Development and application of a multiplex PCR procedure for the detection of DNA methylation in colorectal cancer.
Cancers often exhibit aberrant methylation of gene promoter regions associated with loss of tumor suppressor and/or DNA repair gene function. Such methylation constitutes an excellent marker for the molecular detection of micro-metastases and the diagnosis of tumor recurrences. We have developed a multiplex methylation-specific PCR (MSP) procedure for rapid and simultaneous assessment of the methylation of 5 loci: the tumor suppressor genes p16INK4a, death-associated protein kinase (DAPK) and p14ARF, and the DNA repair genes hMLH1 and O6-methylguanine-DNA-methyltransferase (MGMT). This multiplex test uses one single PCR reaction and only one electrophoretic run. In 98 samples of colorectal cancer studied, methylation of MGMT, DAPK, p16, hMLH1 and p14 was present in 31, 20, 17, 16 and 14% of tumors, respectively. In 58% of the tumors at least one methylated gene was found. This multiplex MSP constitutes a simple and inexpensive method for screening of molecular signatures in colorectal cancer and can be used profitably before employing more expensive and complex techniques such as microarray testing.
Anacleto
Charles
C
Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Rossi
Benedito
B
Lopes
Ademar
A
Soares
Fernando A
FA
Rocha
José Cláudio C
JC
Caballero
Otávia
O
Camargo
Anamaria A
AA
Simpson
Andrew J G
AJ
Pena
Sérgio D J
SD
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics; D002352:Carrier Proteins; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics; D019175:DNA Methylation; D064550:Death-Associated Protein Kinases; D019942:Genes, p16; D006801:Humans; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000070957:MutL Protein Homolog 1