ANTICANCER RESEARCH 28 : 2807-2812 (2008)

Methylenetetrahy drofolate Reductase (MTHFR) and Synthase Reductase (MTRR) 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 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 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 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 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. For nucleotide 66 of MTRR , an undigested subgroup was a risk for the disease. 150 bp PCR product indicated a homozygous wild-type, three bands of 150, 123 and 27 bp indicated the heterozygous, and two bands of 123 and 27 bp indicated the homozygous genotype. Discussion Statistical analysis. Allele and genotype frequencies between the case and control groups were compared using the Chi-square test. When the genotype frequencies of the three Odds ratios (ORs) and 95% confidence interval (95% CI) were polymorphisms in the MTHFR and MTRR genes were used as a measure of the strength of the association between the compared between the case and control groups, the MTRR

2808 Kwak et al : MTHFR and MTRR Polymorphisms and HCC Risk

Table I. Genotype and allele frequencies of MTHFR and MTRR gene Table III. Odds ratios (OR) and 95% confidence interval (CI) of three polymorphisms in HCC cases and controls. polymorphisms in controls and HCC cases classified by age.

Genotype Control (%) Cases (%) AOR (95% CI) a Genotype Control (%) Cases (%) OR (95% CI) P

MTHFR 677C>T ≥65 Years 677CC 64 (31.84) 32 (33.33) 1.0 (-) MTRR 677CT 106 (52.74) 46 (47.92) 0.836 (0.477-1.464) 66AA 24 (70.59) 9 (36.00) 1.0 (-) - 677TT 31 (15.42) 18 (18.75) 1.154 (0.553-2.407) 66AG 9 (26.47) 13 (48.00) 3.852 (1.226-12.10) 0.026 677CT+TT 137 (68.16) 64 (66.67) 0.916 (0.541-1.554) 66AG+GG 10 (29.41) 16 (64.00) 4.267 (1.419-12.83) 0.016 T allele 0.418 0.427 MTHFR 1298/ MTHFR 1298A>C MTRR 66 1298AA 155 (77.11) 67 (69.79) 1.0 (-) AA/AA 16 (47.06) 6 (24.00) 1.0 (-) - 1298AC 41 (20.40) 28 (29.17) 1.502 (0.851-2.651) AA/AG+GG 6 (17.65) 13 (48.00) 5.778 (1.501-22.24) 0.013 1298CC 5 (2.49) 1 (1.04) 0.455 (0.050-4.180) 1298AC+CC 46 (22.89) 29 (30.21) 1.389 (0.797-2.420) <65 Years C allele 0.127 0.156 MTHFR MTRR 66A>G 1298AA 133 (79.64) 48 (67.61) 1.0 (-) - 66AA 111 (55.22) 40 (42.55) 1.0 (-) 1298AC 29 (17.37) 23 (32.39) 2.198 (1.160-4.164) 0.017 66AG 78 (38.81) 45 (47.87) 1.612 (0.956-2.719) MTHFR 1298/ 66GG 12 (5.97) 9 (9.57) 2.118 (0.823-5.454) MTRR 66 66AG+GG 90 (44.78) 54 (57.45) 1.687 (1.022-2.787) b AA/AA 67 (40.12) 19 (26.76) 1.0 (-) - G allele 0.254 0.335 AC+CC/AG+GG 14 (8.38) 11 (15.49) 2.771 (1.802-7.093) 0.041 aAdjusted odds ratio (adjusted by age and gender); bp<0.05.

Table IV. Odds ratios (OR) and 95% confidence interval (CI) of three polymorphisms in controls and HBV subgroup patients. Table II. Combination analysis of MTHFR 677C>T, 1298A>C and MTRR 66A>G polymorphisms in HCC cases and controls. Genotype Control HBV OR (95% CI)

a Genotype Control (%) Cases (%) AOR (95% CI) MTHFR 677C>T 677CC 64 (31.84) 23 (34.85) 1.0 (-) MTHFR 677/1298 677CT 106 (52.74) 29 (43.94) 0.761 (0.406-1.428) CC/AA 39 (19.40) 19 (19.79) 1.0 (-) 677TT 31 (15.42) 14 (21.21) 1.257 (0.570-2.772) CC/AC 20 (9.95) 12 (12.50) 1.197 (0.476-3.009) 677CT+TT 137 (68.16) 43 (65.15) 0.873 (0.486-1.571) CC/CC 5 (2.49) 1 (1.04) 0.338 (0.028-4.081) MTHFR 1298A>C CT/AA 85 (42.29) 30 (31.25) 0.696 (0.341-1.421) 1298AA 155 (77.11) 52 (78.79) 1.0 (-) CT/AC 21 (10.45) 16 (16.67) 1.361 (0.564-3.285) 1298AC 41 (20.40) 14 (21.21) 1.018 (0.514-2.016) TT/AA 31 (15.42) 18 (18.75) 1.172 (0.514-2.672) 1298CC 5 (2.49) 0 (0) 0.269 (0.015-4.956) MTHFR 677/ 1298AC+CC 46 (22.89) 14 (21.21) 0.907 (0.462-1.783) MTRR 66 MTRR 66A>G CC/AA 33 (16.42) 11 (11.46) 1.0 (-) 66AA 111 (52.22) 26 (39.39) 1.0 (-) CC/AG+GG 31 (15.42) 20 (20.83) 2.520 (0.973-6.529) 66AG 78 (38.81) 31 (46.97) 1.697 (0.935-3.081) CT+TT/AA 78 (38.81) 29 (30.21) 1.142 (0.507-2.572) 66GG 12 (5.97) 7 (10.61) 2.490 (0.893-6.945) CT+TT/AG+GG 59 (29.35) 34 (35.42) 1.780 (0.792-4.004) 66AG+GG 90 (44.78) 38 (57.58) 1.803 (1.018-3.191) a MTHFR 1298/ MTRR 66 HBV; Hepatitis B virus; ap<0.05. AA/AA 83 (41.29) 27 (28.13) 1.0 (-) AA/AG+GG 72 (35.82) 38 (39.58) 1.854 (1.005-3.420) b AC+CC/AA 28 (13.93) 13 (13.54) 1.428 (0.645-3.164) AC+CC/AG+GG 18 (8.96) 16 (16.67) 2.733 (1.195-6.249) b risk factors for HCC. Considering the fact that the mean aAdjusted odds ratio (adjusted by age and gender); bp<0.05. age of the patients in this study was 57.63 years and that 71 out of 96 (73.96%) were under 65 years old, the MTHFR 1298A>C variant in fact has more effect on the occurrence of HCC than does MTRR 66A>G. In addition, 66AG+GG, MTHFR 1298AA/ MTRR 66AG+GG and the combinations of these polymorphisms also showed an MTHFR 1298AC+CC/ MTRR 66AG+GG genotypes were association in each age group. Interestingly, the MTHFR associated with HCC risk in this study. In the data 1298AA/ MTRR 66AG+GG genotype was associated with classified by age, MTRR 66A>G over the age of 65 and the disease over the age of 65 and the MTHFR MTHFR 1298A>C under the age of 65 were an increased 1298AC+CC/ MTRR 66AG+GG genotype showed an

2809 ANTICANCER RESEARCH 28 : 2812 (2008) increase for HCC under the age of 65. This suggested that 3 Duthie SJ: Folic acid deficiency and cancer: mechanisms of these polymorphisms have different effects on the DNA instability. Br Med Bull 55 : 578-592, 1999. occurrence of the disease at different ages. 4 Wei Q, Shen H, Wang LE, Duphorne, CM, Pillow PC, Guo Z, Qiao Y and Spitz MR: Association between low dietary folate MTRR The 66A>G genotype in relation to the risk of intake and suboptimal cellular DNA repair capacity. Cancer acute lymphoblastic leukemia, non-Hodgkin’s lymphoma, Epidemiol Biomarkers Prev 12 : 963-969, 2003. gastric cardia, esophageal and colorectal cancer has been 5 Bailey LB and Gregory JF III: Polymorphisms of methylene- investigated in a few studies, most of which showed no tetrahydrofolate reductase and other : metabolic association except for increased esophageal cancer risk significance, risks and impact on folate requirement. J Nutr 129 : among those who did not consume alcohol, and for 919-922, 1999. colorectal cancer in Japanese patients (13, 21, 22). Since 6 Frosst P, Blom HJ, Milos R, Goyette, P, Sheppard CA, Matthews RG, Boers GJH, den Heijer M, Kluijtmans LAJ, van den Heuve the major cause of HCC in Korea was hepatitis B virus LP and Rozen R: A candidate genetic risk factor for vascular (HBV) infection, we examined the correlation of three disease: a common mutation in methylenetetrahydrofolate polymorphisms and HCC in patients caused by chronic reductase. Nat Genet 10 : 111-113, 1995. HBV and found the association with the MTRR 66AG+GG 7 van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels genotype. Moreover, the results of previous studies of the FJ, Eskes TK, van den Heuvel LP and Blom HJ: A second effect of the allelic variants of this polymorphism on common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J homocysteine status have not been consistent (12, 23-26). Hum Genet 62 : 1044-1051, 1998. The correlation of homocysteine concentration with this 8 Chango A, Boisson F, Barbe F, Quilliot D, Droesch S, Pfister M, polymorphism and the risk of HCC was also investigated Fillon-Emery N, Lambert D, Frémont S, Rosenblatt DS and in the present study, but there was no difference in Nicolas JP: The effect of 677C>T and 1298A>C mutations on homocysteine concentration among the MTRR 66A>G plasma homocysteine and 5,10-methylenetetrahydrofolate genotypes, which was consistent with the finding of reductase activity in healthy subjects. Br J Nutr 83 : 593-596, 2000. Vaughn et al. (26) (data not shown). 9 Weisberg I, Tran P, Christensen B, Sibani S and Rozen R: A second genetic polymorphism in methylenetetrahydrofolate MTHFR The 677C>T polymorphism has been reported to reductase ( MTHFR ) associated with decreased enzyme activity. significantly reduce the risk of colorectal cancer (14, 27, 28) Mol Genet Metabol 64 : 169-172, 1998. and acute lymphocytic cancer (15, 29-31). In contrast, other 10 Matsuo K, Suzuki R, Hamajima N, Ogura M, Kagami Y, Taji H, studies reported elevated risks of esophageal (32), gastric (33, Kondoh E, Maeda S, Asakura S, Kaba S, Nakamura S, Seto M, 34), ovarian (35), cervical (36), bladder (37) and lung cancer Morishima Y and Tajima K: Association between polymorphisms (38) as well as HCC (14). Moreover, Saffroy et al. (39) of folate- and methionine-metabolizing enzymes and susceptibility to malignant lymphoma. Blood 97 : 3205-3209, 2001. reported that the MTHFR 677CC genotype was significantly 11 Ko KH, Kim NK, Yim DJ, Hong SP, Park PW, Rim KS, Kim S higher in patients who had developed HCC from alcoholic and Hwang SG: Polymorphisms of 5,10-methylenetetra- cirrhosis. But, no statistically significant associations between hy drofolate reductase ( MTHFR C677T) and thymidylate synthase this variant and HCC were found in the present study. enhancer region ( TSER ) as a risk factor of cholangiocarcinoma In summary, we found that the MTHFR 1298A>C and the in a Korean population. Anticancer Res 26 : 4229-4233, 2006. MTRR 66A>G genotypes were associated with an increased 12 Gaughan DJ, Kluijtmans LA, Barbaux S, McMaster D, Young risk of HCC in th is Korean population. Further studies IS, Yarnell JWG, Evans A and Whitehead AS: The methionine synthase reductase ( MTRR ) A66G polymorphism is a novel involving larger and varied populations would be of a great genetic determinant of plasma homocysteine concentrations. value to firmly validate the association with HCC and to Atherosclerosis 157 : 451-456, 2001. provide a potential tool for cancer risk assessment in patients 13 Matsuo K, Hamajima N, Hirai T, Kato T, Inoue M, Takezaki T who are at risk of developing HCC. and Tajima K: Methionine synthase reductase gene A66G polymorphism is associated with risk of colorectal cancer. Asian Acknowledgements Pac J Cancer Prev 3: 353-359, 2002. 14 Slattery ML, Potter JD, Samowitz W, Schaffer D and Leppert M: This work was supported by the Korea Research Foundation Grant Methylenetetrahydrofolate reductase, diet, and risk of colon funded by the Korean Government (MOEHRD) (KRF-2006-331- cancer. Cancer Epidemiol Biomarkers Prev 8: 513-518, 1999. E00304) and the Korean Science and Engineering Foundation 15 Skibola CF, Smith MT, Kane E, Roman E, Rollinson S, (KOSEF), funded by the Korean government (MOST; R01-2007- Cartwright RA and Morgan G: Polymorphisms in the 000-20459-0), Daejeon, Republic of Korea. methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults. Proc Natl Acad Sci References USA 96 : 12810-12815, 1999. 16 Keku T, Millikan R, Worley K, Winkels S, Eaton A, Biscocho L, 1 Annual Report for the Cause of Mortality in 2005, Korea Martin C and Sandler R: 5,10- Methylenetetrahydrofolate National Statistical Office. Seoul, 2006. reductase codon 677 and 1298 polymorphisms and colon cancer 2 Parkin DM: Global cancer statistics in the year 2000. Lancet in African-Americans and whites. Cancer Epidemiol Biomarkers Oncol 2: 533-543, 2001. Prev 11 : 1611-1621, 2002.

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Cancer Res 57 : 1098- Revised July 19, 2008 1102, 1997. Accepted August 5, 2008

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