Gene-body hypermethylation of ATM in peripheral blood DNA of bilateral breast cancer patients

Abstract

Bilaterality of breast cancer is an indicator of constitutional cancer susceptibility; however, the molecular causes underlying this predisposition in the majority of cases is not known. We hypothesize that epigenetic misregulation of cancer-related genes could partially account for this predisposition. We have performed methylation microarray analysis of peripheral blood DNA from 14 women with bilateral breast cancer compared with 14 unaffected matched controls throughout 17 candidate breast cancer susceptibility genes including BRCA1, BRCA2, CHEK2, ATM, ESR1, SFN, CDKN2A, TP53, GSTP1, CDH1, CDH13, HIC1, PGR, SFRP1, MLH1, RARB and HSD17B4. We show that the majority of methylation variability is associated with intragenic repetitive elements. Detailed validation of the tiled region around ATM was performed by bisulphite modification and pyrosequencing of the same samples and in a second set of peripheral blood DNA from 190 bilateral breast cancer patients compared with 190 controls. We show significant hypermethylation of one intragenic repetitive element in breast cancer cases compared with controls (P = 0.0017), with the highest quartile of methylation associated with a 3-fold increased risk of breast cancer (OR 3.20, 95% CI 1.78-5.86, P = 0.000083). Increased methylation of this locus is associated with lower steady-state ATM mRNA level and correlates with age of cancer patients but not controls, suggesting a combined age-phenotype-related association. This research demonstrates the potential for gene-body epigenetic misregulation of ATM and other cancer-related genes in peripheral blood DNA that may be useful as a novel marker to estimate breast cancer risk. ACCESSION NUMBERS: The microarray data and associated .BED and .WIG files can be accessed through Gene Expression Omnibus accession number: GSE14603.

Figure 1.

Methylation microarray analysis of 17 genes reveals methylation variability in repetitive elements. (A) The distance from each of the MVP to the nearest repetitive element was calculated for each of the 181 distinct MVPs revealing 44% of peaks within 100 bp of the nearest repetitive element. Random placement of 181 simulated MVP peaks reveals a distribution containing only 26% of peaks within 100 bp of the nearest repetitive element. Kolmogorov–Smirnov test was used to determine the significance of the difference in distribution. (B) The smoothed average of MATScores (±2 SD in red) for genes aligned at the TSS reveals periodic increase in methylation variability across the first 10 kb (of genes >10 kb) or across the first 5 kb (of shorter genes, median 4.5 kb).

Figure 2.

Investigation of ATM gene methylation. (A) Methylation microarray data for the ATM gene locus. Seven methylation variable peaks were detected across the tiled region surrounding the ATM gene. Data are presented as a custom WIG file track on the UCSC genome browser. (B) Pair-wise comparison of ATM mvp2a methylation reveals increased methylation in 5/14 bilateral breast cancer patients (blue) compared with matched controls (yellow). Methylation of six CpG dinucleotides within the repetitive element mvp2a was determined by pyrosequencing. Pair-wise Wilcoxon signed rank sum test was used to determine statistical significance (# indicates P < 0.05). (C) DNA methylation analysis of MVPs reveals significant variability detected within the ATM gene. LINE 1 assay shows no significant differences in methylation of LINE1 repetitive elements in peripheral blood DNA of cases (blue) compared with controls (yellow). Methylation variability was detected in mvp1b, mvp2a, mvp2b, mvp3 and mvp4. Box and whisker plots represent median (centre line), inter-quartile range (box) and 95th percentiles (whisker), and samples outwith this range are represented as points.

Figure 3.

Methylation of LINE1, ATM CpG island, mvp2a and mvp2b in 190 bilateral breast cancer cases compared with 190 controls. (A) Schematic of the genomic location of the ATM CpG island (CGI) at the TSS and at mvp2a and mvp2b within the second intron of ATM. (B) Pyrosequencing-based methylation analysis of 190 bilateral breast cancer cases compared with 190 matched controls reveals no methylation in the promoter CpG island and significant inter-individual methylation variability in the intronic mvp2a and mvp2b. Significant hypermethylation of mvp2b is detected in bilateral breast cancer cases compared with controls (P = 0.0017, Wilcoxon signed rank sum test). Box and whisker plots represent median (centre line), inter-quartile range (box) and 95th percentiles (whisker), and samples outwith this range are represented as points. (C) Kernel density plot of methylation values in cases (solid line) compared with controls (dotted lines) showing overlapping distributions for LINE1, ATM CpG island and ATM mvp2a and a skewed distribution of methylation at ATM mvp2b in the cases.

Figure 4.

Correlation between methylation of ATM mvp2b and expression of ATM in cancer cell lines. (A) Methylation analysis in 62 cancer cell lines reveals a similar range of methylation as in PBMCs of breast cancer patients (77–99%); however, the distribution is skewed towards increased methylation in the cancer cell lines (green). Methylation distributions of breast cancer patients (red) and controls (black) are shown for comparison. (B) ATM expression was determined by qRT–PCR in the five breast cancer cell lines and by gene expression microarray data (C) for a panel of sarcoma cell lines. Error bars represent SEM from triplicate qRT–PCR experiments. Correlation between methylation and expression is shown using Spearmans rank correlation coefficient.