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.
Kang
Mi Yeon
MY
Center for Genome Research, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Kangnam-Ku, Seoul, South Korea.
Lee
Bo Bin
BB
Ji
Yong Ick
YI
Jung
Eun Hyun
EH
Chun
Ho-Kyung
HK
Song
Sang Yong
SY
Park
Seong-Eun
SE
Park
Joobae
J
Kim
Duk-Hwan
DH
D000225:Adenine; D003110:Colonic Neoplasms / Q000235:genetics*; D018899:CpG Islands / Q000235:genetics; D003596:Cytosine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D019175:DNA Methylation; D005260:Female; D014644:Genetic Variation / Q000235:genetics; D005838:Genotype; D006147:Guanine; D006239:Haplotypes; D006720:Homozygote; D006801:Humans; D015810:Linkage Disequilibrium / Q000235:genetics; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012004:Rectal Neoplasms / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013941:Thymine; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics*; D000097584:DNA Methyltransferase 3B
D000225:Adenine; D003596:Cytosine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D006147:Guanine; D012333:RNA, Messenger; D013941:Thymine; D025803:Tumor Suppressor Protein p14ARF; D000097584:DNA Methyltransferase 3B; C000619380:DNMT1 protein, human