Assessing combined methylation–sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

Abstract

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.

Association of aberrant DNA methylation with clinicopathological features in breast cancer

“…It is considered as a very sensitive (detects 5% levels of DNA methylation in an unmethylated background) and quantitative approach for the examination of heterogeneously methylated promoters as well as capable of detecting the methylation levels of individual CpGs within an amplicon (23). For these reasons it has been described as a complementary method of MS-HRM in DNA methylation studies (9). Furthermore, it comprises control positions for the efficiency of bisulfite treatment.…”

“…MS-HRM is a novel approach to identify aberrant methylation of gene promoter regions using sequence-dependent melting profiles of each amplicon (22). It has been considered as the most rapid and sensitive in-tube method capable of detecting even 0.1-1% of DNA methylation in an unmethylated background, minimizing possible sample contamination, and requiring only low amounts of DNA template (9,21). Furthermore, MS-HRM is a semi-quantitative method that has been claimed to distinguish homogeneous from heterogeneous methylation (9).…”

“…In order to validate the sensitivity of the newly established MS-HRM analysis for methylation detection we performed pyrosequencing analysis for MGMT gene promoter methylation status in most of the cases. Bisulfite pyrosequencing analyses short genomic regions (<100 bp), therefore, it is suitable for methylation studies of degraded genomic material extracted from formalin-fixed paraffin embedded tissues (FFPE) (9). It is considered as a very sensitive (detects 5% levels of DNA methylation in an unmethylated background) and quantitative approach for the examination of heterogeneously methylated promoters as well as capable of detecting the methylation levels of individual CpGs within an amplicon (23).…”

“…It has been considered as the most rapid and sensitive in-tube method capable of detecting even 0.1-1% of DNA methylation in an unmethylated background, minimizing possible sample contamination, and requiring only low amounts of DNA template (9,21). Furthermore, MS-HRM is a semi-quantitative method that has been claimed to distinguish homogeneous from heterogeneous methylation (9). In order to validate the sensitivity of the newly established MS-HRM analysis for methylation detection we performed pyrosequencing analysis for MGMT gene promoter methylation status in most of the cases.…”

“…Furthermore, in order to validate results obtained by the MS-HRM method, pyrosequencing was also used for the quantitative assessment of MGMT gene methylation status (9).…”