Evaluation of a potential epigenetic biomarker by quantitative methyl‐single nucleotide polymorphism analysis

Abstract

Tumorigenesis is characterized by alterations of methylation profiles including loss and gain of 5-methylcytosine. Recently, we identified a single CpG, which seemed to be consistently hypomethylated in pilocytic astrocytomas but not in other gliomas. To evaluate its applicability as a biomarker, we examined its methylation status in a large panel of gliomas (n = 97). Methylation-dependent DNA sequence variation may be considered a kind of single nucleotide polymorphism (methylSNP). MethylSNPs can be easily converted into common SNPs of the C/T type by sodium bisulfite treatment of the DNA and afterwards subjected to conventional SNP typing. We adapted SnaPshot trade mark and Pyrosequencing trade mark to determine the methylation of our test CpG in a quantitative manner. The adapted methods, called SNaPmeth and PyroMeth, respectively, gave nearly identical results, however data obtained with PyroMeth showed less scattering. Furthermore, the integrated software for allele frequency determination from Pyrosequencing could be used directly for data analysis while SnaPmeth data had to be exported and processed manually. Although data did not confirm our previous result of a preferential hypomethylation of the tested CpG in pilocytic astrocytomas, we consider quantitative methylSNP analysis by SNaPmeth or PyroMeth a favorable alternative to existing high-throughput methylation assays. It combines single CpG analysis with accurate quantitation and is amenable to high throughput.

446 Detection of EGFR and KRAS Mutation by Pyrosequencing Analysis in Cytologic Samples of Non-Small Cell Lung Cancer

“…Subsequently, the released pyrophosphate is converted to adenosine triphosphate, which is utilized to produce light (15,17). Pyrosequencing is being increasingly used in various conditions, such as bacterial strain typing, mutation detection, SNP genotyping, and quantitative CpG island methylation analysis (15,(19)(20)(21)(22)(23)(24).…”

“…There are various and newly developed techniques for detecting mutations in lung cancer such as Amplification Refractory Mutation System (ARMS), cationic conjugated polymerbased fluorescence resonance energy transfer (CCP-RFLP), Smart Amplification Process (SmartAMP), pyrosequencing, etc (24). Standard method for detecting mutation of EGFR or KRAS is still direct DNA sequencing method.…”

CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics

“…Methods for detecting CpG methylation after methylation-independent PCR using bisulfite-modified DNA include combined bisulfite restriction analysis, 31 pyrosequencing, 8,[32][33][34] matrix-assisted laser desorption/ ionization-time of flight, 9 and high performance liquid chromatography. 10,35,36 …”

Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS)

“…2; Ronaghi et al 1998). Pyrosequencing has gained enormous popularity in DNA methylation analysis (Uhlmann et al 2002;Tost and Gut 2007), microbial identification (Clarke 2005), detection of allelic expression imbalance (Wang and Elbein 2007), and next-generation sequencing (454 Life Sciences (Roche)/FLX; http://www.454.com/). In particular, pyrosequencing has been widely used for SNP detection (Fakhrai-Rad et al 2002;Langaee and Ronaghi 2005;Ronaghi et al 2007) because it offers an easy, quantitative, and accurate method to detect the relative abundance of alleles in a mixture, provided they differ by a single nucleotide and that their relative fraction is between 10% and 90% (Ronaghi et al 1998;Wasson et al 2002).…”