Heritable DNA methylation marks associated with susceptibility to breast cancer

Abstract

Mendelian-like inheritance of germline DNA methylation in cancer susceptibility genes has been previously reported. We aimed to scan the genome for heritable methylation marks associated with breast cancer susceptibility by studying 25 Australian multiple-case breast cancer families. Here we report genome-wide DNA methylation measured in 210 peripheral blood DNA samples provided by family members using the Infinium HumanMethylation450. We develop and apply a new statistical method to identify heritable methylation marks based on complex segregation analysis. We estimate carrier probabilities for the 1000 most heritable methylation marks based on family structure, and we use Cox proportional hazards survival analysis to identify 24 methylation marks with corresponding carrier probabilities significantly associated with breast cancer. We replicate an association with breast cancer risk for four of the 24 marks using an independent nested case-control study. Here, we report a novel approach for identifying heritable DNA methylation marks associated with breast cancer risk.

Fig. 1

Predicting genetic variation with ∆l. The proportion of methylation sites with nearby SNPs as a function of ∆l, both by categories of ∆l (horizontal lines with error bars representing 95% confidence intervals) and as a polynomial function fitted by logistic regression (curvilinear line)

Fig. 2

DNA methylation at the VTRNA2-1 promoter. a Genomic locations of 6 HM450K probes associated with VTRNA2-1 promoter region. b DNA methylation levels (β-values) of these 6 probes labelled by breast cancer status. β-values for each individual are shown on y axis for 6 VTRNA2-1 probes. c Average DNA methylation levels across all six probes shown separately for individual families and labelled by breast cancer status. β-values are shown on y axis for members from each family (y axis)

Fig. 3

Analytical study approach. An overview of the analytical approach for each of the 1000 most-Mendelian probes in the multiple-case family-based analyses (a) and for the replication study of 24 probes in the population-based, case–control analyses (b). A measure of Mendelian heritability was calculated for all probes not on a sex chromosome or within 10 base pairs of a SNP (not depicted). For each of the 1000 most-Mendelian probes, a Mendelian model was fitted to the probe’s M-values and this was used to calculate carrier probabilities (e.g., for a hypothetical genetic variant that causes aberrant DNA methylation at the probe), then these carrier probabilities were tested for association with breast cancer (note that unbiased p-values could be calculated but unbiased risks could not because we could not adjust for ascertainment). This gave 24 highly heritable methylation marks that were associated with breast cancer, and a nested case–control study was used to test the M-values of each of these probes for association with breast cancer and to estimate the corresponding odds ratios (ORs)