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. 2013 Jul;8(3):167-75.

Quantitative evaluation of DNMT3B promoter methylation in breast cancer patients using differential high resolution melting analysis

M Naghitorabi  1 , J Mohammadi Asl, H Mir Mohammad Sadeghi, M Rabbani, A Jafarian-Dehkordi, Haghjooye S Javanmard
Affiliations

Quantitative evaluation of DNMT3B promoter methylation in breast cancer patients using differential high resolution melting analysis

M Naghitorabi et al. Res Pharm Sci. 2013 Jul.

Abstract

DNA methylation plays an important role in carcinogenesis through epigenetic silencing of tumor suppressor genes. Aberrant methylation usually results from changes in the activity of DNA methyltransferases (DNMTs). Some studies show that the overexpression of the DNMTs may lead to aberrant methylation of tumor suppressor genes. Also the overexpression of DNMTs may be related to methylation status of their genes. Due to limited number of studies on DNMT3B promoter methylation, this study was performed to quantitatively measure the methylation level of DNMT3B gene in archival formalin fixed paraffin embedded (FFPE) tissues from breast cancer patients. Using differential high resolution melting analysis (D-HRMA) technology, the methylation level of DNMT3B gene promoter was quantified in 98 breast cancer FFPE tissues and also 10 fresh frozen normal tissue samples. Statistical analyses used for analyzing the correlation between the methylation and clinical variables. All the normal samples were found to be methylated at the DNMT3B promoter (the average methylation level 3.34%). Patients were identified as hypo-methylated (mean methylation level 0.8%), methylated (mean methylation level 2.48%) and hyper-methylated (mean methylation level 10.5%). Statistical analysis showed a significant correlation between the methylation status and the sample type, cancer type and tumor size. Also the methylation level was significantly associated with histologic grade. It is concluded that quantification of DNMT3B promoter methylation might be used as a reliable and sensitive diagnostic and prognostic tool in breast cancer. Also D-HRMA is demonstrated as a rapid and cost effective method for quantitative evaluation of promoter methylation.

Keywords: Breast cancer; DNA methylation; DNMT3B; FFPE; High resolution melting analysis.

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Figures

Fig. 1
Fig. 1
DNMT3B D-HRMA graphs using serial dilutions of methylated DNA (from 100% to 0%). (A) The amplification plots were obtained for all standard dilutions (from 100% to 0% methylated DNA) as the template, with comparable Ct values. (B) The normalized fluorescence HRM profiles of various amplicons amplified from each standard diluted methylated DNA. (C) The differential fluorescence plots were obtained by normalizing HRM profiles against the unmethylated DNA. (D) The melting curve of the standard dilutions identified the specificity of the assay.
Fig. 2
Fig. 2
Differential fluorescence values and the standard curve of the serial dilutions of methylated DNA (from 100% to 0%). (A) Differential fluorescence values obtained at the melting point of each standard dilution. (B) The standard curve generated by plotting differential fluorescence values against the percentage of methylation. All the dilutions were tested in duplicate.
Fig. 3
Fig. 3
Association between DNMT3B methylation level and cancer type (A), histologic grade (B,) and tumor size (C).
See this image and copyright information in PMC

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