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Association between polymorphisms of folate-metabolizing enzymes and hematological malignancies
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Leukemia Research 33 (2009) 82–87
Association between polymorphisms of folate-metabolizing enzymes and hematological malignancies Hee Nam Kim a , Yeo-Kyeoung Kim b , Il-Kwon Lee a , Deok-Hwan Yang b , Je-Jung Lee b , Min-Ho Shin c , Kyeong-Soo Park d , Jin-Su Choi c , Moo Rim Park e , Deog Yeon Jo f , Jong Ho Won g , Jae-Yong Kwak h , Hyeoung-Joon Kim a,b,∗ a
Genome Research Center for Hematopoietic Diseases, Chonnam National University Hwasun Hospital, Jeollanamdo,Republic of Korea b Department of Hematology/Oncology, Chonnam National University Hwasun Hospital, Jeollanamdo, Republic of Korea c Department of Preventive Medicine, Chonnam National University Medical School, Gwangju, Republic of Korea d Department of Preventive Medicine, College of Medicine, Seonam University, Namwon, Republic of Korea e Department of Internal Medicine, Wonkwang University School of Medicine, Iksan, Republic of Korea f Division of Hematology/Oncology, Department of Internal Medicine, College of Medicine, Chungnam National University, Daejon, Republic of Korea g Department of Internal Medicine, Soonchunhyang University, College of Medicine, Seoul, Republic of Korea h Department of Internal Medicine, Chonbuk National University Medical School, Jeonju, Republic of Korea Received 8 April 2008; received in revised form 25 July 2008; accepted 25 July 2008 Available online 6 September 2008
Abstract Several genetic polymorphisms in the genes coding folate-metabolizing enzymes have been associated with susceptibility to hematology malignancies. We conducted a Korean population-based case–control study to examine the relationship between the polymorphisms of folatemetabolizing enzymes and the risk of AML (acute myelogenous leukemia), CML (chronic myelogenous leukemia), MDS (myelodyspastic syndrome), and ALL (acute lymphoblastc leukemia). The MTHFR 677TT genotype was associated with an increased risk for ALL (odds ratios (OR) = 1.77; 95% confidence intervals (CI) = 1.02–3.09, p = .044). The MTRR 66 AG genotype was associated with an increased risk for MDS (OR = 1.59; 1.06–2.38, p = .026) and the MTRR 66 GG genotype was associated with increased risk for AML (OR = 1.51; 1.03–2.23, p = .037). The TYMS 2R3R genotype was associated with a decreased risk for AML (OR = 0.76; 0.60–0.96, p = .022). The TYMS hap3 (2R-6bp) and hap4 (2R-0bp) were associated with decreased risk (OR = 0.69; 0.53–0.90, p = .006) and increased risk (OR = 1.65; 1.20–2.27, p = .002), respectively for AML. Hap C (677T1298A) was associated with an increased risk (OR = 1.40; 1.02–1.92, p = .04) for ALL. The risk for ALL appears to be associated with the MTHFR 677 polymorphism. The results are supportive of a risk modification by folate polymorphisms in several hematologic malignancies in Korea. The pattern of results suggests that MDS was associated with the DNA methylation status and the risk for AML was associated with both the DNA synthesis and DNA methylation status. © 2008 Elsevier Ltd. All rights reserved. Keywords: AML; CML; MDS; ALL; Polymorphism; Folate-metabolizing enzymes; Association
1. Introduction ∗
Corresponding author at: Department of Hematology/Oncology, Chonnam National University Hwasun Hospital, 160 Ilsimri, Hwasun-gun, Jeollanam-do 519-809, Republic of Korea. Tel.: +82 61 379 7637; fax: +82 61 379 7736. E-mail address: [email protected] (H.-J. Kim). 0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.07.026
Folic acid and methionine metabolism play an essential role in both DNA synthesis and cellular methylation reactions. At least 30 enzymes have been identified in the folate pathway [1]. Several functional polymorphisms in the genes encoding these enzymes have been associated with various
H.N. Kim et al. / Leukemia Research 33 (2009) 82–87
malignancies, including adult and childhood acute lymphocytic leukemia (ALL) [2–4], AML [5,6] and CML [5,6]. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methyl-tetrahydrofolate (THF) to 5methyl-THF, the major form of circulating folate, which acts as the methyl donor for the transformation of homocysteine to methionine. Two known polymorphisms in the MTHFR gene, C677T and A1298C, lead to a 30–60% reduction in enzyme activity [1,7,8]. Methionine synthase (MS) catalyzes the transfer of the methyl group from 5-MeTHF to homocysteine, producing tetrahydrofolate and methionine [9]. The MTR A2756G polymorphism is thought to reduce enzyme activity [10] with the GG genotype and to increase homocysteine levels and DNA hypomethylation with the AA genotype [11]. Methionine synthase reductase (MTRR) is required for the reductive methylation of cobalamin, which is the activated cofactor for MS in the remethylation of homocysteine to methionine. Thymidylate synthase (TS) catalyzes the conversion of dUMP to dTMP using 5,10-methylenetetrahydrofolate during DNA synthesis. One polymorphism is a unique variable nucleotide tandem repeat (VNTR) sequence, with either two or three 28-bp repeats in the TYMS 5 UTR [12]. These polymorphic tandem repeats influence protein expression in cancer cells [13,14]. A 6-bp deletion/insertion (0-bp/6-bp), at 1494 in the 3 -UTR of the TYMS, is thought to influence TYMS mRNA stability in vitro and its expression in vivo [15,16]. Many studies have reported the association between the polymorphisms in these genes and risk for ALL [2,4,17–20]. However, few studies have been reported for AML and CML and there have been no reports on association with MDS. Besides, the results are inconsistent by different populations. In addition, there is no prior report on MTRR A66G, TYMS 2R → 3R and the 6-bp insertion/deletion and AML, CML, MDS or ALL risk. Therefore, we conducted a Korean largescale population-based, case–control study to examine the relationship between the MTHFR C677T and A1298C, MTR A2756G, MTRR A66G, TYMS 2R → 3R and 6-bp insertion/deletion genotypes and the risk for AML, CML, MDS, and ALL.
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a defined geographical area served by the hospital, Jeollanam-do province, Korea, we estimate that the case is approximately 70% of all leukemia cases in one area. The control group (n = 1700) consisted of participants in the Thyroid Disease Prevalence Study. This study was performed in the Yeonggwang and Muan counties of Jeollanam-do Province and Namwon city of Jeollabuk-do province, Korea, from July 2004 to January 2006. There were 4018 subjects, mean age 50.6 ± 14.6 years (range 20–74 years), who were randomly selected by a 5-year age strata and gender. Among the total number of patients, 3486 were eligible for the study. Among the eligible patients, 1700 (48.8% of the eligible subjects; 821 men and 879 women), mean age 52.2 ± 14.3 years, underwent clinical examinations. At the time of their peripheral blood collection, all cases and control subjects provided their informed consent to participate in this study. The Institutional Review Board of the Chonnam National University Hwasun Hospital in Hwasun, Korea approved this study. 2.2. Genotyping Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s protocol. The genotyping protocol for MTHFR C677T polymorphism detection was adapted from Frosst et al. [7]. After HinfI (Takara, Tokyo, Japan) restriction enzyme digestion, the samples were run on a 10% polyacrylamide gel (19:1) using the MADGE system (MadgeBio, Grantham and Southampton, UK). Genotyping for MTHFR A1298C was performed by allelic discrimination, using dual-labeled probes containing locked nucleic acids (LNA), in a real-time PCR assay. PCR primers and LNA probes were designed and synthesized by IDT (Coralville, IA, USA) [21]. Genotyping for the detection of the MTR A2756G polymorphism was performed using specific oligonucleotide primers [10]. Digestion products with HaeIII (Takara) were visualized after electrophoresis in a 10% polyacrylamide gel (19:1) using the MADGE system. MTRR A66G genotyping was performed by Pyrosequencing as simplex assays. PCR primers and sequencing primer were designed by the Pyrosequencing SNP Primer Design Software (version v1.0.6) [21]. Genotyping for 28-bp tandem repeat (2R → 3R) genotypes in the 5 -UTR of the TYMS gene was conducted using a protocol described by Horie et al. [13]. The 3 -UTR 6-bp deletion/insertion genotyping was performed using a 6% PAGE electrophoresis with the previously described primers [15].
2. Materials and methods
2.3. Statistical analysis
2.1. Study population
The statistical significance of the differences between the patient and control groups was estimated by logistic regression analysis. Adjusted odds ratios (ORs) were calculated with a logistic regression model that controlled for gender and age and are reported at 95% confidence intervals (CI). Subjects with the wild type genotypes (MTHFR 677CC, MTHFR 1298AA, MTR 2756AA, MTRR 66AA, TYMS 3R3R, and TYMS 0-bp/0-bp genotype) were considered to be at baseline risk. The expected frequency of control genotypes was evaluated by the Hardy–Weinberg equilibrium test. Haplotype frequencies for MTHFR and TYMS were estimated using the SNPAnalyzer program (Istech, Ltd., Korea) based on the expectation–maximization algorithm. All analyses were performed using SPSS software version 11.0 (SPSS, Chicago, IL, USA).
The patients enrolled in the study were adults aged ≥15 years, diagnosed with AML, CML, MDS and ALL at Chonnam National University Hospital between January 1997 and December 2006. The cases consisted of 389 AML (211 men and 184 women; mean aged 49.0 ± 17.4 years; range: 15–85 years), 149 CML (94 men and 55 women; mean aged 50.4 ± 17.1 years; range: 16–87 years), 109 MDS (65 men and 44 women; mean aged 57.8 ± 14.5 years; range: 15–81 years) and 110 ALL (61 men and 47 women; mean aged 40.9 ± 18.2 years; range: 15–77 years). The cases were most of the cases that come to the hospital without regard to sociologic variables. Since our hospital is tertiary national hospital in
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Table 1 Distribution of polymorphisms involved in the folate-metabolizing pathway in patients and controls Genotype MTHFR 677 CC CT TT
Control n (%)
AML n (%)
ORa (95% CI)
p
CML n (%)
ORa (95% CI)
p
MDS n (%)
ORa (95% CI)
ALL n (%)
ORa (95% CI)
p
121 (31) 202 (51) 76 (19)
1 1.02 (0.80–1.32) 1.1 (0.80–1.54)
.85 .53
54 (36) 72 (47) 26 (17)
1 0.81 (0.55–1.17) 0.90 (0.55–1.47)
.26 .67
34 (31) 58 (53) 17 (16)
1 1.04 (0.67–1.61) 0.90 (0.49–1.63)
.30 .37
29 (27) 51 (48) 27 (25)
1 1.11 (0.68–1.79) 1.77 (1.02–3.09)
.67 .044
MTHFR 1298 AA 1147 (68) AC 500 (29) CC 53 (3)
275 (69) 112 (28) 12 (3)
1 0.93 (0.72–1.19) 0.96 (0.51–1.84)
.55 .91
97 (64) 49 (33) 5 (3)
1 1.15 (0.80–1.66) 1.11 (0.43–2.85)
.44 .83
73 (67) 32 (29) 4 (4)
1 1.03 (0.67–1.58) 1.12 (0.39–3.20)
.90 .84
67 (63) 38 (36) 1 (1)
1 1.24 (0.81–1.89) 0.31 (0.04–2.32)
.32 .25
MTR 2756 AA AG GG
1282 (75) 392 (23) 26 (2)
308 (77) 86 (22) 5 (1)
1 0.90 (0.70–1.20) 0.66 (0.23–1.92)
.46 .44
107 (71) 43 (28) 2 (1)
1 1.30 (0.89–1.90) 0.92 (0.22–4.00)
.18 .92
80 (73) 26 (24) 3 (3)
1 1.06 (0.67–1.69) 1.74 (0.36–5.94)
.79 .37
77 (71) 28 (26) 3 (3)
1 1.24 (0.78–1.96) 2.42 (0.70–8.39)
.36 .17
MTRR 66 AA AG GG
857 (50) 718 (43) 125 (7)
195 (49) 162 (41) 42 (10)
1 0.97 (0.76–1.22) 1.51 (1.03–2.23)
.81 .037
73 (48) 68 (45) 11 (7)
1 1.15 (0.81–1.64) 1.04 (0.54–2.03)
.56 .94
44 (40) 58 (53) 7 (7)
1 1.59 (1.06–2.38) 0.74 (0.45–2.34)
58 (54) .026 34 (32) .95 15 (14)
1 0.69 (0.44–1.08) 1.72 (0.93–3.26)
.09 .087
TYMSb 2R - > 3R 3R3R 1129 (67) 2R3R 520 (31) 2R2R 48 (3)
274 (69) 105 (27) 16 (4)
1 0.84 (0.66–1.09) 1.28 (0.69–2.37)
.17 .43
103 (69) 39 (26) 7 (5)
1 0.82 (0.56–1.22) 1.72 (0.75–3.92)
.33 .20
73 (68) 32 (30) 2 (2)
1 0.99 (0.64–1.53) 0.60 (0.14–2.52)
.97 .48
71 (67) 31 (29) 4 (4)
1 0.93 (0.60–1.45) 1.52 (0.52–4.46)
.74 .45
TYMS 6bp 0bp/0bp 0bp/6bp 6bp/6bp
220 (55) 143 (36) 34 (9)
1 0.76 (0.60–0.96) 0.79 (0.52–1.19)
.022 .26
80 (53) 59 (39) 11 (7)
1 0.85 (0.59–1.21) 0.77 (0.40–1.49)
.36 .44
56 (51) 41 (38) 12 (11)
1 0.85 (0.56–1.29) 1.25 (0.65–2.40)
.45 .51
60 (56) 42 (39) 5 (5)
1 0.79 (0.52–1.20) 0.43 (0.17–1.11)
.26 .08
a b
824 (49) 723 (43) 151 (90)
ORs and 95% CIs were estimated using multiple logistic regression and adjusted for sex and age Unclassifiable cases (n = 32) were excluded in subgroup analysis. The rare TYMS genotypes (2R5R, 3R4R, 3R5R and 3R6R) were excluded in TYMS genotype analysis.
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540 (32) 863 (51) 297 (17)
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a ORs and 95% CIs were estimated using multiple logistic regression and adjusted for sex and age.
b The rare TYMS genotypes (2R5R, 3R4R, 3R5R and 3R6R) were excluded in TYMS genotype analysis.
1 0.85 (0.60–1.21) 0.90 (0.62–1.30) 1.11 (0.64–1.93)
0.366 0.559 0.713
141 (66) 37 (17) 27 (13) 9 (4)
1 1.05 (0.72–1.53) 0.95 (0.62–1.46) 0.883 (0.44–2.46)
0.797 0.827 0.726
150 (69) 27 (13) 24 (11) 15 (7)
1 0.71 (0.46–1.09) 0.76 (0.48–1.19) 1.60 (0.90–2.85)
0.113 0.222 0.108
3. Results
200 (68) 44 (15) 36 (12) 16 (5) 0.735 0.006 0.002 522 (66) 128 (16) 77 (10) 59 (8) 2215 (65) 562 (17) 462 (13) 153 (5)
1 0.96 (0.78–1.20) 0.69 (0.53–0.90) 1.65 (1.20–2.27)
0.264 0.040 1 1.26 (0.84–1.90) 1.40 (1.02–1.92) ND 70 (32) 41 (19) 105 (49) ND 0.918 0.835 0.924 1 1.02 (0.69–1.51) 0.97 (0.71–1.31) 1.11 (0.14–8.71) 87 (40) 39 (18) 91 (42) 1 (0.5) 0.526 0.769 ND 1 1.11 (0.80–1.55) 0.96 (0.74–1.26) ND 120 (40) 59 (19) 123 (41) ND
OR (95% CI) ALL n (%) p OR (95% CI) MDS n (%) p OR (95% CI) CML n (%) p
0.564 0.833 0.179 1 0.94 (0.74–1.18) 1.02 (0.86–1.21) 2.08 (0.72–6.07) 313 (39) 131 (16) 349 (44) 5 (0.6) 1348 (40) 595 (18) 1446 (42) 11 (0.3)
1298 A > C A C A C Del 6 bp 0 bp 6 bp 6 bp 0 bp 677 C > T C C T T 28 bp repeat 3R 3R 2R 2R
ORa (95% CI) AML n (%) Control n (%)
Table 2 Distribution of haplotype frequencies for MTHFR and TYMS in patients and controls
MTHFR Hap A Hap B Hap C Hap D TYMSb Hap 1 Hap 2 Hap 3 Hap 4
p
H.N. Kim et al. / Leukemia Research 33 (2009) 82–87
The distribution of the MTHFR, MTR, MTRR, and TYMS polymorphisms in the controls was in Hardy–Weinberg equilibrium (MTHFR 677 and 1298; p = 0.132 and 0.867, MTR 2756; p = 0.522, TYMS 66; p = 0.127, TYMS 2R3R; p = 0.196, TYMS 6 bp; p = 0.671), and not significantly different in the comparisons between men and women (data not shown). The genotype distributions among all cases and controls for the MTHFR C677T, A1298C, MTR A2756G, MTRR A66G, TYMS 2R → 3R, and 6-bp insertion/deletion polymorphisms are shown in Table 1. For the TYMS genotype, we observed rare TYMS alleles with 4R (four tandem repeats), 5R and 6R in addition to the major 2R and 3R alleles. The frequencies of TYMS 2R4R, 3R4R and 3R5R were 0.15%, 0.45% and 0.08%, respectively, in the controls; the frequencies of TYMS 3R4R and 3R5R were 0.26% and 0.78%, respectively, in all cases. The three major TYMS genotypes (3R3R, 2R3R, 2R2R) were used in this analysis and the rare TYMS genotypes (2R5R, 3R4R, 3R5R, 3R6R) were excluded because of the extremely low frequencies (<1%). The MTHFR 677TT genotype was significantly associated with an increased risk for ALL (OR = 1.77; 95% CI = 1.02–3.09), but not for AML, CML and MDS. The MTRR 66AG was associated with an increased risk for MDS (OR = 1.59; 95% CI = 1.06–2.38), but not for AML, CML and ALL. The MTRR 66GG genotype was associated with an increased risk for AML (OR = 1.51; 95% CI = 1.03–2.23), but not for CML and MDS. A marginal increased risk was also observed for ALL and the MTRR 66GG genotype (OR = 1.77; 95% CI = 0.96–3.26). The TYMS 0-bp/6-bp was associated with a decreased risk for only AML (OR = 0.76; 95% CI = 0.60–0.96). However, the MTHFR 677CT and TYMS 6-bp/6-bp genotype had no affect on the risk for AML, CML, MDS, and ALL. There was no significant association of MTHFR A1298C, MTR A2756G and TYMS 2R3R with AML, CML, MDS, and ALL. Although the OR of the MTR 2756 GG genotype for AML, MDS and ALL was 0.66, 1.74 and 2.43, respectively, and the OR of the TYMS 2R2R genotype for CML, MDS and ALL was 1.72, 0.60 and 1.52, none of the results reached statistical significance. The distribution of the haplotype frequencies of MTHFR and TYMS for the patients and controls are shown in Table 2. The TYMS hap3 (2R-6bp) and hap4 (2R-0bp) were associated with a decreased risk (OR = 0.69; 0.53–0.90, p = .006) and increased risk (OR = 1.65; 1.20–2.27, p = .002), respectively, for AML. The MTHFR Hap C (677T-1298A) was associated with an increased risk (OR = 1.40; 1.02–1.92, p = .04) for ALL.
4. Discussion In this population-based, case–control study, we investigated the association between folate-metabolizing gene polymorphisms and susceptibility to AML, CML, MDS, and ALL in the Korean population. We found an association
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between the MTHFR 677TT genotype and ALL, the MTRR 66 AG genotype and MDS, the MTRR 66 GG genotype and AML, and the TYMS 2R3R genotype and AML. As shown Table 1, the frequencies of the MTHFR C677T and A1298C, MTR A2756G, MTRR A66G and TYMS 2R → 3R genotypes identified in this study were similar to those reported in previous studies [22,23] as well as the HapMap project. However, the frequency of the MTHFR 677TT genotype, in our control group, was high compared to prior reports on Caucasian patients (17% versus 11–12%) [24–26]. Conversely, the frequency of the MTHFR 1298CC, in our control group, was lower than that of previous reports on Caucasian patients (3% versus 7–12%) as well as the HapMap project (data not shown). The frequencies of MTRR 66 GG (7%) and TYMS 28-bp tandem repeat 2R2R (3%) genotypes in our controls were significantly lower than the Caucasian data (for MTRR 66 GG, 30% and for TYMS 28-bp 2R2R, 22–38%) [24–26]. The reduced enzyme activities of the MTHFR 677TT and MTHFR 1298CC genotypes lead to increased 5,10methylene THF availability for DNA synthesis by thymidylate synthase [7,8], which may protect cells from DNA damage. Our findings for MTHFR C677T and A1298C in adult ALL were different from previously published reports [2,5], where MTHFR 677TT, 1298AC and 1298CC (Skibola et al.) and MTHFR 1298AC genotypes (Hur et al.) were reported to be associated with a decreased risk for adult ALL. In the present study, the MTHFR 677TT genotype was associated with a significantly increased risk for ALL. In addition, our results showed no association between MTHFR C677T and A1298C with AML, CML and MDS. The findings for AML were consistent with two prior reports [5,6]. However, for CML, previous studies have shown that the MTHFR 1298AC genotype was associated with a decreased risk by Moon et al. [6] and the MTHFR 1298CC genotype was associated with an increased risk by Hur et al. [5], in contrast to our results. Additionally, the MTHFR Hap C (677T-1298A) was associated with an increased risk for ALL, but not for other hematological malignancies. These results suggest that the ALL risk is associated with the MTHFR polymorphism and haplotypes. The MTR and MTRR genes are involved in the same pathway of remethylation of homocysteine to methionine. The MTR A2756G and the MTRR A66G polymorphism contribute to the alteration of plasma levels of homocysteine and folate [27,28]. The MTR 2756GG genotype reduces enzyme activity, which results in a decrease of the DNA methylation and the methionine levels [29]. There are no prior reports on the risk for leukemia with the MTR A2756G and MTRR A66G polymorphisms; our results showed no association with these polymorphisms and the risk for AML, CML, MDS, and ALL. However, we found an increase risk for MDS with MTRR 66AG and an increased risk for AML with the MTRR 66GG genotype. The presence of the triple versus the double 28-bp repeat in the 5 UTR of the TYMS gene is associated
higher mRNA translation efficiency [14], elevated intratumor TYMS mRNA [30] and protein expression levels [14]. This enhanced expression may increase the conversion of dUMP to dTMP, reducing the level of uracil that might otherwise be misincorporated into DNA. Impairment of the TYMS enzyme has been associated with chromosomal damage and fragile site induction [31,32]. The role of the TYMS 6-bp deletion/insertion (0-bp/6-bp) polymorphism remains unclear. This polymorphism may influence TYMS mRNA stability in vitro and its expression in vivo [15]. The TYMS 2R3R and 6bp (0-bp/6-bp) polymorphisms have not been associated with leukemia risk. We found no significant association between the TYMS 2R3R polymorphism and AML, CML, MDS, and ALL. The TYMS 0-bp/6-bp was associated with a decreased risk for only AML (OR = 0.76; 95% CI = 0.60–0.96). TYMS hap3 (2R-6 bp) and hap4 (2R-0 bp) were associated with a decreased risk (OR = 0.69; 0.53–0.90, p = .006) and an increased risk (OR = 1.65; 1.20–2.27, p = .002), respectively for AML. These results suggest that the risk for AML is associated with DNA synthesis. These apparent inconsistencies between our results and those of previous studies, in different populations, may be due to the diverse number of patients studied, different genotype frequencies in the polymorphisms, different ethnicities, and the absence of information on dietary folate intake. Several studies have suggested that cancer risk is associated with genes and dietary folate intake or plasma folate levels [33–36]. Moreover, one report showed that folate supplementation during pregnancy reduced ALL risk, and that the protective effects for the prevention of ALL, with MTHFR polymorphisms, was dependent on adequate folate intake [37]. In conclusion, our results showed that the MTRR and TYMS polymorphisms were involved in the etiopathogenesis of AML and that both DNA synthesis and methylation play important roles in the pathogenesis of AML. MTRR polymorphisms appear to be involved the risk associated with MDS based on the DNA methylation status. The risk for ALL was associated with MTHFR polymorphisms. In addition, multiple comparisons were not considered in this analysis, so, although some genotypes showed associations, these may be false positive. Because typical daily Korean diet is composed mainly of vegetables, grains and cereals, we think folate intake of Korean may be higher than other cultures. Additional studies, with a larger sample size in various geographical areas and evaluation of environmental factors such as folate and vitamin intake, should be conducted to explore the associations between the genetic polymorphisms of the folate-metabolizing enzyme genes and leukemogenesis.
Acknowledgements This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (01-PJ10-PG6-01GN16-0005.
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Association between polymorphisms of folate-metabolizing enzymes and hematological malignancies Hee Nam Kim a , Yeo-Kyeoung Kim b , Il-Kwon Lee a , Deok-Hwan Yang b , Je-Jung Lee b , Min-Ho Shin c , Kyeong-Soo Park d , Jin-Su Choi c , Moo Rim Park e , Deog Yeon Jo f , Jong Ho Won g , Jae-Yong Kwak h , Hyeoung-Joon Kim a,b,∗ a
Genome Research Center for Hematopoietic Diseases, Chonnam National University Hwasun Hospital, Jeollanamdo,Republic of Korea b Department of Hematology/Oncology, Chonnam National University Hwasun Hospital, Jeollanamdo, Republic of Korea c Department of Preventive Medicine, Chonnam National University Medical School, Gwangju, Republic of Korea d Department of Preventive Medicine, College of Medicine, Seonam University, Namwon, Republic of Korea e Department of Internal Medicine, Wonkwang University School of Medicine, Iksan, Republic of Korea f Division of Hematology/Oncology, Department of Internal Medicine, College of Medicine, Chungnam National University, Daejon, Republic of Korea g Department of Internal Medicine, Soonchunhyang University, College of Medicine, Seoul, Republic of Korea h Department of Internal Medicine, Chonbuk National University Medical School, Jeonju, Republic of Korea Received 8 April 2008; received in revised form 25 July 2008; accepted 25 July 2008 Available online 6 September 2008
Abstract Several genetic polymorphisms in the genes coding folate-metabolizing enzymes have been associated with susceptibility to hematology malignancies. We conducted a Korean population-based case–control study to examine the relationship between the polymorphisms of folatemetabolizing enzymes and the risk of AML (acute myelogenous leukemia), CML (chronic myelogenous leukemia), MDS (myelodyspastic syndrome), and ALL (acute lymphoblastc leukemia). The MTHFR 677TT genotype was associated with an increased risk for ALL (odds ratios (OR) = 1.77; 95% confidence intervals (CI) = 1.02–3.09, p = .044). The MTRR 66 AG genotype was associated with an increased risk for MDS (OR = 1.59; 1.06–2.38, p = .026) and the MTRR 66 GG genotype was associated with increased risk for AML (OR = 1.51; 1.03–2.23, p = .037). The TYMS 2R3R genotype was associated with a decreased risk for AML (OR = 0.76; 0.60–0.96, p = .022). The TYMS hap3 (2R-6bp) and hap4 (2R-0bp) were associated with decreased risk (OR = 0.69; 0.53–0.90, p = .006) and increased risk (OR = 1.65; 1.20–2.27, p = .002), respectively for AML. Hap C (677T1298A) was associated with an increased risk (OR = 1.40; 1.02–1.92, p = .04) for ALL. The risk for ALL appears to be associated with the MTHFR 677 polymorphism. The results are supportive of a risk modification by folate polymorphisms in several hematologic malignancies in Korea. The pattern of results suggests that MDS was associated with the DNA methylation status and the risk for AML was associated with both the DNA synthesis and DNA methylation status. © 2008 Elsevier Ltd. All rights reserved. Keywords: AML; CML; MDS; ALL; Polymorphism; Folate-metabolizing enzymes; Association
1. Introduction ∗
Corresponding author at: Department of Hematology/Oncology, Chonnam National University Hwasun Hospital, 160 Ilsimri, Hwasun-gun, Jeollanam-do 519-809, Republic of Korea. Tel.: +82 61 379 7637; fax: +82 61 379 7736. E-mail address: [email protected] (H.-J. Kim). 0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.07.026
Folic acid and methionine metabolism play an essential role in both DNA synthesis and cellular methylation reactions. At least 30 enzymes have been identified in the folate pathway [1]. Several functional polymorphisms in the genes encoding these enzymes have been associated with various
H.N. Kim et al. / Leukemia Research 33 (2009) 82–87
malignancies, including adult and childhood acute lymphocytic leukemia (ALL) [2–4], AML [5,6] and CML [5,6]. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methyl-tetrahydrofolate (THF) to 5methyl-THF, the major form of circulating folate, which acts as the methyl donor for the transformation of homocysteine to methionine. Two known polymorphisms in the MTHFR gene, C677T and A1298C, lead to a 30–60% reduction in enzyme activity [1,7,8]. Methionine synthase (MS) catalyzes the transfer of the methyl group from 5-MeTHF to homocysteine, producing tetrahydrofolate and methionine [9]. The MTR A2756G polymorphism is thought to reduce enzyme activity [10] with the GG genotype and to increase homocysteine levels and DNA hypomethylation with the AA genotype [11]. Methionine synthase reductase (MTRR) is required for the reductive methylation of cobalamin, which is the activated cofactor for MS in the remethylation of homocysteine to methionine. Thymidylate synthase (TS) catalyzes the conversion of dUMP to dTMP using 5,10-methylenetetrahydrofolate during DNA synthesis. One polymorphism is a unique variable nucleotide tandem repeat (VNTR) sequence, with either two or three 28-bp repeats in the TYMS 5 UTR [12]. These polymorphic tandem repeats influence protein expression in cancer cells [13,14]. A 6-bp deletion/insertion (0-bp/6-bp), at 1494 in the 3 -UTR of the TYMS, is thought to influence TYMS mRNA stability in vitro and its expression in vivo [15,16]. Many studies have reported the association between the polymorphisms in these genes and risk for ALL [2,4,17–20]. However, few studies have been reported for AML and CML and there have been no reports on association with MDS. Besides, the results are inconsistent by different populations. In addition, there is no prior report on MTRR A66G, TYMS 2R → 3R and the 6-bp insertion/deletion and AML, CML, MDS or ALL risk. Therefore, we conducted a Korean largescale population-based, case–control study to examine the relationship between the MTHFR C677T and A1298C, MTR A2756G, MTRR A66G, TYMS 2R → 3R and 6-bp insertion/deletion genotypes and the risk for AML, CML, MDS, and ALL.
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a defined geographical area served by the hospital, Jeollanam-do province, Korea, we estimate that the case is approximately 70% of all leukemia cases in one area. The control group (n = 1700) consisted of participants in the Thyroid Disease Prevalence Study. This study was performed in the Yeonggwang and Muan counties of Jeollanam-do Province and Namwon city of Jeollabuk-do province, Korea, from July 2004 to January 2006. There were 4018 subjects, mean age 50.6 ± 14.6 years (range 20–74 years), who were randomly selected by a 5-year age strata and gender. Among the total number of patients, 3486 were eligible for the study. Among the eligible patients, 1700 (48.8% of the eligible subjects; 821 men and 879 women), mean age 52.2 ± 14.3 years, underwent clinical examinations. At the time of their peripheral blood collection, all cases and control subjects provided their informed consent to participate in this study. The Institutional Review Board of the Chonnam National University Hwasun Hospital in Hwasun, Korea approved this study. 2.2. Genotyping Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s protocol. The genotyping protocol for MTHFR C677T polymorphism detection was adapted from Frosst et al. [7]. After HinfI (Takara, Tokyo, Japan) restriction enzyme digestion, the samples were run on a 10% polyacrylamide gel (19:1) using the MADGE system (MadgeBio, Grantham and Southampton, UK). Genotyping for MTHFR A1298C was performed by allelic discrimination, using dual-labeled probes containing locked nucleic acids (LNA), in a real-time PCR assay. PCR primers and LNA probes were designed and synthesized by IDT (Coralville, IA, USA) [21]. Genotyping for the detection of the MTR A2756G polymorphism was performed using specific oligonucleotide primers [10]. Digestion products with HaeIII (Takara) were visualized after electrophoresis in a 10% polyacrylamide gel (19:1) using the MADGE system. MTRR A66G genotyping was performed by Pyrosequencing as simplex assays. PCR primers and sequencing primer were designed by the Pyrosequencing SNP Primer Design Software (version v1.0.6) [21]. Genotyping for 28-bp tandem repeat (2R → 3R) genotypes in the 5 -UTR of the TYMS gene was conducted using a protocol described by Horie et al. [13]. The 3 -UTR 6-bp deletion/insertion genotyping was performed using a 6% PAGE electrophoresis with the previously described primers [15].
2. Materials and methods
2.3. Statistical analysis
2.1. Study population
The statistical significance of the differences between the patient and control groups was estimated by logistic regression analysis. Adjusted odds ratios (ORs) were calculated with a logistic regression model that controlled for gender and age and are reported at 95% confidence intervals (CI). Subjects with the wild type genotypes (MTHFR 677CC, MTHFR 1298AA, MTR 2756AA, MTRR 66AA, TYMS 3R3R, and TYMS 0-bp/0-bp genotype) were considered to be at baseline risk. The expected frequency of control genotypes was evaluated by the Hardy–Weinberg equilibrium test. Haplotype frequencies for MTHFR and TYMS were estimated using the SNPAnalyzer program (Istech, Ltd., Korea) based on the expectation–maximization algorithm. All analyses were performed using SPSS software version 11.0 (SPSS, Chicago, IL, USA).
The patients enrolled in the study were adults aged ≥15 years, diagnosed with AML, CML, MDS and ALL at Chonnam National University Hospital between January 1997 and December 2006. The cases consisted of 389 AML (211 men and 184 women; mean aged 49.0 ± 17.4 years; range: 15–85 years), 149 CML (94 men and 55 women; mean aged 50.4 ± 17.1 years; range: 16–87 years), 109 MDS (65 men and 44 women; mean aged 57.8 ± 14.5 years; range: 15–81 years) and 110 ALL (61 men and 47 women; mean aged 40.9 ± 18.2 years; range: 15–77 years). The cases were most of the cases that come to the hospital without regard to sociologic variables. Since our hospital is tertiary national hospital in
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Table 1 Distribution of polymorphisms involved in the folate-metabolizing pathway in patients and controls Genotype MTHFR 677 CC CT TT
Control n (%)
AML n (%)
ORa (95% CI)
p
CML n (%)
ORa (95% CI)
p
MDS n (%)
ORa (95% CI)
ALL n (%)
ORa (95% CI)
p
121 (31) 202 (51) 76 (19)
1 1.02 (0.80–1.32) 1.1 (0.80–1.54)
.85 .53
54 (36) 72 (47) 26 (17)
1 0.81 (0.55–1.17) 0.90 (0.55–1.47)
.26 .67
34 (31) 58 (53) 17 (16)
1 1.04 (0.67–1.61) 0.90 (0.49–1.63)
.30 .37
29 (27) 51 (48) 27 (25)
1 1.11 (0.68–1.79) 1.77 (1.02–3.09)
.67 .044
MTHFR 1298 AA 1147 (68) AC 500 (29) CC 53 (3)
275 (69) 112 (28) 12 (3)
1 0.93 (0.72–1.19) 0.96 (0.51–1.84)
.55 .91
97 (64) 49 (33) 5 (3)
1 1.15 (0.80–1.66) 1.11 (0.43–2.85)
.44 .83
73 (67) 32 (29) 4 (4)
1 1.03 (0.67–1.58) 1.12 (0.39–3.20)
.90 .84
67 (63) 38 (36) 1 (1)
1 1.24 (0.81–1.89) 0.31 (0.04–2.32)
.32 .25
MTR 2756 AA AG GG
1282 (75) 392 (23) 26 (2)
308 (77) 86 (22) 5 (1)
1 0.90 (0.70–1.20) 0.66 (0.23–1.92)
.46 .44
107 (71) 43 (28) 2 (1)
1 1.30 (0.89–1.90) 0.92 (0.22–4.00)
.18 .92
80 (73) 26 (24) 3 (3)
1 1.06 (0.67–1.69) 1.74 (0.36–5.94)
.79 .37
77 (71) 28 (26) 3 (3)
1 1.24 (0.78–1.96) 2.42 (0.70–8.39)
.36 .17
MTRR 66 AA AG GG
857 (50) 718 (43) 125 (7)
195 (49) 162 (41) 42 (10)
1 0.97 (0.76–1.22) 1.51 (1.03–2.23)
.81 .037
73 (48) 68 (45) 11 (7)
1 1.15 (0.81–1.64) 1.04 (0.54–2.03)
.56 .94
44 (40) 58 (53) 7 (7)
1 1.59 (1.06–2.38) 0.74 (0.45–2.34)
58 (54) .026 34 (32) .95 15 (14)
1 0.69 (0.44–1.08) 1.72 (0.93–3.26)
.09 .087
TYMSb 2R - > 3R 3R3R 1129 (67) 2R3R 520 (31) 2R2R 48 (3)
274 (69) 105 (27) 16 (4)
1 0.84 (0.66–1.09) 1.28 (0.69–2.37)
.17 .43
103 (69) 39 (26) 7 (5)
1 0.82 (0.56–1.22) 1.72 (0.75–3.92)
.33 .20
73 (68) 32 (30) 2 (2)
1 0.99 (0.64–1.53) 0.60 (0.14–2.52)
.97 .48
71 (67) 31 (29) 4 (4)
1 0.93 (0.60–1.45) 1.52 (0.52–4.46)
.74 .45
TYMS 6bp 0bp/0bp 0bp/6bp 6bp/6bp
220 (55) 143 (36) 34 (9)
1 0.76 (0.60–0.96) 0.79 (0.52–1.19)
.022 .26
80 (53) 59 (39) 11 (7)
1 0.85 (0.59–1.21) 0.77 (0.40–1.49)
.36 .44
56 (51) 41 (38) 12 (11)
1 0.85 (0.56–1.29) 1.25 (0.65–2.40)
.45 .51
60 (56) 42 (39) 5 (5)
1 0.79 (0.52–1.20) 0.43 (0.17–1.11)
.26 .08
a b
824 (49) 723 (43) 151 (90)
ORs and 95% CIs were estimated using multiple logistic regression and adjusted for sex and age Unclassifiable cases (n = 32) were excluded in subgroup analysis. The rare TYMS genotypes (2R5R, 3R4R, 3R5R and 3R6R) were excluded in TYMS genotype analysis.
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540 (32) 863 (51) 297 (17)
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a ORs and 95% CIs were estimated using multiple logistic regression and adjusted for sex and age.
b The rare TYMS genotypes (2R5R, 3R4R, 3R5R and 3R6R) were excluded in TYMS genotype analysis.
1 0.85 (0.60–1.21) 0.90 (0.62–1.30) 1.11 (0.64–1.93)
0.366 0.559 0.713
141 (66) 37 (17) 27 (13) 9 (4)
1 1.05 (0.72–1.53) 0.95 (0.62–1.46) 0.883 (0.44–2.46)
0.797 0.827 0.726
150 (69) 27 (13) 24 (11) 15 (7)
1 0.71 (0.46–1.09) 0.76 (0.48–1.19) 1.60 (0.90–2.85)
0.113 0.222 0.108
3. Results
200 (68) 44 (15) 36 (12) 16 (5) 0.735 0.006 0.002 522 (66) 128 (16) 77 (10) 59 (8) 2215 (65) 562 (17) 462 (13) 153 (5)
1 0.96 (0.78–1.20) 0.69 (0.53–0.90) 1.65 (1.20–2.27)
0.264 0.040 1 1.26 (0.84–1.90) 1.40 (1.02–1.92) ND 70 (32) 41 (19) 105 (49) ND 0.918 0.835 0.924 1 1.02 (0.69–1.51) 0.97 (0.71–1.31) 1.11 (0.14–8.71) 87 (40) 39 (18) 91 (42) 1 (0.5) 0.526 0.769 ND 1 1.11 (0.80–1.55) 0.96 (0.74–1.26) ND 120 (40) 59 (19) 123 (41) ND
OR (95% CI) ALL n (%) p OR (95% CI) MDS n (%) p OR (95% CI) CML n (%) p
0.564 0.833 0.179 1 0.94 (0.74–1.18) 1.02 (0.86–1.21) 2.08 (0.72–6.07) 313 (39) 131 (16) 349 (44) 5 (0.6) 1348 (40) 595 (18) 1446 (42) 11 (0.3)
1298 A > C A C A C Del 6 bp 0 bp 6 bp 6 bp 0 bp 677 C > T C C T T 28 bp repeat 3R 3R 2R 2R
ORa (95% CI) AML n (%) Control n (%)
Table 2 Distribution of haplotype frequencies for MTHFR and TYMS in patients and controls
MTHFR Hap A Hap B Hap C Hap D TYMSb Hap 1 Hap 2 Hap 3 Hap 4
p
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The distribution of the MTHFR, MTR, MTRR, and TYMS polymorphisms in the controls was in Hardy–Weinberg equilibrium (MTHFR 677 and 1298; p = 0.132 and 0.867, MTR 2756; p = 0.522, TYMS 66; p = 0.127, TYMS 2R3R; p = 0.196, TYMS 6 bp; p = 0.671), and not significantly different in the comparisons between men and women (data not shown). The genotype distributions among all cases and controls for the MTHFR C677T, A1298C, MTR A2756G, MTRR A66G, TYMS 2R → 3R, and 6-bp insertion/deletion polymorphisms are shown in Table 1. For the TYMS genotype, we observed rare TYMS alleles with 4R (four tandem repeats), 5R and 6R in addition to the major 2R and 3R alleles. The frequencies of TYMS 2R4R, 3R4R and 3R5R were 0.15%, 0.45% and 0.08%, respectively, in the controls; the frequencies of TYMS 3R4R and 3R5R were 0.26% and 0.78%, respectively, in all cases. The three major TYMS genotypes (3R3R, 2R3R, 2R2R) were used in this analysis and the rare TYMS genotypes (2R5R, 3R4R, 3R5R, 3R6R) were excluded because of the extremely low frequencies (<1%). The MTHFR 677TT genotype was significantly associated with an increased risk for ALL (OR = 1.77; 95% CI = 1.02–3.09), but not for AML, CML and MDS. The MTRR 66AG was associated with an increased risk for MDS (OR = 1.59; 95% CI = 1.06–2.38), but not for AML, CML and ALL. The MTRR 66GG genotype was associated with an increased risk for AML (OR = 1.51; 95% CI = 1.03–2.23), but not for CML and MDS. A marginal increased risk was also observed for ALL and the MTRR 66GG genotype (OR = 1.77; 95% CI = 0.96–3.26). The TYMS 0-bp/6-bp was associated with a decreased risk for only AML (OR = 0.76; 95% CI = 0.60–0.96). However, the MTHFR 677CT and TYMS 6-bp/6-bp genotype had no affect on the risk for AML, CML, MDS, and ALL. There was no significant association of MTHFR A1298C, MTR A2756G and TYMS 2R3R with AML, CML, MDS, and ALL. Although the OR of the MTR 2756 GG genotype for AML, MDS and ALL was 0.66, 1.74 and 2.43, respectively, and the OR of the TYMS 2R2R genotype for CML, MDS and ALL was 1.72, 0.60 and 1.52, none of the results reached statistical significance. The distribution of the haplotype frequencies of MTHFR and TYMS for the patients and controls are shown in Table 2. The TYMS hap3 (2R-6bp) and hap4 (2R-0bp) were associated with a decreased risk (OR = 0.69; 0.53–0.90, p = .006) and increased risk (OR = 1.65; 1.20–2.27, p = .002), respectively, for AML. The MTHFR Hap C (677T-1298A) was associated with an increased risk (OR = 1.40; 1.02–1.92, p = .04) for ALL.
4. Discussion In this population-based, case–control study, we investigated the association between folate-metabolizing gene polymorphisms and susceptibility to AML, CML, MDS, and ALL in the Korean population. We found an association
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between the MTHFR 677TT genotype and ALL, the MTRR 66 AG genotype and MDS, the MTRR 66 GG genotype and AML, and the TYMS 2R3R genotype and AML. As shown Table 1, the frequencies of the MTHFR C677T and A1298C, MTR A2756G, MTRR A66G and TYMS 2R → 3R genotypes identified in this study were similar to those reported in previous studies [22,23] as well as the HapMap project. However, the frequency of the MTHFR 677TT genotype, in our control group, was high compared to prior reports on Caucasian patients (17% versus 11–12%) [24–26]. Conversely, the frequency of the MTHFR 1298CC, in our control group, was lower than that of previous reports on Caucasian patients (3% versus 7–12%) as well as the HapMap project (data not shown). The frequencies of MTRR 66 GG (7%) and TYMS 28-bp tandem repeat 2R2R (3%) genotypes in our controls were significantly lower than the Caucasian data (for MTRR 66 GG, 30% and for TYMS 28-bp 2R2R, 22–38%) [24–26]. The reduced enzyme activities of the MTHFR 677TT and MTHFR 1298CC genotypes lead to increased 5,10methylene THF availability for DNA synthesis by thymidylate synthase [7,8], which may protect cells from DNA damage. Our findings for MTHFR C677T and A1298C in adult ALL were different from previously published reports [2,5], where MTHFR 677TT, 1298AC and 1298CC (Skibola et al.) and MTHFR 1298AC genotypes (Hur et al.) were reported to be associated with a decreased risk for adult ALL. In the present study, the MTHFR 677TT genotype was associated with a significantly increased risk for ALL. In addition, our results showed no association between MTHFR C677T and A1298C with AML, CML and MDS. The findings for AML were consistent with two prior reports [5,6]. However, for CML, previous studies have shown that the MTHFR 1298AC genotype was associated with a decreased risk by Moon et al. [6] and the MTHFR 1298CC genotype was associated with an increased risk by Hur et al. [5], in contrast to our results. Additionally, the MTHFR Hap C (677T-1298A) was associated with an increased risk for ALL, but not for other hematological malignancies. These results suggest that the ALL risk is associated with the MTHFR polymorphism and haplotypes. The MTR and MTRR genes are involved in the same pathway of remethylation of homocysteine to methionine. The MTR A2756G and the MTRR A66G polymorphism contribute to the alteration of plasma levels of homocysteine and folate [27,28]. The MTR 2756GG genotype reduces enzyme activity, which results in a decrease of the DNA methylation and the methionine levels [29]. There are no prior reports on the risk for leukemia with the MTR A2756G and MTRR A66G polymorphisms; our results showed no association with these polymorphisms and the risk for AML, CML, MDS, and ALL. However, we found an increase risk for MDS with MTRR 66AG and an increased risk for AML with the MTRR 66GG genotype. The presence of the triple versus the double 28-bp repeat in the 5 UTR of the TYMS gene is associated
higher mRNA translation efficiency [14], elevated intratumor TYMS mRNA [30] and protein expression levels [14]. This enhanced expression may increase the conversion of dUMP to dTMP, reducing the level of uracil that might otherwise be misincorporated into DNA. Impairment of the TYMS enzyme has been associated with chromosomal damage and fragile site induction [31,32]. The role of the TYMS 6-bp deletion/insertion (0-bp/6-bp) polymorphism remains unclear. This polymorphism may influence TYMS mRNA stability in vitro and its expression in vivo [15]. The TYMS 2R3R and 6bp (0-bp/6-bp) polymorphisms have not been associated with leukemia risk. We found no significant association between the TYMS 2R3R polymorphism and AML, CML, MDS, and ALL. The TYMS 0-bp/6-bp was associated with a decreased risk for only AML (OR = 0.76; 95% CI = 0.60–0.96). TYMS hap3 (2R-6 bp) and hap4 (2R-0 bp) were associated with a decreased risk (OR = 0.69; 0.53–0.90, p = .006) and an increased risk (OR = 1.65; 1.20–2.27, p = .002), respectively for AML. These results suggest that the risk for AML is associated with DNA synthesis. These apparent inconsistencies between our results and those of previous studies, in different populations, may be due to the diverse number of patients studied, different genotype frequencies in the polymorphisms, different ethnicities, and the absence of information on dietary folate intake. Several studies have suggested that cancer risk is associated with genes and dietary folate intake or plasma folate levels [33–36]. Moreover, one report showed that folate supplementation during pregnancy reduced ALL risk, and that the protective effects for the prevention of ALL, with MTHFR polymorphisms, was dependent on adequate folate intake [37]. In conclusion, our results showed that the MTRR and TYMS polymorphisms were involved in the etiopathogenesis of AML and that both DNA synthesis and methylation play important roles in the pathogenesis of AML. MTRR polymorphisms appear to be involved the risk associated with MDS based on the DNA methylation status. The risk for ALL was associated with MTHFR polymorphisms. In addition, multiple comparisons were not considered in this analysis, so, although some genotypes showed associations, these may be false positive. Because typical daily Korean diet is composed mainly of vegetables, grains and cereals, we think folate intake of Korean may be higher than other cultures. Additional studies, with a larger sample size in various geographical areas and evaluation of environmental factors such as folate and vitamin intake, should be conducted to explore the associations between the genetic polymorphisms of the folate-metabolizing enzyme genes and leukemogenesis.
Acknowledgements This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (01-PJ10-PG6-01GN16-0005.
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