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. 2018 Sep 15;143(6):1416-1425.
doi: 10.1002/ijc.31526. Epub 2018 Apr 25.

Single CpG Hypermethylation, Allele Methylation Errors, and Decreased Expression of Multiple Tumor Suppressor Genes in Normal Body Cells of Mutation-Negative Early-Onset and High-Risk Breast Cancer Patients

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Single CpG Hypermethylation, Allele Methylation Errors, and Decreased Expression of Multiple Tumor Suppressor Genes in Normal Body Cells of Mutation-Negative Early-Onset and High-Risk Breast Cancer Patients

Julia Böck et al. Int J Cancer. .
Free PMC article

Abstract

To evaluate the role of constitutive epigenetic changes in normal body cells of BRCA1/BRCA2-mutation negative patients, we have developed a deep bisulfite sequencing assay targeting the promoter regions of 8 tumor suppressor (TS) genes (BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, RB1) and the estrogene receptor gene (ESR1), which plays a role in tumor progression. We analyzed blood samples of two breast cancer (BC) cohorts with early onset (EO) and high risk (HR) for a heterozygous mutation, respectively, along with age-matched controls. Methylation analysis of up to 50,000 individual DNA molecules per gene and sample allowed quantification of epimutations (alleles with >50% methylated CpGs), which are associated with epigenetic silencing. Compared to ESR1, which is representative for an average promoter, TS genes were characterized by a very low (< 1%) average methylation level and a very low mean epimutation rate (EMR; < 0.0001% to 0.1%). With exception of BRCA1, which showed an increased EMR in BC (0.31% vs. 0.06%), there was no significant difference between patients and controls. One of 36 HR BC patients exhibited a dramatically increased EMR (14.7%) in BRCA1, consistent with a disease-causing epimutation. Approximately one third (15 of 44) EO BC patients exhibited increased rates of single CpG methylation errors in multiple TS genes. Both EO and HR BC patients exhibited global underexpression of blood TS genes. We propose that epigenetic abnormalities in normal body cells are indicative of disturbed mechanisms for maintaining low methylation and appropriate expression levels and may be associated with an increased BC risk.

Keywords: allele methylation error; breast cancer susceptibility gene; deep bisulfite sequencing; early onset breast cancer; epimutation; familial breast cancer; single CpG hypermethylation; tumor suppressor gene.

Figures

Figure 1
Figure 1
Samples with epimutations (top panel) and single CpG hypermethylation (bottom) in multiple TS genes. Lines represent samples, lanes genes. Epimutations and single CpG hypermethylation, respectively, in a given sample are indicated by gray bars. For example HR BC27 exhibits epimutations in BRCA1, RAD51C, PTEN, and TP53 (region 2); EO BC96 single CpG hypermethylation in BRCA1, ATM (region 1 and 2), TP53 (region 2), and RB1.
Figure 2
Figure 2
Variation in epimutation rates in BC cases and controls. For each of the 8 TS genes (BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, and RB1) EMRs in BC patients and controls are indicated by filled circles. Colored diamonds indicate multiple samples with the same EMR. Colored arrows indicate samples of BC patients with a tumor at the time of analysis. Most epimutations are seen in BRCA1 and RAD51C. Most EMRs are below the 1% threshold. Relatively few EMRs, mainly in BC samples are in the intermediated range from 1% to 2.5%. Only one sample shows an EMR > 10%. Samples without epimutations are not shown.
Figure 3
Figure 3
Single CpG methylation profiles of four TS genes (BRCA1, BRCA2, PTEN, and TP53 region 1) in control and BC samples (without epimutations). The x‐axis indicates the number of CpGs (in 5'→3' direction) for a given amplicon, the y‐axis mean methylation of each CpG. The bold black dotted line indicates the threshold for single CpG hypermethylation (5 IQRs away from the 75th percentile in controls). Each sample is indicated by a gray (in different shades) line. Samples with single CpG hypermethylation are indicated by colored lines and sample ID. There are very few hypermethylated single CpGs in controls. Asterisks indicate samples from BC patients with a tumor at the time of analysis. A subset of BC, in particular EO BC samples displays excessive hypermethylation in different individual CpGs.
Figure 4
Figure 4
Expression levels of tumor suppressor genes BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, and RB1 (indicated by different colors) in blood samples of controls (CTRL81, 82, and 83), BC samples with normal methylation patterns (EO BC49), with single CpG hypermethylation (EO BC96 and 53), and epimutations (EO BC74, HR BC8, 11, and 17), respectively. Diamonds represent relative expression of the color‐coded gene in a given sample. Circles indicate genes with single CpG hypermethylation or epimutation in the analyzed sample. A log2 RQ values of 1 correspond to an expression doubling and of −1 to a division in half (compared to sample CTRL83, which was used as a reference).

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