A second-generation profiling system for quantitative methylation analysis of multiple gene promoters: application to lung cancer

• Published in: Oncogene Vol. 26; no. 45; pp. 6518 - 6525
• Main Authors: SANO, A, KAGE, H, YATOMI, Y, OHISHI, N, TAKAI, D, SUGIMOTO, K, KITAGAWA, H, AKI, N, GOTO, A, FUKAYAMA, M, NAKAJIMA, J, TAKAMOTO, S, NAGASE, T
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing 04.10.2007; Nature Publishing Group
• Subjects: Biological and medical sciences; Bisulfite; Care and treatment; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Cluster Analysis; CpG Islands; DNA fingerprinting; DNA Methylation; DNA Probes; Epidermal growth factor receptors; Fluorescent indicators; Fundamental and applied biological sciences. Psychology; Genes, erbB-1; Genetic aspects; Health aspects; Humans; Lung cancer; Lung Neoplasms - genetics; Medical sciences; Metastases; Methylation; Molecular and cellular biology; Molecular Probe Techniques; Mutation; Oncology; Pneumology; Promoter Regions, Genetic; Promoters; Quantitative genetics; Reverse Transcriptase Polymerase Chain Reaction - methods; Risk factors; Tumors of the respiratory system and mediastinum; Japan; Lung disease; Respiratory disease; Lung cancer; epigenetic change; Time; Malignant tumor; real-time PCR; Carcinogenesis; Polymerase chain reaction; EGFR mutation; Gene; DNA; Epigenetics; DNA methylation; Bronchus disease; Mutation; Methylation; Quantitative analysis; CpG islands
• ISSN: 0950-9232; 1476-5594
• DOI: 10.1038/sj.onc.1210483

Cancer-specific gene promoter methylation has been described in many types of cancers, and various semi-quantified results have shown their usefulness. Here, we show a more sensitive and specific second-generation system for profiling the DNA methylation status. This method is based on bisulfite reaction of DNA and real-time PCR using two TaqMan MGB probes labeled with different fluorescence, followed by clustering analysis. Primers were designed with CpG-less sequences, and TaqMan MGB probes were designed to contain three or four CpG sites and to be shorter than conventional TaqMan probes. We have added new criteria for primer and probe design for further specificity. We confirmed the reliability of this system and applied it to analysis of lung cancers. Using 10 promoters, 90 primary lung cancers were clustered into six groups consisting of cases having similar smoking status and pathological findings. EGFR mutation and p16 promoter DNA methylation were exclusive, as previously reported; however, DNA methylation in other genes was unrelated to EGFR mutation. This system was also useful to distinguish double primary lung cancers from a single cancer with intrapulmonary metastasis. As above, our system has widespread availability in clinical use and biological research.