Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorectal cancer risk in Thais: a case-control study

Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorectal cancer risk in Thais: a case-control study

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NTR-08063; No of Pages 12 NUT RI TI ON R E SE ARC H X X (X XXX ) XX X

Available online at www.sciencedirect.com

ScienceDirect www.nrjournal.com

Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorectal cancer risk in Thais: a case-control study Pornpimol Panprathip a , Songsak Petmitr b , Rungsunn Tungtrongchitr a , Jaranit Kaewkungwal c , Karunee Kwanbunjan a,⁎ a b c

Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, 10400 Bangkok, Thailand Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, 10400 Bangkok, Thailand Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, 10400 Bangkok, Thailand

ARTI CLE I NFO

A BS TRACT

Article history:

Folate plays essential roles in DNA synthesis, repair, and methylation; thus, folate status may

Received 5 March 2019

affect carcinogenesis. Genetics polymorphisms involved in folate metabolisms have been linked

Revised 17 October 2019

with colorectal cancer (CRC) development. Therefore, we hypothesized that low folate status and

Accepted 22 October 2019

related genetic polymorphisms are associated with higher risk of CRC. This case-control study enrolled 105 new cases of CRC, 101 of colorectal adenoma (CRA), and 182 controls from hospitals

Keywords:

in Bangkok, Thailand, to examine the association between folate status and methylenetetrahy-

Folate

drofolate reductase (MTHFR) 677C > T, methionine synthase (MTR) 2756A > G, and methionine

Colorectal cancer

synthase reductase (MTRR) 66A > G with the risk of CRC and CRA. Regarding CRC risk, the lowest

Risk factors

quartile group of serum folate and folate intake had higher risk of CRC than the highest quartile

Genetic polymorphisms

group (odds ratio [OR] = 11.45, 95% confidence interval [CI] = 4.43-29.59) and (OR = 10.29, 95%

Case-control Study

CI = 4.17-25.41). The lowest quartile group of folate intake also had a higher risk of CRA (OR = 5.22, 95% CI = 2.19-6.09). Low red blood cell folate combined with MTHFR 677C > T polymorphism statistically increased CRC risk (OR = 10.00, 95% CI = 1.36-73.42). Low folate status combined with MTR 2756A > G significantly increased CRA risk (OR = 6.43, 95% CI = 1.38-29.94). Moreover, the risk of CRC was elevated with alcohol consumption and low exercise activity when combined with low folate status (P < .05). This study supported the hypothesis that, in Thais, low folate status is associated with a higher risk of CRC, particularly among those with polymorphisms of the MTHFR 677C > T and MTR 2756 A > G genes. © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.

Introduction

Folate is a water-soluble B vitamin and has a central role in 1-carbon metabolism. Folate is a crucial coenzyme for

nucleotide synthesis and repair and for methylation. These functions are essential because disruptions in DNA synthesis, repair, and methylation are considered to play major roles in carcinogenesis [1]. Several studies have demonstrated that

Abbreviations: CI, confidence intervals; CRA, colorectal adenoma; CRC, colorectal cancer; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; OR, odds ratio; PCR, polymerase chain reaction; RBC, red blood cell; SAM, S-adenosylmethionine; SNPs, single nucleotide polymorphisms; THF, tetrahydrofolate. ⁎ Corresponding author at: 420/6 Ratchawithi Rd, Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, 10400 Bangkok, Thailand. E-mail address: [email protected] (K. Kwanbunjan).

Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

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insufficient folate intake can result in DNA damage via uracil misincorporation or aberrant DNA repair. Low consumption of folate can also induce DNA hypomethylation, which has been associated with colorectal carcinogenesis [2]. Intracellular folate metabolism is regulated by several enzymes, including methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and methionine synthase reductase (MTRR). The activity of these enzymes may vary owing to different genetic variations, commonly referred to as functional polymorphisms [3]. A folate metabolite, 5-methyl tetrahydrofolate (THF), provides the methyl group in the reaction catalyzed by MTR to convert homocysteine to methionine, the precursor of Sadenosylmethionine (SAM). SAM is the universal methylgroup donor for the methylation of a wide variety of biological substrates. Thus, folate/methyl depletion results in aberrant DNA methylation, that is, global genomic hypomethylation and specific methylation of CpG clusters in the promoters of tumor suppressor and DNA repair genes [4]. MTHFR converts 5,10-methylenetetrahydrofolate into 5methyltetrahydrofolate and provides methyl groups for DNA methylation via the remethylation of homocysteine, whereas this enzyme and its derivatives are essential cofactors for thymidylate and de novo purine synthesis [5]. This enzyme has a genetic polymorphism of C to T at the 677 base pair (bp), which results in a substitution in codon 222, alanine to valine [6]. This single nucleotide polymorphism (SNP) produces a thermolabile enzyme with reduced functional activity, which results in an altered intracellular distribution of folate substrates [7]. A previous meta-analysis reported an association between the 677C > T polymorphism and decreased colorectal cancer (CRC) risk [8]. MTR plays a central role in maintaining adequate intracellular folate. It is essential for the provision of SAM, the universal donor of methyl groups, as well as the provision of THF for use in nucleotide synthesis. A common MTR variant comprises an A-to-G transition at bp 2756 and leads to a change from aspartic acid to glycine at codon 919 [9]. An association between MTR 2756A > G polymorphism and genetic susceptibility to CRC and colorectal adenoma (CRA) has been widely documented but with inconsistent outcomes. MTRR maintains methionine synthase in an active form. It catalyzes the reduction of oxidized vitamin B12, releasing an activated MTR. MTRR has a common genetic polymorphism of A-to-G in the 66-bp region that leads to a change from isoleucine to methionine codon 22 [10]. This polymorphism may modify the effect of folate, vitamin B6, or vitamin B12 on the development of CRC [11]; however, the function of the polymorphism remains unknown. CRC is a serious health concern in Thailand, where it is one of the fifth most common cancers in the last 2 decades. Recently, studies in many different parts of the world have reported a relationship between SNPs involved in folaterelated metabolism and the risk of CRC. However, there have been limited reports of studies on folate status, gene polymorphisms, and CRC in the Thai population. We hypothesized that low folate status and related genetic polymorphisms are associated with a higher risk of CRC in Thais. Therefore, this case-control study aimed to investigate the associations between folate status, gene

polymorphisms, and the risk of CRC in Thais and to identify the interactions of folate-gene polymorphism and their impact on CRC risk. We studied the role of nutrition and genetic factors affecting CRC risk in Thais. The result of this study might be beneficial for CRC prevention by improving nutritional behavior.

2.

Methods and materials

2.1.

Subjects

The participants were outpatients, aged 30-85 years, recruited from Phramongkutklao Hospital and Vajira Hospital, Bangkok. Colonoscopies and biopsies were performed to confirm histological diagnosis. Subjects were classified into 3 groups: group 1 (control group) subjects were not diagnosed with cancer or any other neoplastic conditions, as confirmed by normal colonoscopy. Control subjects did not have any other diseases or serious conditions. Subjects with anemia were excluded. Group 2 (CRA group) patients had a diagnosis of adenoma or polyp of size ≥1 cm or ≥ 1 polyps in the colon and/or rectum. Group 3 (CRC group) patients were diagnosed with a new case of colon and/or rectal cancer. The medical records of all participants were obtained from the hospital and reviewed for disease history. Patients with a history of benign tumors and/or neoplasm were excluded from the study. Written informed consent was obtained from all participants. The study was approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (TMEC 17-020). Study subject selection is shown in Fig. 1.

2.2.

Socioeconomic and demographic information

Socioeconomic and demographic information, including education, income, frequency of exercise, alcohol consumption, cigarette smoking, family history of CRC, and other possible risk factors, was recorded by a trained interviewer.

2.3.

Dietary assessment

Food intake over the past year was investigated using a semiquantitative food frequency questionnaire to determine normal dietary intake, with particular emphasis on folate intake. The semiquantitative food frequency questionnaire was designed to include 66 high-folate Thai food items. The frequency of consumption of each food varied from high to low, with the following options: 6 times a day, 4 or 5 times a day, 2 or 3 times a day, once a day, 5 or 6 times a week, 2 to 4 times a week, once a week, once to thrice a month, and never or rarely. The amount of food consumed was categorized, on the basis of portion size, as small, medium, or large; consumption was aided with photographs of the foods to ensure that information provided by the patient was consistent. The information obtained was used to convert the amount of food eaten into grams per day, which was then entered into a food calculation program “Nutrisurvey” (http://www.nutrisurvey.de/) equipped with a folic acid and vitamin B12 Thai food database (Areekul, 1986) to determine total intake.

Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

NUT RI TI ON R E SE ARC H X X (X XXX ) XX X

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Fig. 1 – The Illustration of the study subject selection.

2.4.

Laboratory assessment

Six milliliters of fasting blood was drawn from the subjects and added into clotted blood tubes, heparin-treated blood tubes, and EDTA-treated blood tubes for serum folate level, red blood cell (RBC) folate level, and genotyping, respectively. Serum and red cell folate levels were measured in duplicates by a microbiological assay using Lactobacillus casei ATCC no. 7469. Genomic DNA was extracted from leukocytes in EDTAtreated blood sample using the FlexiGene DNA Kit (Qiagen, Hilden, Germany). MTHFR 677C > T (rs1801133), MTR 2756A > G (rs1805087), and MTRR 66A > G (rs1801394) were identified by the polymerase chain reaction (PCR)–restriction fragment length polymorphism method. The Primer3Plus Program (http://www.bioinformatics.nl/cgi-bin/primer3plus/ primer3plus.cgi) was used to design 3 pairs of PCR primers.

NEB cutter v2.0 (http://www.labtools.us/nebcutter-v2-0/) was used to select restriction endonuclease enzymes. PCRs were performed in 50-μL reactions containing forward and reverse primers, dNTPs, buffer and MgCl2, Taq DNA polymerase, and DNA template using a C1000TM Thermal Cycler (Bio-Rad, Hercules, California, USA). Initially, samples were heated for 5 minutes at 95°C, followed by 34 cycles for 30 seconds at 95°C, 30 seconds at 55°C, and 50 seconds at 72°C, with an extension step of 5 minutes at 72°C. Forward and reverse primers of the target SNPs were as follows: (5′tcc ctg tgg tct ctt cat cc 3′) and (5′ctg gga aga act cag cga ac 3′) for MTHFR 677C > T, (5′tcc cag aaa cca gtc aaa gg 3′) and (5′cct gcc tca tgt ctc cat tt 3′) for MTR 2756A > G, and (5′cag gca aag gcc atc gca gaa gac at 3′) and (5′cac ttc cca acc aaa att ctt caa ag 3′) for MTRR 66A > G, respectively. PCR products of MTHFR 677C > T, MTR 2756A > G, and MTRR 66A > G were digested with the

Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

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restriction endonucleases TaqI (product sizes: 379, 227, and 152 bp), Eco47I (product sizes: 725, 416, and 309 bp), and NdeI (product sizes: 152, 123, and 29 bp) for 3 hours at 65°C, 37°C, and 37°C, respectively. The digested fragments were analyzed by gel electrophoresis. Finally, approximately 10% of the study population was randomly selected to confirm using DNA sequencing (Macrogen Inc, Seoul, South Korea).

2.5.

many factors may influence the etiology of disease, particularly gene polymorphisms, multivariable analysis was considered. Logistic regression was used to demonstrate risk factors for CRC and CRA. ORs and 95% confidence intervals (CIs) were calculated. P < .05 was considered statistically significant (power = 0.80) and was performed to determine all sample sizes.

Statistical analyses

Statistical analyses were conducted using Statistical Package for Social Science (SPSS, Chicago, IL, USA), version 18. The χ2 test was used to determine any significant differences in socioeconomic/demographic information between CRC, CRA, and control groups. The folate statuses of the control and other groups were compared by Mann-Whitney U test. The genotypic distribution of MTHFR 677C > T, MTR 2756A > G, and MTRR 66A > G between the CRC/CRA and control groups was used to determine the odd ratios (ORs) by χ2 test. Because

3.

Results

3.1.

Characteristics of the study population

The demographic and socioeconomic information of the study groups is shown in Table 1. The number of men was higher than women in the CRC and CRA groups. The mean ages of individuals in the CRC and control groups did not differ; however, the mean age of those in the CRA group was slightly higher than the control. The body mass indexes of

Table 1 – Characteristics and potential risk factors of the study groups Variables

Control n

Colorectal adenoma %

Sex Female 101 55.5 Male 81 44.5 Age (y) <50 25 13.7 50-59 44 24.2 60-69 66 36.3 ≥70 47 25.8 Body mass index (kg/m2) 18.50-22.99 78 42.9 23.00-24.99 45 24.7 ≥25.00 59 32.4 Education University 91 50.0 High school 43 23.6 Primary school 47 25.8 Illiterate 1 0.6 Income (THB/mo) >50 000 44 24.2 20 001-50 000 63 34.6 5000-20 000 68 37.4 <5000 7 3.8 First-degree family history of colorectal cancer No 160 87.9 Yes 22 12.1 Frequency of exercise c >1 time/wk 76 41.8 ≤1 time/wk 106 58.2 Alcohol consumption Never 100 54.9 Ever 82 45.1 Cigarette smoking Never 127 69.8 Ever 55 30.2

n

%

33 68

32.7 67.3

4 26 27 44

4.0 25.7 26.7 43.6

42 23 36

42.6 22.8 35.6

45 34 20 2

44.6 33.6 19.8 2.0

19 35 40 7

18.8 34.7 39.6 6.9

90 11

89.1 10.9

26 75

25.7 74.3

43 58

42.6 57.4

55 46

54.5 45.5

P value

Colorectal cancer a

n

%

36 69

34.3 65.7

11 25 33 36

10.5 23.8 31.4 34.3

45 17 43

42.9 16.1 41.0

28 26 38 1

30.1 28.0 40.8 1.1

9 20 48 16

9.7 21.5 51.6 17.2

82 11

88.2 11.8

11 82

11.8 88.2

24 69

25.8 74.2

38 55

40.9 59.1

.000

P value b .001

.003

.446

.848

.164

.168

.013

.537

.000

.764

.950

.007

.000

.046

.000

.014

.000

Values are P values of χ2 test. P < .05 is considered to be statistically significant. P for comparison between control and colorectal adenoma group. b P for comparison between control and colorectal cancer group. c Common exercise, that is, aerobic dance, jogging, swimming, football, basketball badminton, and tennis. Duration of exercise ≥30 min/time. a

Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

NUT RI TI ON R E SE ARC H X X (X XXX ) XX X

individuals in both CRC and CRA groups were not significantly different compared with the control group. The educational background of the control group was significantly higher than that of the CRC group. Half of the individuals in the control group had a university degree, whereas nearly half of the cancer group had only finished primary school. There were no statistical differences in educational background between CRA and control groups. There was no difference between the study groups for family history of CRC and CRA. Individuals with CRC and CRA were more likely to drink alcohol and smoke than those in the control group. Individuals in the control group exercised more than those in the other groups.

3.2. Folate status of the study groups and association with colorectal adenoma and cancer risk Folate status was assessed, including serum folate levels, RBC folate levels, and folate intake. Median serum folate levels of the control, CRA, and CRC groups were 70.73, 57.59, and 25.01 nmol/L, respectively. Median RBC folate levels of control,

5

CRA, and CRC groups were 577.71, 591.30, and 570.91 nmol/L, respectively. Median dietary folate intake levels were 127.95, 103.48, and 69.71 μg/d, respectively. P values were calculated by the Mann-Whitney U test (Fig. 2). After adjustment for sex, age, cigarette smoking, alcohol consumption, and frequency of exercise, low folate status was significantly associated with increased risk of CRC (Table 2). Comparing the lowest quartile and highest quartile, serum folate (OR = 11.45, 95% CI = 4.43-29.59, P < .001) and folate intake (OR = 10.29, 95% CI = 4.17-25.41, P < .001) indicated increased risk, as well as relationship between CRA and folate intake (OR = 5.22 (95% CI = 2.19-6.09, P < .001).

3.3. Genotype frequency and association with colorectal adenoma and cancer risk The distributions of genotypes and the adjusted ORs for each genetic polymorphism of CRA and CRC are shown in Table 3. Hardy-Weinberg test results confirmed that all genotypes were in equilibrium (P > .05). Overall, the distribution of the MTHFR, MTR, and MTRR gene polymorphisms among CRA

Fig. 2 – Comparison of folate levels between control and cases groups, CRA and CRC.A, Serum folate, (B) RBC folate, and (C) folate intake. Solid line represents the median of folate status. Values are P values. P values are calculated by Mann-Whitney U test. P < .05 is considered to be statistically significant.

Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

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NUT RI TI ON R ES E ARC H X X (X XXX ) XXX

Table 2 – Colorectal adenoma and cancer risk regarding serum folate, RBC folate, and folate intake Folate status

Quartiles of folate status Q4

Q3

Q2 P value

Serum folate (nmol/L) Colorectal adenoma

Colorectal cancer

RBC folate (nmol/L) Colorectal adenoma

Colorectal cancer

Folate intake (μg/d) Colorectal adenoma

Colorectal cancer

Median na n a (%) OR (95% na n a (%) OR (95% Median na n a (%) OR (95% na n a (%) OR (95% Median na n a (%) OR (95% na n a (%) OR (95%

CI)

CI)

CI)

CI)

CI)

CI)

118.50 23/63 22.8/34.6 1.00 11/63 10.5/34.6 1.00 1017.22 30/48 29.7/26.4 1.00 19/48 18.1/26.4 1.00 230.22 16/65 15.8/35.7 1.00 13/65 14.0/35.7 1.00

72.72 28/49 27.7/26.9 1.78 (0.87-3.65) 19/49 18.1/26.9 2.41 (0.92-6.28) 675.12 23/43 22.7/23.6 0.92 (0.44-1.94) 32/43 30.5/23.6 2.62 (1.13-6.06) 128.78 27/53 26.7/29.1 2.01 (0.94-4.30) 14/53 15.1/29.1 1.38 (0.56-3.43)

.113

.073

.835

.024

.073

.486

Q1 P value

42.48 30/46 29.7/25.3 2.10 (1.01-4.35) 22/46 21.0/25.3 2.78 (1.06-7.30) 507.48 24/46 23.8/25.3 0.91 (0.43-1.94) 26/46 24.8/25.3 1.76 (0.72-4.20) 89.21 30/45 29.7/24.7 2.83 (1.32-6.09) 19/45 20.4/29.7 1.72 (0.71-4.20)

.046

.038

.792

.199

.008

.231

P value 14.57 20/24 19.8/13.2 2.23 (0.94-5.26) 53/24 50.5/13.2 11.45 (4.43-29.59) 339.83 24/45 23.8/24.7 0.86 (0.40-1.85) 28/45 26.7/24.7 2.30 (0.95-5.53) 47.02 28/19 27.7/10.4 5.22 (2.19-6.09) 47/19 50.5/10.4 10.29 (4.17-25.41)

.068

.000

.700

.064

.000

.000

Data are analyzed by logistic regression model adjusted for sex, age (10-year class), cigarette smoking, alcohol consumption, and exercise. Value are ORs (95% CIs). P < .05 is considered to be statistically significant. a Number (%) of cases/control.

cases, CRC cases, and controls was not different. There was no statistically significant association between each SNP and the risk of CRA and CRC.

3.4. Impact of folate status–gene polymorphisms interaction on colorectal adenoma and cancer risk The combined effects of folate status and genotype on CRA and CRC risk were examined. The results are presented in Tables 4 and 5, respectively. Individuals with MTHFR 677CT + TT, MTR 2756AA + AG, and MTRR 66AA + AG genotypes with low serum folate levels (≤25.02 nmol/L) had significantly higher odds of developing CRC than those with high serum folate level; however, significantly higher ORs were also found among the wild-type group. The significant differences in these results derived from the effect of low serum folate, not genetic variation. Individuals with MTHFR 677CT + TT and low RBC folate level (≤424.77 nmol/L) had significantly higher odds of developing CRC (OR = 10.00, 95% CI = 1.36-73.42), whereas statistical significance was not found among the wild-type group. This significantly different result derived from the exacerbated effect of low RBC folate and variants of MTHFR 677C > T gene polymorphisms. Individuals with MTHFR, MTR and MTRR variants with low folate intake (≤69.78 μg/d) had significantly higher odds of developing CRC than those with high folate intake. Significances were found in the wild-type group as well; therefore, significant differences in these results derived from the effect of low folate intake, not genetic variation. Individuals with wild-type MTHFR, MTR, and MTRR with low folate intake showed

significantly higher risk of CRA; the results are shown in Table 5. Therefore, the significant differences in these results derived from the effect of low folate intake, not genetic variation. To determine the risk of CRC and CRA stratified by folate status, which combined all folate indicators, with OR and 95% CI for CRC and CRA, are presented in Table 6. Serum, RBC levels, and dietary folate intake were combined and classified into 3 groups: low, intermediate, and high folate status. Individuals with low folate status had significantly higher odds of developing CRC and CRA, especially those with MTR gene variances. Individuals with MTR 2756AA + AG and low folate status had significantly higher odds of developing CRA (OR = 6.43, 95% CI = 1.38-29.94) than those with high folate status, whereas no statistical significance was found among the wild-type group. The significant differences derived from the exacerbated effect of low folate status and variant MTR 2756A > G gene polymorphisms. Furthermore, the combined effects of folate status and environmental factors (cigarette smoking, alcohol consumption, and exercise frequency) on CRA and CRC risk were examined. Individuals who reported alcohol consumption (OR = 6.33, 95% CI = 1.79-22.37) and low exercise (OR = 5.02, 95% CI = 1.79-14.06) and had low folate status showed the highest risk of CRC (Table 7).

4.

Discussion

CRC is well known as a complex disorder influenced by both genetic and environmental factors. Differences in age, sex,

Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

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Table 3 – Prevalence of MTHFR, MTR, and MTRR polymorphisms in the study groups Genotype

MTHFR 677C > T a: CC CT TT b: CC CT + TT c: CC + CT TT MTR 2756A > G a: AA AG GG b: AA AA+AG c: AA+AG GG MTRR 66A > G a: AA AG GG b: AA AA+AG c: AA+AG GG

Control (n = 182)

Colorectal adenoma (n = 101)

n (%)

n (%)

OR (95% CI)

140 (76.9) 39 (21.4) 3 (1.7) 140 (76.9) 42 (23.1) 179 (98.4) 3 (1.6)

78 (77.2) 21 (20.8) 2 (2.0) 78 (77.2) 23 (22.8) 99 (98.0) 2 (2.0)

1.00 0.89 1.32 1.00 0.91 1.00 1.36

138 (75.8) 39 (21.4) 5 (2.8) 138 (75.8) 44 (24.2) 177 (97.2) 5 (2.8)

71 (70.3) 28 (27.7) 2 (2.0) 71.(70.3) 30 (29.7) 99 (98.0) 2 (2.0)

1.00 1.40 1.21 1.00 1.38 1.00 1.12

91 (50.0) 74 (40.7) 17 (9.3) 91 (50.0) 91 (50.0) 165 (90.7) 17 (9.3)

44 (43.6) 48 (47.5) 9 (8.9) 44 (43.6) 57 (56.4) 92 (91.1) 9 (8.9)

1.00 1.29 1.03 1.00 1.24 1.00 0.92

P value

(0.46-1.70) (0.16-11.12)

.720 .797

(0.49-1.72)

.775

(0.16-11.37)

.778

(0.75-2.59) (0.21-7.07)

.292 .836

(0.76-2.51)

.295

(0.19-6.47)

.908

(0.74-2.25) (0.39-2.74)

.371 .946

(0.73-2.11)

.426

(0.36-2.35)

.863

Colorectal cancer (n = 105) n (%)

OR (95% CI)

73 (69.5) 31 (29.5) 1 (1.0) 73 (69.5) 32 (30.5) 104 (99.0) 1 (1.0)

1.00 1.17 1.04 1.00 1.16 1.00 1.01

79 (75.2) 25 (23.8) 1 (1.0) 79 (75.2) 26 (24.8) 104 (99.0) 1 (1.0)

1.00 0.77 0.34 1.00 0.71 1.00 0.34

47 47 11 47 58 94 11

(44.8) (44.8) (10.5) (44.8) (55.2) (89.5) (10.5)

P value

(0.60-2.27) (0.10-11.23)

.652 .974

(0.60-2.22)

.659

(0.09-10.84)

.994

(0.38-1.61) (0.04-3.25)

.511 .352

(0.35-1.44)

.339

(0.36-3.22)

.348

1.00 1.18 (0.64-2.16) 1.63 (0.60-4.44)

.597 .335

1.25 (0.71-2.23) 1.00 1.52 (0.58-3.95)

.442 .396

Data are analyzed by logistic regression model adjusted for sex, age (10-year class), cigarette smoking, alcohol consumption, and exercise. Value are ORs (95% CI). P < .05 is considered to be statistically significant. There is no statistically significant association between each SNP and the risk of colorectal adenoma and cancer. a: Codominant model analysis (wild-type vs heterozygous variants and wild-type vs homozygous variants). b: Dominant model analysis (wild type vs variants). c: Recessive model analysis (wild-type + heterozygous variants vs homozygous variants).

socioeconomic status, and lifestyle may lead to different outcomes. This case-control study found that the proportions of subjects with low education, who lack of exercise, who drank alcohol, and who smoked cigarettes were higher in the 2 case groups than the control group. Overall, individuals in the control group were living healthier lifestyles than those in the patient groups. Individuals with higher education levels may have greater concern for their health and greater access to resources that supported a healthier lifestyle. The American Cancer Society recommends screening examinations to prevent cancer. Screening tests are useful for cancer prevention because polyps or suspect cells can be found and removed before they progress unfavorably. A healthy lifestyle may reduce the risk of CRC, that is, by increasing the intensity and frequency of physical activity; consuming more vegetables and fruits; and avoiding obesity, smoking, and heavy alcohol consumption [12]. Folate status assessment showed that the control group had significantly higher serum folate and dietary folate intake than both CRC and CRA groups. Our results agree with those previously reported [13] from the New York University Women's Health Study, that is, that subjects with CRC have significantly lower serum folate levels than control subjects. Similarly, a study of male and female Korean subjects found lower dietary folate intake in CRC patients compared with subjects without CRC [14]. Our results concur with previous

studies that found increased risk of CRC among individuals with both low serum folate levels and low dietary folate intake, and that the risk of CRA is increased with low folate intake [15-17]. In a meta-analysis, Park et al [18] found that low circulating folate levels were associated with a higher risk of the formation of recurrent adenoma. Evidence from experimental and epidemiological studies suggests that folate depletion might lead to carcinogenesis possibly by the misincorporation of uracil into DNA, impaired DNA repair processes, decreased DNA stability [19,20], and decreased DNA methylation [21]. The polymorphisms alone showed no relationship to CRC and CRA. However, other studies found some relationship between SNPs and CRC/CRA [22-24]. Understanding the interaction between nutrition and genetic variation is useful to distinguish between individuals who will and who will not benefit from diet intervention strategies. Folate and related genetic polymorphisms are of special interest to CRC risk. Therefore, we investigated the interaction between folate status and individual gene polymorphisms. Although polymorphisms were not independent risk factors for CRC and CRA, a number of significant associations were identified in subsets of the population, especially in the mutant group. We applied the criteria of the dominant model, which combined heterozygous and homozygous variants together, to observe the effect of mutation. Our findings suggest that individuals

Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

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Table 4 – Impact of serum folate, RBC folate, and folate intake–gene polymorphisms interaction on colorectal cancer risk Genotype

Folate status OR (95% CI)

Quartile of serum folate (nmol/L) n (%) a Overall MTHFR 677C > T CC CT + TT MTR 2756A > G AA AG + GG MTRR 66A > G AA AG + GG Quartile of RBC folate (nmol/L) n (%) a Overall MTHFR 677C > T CC CT + TT MTR 2756A > G AA AG + GG MTRR 66A > G AA AG + GG Quartile of folate intake (μg/d) n (%) a Overall MTHFR 677C > T CC CT + TT MTR 2756A > G AA AG + GG MTRR 66A > G AA AG + GG

P value

OR (95% CI)

P value

OR (95% CI)

P value

.038

Q1 (≤25.02) 50.4/13.2 11.45 (4.43-29.59)

.000

Q4 (≥94.29) 34.6/10.5 1.00

Q3 (57.30-94.28) 18.1/26.9 2.41 (0.92-6.28)

.073

Q2 (25.03-57.27) 21.0/25.3 2.78 (1.06-7.30)

1.00 1.00

3.15 (0.94-10.60) 3.40 (0.45-25.72)

.064 .237

3.66 (1.07-12.52) 2.28 (0.35-14.82)

.039 .389

14.79 (4.51-48.54) 12.73 (1.73-93.59)

.000 .012

1.00 1.00

3.09 (1.03-9.26) 0.72 (0.06-8.84)

.044 .796

2.97 (0.98-8.94) 1.43 (0.12-17.34)

.053 .781

10.33 (3.51-30.38) 40.10 (2.58-622.39)

.000 .008

1.35 (0.32-5.70) 5.38 (1.28-22.59) Q3 (579.97-790.09) 30.5/23.6 2.62 (1.13-6.06)

.681 .022

.339 .035

.199

8.43 (2.16-32.90) 23.62 (5.24-106.44) Q1 (≤424.77) 26.7/24.7 2.30 (0.95-5.53)

.002 .000

.024

2.02 (0.48-8.52) 4.92 (1.12-21.61) Q2 (424.78-579.96) 24.8/25.3 1.76 (0.74-4.20)

1.00 1.00

2.63 (0.96-7.18) 4.17 (0.68-25.57)

.059 .122

2.56 (0.87-7.56) 1.04 (0.20-5.44)

.088 .965

1.75 (0.61-5.04) 10.00 (1.36-73.42)

.303 .024

1.00 1.00

2.11(0.81-5.47) 9.50 (0.98-92.39)

.125 .052

1.29 (0.50-3.32) 23.78 (1.11-507.85)

.601 .042

2.28 (0.85-6.10) 2.91 (0.28-30.14)

.100 .369

3.73 (0.96-14.48) 2.24 (0.74-6.84) Q3(106.46-171.78) 15.1/29.1 1.38 (0.56-3.43)

.057 .156

.184 .551

.231

1.88 (0.45-7.89) 3.16 (0.96-10.36) Q1 (≤69.78) 50.5/10.5 10.29 (4.17-25.41)

.387 .057

.486

2.44 (0.66-9.10) 1.46 (0.42-4.99) Q2 (69.79-106.45) 20.4/24.7 1.72 (0.71-4.20)

.000

1.00 1.00

1.94 (0.65-5.85) 0.55 (0.09-3.57)

.238 .534

1.68 (0.56-4.99) 2.13 (0.38-11.91)

.354 .391

13.02 (4.30-39.44) 6.42 (1.13-36.60)

.000 .036

1.00 1.00

1.39 (0.49-3.95) 0.58 (0.07-5.03)

.536 .622

1.68 (0.60-4.69) 1.24 (0.16-9.55)

.320 .834

9.55 (3.46-26.36) 13.69 (1.16-161.78)

.000 .038

1.00 1.00

2.73 (0.70-10.58) 0.69 (0.17-2.75)

.148 .600

5.72 (1.30-25.17) 0.84 (0.25-2.77)

.021 .768

21.18 (4.29-104.51) 7.97 (2.44-26.04)

.000 .001

1.00 1.00 Q4 (≥790.10) 18.1/26.4 1.00

1.00 1.00 Q4 (≥171.79) 14.0/35.7 1.00

.064

Data are analyzed by logistic regression model adjusted for sex, age (10-year class), cigarette smoking, alcohol consumption, and exercise. Value are ORs (95% CI). P < .05 is considered to be statistically significant. a Number (%) of cases/control.

with MTHFR 677CT + TT with low RBC folate levels had significantly higher risk of developing CRC. In a previous study with an Asian population, the MTHFR 677 T allele was associated with a lower risk of CRC, and an effect was modified by population plasma folate [25]. However, the association between MTHFR polymorphisms and CRC risk has been inconsistent across populations, and the statistical significance of such associations appears to depend on folate supplementation status [8,26]. Keku et al reported interactions between folate intake and 2 MTHFR polymorphisms. In their study, individuals with low dietary folate intake who carried the MTHFR 677CT + TT genotype associated with increased CRC risk compared with the wild-type (CC) group [26]. Kim et al (2015) studied the combination of SNPs and folate status and the effect on risk of CRC. They found the interaction of low plasma folate and combination of SNPs; MTHFR 677CT/1298 AC and MTHFR 1298 AC + CC/TSER 2R3R genotypes with low plasma folate levels ≤9.33 nmol/L were

significantly associated with CRC risk [27]. Therefore, multiple SNPs and diet synergistically contribute to cancer risk. An association between the MTR 2756A > G polymorphism and genetic susceptibility to CRC and CRA has been widely documented but with inconsistent results. Goode et al suggest that the MTR 2756A > G polymorphism may have a modest role in CRA development, particularly among women (for AG/ GG vs AA, OR = 1.4, 95% CI, 0.9-2.1) [28]. Previous case-control studies have suggested that MTR 2756A > G increased risk for CRA [23] and CRC [24] without sex differences. The other study found no results suggestive of CRC association [29]. De Vogel et al found that the MTR 2756GG genotype was associated with increased CRC risk (incidence rate ratio, 1.58; P = .04) [24]. However, Ma et al showed that the MTR 2756GG genotype was associated with decreased risk of CRC in individuals with high plasma folate or low alcohol consumption [30]. Our results indicate that subjects with the MTR2756AG + GG mutation and low folate status are at increased risk of CRA (OR = 6.43,

Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

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Table 5 – Impact of folate status–gene polymorphisms interaction on colorectal adenoma risk Genotype

Folate status OR (95% CI)

Quartile of serum folate (nmol/L) n (%) a Overall MTHFR 677C > T CC CT + TT MTR 2756A > G AA AG + GG MTRR 66A > G AA AG + GG Quartile of RBC folate (nmol/L) n (%) a Overall MTHFR 677C > T CC CT + TT MTR 2756A > G AA AG + GG MTRR 66A > G AA AG + GG Quartile of folate intake (μg/d) n (%) a Overall MTHFR 677C > T CC CT + TT MTR 2756A > G AA AG + GG MTRR 66A > G AA AG + GG

P value

OR (95% CI)

P value

OR (95% CI)

P value

.046

Q1 (≤25.02) 19.8/13.2 2.23 (0.94-5.26)

.068

Q4 (≥94.29) 22.8/34.6 1.00

Q3 (57.30-94.28) 27.7/26.9 1.78 (0.87-3.65)

.113

Q2 (25.03-57.27) 29.7/25.3 2.10 (1.01-4.35)

1.00 1.00

1.65 (0.73-3.76) 7.31 (0.94-56.80)

.231 .057

2.00 (0.86-4.65) 12.41 (1.31-117.16)

.106 .028

2.65 (1.01-6.95) 6.46 (0.41-102.22)

.049 .185

1.00 1.00

1.46 (0.63-3.40) 4.95 (0.94-26.03)

.381 .059

1.33 (0.56-3.20) 8.97 (1.66-48.42)

.520 .011

1.70 (0.65-4.45) 10.64 (0.94-120.23)

.283 .056

4.18 (1.18-14.87) 1.17 (0.42-3.16) Q3 (579.97-790.09) 22.7/23.6 0.92 (0.44-1.94)

.027 .759

.107 .110

.792

5.36 (1.26-22.92) 1.67 (0.51-5.46) Q1 (≤424.77) 23.8/24.7 0.86 (0.40-1.85)

.023 .396

.835

3.04 (0.79-11.74) 2.10 (0.85-5.20) Q2 (424.78-579.96) 23.8/25.3 0.91 (0.44-1.94)

1.00 1.00

1.08 (0.46-2.56) 0.51 (0.08-3.28)

.858 .475

1.24 (0.51-3.06) 0.37 (0.07-1.94)

.635 .238

1.01 (0.42-2.43) 0.27 (0.02-3.47)

.992 .316

1.00 1.00

0.92 (0.38-2.24) 1.02 (0.23-4.46)

.847 .984

0.68 (0.29-1.59) 4.76 (0.78-29.05)

.371 .091

0.55 (0.21-1.40) 2.28 (0.60-12.87)

.209 .192

0.78 (0.26-2.42) 1.10 (0.40-2.99) Q3(106.46-171.78) 26.7/29.1 2.01 (0.94-4.30)

.672 .857

.234 .418

.008

0.69 (0.20-2.30) 1.03 (0.37-2.83) Q1 (≤69.78) 27.8/10.5 5.22 (2.19-12.48)

.540 .959

.073

0.49 (0.15-1.59) 1.50 (0.56-4.02) Q2 (69.79-106.45) 29.7/24.7 2.84 (1.32-6.09)

1.00 1.00

2.31 (0.96-5.56) 2.05 (0.29-14.59)

.062 .473

2.27 (0.94-5.45) 8.33 (1.10-63.06)

.067 .040

5.89 (2.11-16.43) 5.01 (0.65-38.51)

.001 .122

1.00 1.00

1.96 (0.76-5.05) 2.37 (0.58-9.68)

.162 .228

3.86 (1.48-10.08) 1.70 (0.44-6.51)

.006 .441

5.69 (2.04-15.88) 4.45 (0.56-35.54)

.001 .160

1.00 1.00

4.61 (1.08-19.72) 1.35 (0.50-3.62)

.040 .554

15.88 (3.52-71.58) 1.08 (0.41-2.860

.000 .873

28.63 (5.37-152.69) 2.46 (0.82-7.39)

.000 .108

1.00 1.00 Q4 (≥790.10) 29.7/26.4 1.00

1.00 1.00 Q4 (≥171.79) 15.8/35.7 1.00

.700

.000

Data are analyzed by logistic regression model adjusted for sex, age (10-year class), cigarette smoking, alcohol consumption, and exercise. Value are ORs (95% CI). P < .05 is considered to be statistically significant. a Number (%) of cases/control.

95% CI = 1.38-29.94). In a meta-analysis, the combined evidence suggested that MTR 2756A > G polymorphism did not contribute to the development of CRC or CRA. Most of those studies did not consider important environmental factors. It is possible that variation at this locus has modest effects on CRC or CRA; however, environmental factors may predominate in the progress of diseases and mask the effects of this variation [31]. Matsuo et al founded that the GG genotype of MTRR 66A > G is a risk factor for CRC in the Japanese population [29]. Le Marchand et al reported that the MTRR 66A > G polymorphism was associated with CRC and did not interact with any nutrient, including folate [11]. However, in our study, a gene-nutrient intake was evident between the MTRR 66 (AG + GG) variant and RBC folate level, which was associated with increased CRC risk (OR = 3.16, P = .057). MTRR triggers the reactivation of MTR via reductive methylation and thus acts at the junction between the folate and methionine cycles.

Kinetic investigations of the functional properties of the MTRR variant enzyme revealed a decreased affinity of the protein toward MTR [32]. The interaction effects of folate status and environmental factors on CRA and CRC risk were examined in this study. We found that subjects with low folate status had increased risk of CRC. CRC risk was higher among subjects who reported low exercise and alcohol consumption; these findings agree with previous studies [16,33-35]. Alcohol consumption inhibits folate absorption from the intestine, interrupts folate release from the liver, and promotes urinary excretion of folate from the body, all of which result in reduced levels of circulating blood folate [31]. As a folate antagonist, alcohol inhibits folate-mediated methionine synthesis, inducing abnormal DNA methylation, which is frequently observed in colorectal neoplasia [36-38]. CRC is complicated, and environmental and genetic factors are involved in the development of malignancy. We

Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

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NUT RI TI ON R ES E ARC H X X (X XXX ) XXX

Table 6 – Impact of folate status–gene polymorphisms interaction on colorectal adenoma and cancer risk Genotype

Folate status High folate status

Colorectal adenoma n (%) a Overall MTHFR 677C > T CC CT + TT MTR 2756A > G AA AG + GG MTRR 66A > G AA AG + GG Colorectal cancer n (%) a Overall MTHFR 677C > T CC CT + TT MTR 2756A > G AA AG + GG MTRR 66A > G AA AG + GG

Intermediate folate status

Low folate status

OR (95% CI)

P value

OR (95% CI)

P value

16.8/26.9 1.00

29.7/34.1 1.71 (0.84-3.65)

.16

53.5/39.0 2.27 (1.11-4.64)

.024

1.00 1.00

1.95 (0.82-4.64) 0.83 (0.13-5.52)

.132 .848

2.28 (1.00-5.20) 3.95 (0.59-26.61)

.049 .158

1.00 1.00

1.71 (0.70-4.20) 2.06 (0.46-9.38)

.242 .346

1.74 (0.75-4.040 6.43 (1.38-29.94)

.196 .018

1.00 1.00

2.85 (0.85-9.59) 1.14 (0.41-3.17)

.909 .799

3.02 (1.03-8.82) 1.80 (0.67-4.83)

.044 .242

7.5/26.9 1.00

25.8/66.7 2.66 (0.99-7.11)

.052

66.7/39.0 4.98 (1.96-12.67)

.001

1.00 1.00

3.70 (1.15-11.92) 1.2 (0.18-8.14)

.028 .852

5.20 (1.71-15.85) 4.07 (0.71-23.41)

.004 .116

1.00 1.00

2.84 (0.89-9.05) 3.36 (0.41-27.32)

.077 .256

4.53 (1.51-13.59) 8.84 (1.09-71.69)

.007 .041

1.00 1.00

3.27 (0.79-13.57) 2.06 (0.48-8.92)

.102 .335

5.22 (1.41-19.31) 4.99 (1.19-20.86)

.013 .028

Data are analyzed by logistic regression model adjusted for sex, age (10-year class), cigarette smoking, alcohol consumption, and exercise. Value are ORs (95% CI). P < .05 is considered to be statistically significant. a Number (%) of cases/control.

Table 7 – Impact of folate status–environmental factors interaction on colorectal adenoma and cancer risk Environmental factors

Folate status High folate status

Colorectal adenoma n (%) a Overall Frequency of exercise >1 time/wk ≤1 time/wk Alcohol consumption Never Ever Cigarette smoking Never Ever Colorectal cancer n (%) a Overall Frequency of exercise >1 time/wk ≤1 time/wk Alcohol consumption Never Ever

Intermediate folate status

Low folate status

OR (95% CI)

P value

OR (95% CI)

P value

16.8/26.9 1.00

29.7/34.1 1.71 (0.84-3.65)

.16

53.5/39.0 2.27 (1.11-4.64)

.024

1.00 1.00

1.01 (0.25-4.16) 0.40 (0.94-6.09)

.99 .067

2.24 (0.61-8.22) 2.15 (0.90-5.15)

.222 .084

1.00 1.00

2.46 (0.81-7.50) 1.43 (0.49-4.18)

.114 .519

2.83 (0.97-8.24) 2.11 (0.757-5.873)

.056 .153

1.00 1.00

2.53 (0.91-7.01) 1.04 (0.29-3.72)

.074 .952

3.40 (1.29-8.95) 1.25 (0.38-4.13)

.013 .717

7.5/26.9 1.00

25.8/66.7 2.66 (0.99-7.11)

.052

66.7/39.0 4.98 (1.96-12.67)

.001

1.00 1.00

0.48 (0.02-9.39) 3.73 (1.26-11.01)

.626 .017

6.11 (0.59-63.45) 5.02 (1.79-14.06)

.130 .002

1.00 1.00

3.33 (0.78-14.21) 2.28 (0.60-8.67)

.104 .26

2.93 (0.69-12.41) 6.33 (1.79-22.37)

.144 .004

Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

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NUT RI TI ON R E SE ARC H X X (X XXX ) XX X

Table 7 (continued) Environmental factors

Folate status High folate status

Cigarette smoking Never Ever

1.00 1.00

Intermediate folate status

Low folate status

OR (95% CI)

P value

OR (95% CI)

P value

3.93 (1.08-14.29) 1.81 (0.36-9.09)

.037 .474

4.85 (1.38-17.05) 4.96 (1.09-22.48)

.014 .038

Data are analyzed by logistic regression model adjusted for sex, age (10-year class), cigarette smoking, alcohol consumption, and exercise. Value are ORs (95% CI). P < .05 is considered to be statistically significant. a Number (%) of cases/control.

suggest that individuals with genetic variations in MTHFR 677C > T and MTR 2756A > G with low folate status, a sedentary lifestyle, and high alcohol consumption are at highest risk of CRC. Cancer development is a multistage process. The limitation of our study is that we studied only 1 cancer time point: when it was first found. We could not know for how long the patients had been developing cancer or when cell changes began. The strength of this association will be better quantified by a prospective study. In conclusion, the present study suggests that low serum folate and folate intake are associated with increased risk of CRC among Thais. This study further supports the existing hypothesis of genetic-nutrient interactions in colorectal carcinogenesis. We confirm that folate status and related genetic polymorphisms may be related with CRC development in Thais. Low folate status may be a serious risk, particularly for those with MTHFR 677C > T and MTR 2756A > G polymorphisms.

Acknowledgment We thank the staff at Phramongkutklao Hospital, Vajira Hospital, and the Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University. We also thank our subjects for their participation in our study, Anette from ENAGO Group (www.enago.com), and Paul Adams for editing a draft of this manuscript. We have no conflicts of interest to disclose. This research was supported by Mahidol University. The finding is part of a PhD program at Mahidol University.

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Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008

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Please cite this article as: P. Panprathip, S. Petmitr, R. Tungtrongchitr, et al., Low folate status, and MTHFR 677C > T and MTR 2756A > G polymorphisms associated with colorec..., Nutr Res (2019), https://doi.org/10.1016/j.nutres.2019.10.008