Methylation analysis by restriction endonuclease digestion and real-time PCR.

DNA methylation is a highly characterized epigenetic modification of the human genome that is critical for normal cell processes, including tissue regulation, genetic expression development, genomic imprinting, X-chromosome inactivation, and DNA repair. DNA methylation in humans occurs almost exclusively at the C5 position of CpG dinucleotides in genomic promoter regions. Alterations in DNA methylation cause developmental diseases, such as genetic imprinting defects, cancer, and potentially a wide range of adultonset chronic diseases. Therefore, sensitive techniques that detect methylation are critical for epigenetic research, clinical diagnostics, cancer prognosis, and therapeutics (1, 2 ). Methylation-specific PCR is a commonly used rapid technique for methylation screening that replaces the laborious Southern blot assay, the standard procedure on which most DNA analysis is based. Methylation-specific PCR and other PCR-based methylation methods that use bisulfite-treated DNA as a template are generally accepted as the most analytically sensitive and specific techniques for analyzing DNA methylation at a single locus. The protocol described by Frommer et al. has been widely used for sodium bisulfite modification, and a variety of commercial kits are available for this purpose (3). Sodium bisulfite deaminates unmethylated cytosine to uracil, and the methylation is accessed by the PCR with either methylationspecific primers or methylation-independent primers (MIPs). Methylation-specific primers amplify either methylated or unmethylated alleles. These primers are highly sensitive and able to detect the presence of a methylated allele at a frequency as low as 0.1% in an unmethylated population (4 ). PCR with methylationspecific primers is not quantitative, however. Alternatively, the MethyLight method is a sensitive, fluorescence-based real-time PCR technique capable of quantifying DNA methylation at a specific locus (5, 6 ). MethyLight can detect completely methylated or unmethylated alleles but is oblivious to partially methylated CpGs. Considering the random conversion of cytosine to uracil, it is possible that a subset of DNA copies have a substantially lower conversion rate and that some promoter regions might prove more prone to incomplete conversion than others, thereby leading to the disregard of several partially methylated alleles. Therefore, MethyLight, despite being a quantitative assay, cannot distinguish different methylation patterns. MIP-PCR uses primers designed for universally amplifying proportional methylated and unmethylated alleles that include a number of CpG sites. Because of their GC content, the methylated and unmethylated alleles are distinguished by high-resolution melting analysis, which exploits differences in thermal stability (7 ). This PCR has a tendency to amplify unmethylated DNA, because of the low GC content after bisulfite modification of the DNA. The sensitivity of MIP-based methods is relatively low, but it can be increased by introducing CpG sites into the primers or by using oligonucleotide blockers. Nonetheless, these assays can measure methylation quantitatively and estimate the mosaic situation. Several other MIP-based methods also have been successfully used for methylation analysis, such as methylation-sensitive single-nucleotide primer extension and combined bisulfite restriction analysis (8 ). Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) uses multiple probes that are hybridized to their targeted methylation sites. The methylation sites are recognized by a methylationsensitive restriction enzyme (HhaI), and the probe pair is ligated in the ligation reaction (9 ). This method not only permits simultaneous analysis of multiple methylation sites but also allows quantification of copy number changes by comparing target-specific probes to the control probes (10 ). Bisulfite Sanger sequencing, considered the gold standard for methylation analysis, provides information about individual CpG sites. In addition to requiring laborious processes to implement, the Sanger sequencing approach has a sensitivity for detecting low-level mosaicism that is limited to 20%. An attractive alternative is pyrosequencing. Pyrosequencing is 1 Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT; 2 ARUP Laboratories, Salt Lake City, UT; 3 Department of Pathology, University of Utah Health Science Center, Salt Lake City, UT. * Address correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108-1221. E-mail [email protected]. Received April 12, 2010; accepted April 27, 2010. Previously published online at DOI: 10.1373/clinchem.2010.146654 4 Nonstandard abbreviations: MIP, methylation-independent primer; MS-MLPA, methylation-specific multiplex ligation-dependent probe amplification; qPCR, quantitative PCR; Cq, quantification cycle. Clinical Chemistry 56:7 1050–1052 (2010) Editorials