequencing designs into mapping efforts presents an opportunity for the field to capitalize on the potential of association loci and accelerate translation of association signals to their underlying FSVs. Cancer Res; 76(7); 1916-25. ©2016 AACR.

Figure 1. DNA sequencing coverage and variants in the 1 Mb region

Eighteen genes reside in the region defined by chromosome 2 201,566,128 to 202,566,128 bp (hg19). Upper Panel: A graph of the number of variant positions identified in the DNA sequencing across the region. Lower Panel: A graph of the average depth of sequencing coverage in the 38 sequenced individuals.

Figure 2. Association and eQTL evidence and regulatory annotations for 17 candidate functional sequence variants

Seventeen cFSVs are illustrated (16 selected from DNA sequencing, plus rs10197246). These reside within 100 kb region at chromosome 2 202,110,000 – 202,210,000 bp (hg19). Two genes, CASP8 and ALS2CR12, reside in this genomic region. Upper Panel: A graph showing the association evidence (−log₁₀p) for each of the 17 cFSVs in the discordant haplotype design. Middle Panel: A graph showing eQTL evidence (−log₁₀p) based on RNA sequencing data in the local tissue panel for expression of three genes: CASP8 (red), CASP10 (blue) and CFLAR (green) in normal (circle symbol) and tumor (triangle symbol) breast tissues. Lower Panel: Five regulatory annotation tracks from UCSC Genome Browser using ENCODE data(22, 29). In order, from top to bottom: (1) DNAseIHS, DNAse I hypersensitivity clusters in 125 cell types; (2) Tfbs, Transcription Factor ChIP-seq (161 factors); (3) H3K4Me1, Layered H3K4Me1 histone modification marks (often found near Regulatory Elements) on 7 cell lines; (4) H3K4Me3, Layered H3K4Me3 histone modification marks (often found near Promoters) on 7 cell lines; (5) H3K27Ac, Layered H3K27Ac histone modification marks (often found near active regulatory elements) on 7 cell lines.

Figure 3. Expression levels of CASP8 in normal breast tissue

The three categories on the x-axis indicate genotype at rs10931936, rs3769818, rs700635, rs6714430, or rs6743068 (all 5 of these cFSV are identical for genotype in our RNAseq panel of women). These cFSVs exhibited the most significant evidence for association with CASP8 expression in normal breast tissue (ratio=0.85, p=5×10⁻⁴) Genotype=1 includes women homozygous for the common allele; Genotype=2 includes heterozygous women; and Genotype=3 includes women homozygous for the risk allele.

Figure 4. Allele specific enhancer activity for rs3769821/rs3769823 and rs10197246

The bar graph shows the log₂ of the relative expression of the risk allele to the neutral allele, within a cell type, as measured by luciferase output in an enhancer assay. Expression of the neutral allele was used to normalize each pair of experiments. Expression measured in MCF10A is shown in blue and expression measured in T-47D is shown in red. Significance level is indicated above each pair. ‘ns’ indicates not significant. Neutral/risk alleles as follows: rs3769821, A/G; rs3769823, C/T; and rs10197246, C/T