CpG methylation analysis--current status of clinical assays and potential applications in molecular diagnostics: a report of the Association for Molecular Pathology

Abstract

Methylation of CpG islands in gene promoter regions is a major molecular mechanism of gene silencing and underlies both cancer development and progression. In molecular oncology, testing for the CpG methylation of tissue DNA has emerged as a clinically useful tool for tumor detection, outcome prediction, and treatment selection, as well as for assessing the efficacy of treatment with the use of demethylating agents and monitoring for tumor recurrence. In addition, because CpG methylation occurs early in pre-neoplastic tissues, methylation tests may be useful as markers of cancer risk in patients with either infectious or inflammatory conditions. The Methylation Working Group of the Clinical Practice Committee of the Association of Molecular Pathology has reviewed the current state of clinical testing in this area. We report here our summary of both the advantages and disadvantages of various methods, as well as the needs for standardization and reporting. We then conclude by summarizing the most promising areas for future clinical testing in cancer molecular diagnostics.

Figure 1

Bisulfite modification of DNA for methylation assays. Bisulfite modification converts unmethylated cytosine to uracil, while methylated cytosine is not modified. After PCR, uracil is replaced by thymine on the newly synthesized DNA strands.

Figure 2

Methylation of the MLH1 gene CpG island promoter region detected by methylation specific PCR (MSP). After sodium bisulfite conversion of genomic DNA from colon cancer tumor samples (T1 and T2) or positive control DNA (Pos. Cont.), PCR was performed with the primer pair specific for methylated MLH1 DNA (M) or with the primer pair specific for the unmethylated MLH1 sequence (U). The negative control is a PCR reaction without DNA template. The DNA size ladder (Bp) is indicated. The presence of a PCR product in the lanes labeled (M) indicates the presence of CpG methylation in the sample T1 and in the positive control. For sample T2 no MLH1 CpG methylation is detected.

Figure 3

Methylation-sensitive restriction enzymes for detection of CpG Methylation without bisulfite conversion. DNA is first treated with HhaI methylation-sensitive restriction endonuclease and then used for PCR. When the CpG locus being amplified is not methylated, HhaI cleaves its restriction site, resulting in lack of PCR amplification; whereas, if it is methylated, the HhaI restriction sites are protected from restriction enzyme digestion, allowing for PCR amplification.