Association of interindividual differences in p14ARF promoter methylation with single nucleotide polymorphism in primary colorectal cancer.

Background: CpG island hypermethylation has been reported at the promoter region of many tumor suppressor genes in colorectal cancers. However, there are significant interindividual differences in the degree of DNA methylation in colorectal cancers. The objective of the current study was to understand whether single nucleotide polymorphisms (SNPs) around the promoter of a gene are implicated in the interindividual differences of CpG island hypermethylation.

Methods: Promoter methylation of the p14(ARF) gene and messenger RNA (mRNA) expression levels of p14(ARF), DNA methyltransferase 1 (DNMT1), and DNMT3b were investigated by using methylation-specific polymerase chain reaction (PCR) analysis (MSP) and quantitative real-time PCR analysis in fresh tissues from 188 patients with colorectal cancer. SNPs around the p14(ARF) promoter were genotyped in DNA from peripheral blood lymphocytes in 300 healthy individuals and in 188 patients with colorectal cancer by using matrix-assisted laser desorption/ionization mass spectrometry.

Results: p14(ARF) methylation was present in 61 of 188 colorectal cancers (32%). Fourteen SNPs among the 20 candidate SNPs were identified as monomorphic in the Korean population studied. Two individual SNPs (-4256 thymine to cytosine [T-->C] and -1477 guanine to adenine [G-->A]), which were in strong linkage disequilibrium (|D'|=0.99; correlation coefficient [r(2)]=0.95), were associated significantly with p14(ARF) methylation. Patients who had the CC variant at the-4256 locus or the AA variant at the -1477 locus had 2.42 times (95% confidence interval [95% CI], 1.07-5.46; P = .03) and 2.47 times (95% CI, 1.09-5.56; P= .03) greater risk of p14(ARF) methylation than patients who had the TT or GG homozygote, respectively, after adjusting for mRNA levels of DNMTs. Four major haplotypes were identified within a block (-4256 T-->C, -3631 T-->C, -1477 G-->A, and +20,188 T-->C). p14(ARF) promoter methylation also was associated significantly with the CCAT haplotype (odds ratio [OR], 8.31; 95% CI, 2.43-28.41; P= .0007) and the CTAC haplotype (OR, 9.71; 95% CI, 1.09-86.24; P= .04).

Conclusions: The current results suggested that SNPs around the p14(ARF) promoter region may be responsible for the interindividual susceptibility to p14(ARF) promoter methylation among individuals with colorectal cancer.