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Gene promoter hypermethylation in ductal lavage fluid from healthy BRCA gene mutation carriers and mutation-negative controls.

Locke I, Kote-Jarai Z, Fackler MJ, Bancroft E, Osin P, Nerurkar A, Izatt L, Pichert G, Gui GP, Eeles RA - Breast Cancer Res. (2007)

Bottom Line: The pattern of CpG island hypermethylation within the promoter region of a panel of four genes (RAR-beta, HIN-1, Twist and Cyclin D2) was assessed by methylation-specific polymerase chain reaction using free DNA extracted from DL fluid.Two BRCA mutation carriers, in whom aberrant methylation was found, also had duct epithelial cell atypia identified.No hypermethylation was found in DL samples from 5 negative controls (p = 0.13).

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Affiliation: Translational Cancer Genetics Team, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK. imogen.locke@icr.ac.uk

ABSTRACT

Introduction: Female germline BRCA gene mutation carriers are at increased risk for developing breast cancer. The purpose of our study was to establish whether healthy BRCA mutation carriers demonstrate an increased frequency of aberrant gene promoter hypermethylation in ductal lavage (DL) fluid, compared with predictive genetic test negative controls, that might serve as a surrogate marker of BRCA1/2 mutation status and/or breast cancer risk.

Methods: The pattern of CpG island hypermethylation within the promoter region of a panel of four genes (RAR-beta, HIN-1, Twist and Cyclin D2) was assessed by methylation-specific polymerase chain reaction using free DNA extracted from DL fluid.

Results: Fifty-one DL samples from 24 healthy women of known BRCA mutation status (7 BRCA1 mutation carriers, 12 BRCA2 mutation carriers and 5 controls) were available for methylation analysis. Eight of 19 (42.1%) BRCA mutation carriers were found to have at least one hypermethylated gene in the four-gene panel. Two BRCA mutation carriers, in whom aberrant methylation was found, also had duct epithelial cell atypia identified. No hypermethylation was found in DL samples from 5 negative controls (p = 0.13).

Conclusion: We found substantial levels of aberrant methylation, with the use of a four-gene panel, in the fluid from the breasts of healthy BRCA mutation carriers compared with controls. Methylation analysis of free DNA in DL fluid may offer a useful surrogate marker for BRCA1/2 mutation status and/or breast cancer risk. Further studies are required for the evaluation of the specificity and predictive value of aberrant methylation in DL fluid for future breast cancer development in BRCA1/2 mutation carriers.

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Representative methylation analysis of DL samples from BRCA carriers in whom aberrant methylation was found. (a) Hypermethylation of RAR-β found in ductal lavage (DL) samples from subjects 17, 11, 3 and 6. Replicate PCR experiments for subject 11, using DNA from the same duct, are shown to illustrate reproducibility. (b) Methylation analysis of HIN-1. Two different ducts for subject 1 are shown: the first is unmethylated (duct right 2a; see Figure 2) and the second shows hypermethylation of HIN-1 (duct right 1a). Subjects 11, 13 and 8 show aberrant methylation of HIN-1. In both panels, unmethylated control samples consisted of 1 mg/ml solutions of human sperm DNA (HSD), and methylated controls were 1 mg/ml solutions of DNA extracted from the MDA-MB-231 breast cancer cell line (231). Marker used is a 123bp DNA ladder - rungs represent 123bp, 246bp and 369bp. M, methylated; U, unmethylated.
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Figure 1: Representative methylation analysis of DL samples from BRCA carriers in whom aberrant methylation was found. (a) Hypermethylation of RAR-β found in ductal lavage (DL) samples from subjects 17, 11, 3 and 6. Replicate PCR experiments for subject 11, using DNA from the same duct, are shown to illustrate reproducibility. (b) Methylation analysis of HIN-1. Two different ducts for subject 1 are shown: the first is unmethylated (duct right 2a; see Figure 2) and the second shows hypermethylation of HIN-1 (duct right 1a). Subjects 11, 13 and 8 show aberrant methylation of HIN-1. In both panels, unmethylated control samples consisted of 1 mg/ml solutions of human sperm DNA (HSD), and methylated controls were 1 mg/ml solutions of DNA extracted from the MDA-MB-231 breast cancer cell line (231). Marker used is a 123bp DNA ladder - rungs represent 123bp, 246bp and 369bp. M, methylated; U, unmethylated.

Mentions: Free DNA sufficient for PCR amplification was obtained from the supernatant of 49 of 51 DL samples. Eight of 19 (42.1%) mutation carriers were found to have at least one hypermethylated gene in the four-gene panel, in comparison with none of the 7 DL samples obtained from 5 negative controls (p = 0.13). We found HIN-1 to be the most frequently methylated gene and CyclinD2 the least frequently methylated gene in the panel (Table 1). Representative examples of methylated DL samples are shown in Figure 1. Four of the eight BRCA mutation carriers, in whom aberrant methylation was found, demonstrated simultaneous methylation of two different genes in the four-gene panel – two BRCA mutation carriers in the same DL sample (subjects 1 and 13) and two BRCA mutation carriers in different ducts (subjects 3 and 11). A further BRCA mutation carrier (subject 6) was found to have hypermethylation of RAR-β in a DL sample from the left breast at her first visit and hypermethylation of HIN-1 in a repeat sample taken from the same duct 1 year later (Figure 2).


Gene promoter hypermethylation in ductal lavage fluid from healthy BRCA gene mutation carriers and mutation-negative controls.

Locke I, Kote-Jarai Z, Fackler MJ, Bancroft E, Osin P, Nerurkar A, Izatt L, Pichert G, Gui GP, Eeles RA - Breast Cancer Res. (2007)

Representative methylation analysis of DL samples from BRCA carriers in whom aberrant methylation was found. (a) Hypermethylation of RAR-β found in ductal lavage (DL) samples from subjects 17, 11, 3 and 6. Replicate PCR experiments for subject 11, using DNA from the same duct, are shown to illustrate reproducibility. (b) Methylation analysis of HIN-1. Two different ducts for subject 1 are shown: the first is unmethylated (duct right 2a; see Figure 2) and the second shows hypermethylation of HIN-1 (duct right 1a). Subjects 11, 13 and 8 show aberrant methylation of HIN-1. In both panels, unmethylated control samples consisted of 1 mg/ml solutions of human sperm DNA (HSD), and methylated controls were 1 mg/ml solutions of DNA extracted from the MDA-MB-231 breast cancer cell line (231). Marker used is a 123bp DNA ladder - rungs represent 123bp, 246bp and 369bp. M, methylated; U, unmethylated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851387&req=5

Figure 1: Representative methylation analysis of DL samples from BRCA carriers in whom aberrant methylation was found. (a) Hypermethylation of RAR-β found in ductal lavage (DL) samples from subjects 17, 11, 3 and 6. Replicate PCR experiments for subject 11, using DNA from the same duct, are shown to illustrate reproducibility. (b) Methylation analysis of HIN-1. Two different ducts for subject 1 are shown: the first is unmethylated (duct right 2a; see Figure 2) and the second shows hypermethylation of HIN-1 (duct right 1a). Subjects 11, 13 and 8 show aberrant methylation of HIN-1. In both panels, unmethylated control samples consisted of 1 mg/ml solutions of human sperm DNA (HSD), and methylated controls were 1 mg/ml solutions of DNA extracted from the MDA-MB-231 breast cancer cell line (231). Marker used is a 123bp DNA ladder - rungs represent 123bp, 246bp and 369bp. M, methylated; U, unmethylated.
Mentions: Free DNA sufficient for PCR amplification was obtained from the supernatant of 49 of 51 DL samples. Eight of 19 (42.1%) mutation carriers were found to have at least one hypermethylated gene in the four-gene panel, in comparison with none of the 7 DL samples obtained from 5 negative controls (p = 0.13). We found HIN-1 to be the most frequently methylated gene and CyclinD2 the least frequently methylated gene in the panel (Table 1). Representative examples of methylated DL samples are shown in Figure 1. Four of the eight BRCA mutation carriers, in whom aberrant methylation was found, demonstrated simultaneous methylation of two different genes in the four-gene panel – two BRCA mutation carriers in the same DL sample (subjects 1 and 13) and two BRCA mutation carriers in different ducts (subjects 3 and 11). A further BRCA mutation carrier (subject 6) was found to have hypermethylation of RAR-β in a DL sample from the left breast at her first visit and hypermethylation of HIN-1 in a repeat sample taken from the same duct 1 year later (Figure 2).

Bottom Line: The pattern of CpG island hypermethylation within the promoter region of a panel of four genes (RAR-beta, HIN-1, Twist and Cyclin D2) was assessed by methylation-specific polymerase chain reaction using free DNA extracted from DL fluid.Two BRCA mutation carriers, in whom aberrant methylation was found, also had duct epithelial cell atypia identified.No hypermethylation was found in DL samples from 5 negative controls (p = 0.13).

View Article: PubMed Central - HTML - PubMed

Affiliation: Translational Cancer Genetics Team, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK. imogen.locke@icr.ac.uk

ABSTRACT

Introduction: Female germline BRCA gene mutation carriers are at increased risk for developing breast cancer. The purpose of our study was to establish whether healthy BRCA mutation carriers demonstrate an increased frequency of aberrant gene promoter hypermethylation in ductal lavage (DL) fluid, compared with predictive genetic test negative controls, that might serve as a surrogate marker of BRCA1/2 mutation status and/or breast cancer risk.

Methods: The pattern of CpG island hypermethylation within the promoter region of a panel of four genes (RAR-beta, HIN-1, Twist and Cyclin D2) was assessed by methylation-specific polymerase chain reaction using free DNA extracted from DL fluid.

Results: Fifty-one DL samples from 24 healthy women of known BRCA mutation status (7 BRCA1 mutation carriers, 12 BRCA2 mutation carriers and 5 controls) were available for methylation analysis. Eight of 19 (42.1%) BRCA mutation carriers were found to have at least one hypermethylated gene in the four-gene panel. Two BRCA mutation carriers, in whom aberrant methylation was found, also had duct epithelial cell atypia identified. No hypermethylation was found in DL samples from 5 negative controls (p = 0.13).

Conclusion: We found substantial levels of aberrant methylation, with the use of a four-gene panel, in the fluid from the breasts of healthy BRCA mutation carriers compared with controls. Methylation analysis of free DNA in DL fluid may offer a useful surrogate marker for BRCA1/2 mutation status and/or breast cancer risk. Further studies are required for the evaluation of the specificity and predictive value of aberrant methylation in DL fluid for future breast cancer development in BRCA1/2 mutation carriers.

Show MeSH
Related in: MedlinePlus