eQTL Colocalization Analyses Identify NTN4 as a Candidate Breast Cancer Risk Gene

Abstract

Breast cancer genome-wide association studies (GWASs) have identified 150 genomic risk regions containing more than 13,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements. However, the genes underlying breast cancer risk associations are largely unknown. Here, we used genetic colocalization analysis to identify loci at which gene expression could potentially explain breast cancer risk phenotypes. Using data from the Breast Cancer Association Consortium (BCAC) and quantitative trait loci (QTL) from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Project (TCGA), we identify shared genetic relationships and reveal novel associations between cancer phenotypes and effector genes. Seventeen genes, including NTN4, were identified as potential mediators of breast cancer risk. For NTN4, we showed the rs61938093 CCV at this region was located within an enhancer element that physically interacts with the NTN4 promoter, and the risk allele reduced NTN4 promoter activity. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. These data provide evidence linking risk-associated variation to genes that may contribute to breast cancer predisposition.

Keywords: GWAS; NTN4; breast cancer; colocalization; eQTL; enhancer; tumor suppressor.

Figure 1

Comparison of BCAC Strong Signals with GTEx v8 Breast Tissue eQTLs LocusCompare plots for 11 high-probability colocalized signals. Gene names and the relevant breast cancer phenotypes are shown in the plot headings. Points are colored based on linkage disequilibrium (LD) bins relative to the candidate SNP prioritized by HyPrColoc (purple diamond labeled with rsID; red, ≥0.8; orange, 0.6–0.8; green, 0.4–0.6; light blue, 0.2–0.4; and dark blue, < 0.2). LD data from 1000 Genomes phase 3, v.5 were retrieved from the LDlink portal. Strong CCVs for breast cancer risk are annotated as small diamonds and moderate CCVs as squares.

Figure 2

Regional Association Plots at the 12q22 Breast Cancer Risk Locus Single variant associations with overall breast cancer risk (top) and with NTN4 expression in normal breast tissue from GTEx v.8 (bottom). Variants are represented by points colored relative to linkage disequilibrium (LD) with the candidate variant detected by HyPrColoc (rs17356907; red, ≥0.8; orange, 0.6–0.8; green, 0.4–0.6; light blue, 0.2–0.4; and dark blue, <0.2).

Figure 3

Breast Cancer CCVs Distally Regulate NTN4 (A) WashU genome browser showing topologically associating domains (TADs) as horizontal gray bars above GENCODE-annotated coding genes (blue). The promoter capture Hi-C (PCHi-C) baits are depicted as black boxes. The putative regulatory element (PRE) containing the CCVs is shown as red colored vertical lines. The ATAC-seq tracks for B80T5 and MCF10A breast cells are shown as blue histograms. PCHi-C chromatin interactions are shown as black arcs. Red arcs depict chromatin looping between CCVs and the NTN4 promoter region. (B) dCAS9-KRAB was targeted to the PRE using two different sgRNAs (sgPRE1 and sgPRE2) in Bre80-TERT1 breast cells. SgCON contains a non-targeting control guide RNA. Gene expression was measured by qPCR and normalized to beta-glucuronidase (GUSB) expression. Error bars, SEM (n = 3). p values were determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (^∗∗p < 0.01). (C) Luciferase reporter assays following transient transfection of MCF10A breast cells. A PRE1 containing the protective (Prot.) or risk allele of rs61938093 and a PRE2 containing the protective (Prot.) or risk allele of rs1735907 were cloned into NTN4-promoter driven luciferase constructs. Error bars, SEM (n = 3). p values were determined by two-way ANOVA followed by Dunnett’s multiple comparisons test (^∗∗∗∗p < 0.0001). (D and E) Left: 3C interaction profiles between the NTN4 promoter and the genomic region containing the PRE in MCF10A (D) and T47D (E) 3C libraries generated with HindIII. A physical map of the region interrogated by 3C is shown above; the blue shading represents the position of the PRE and the anchor point set at the NTN4 promoter. Representative 3C profiles are shown. Error bars, SD (n = 3). Right: Allele-specific qPCR using primer set 1 (Table S2) and Taqman SNP assay to quantify the allelic ratio at CCV rs61938093. Error bars, SEM (n = 3). p values were determined using a Student’s t test (^∗∗∗p < 0.001). (F) EMSA for oligonucleotide duplexes containing CCVs rs61938093 or rs17356907 with the risk allele (R) or protective allele (P) as indicated, assayed using Bre80-TERT1 nuclear extracts. Competitor oligonucleotides are listed above each panel and were used at 100-fold molar excess: (−) no competitor; (Neg) a non-specific competitor; (Self) an identical oligonucleotide with no biotin label. Red arrowheads indicate band mobility differences between alleles.

Figure 4

NTN4 Depletion Promotes Breast Cell Proliferation and Tumor Formation (A) Boxplot showing NTN4 expression in normal breast and paired tumor tissue samples from TCGA. Boxplots indicate median (center line), interquartile range (box limits), and range (whiskers). p value was determined using a two-tailed t test. (B) Boxplot showing NTN4 expression in breast tumors from TCGA stratified by PAM50 molecular subtypes (n = 841). Boxplots indicate median (center line), interquartile range (box limits), and range (whiskers). (C) Proliferation of MCF7 cells transfected with a non-targeting control (siCON) or NTN4 (siNTN4) ON-TARGETplus siRNAs. Cells were grown in 24-well plates and confluency of the wells was measured by the IncuCyte live-cell imaging system. Results represent relative cell growth rates. Error bars, SD (n = 2). p value was determined by Student’s t test comparing confluency at the last time point measured (^∗∗∗p < 0.001). (D) MCF7 cells were transfected with the siCON or siNTN4 and grown over 7 days in ultra low-attachment conditions. Cell growth was assessed using the CellTiter-Glo luminescent cell viability assay. Graph shows fold change in luminescence of siNTN4 treated cells relative to siCON treated cells. Error bar, SEM (n = 3). p value was determined by Student’s t test test (^∗∗p < 0.01). (E) MCF7-control (PgCON) or MCF7-dCas9-KRAB NTN4 repressed cells (SgNTN4-P1/P2) were orthotopically injected into the mammary fat pads of nude mice. Tumor growth curves for each group are shown. Values are shown as average tumor volumes at each time point. Error bars, SEM (n = 6–7 mice per group). (F) Tumors of individual mice were dissected at day 38 post-injection. The scale bars represent 1 cm. (G) Plot of the individual weights of tumors with mean and SEM shown by cross-bar and error bars. Mann-Whitney U test (E and G) was used to compare differences between groups (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001).