Rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylation-sensitive high resolution melting (MS-HRM) analysis: application in colorectal cancer.
BACKGROUND
Colorectal cancer is the third most common form of cancer and hypermethylation has been shown to increase the risk of developing this disease. DNA hypermethylation in the A kinase anchor protein 12 (AKAP12/Gravin) promoter region and the accompanied underexpression of it has been noted in a variety of human cancers.


METHODS
We applied methylation-specific high resolution melting (MS-HRM) technology to detect quantitatively A kinase anchor protein 12 (AKAP12/Gravin) methylation in peripheral blood from 100 colorectal cancer patients and 50 healthy volunteers and in 3 colorectal cancer cell lines.


RESULTS
In this study 48 of the 100 colorectal cancer samples (48%) were found to be methylated at the AKAP12 promoter region. AKAP12 methylation was significantly higher in the colorectal cancer samples with differentiation (p=0.03). We also compared the results generated by MS-HRM with a traditional methylation-specific PCR (MSP) assay. We found that intra-assay variability ranged from 6.14 to 9.90% and inter-assay variability ranged from 14.5 to 17.2%. The AKAP12 MS-HRM assay was able to reproducibly detect 1% methylated DNA, whereas the MSP method was unable to detect less than 5% methylation.


CONCLUSIONS
We demonstrate the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods with excellent detection capabilities have many promising applications in the research and diagnosis of colorectal cancer.
Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, PR China.
Weiwei
W
Liu
Ming
M
Guan
Bing
B
Su
Ji
J
Li
Weizhe
W
Ma
Chunfang
C
Liu
Min
M
Li
Yong
Y
Lin
Yuan
Y
Lu
D054758:A Kinase Anchor Proteins / Q000235:genetics*; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D016022:Case-Control Studies; D018797:Cell Cycle Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000097:blood* / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D006801:Humans; D016014:Linear Models; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D013997:Time Factors
D054758:A Kinase Anchor Proteins; C491162:AKAP12 protein, human; D018797:Cell Cycle Proteins; D000077209:Decitabine; D001374:Azacitidine