ANTICANCER RESEARCH 28 : 2807-2812 (2008) Methylenetetrahy drofolate Reductase (MTHFR) and Methionine Synthase Reductase (MTRR) Gene Polymorphisms as Risk Factors for Hepatocellular Carcinoma in a Korean Population SUN YOUNG KWAK 1,2 , UN KYUNG KIM 3, HYO JIN CHO 2, HEE KEUN LEE 3, HYE JIN KIM 2, NAM KEUN KIM 2 and SEONG GYU HWANG 1,2 1Department of Internal Medicine, 2Institute for Clinical Research, College of Medicine, Pochon CHA University, Seongnam; 3Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, South Korea Abstract. Hepatocellular carcinoma (HCC) is the third were increased risk factors for the disease. The MTHFR most frequent cause of cancer death in South Korea, but 1298A>C and the MTRR 66A>G genotypes were associated genetic susceptibility factors of HCC have not been examined with an increased risk of HCC in th is Korean population. extensively. Methylenetetrahydrofolate reductase (MTHFR) Further studies involving larger and varied populations and methionine synthase reductase (MTRR) play an essential could provide a potential tool for cancer risk assessment in role in both DNA synthesis and methylation and patients who are at risk of developing HCC. polymorphisms in the MTHFR gene, 677C>T, 1298A>C and the MTRR gene, 66A>G, are associated with several types of Hepatocellular carcinoma (HCC) is one of the most common malignancy. In this study, the allelic frequencies and malignant tumors with the third highest mortality rate among genotype distribution of three polymorphisms in the MTHFR malignancies in South Korea. According to a statistical report and MTRR genes from 96 hepatocellular carcinoma (HCC) in 2006, it occurred in 33.8/10,000 men and 11.2/10,000 patients and 201 controls were examined to assess the women in South Korea in 2005 (1). association between these polymorphisms and the The causes of HCC are hepatitis B and C infection, development of HCC in th is Korean population. The alcoholic hepatitis and cirrhosis from any other cause and 66AG+GG (G allele-bearing) genotype of the MTRR gene show a marked difference in geographical distribution (2). was significantly associated with an increased risk of HCC Chronic infection with hepatitis B virus (HBV) is the most (odds ratio, OR, 1.687; 95% confidence interval, CI=1.022- important risk factor for HCC in humans and is the 2.787). Moreover, the combination of MTHFR primary cause of this cancer in high-risk areas including 1298AA/MTRR 66AG+GG (OR=1.854, 95% CI=1.005- Africa and Asia. Chronic infection with hepatitis C virus 3.420) and MTHFR 1298AC+CC/MTRR 66AG+GG (HCV) is another risk factor in countries such as the (OR=2.733, 95% CI=1.195-6.249) showed a significant United States and Japan, where the prevalence of HBV association with HCC risk. In the data classified by age and infection is relatively low. etiology, MTRR 66A>G over the age of 65 years , MTHFR It is known that folate and methionine metabolism play 1298A>C under the age of 65 years and the MTRR essential role s in both DNA methylation and synthesis. 66AG+GG genotype in the hepatitis B virus (HBV) patients Folate deficiency is thought to increase the risk of cancer through impaired DNA repair synthesis and disruption of DNA methylation that may lead to protooncogene activation (3, 4). Methylenetetrahydrofolate reductase (MTHFR) and Correspondence to: Nam Keun Kim, Ph.D., Institute for Clinical methionine synthase reductase (MTRR) play important and Research, College of Medicine, Pochon CHA University, Seongnam interrelated roles in folate metabolism. MTHFR catalyzes the 463-712, South Korea. Tel: +82 31 780 5762, Fax: +82 31 780 regulation of cellular methylation through the conversion of 5766, e-mail: [email protected]/Seong and Gyu Hwang, M.D., 5, 10-methylenetetrahydrofolate (THF) to 5-methyl-THF, the Department of Internal Medicine, College of Medicine, Pochon CHA University, Seongnam 463-712, South Korea. Tel: +82 31 780 primary circulating form of folate (5). MTRR is required for 5213, Fax: +82 31 780 5246, e-mail: [email protected] the reductive methylation of cobalamin, an activated cofactor for methionine synthase (MTR), which catalyzes the Key Words: MTHFR, MTRR, polymorphism, risk factors, HCC. remethylation of homocysteine to methionine. 0250-7005/2008 $2.00+.40 2807 ANTICANCER RESEARCH 28 : 2812 (2008) Two polymorphisms in the MTHFR gene have been shown genotypes and HCC. Stratification analysis was used to study the to lead to a reduction of 30-60% in enzyme activity (6, 7). subgroups of age and etiology. The statistical analysis was Functional studies have demonstrated that individuals performed with GraphPad Prism 4 version 4.03 (GraphPad Software, Inc., San Diego, CA, USA) and StatsDirect statistics possessing both mutations showed the lowest enzyme software version 2.4.4 (StatsDirect Ltd., Altrincham, UK). activities (8, 9). Thus, both DNA methylation and DNA synthesis may be affected, interacting with homocysteine, Results vitamin B12 and folate. MTRR 66A>G also contributed to alternation of plasma levels of homocysteine and folate (10) and has been associated with altered blood or plasma levels. Table I shows the allele frequencies and genotype distributions Many epidemiological studies have provided evidence of for MTHFR and MTRR and their ORs and 95% CIs for HCC. association of p olymorphisms in the MTHFR gene, 677C>T The genotype frequencies for all the polymorphisms were in and 1298 A>C, and the MTRR gene, 66A>G, with several accordance with the Hardy-Weinberg equilibrium in the case types of malignancy (11-18), but few studies have reported an and control groups. In the case of the MTRR 66A>G association between the polymorphisms in these genes and the polymorphism as a risk factor for HCC, the frequency of 66AG risk of HCC (17, 18). In this study, the allelic frequencies and and 66GG genotypes were not different between case and genotype distributions of these three polymorphisms in the control groups, but the 66AG + GG genotype was significantly MTHFR and MTRR genes w ere examined to assess the associated with an increased risk of HCC (OR=1.687, 95% association between these polymorphisms and the CI=1.022-2.787). For the MTHFR 677C>T and 1298A>G development of HCC in a Korean population. polymorphisms, no differences in the frequencies of the genotypes were seen in the patients and controls. When the Patients and Methods three polymorphisms were evaluated together, the combination of MTHFR 1298AA/ MTRR 66AG+GG (OR=1.854, 95% Study population. A total of 96 patients (mean age±SD, CI=1.005-3.420) and MTHFR 1298AC+CC/ MTRR 66AG+GG 57.63±11.17; age range, 28 to 82 years) with HCC diagnosed at (OR=2.733, 95% CI=1.195-6.249) showed a significant Bundang CHA Hospital, Pochon CHA University from July 1999 association with HCC risk (Table II). No association was found to June 2004 were enrolled in this study. The control group consisted of 201 individuals (mean age±SD, 53.58±11.47; age in the other combinations. However, the MTHFR 677CC/ MTRR range, 25 to 81 years) who were randomly selected following health 66AG+GG genotype (OR=2.520, 95% CI=0.973-6.529) screening to exclude those with a history of cancer and other severe showed a higher risk than the MTHFR 677CT+TT/ MTRR diseases. The study was approved by the Institutional Review Board 66AG+GG genotype (OR=1.780, 95% CI=0.792-4.004), (IRB) of Pochon CHA University, South Korea. although the number of mutant alleles in the MTHFR 677CT+TT/ MTRR 66AG+GG genotype was twice as high as DNA analysis . Total genomic DNA was prepared from whole blood in the MTHFR 677CC/ MTRR 66AG+GG genotype. after lysis of red blood cells (19). The areas spanning the When the data were stratified by age (younger than 65 years, polymorphic sites of MTHFR 677C>T, 1298A>C and MTRR 66A>G 65 years and over), the frequencies of MTRR 66AG (OR=3.852, were amplified by polymerase chain reaction (PCR) from genomic DNA using the primers and reaction conditions described previously 95% CI=1.226-12.10) and 66AG+GG (OR=4.267, 95% (6, 20). The polymorphisms were identified following digestion of CI=1.501-22.44) were statistically significant in the group of the amplified DNA with the endonucleases, Hin fI, Fnu 4HI and AfI III over the age of 65 (Table III). Moreover, the combined effect of for the MTHFR 677, 1298 and MTRR 66 genes, respectively. The the MTHFR 1298AA/ MTRR 66AG+GG genotype showed a alleles of each polymorphic site were classified depending on the six-fold higher risk compared with the MTHFR 1298AA/ MTRR presence or absence of a restriction enzyme cutting site. 66AA genotype. In addition, the MTHFR 1298AC genotype Amplification success was monitored by 2.5% agarose (OR=2.198, 95% CI=1.160-4.164) and the combined MTHFR electrophoresis. For the nucleotide 677 of MTHFR , an undigested MTRR PCR product (203 bp) indicated a homozygous wild-type, three 1298AC+CC/ 66AG+GG genotype (OR=2.771, 95% bands of 203, 173 and 30 bp indicated the heterozygous, and two CI=1.802-7.093) were associated with increased risk of HCC in bands of 173 and 30 bp indicated the homozygous genotype. For the group under 65 years of age . We also examined the impact of nucleotide 1298 of MTHFR , a single band of 138 bp indicated a polymorphisms in the subgroups by the clinical causes of HCC. wild-type and two fragments of 119 and 19 bp indicated the As shown in Table IV, MTRR 66AG+GG genotype in the HBV homozygous genotype.