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Investigation of methylenetetrahydrofolate reductase tagging polymorphisms with colorectal cancer in Chinese Han population

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Oncotarget. 2017; 8:63518-63527. https://doi.org/10.18632/oncotarget.18845

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Sheng Zhang, Shuchen Chen, Yu Chen, Mingqiang Kang, Chao Liu, Hao Qiu, Yafeng Wang and Weifeng Tang _

Abstract

Sheng Zhang1,*, Shuchen Chen2,*, Yu Chen3,4,*, Mingqiang Kang2, Chao Liu5, Hao Qiu6, Yafeng Wang7 and Weifeng Tang2,5

1Department of General Surgery, Changzhou No. 3 People’s Hospital, Changzhou, Jiangsu Province, China

2Department of Thoracic Surgery, Affiliated Union Hospital of Fujian Medical University, Fuzhou, Fujian Province, China

3Department of Medical Oncology, Fujian Provincial Cancer Hospital, Fujian Medical University Cancer Hospital, Fuzhou, Fujian Province, China

4Fujian Provincial Key Laboratory of Translational Cancer Medicine, Fuzhou, Fujian Province, China

5Department of Cardiothoracic Surgery, Affiliated People’s Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, China

6Department of Immunology, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu Province, China

7Department of Cardiology, The People's Hospital of Xishuangbanna Dai Autonomous Prefecture, Jinghong, Yunnan Province, China

*These authors have contributed equally to this work

Correspondence to:

Weifeng Tang, email: [email protected]

Yafeng Wang, email: [email protected]

Keywords: polymorphism, MTHFR, colorectal cancer, tagging, susceptibility

Received: January 27, 2017     Accepted: June 04, 2017     Published: June 29, 2017

ABSTRACT

The aim of this case-control study was to assess the relationship between the tagging polymorphisms in methylenetetrahydrofolate reductase (MTHFR) gene and the susceptibility to colorectal cancer (CRC) in a Chinese Han population. A custom-by-design 48-Plex SNPscan Kit was used to determine the genotypes of MTHFR rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A polymorphisms in 387 CRC patients and 1,536 non-cancer controls. The results revealed that MTHFR rs1801133 G>A polymorphism was associated with a decreased risk of overall CRC. While MTHFR rs4845882 G>A polymorphism conferred an increased risk to overall CRC. In a stratified analysis by CRC region, we found MTHFR rs3753584 T>C and rs9651118 T>C polymorphisms were associated with the increased risk of colon cancer. In addition, a significantly increased risk of rectum cancer associated with MTHFR rs3753584 T>C polymorphism was overt. However, MTHFR rs1801133 G>A polymorphism conferred a decreased risk to colon cancer. In conclusion, findings of the present study reveal that the tagging polymorphisms in MTHFR gene (rs3753584 T>C, rs9651118 T>C and rs4845882 G>A) are associated with the increased risk of CRC. However, MTHFR rs1801133 G>A polymorphism confers a decreased risk to CRC. Additional studies with larger sample size are needed to confirm these findings.


INTRODUCTION

Colorectal cancer (CRC) is the fourth most frequent type of malignancy among females and the fifth most frequent type among males in China, accounting for 160,600 and 215,700 cases in 2015, respectively [1]. The CRC morbidity is shooting up in developing countries including China [1, 2]; nevertheless, CRC etiology remains unknown. Risk factors, such as advanced age, family history of CRC, benign adenomatous polyp, inflammatory bowel disease, drinking, smoking, being physically inactive, low intake of fruits and vegetables and high intake of dietary fat, may play important roles in the occurrence and the development of CRC [39]. Accumulating evidences indicated that besides environmental factors and individual lifestyle, some genetic factors might be related to CRC etiology.

Several studies have found association between circulating or dietary folate level and the risk of CRC [1012]. A long induction period deserves close attention to the study of CRC because the course from normal cells of the rectum and colon, to microadenomas, to macroadenomas, and eventually to adenocarcinomas, experiences over a long duration, approximately 30-40 years [13]. The presence of the long induction period between reduced risk of CRC and adequate folate status is evident in both epidemiologic and molecular mechanistic studies [14]. The lower folate levels lead to an increasing rate of DNA hypomethylation and uracil misincorporation even in non-neoplastic, normal-appearing tissue of cases [14]. Folate is reduced to tetrahydrofolate (THF) to participate in one-carbon metabolism [15]. Folate metabolism is accommodated by several enzymes. Methylenetetrahydrofolate reductase (MTHFR) is the key enzyme [1618]. MTHFR is involved in DNA methylation, repair and synthesis [19]. DNA strand break and repair, and impaired DNA methylation have been associated with folate deficiency and CRC.

MTHFR gene locates on 1p36.22. The gene encodes a 74.6-kDa protein containing 656 amino acids. MTHFR is also linked to purine synthesis; therefore, plays a vital role in DNA synthesis by the provision of essential nucleotides [18]. Single nucleotide polymorphisms (SNPs) of MTHFR are considered as a potential biomarker which may influence CRC risk. Therefore, we carried out this case-control study in a Chinese Han population to determine whether MTHFR tagging SNPs (rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A) were associated with the risk of CRC.

RESULTS

Demographic characteristics

The frequency distributions of age, sex, smoking status and drinking habit information for the 387 patients with CRC (mean ± SD age, 60.21 ± 12.48 years) and 1,536 controls (mean ± SD age, 60.82± 8.85 years) are shown in Table 1. The age and sex of the CRC patients and controls were well-matched. The differences of smoking status and drinking habit between CRC and non-cancer controls were not statistically significant (P ≥ 0.05) (Table 1). Tumor grade was defined as high (well differentiated), medium (moderately differentiated); and low (poorly differentiated). Two experienced doctors independently assessed disease stage according to the AJCC criteria (2010). Finally, 196 cases with stage I/II and 191 with stage III/IV were included. Among the CRC patients, 218 were rectum cancer and 169 were colon cancer. And the primary information for MTHFR polymorphisms is summarized in Table 2.

Table 1: Distribution of selected demographic variables and risk factors in colorectal cancer cases and controls

Variable

Cases (n=387)

Controls (n=1,536)

Pa

n

%

n

%

Age (years)

60.21 (±12.48)

60.82 (±8.85)

0.272

Age (years)

0.502

 < 61

186

48.06

709

46.16

 ≥ 61

201

51.94

827

53.84

Sex

0.213

 Male

236

60.98

989

64.39

 Female

151

39.02

547

35.61

Smoking status

0.505

 Never

270

69.77

1098

71.48

 Ever

117

30.23

438

28.52

Alcohol use

0.058

 Never

335

78.55

1381

89.91

 Ever

52

21.45

155

10.09

Site of tumor

 Colon cancer

169

43.67

 Rectum cancer

218

56.33

Degree of differentiationb

 Low

56

16.28

 Medium

261

75.87

 High

27

7.85

Lymph node status

 Positive

177

45.74

 Negative

210

54.26

TMN stage

 I+II

196

50.65

 III+IV

191

49.35

aTwo-sided χ2 test and Student t test.

Table 2: Primary information for MTHFR polymorphisms (rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A)

Genotyped SNPs

rs3753584 T>C

rs9651118 T>C

rs1801133 G>A

rs4846048 A>G

rs4845882 G>A

Chromosome

1

1

1

1

1

Function

NearGene-5

Intron

Missense

Intron

Intron

Chr Pos (Genome Build 36.3)

11787173

11784801

11778965

11768839

11765754

MAFa for Chinese in database

0.093

0.382

0.439

0.105

0.198

MAF in our controls (n = 1,536)

0.109

0.379

0.356

0.096

0.209

P value for HWEb test in our controls

0.164

0.134

0.747

0.082

0.733

Genotyping method

SNPscan

SNPscan

SNPscan

SNPscan

SNPscan

% Genotyping value

99.64%

99.64%

99.64%

99.64%

99.64%

aMAF: minor allele frequency;

bHWE: Hardy–Weinberg equilibrium.

Association of MTHFR rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A polymorphisms with CRC patients

The genotypes of MTHFR rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A polymorphisms in CRC patients and non-cancer controls are summarized in Table 3. The observed genotype frequencies for the five MTHFR tagging SNPs in control group were all in HWE (P = 0.134, 0.082, 0.733, 0.164 and 0.747 for MTHFR rs9651118 T>C, rs4846048 A>G, rs4845882 G>A, rs3753584 T>C and rs1801133 G>A, respectively), which suggested good homogeneity within the present study participants. Four CRC patients (1.03%) and three controls (0.19%) could not be genotyped for poor DNA quantity and/or quality. Overall, we found no statistically significant difference in genotype distribution of MTHFR rs3753584 T>C, rs9651118 T>C and rs4846048 A>G polymorphisms among CRC patients and controls (Table 4).

Table 3: The frequencies of MTHFR rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A polymorphisms in colorectal cancer patients and controls

Genotype

Colorectal cancer (n=387)

Colon cancer

(n=169)

Rectum cancer (n=218)

Controls

(n=1,536)

n

%

n

%

n

%

n

%

MTHFR rs3753584 T>C

TT

287

74.93

131

78.44

156

72.22

1211

79.00

TC

89

23.24

31

18.56

58

26.85

309

20.16

CC

7

1.83

5

2.99

2

0.93

13

0.85

CT+CC

96

25.07

36

21.56

60

27.78

322

21.00

TT+CT

376

98.17

162

97.01

214

99.07

1,520

99.15

CC

7

1.83

5

2.99

2

0.93

13

0.85

C allele

103

13.45

41

12.28

62

14.35

335

10.93

MTHFR rs9651118 T>C

TT

131

34.20

56

33.53

75

34.72

578

37.70

TC

188

49.09

79

47.31

109

50.46

749

48.86

CC

64

16.71

32

19.16

32

14.81

206

13.44

TC+CC

252

65.80

111

66.47

141

65.28

955

62.30

TT+TC

319

83.29

135

80.84

184

85.19

1,327

86.56

CC

64

16.71

32

19.16

32

14.81

206

13.44

C allele

316

41.25

143

42.81

173

40.05

1,161

37.87

MTHFR rs1801133 G>A

GG

177

46.21

82

49.10

95

43.98

639

41.68

GA

175

45.69

73

43.71

102

47.22

697

45.47

AA

31

8.09

12

7.19

19

8.80

197

12.85

GA + AA

206

53.79

85

50.90

121

56.02

894

58.32

GG+ GA

352

91.91

155

92.81

197

91.20

1,336

87.15

AA

31

8.09

12

7.19

19

8.80

197

12.85

A allele

237

30.94

97

29.04

140

32.41

1,091

35.58

MTHFR rs4846048 A>G

AA

308

80.42

136

81.44

172

79.63

1259

82.13

AG

70

18.28

30

17.96

40

18.52

254

16.57

GG

5

1.31

1

0.60

4

1.85

20

1.30

AG+GG

75

19.58

31

18.56

44

20.37

274

17.87

AA+AG

378

98.69

166

99.40

212

98.15

1,513

98.70

GG

5

1.31

1

0.60

4

1.85

20

1.30

G allele

80

10.44

32

9.58

48

11.11

294

9.59

MTHFR rs4845882 G>A

GG

228

59.53

103

61.68

125

57.87

956

62.36

GA

129

33.68

54

32.34

75

34.72

512

33.40

AA

26

6.79

10

5.99

16

7.41

65

4.24

GA+AA

155

40.47

64

38.32

91

42.13

577

37.64

GG+GA

357

93.21

157

94.01

200

92.59

1,468

95.76

AA

26

6.79

10

5.99

16

7.41

65

4.24

A allele

181

23.63

74

22.16

107

24.77

642

20.94

Table 4: Overall and stratified analyses of MTHFR rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A polymorphisms with colorectal cancer

Genotype

Overall colorectal cancer cases (n=387)

Colon cancer (n=169)

Rectum cancer (n=218)

Crude OR (95%CI)

P

Adjusted ORa (95%CI)

P

Crude OR (95%CI)

P

Adjusted ORa (95%CI)

P

Crude OR (95%CI)

P

Adjusted ORa (95%CI)

P

MTHFR rs3753584 T>C

Additive model

1.20 (0.92-1.57)

0.180

1.20 (0.92-1.58)

0.178

0.92(0.61-1.38)

0.674

0.92(0.61-1.38)

0.679

1.44(1.04-2.00)

0.028

1.44(1.04-2.00)

0.030

Homozygote model

2.25 (0.89-5.68)

0.087

2.34 (0.92-5.93)

0.074

3.51(1.23-10.01)

0.019

3.63(1.27-10.38)

0.016

1.18 (0.26-5.29)

0.827

1.20 (0.27-5.38)

0.815

Dominant model

1.26 (0.97-1.63)

0.086

1.26 (0.97-1.64)

0.081

1.03(0.70-1.53)

0.867

1.04(0.70-1.53)

0.852

1.45 (1.05-2.00)

0.025

1.44 (1.04-2.00)

0.026

Recessive model

2.18(0.86-5.49)

0.100

2.26 (0.89-5.73)

0.085

3.61(1.27-10.26)

0.016

3.74(1.31-10.64)

0.014

1.09(0.25-4.88)

0.908

1.11 (0.25-4.97)

0.894

MTHFR rs9651118 T>C

additive model

1.08(0.84-1.38)

0.539

1.08(0.85-1.39)

0.528

1.06(0.74-1.51)

0.762

1.06 (0.75-1.52)

0.734

1.10 (0.80-1.50)

0.556

1.10 (0.80-1.50)

0.552

homozygote model

1.34 (0.95-1.87)

0.091

1.36 (0.97-1.90)

0.077

1.56(0.98-2.47)

0.060

1.56 (0.99-2.48)

0.057

1.17 (0.75-1.82)

0.481

1.22 (0.78-1.90)

0.390

Dominant model

1.16(0.92-1.47)

0.205

1.17(0.93-1.48)

0.190

1.20(0.86-1.68)

0.290

1.21 (0.86-1.69)

0.275

1.14 (0.84-1.53)

0.397

1.15 (0.85-1.55)

0.368

Recessive model

1.29 (0.95-1.76)

0.100

1.31(0.96-1.78)

0.084

1.53(1.01-2.31)

0.044

1.53(1.01-2.31)

0.044

1.12(0.75-1.68)

0.581

1.16 (0.77-1.74)

0.471

MTHFR rs1801133 G>A

additive model

0.89 (0.71-1.13)

0.331

0.89(0.70-1.12)

0.322

0.80(0.58-1.12)

0.188

0.80(0.57-1.11)

0.183

0.97(0.72-1.31)

0.834

0.97(0.72-1.30)

0.817

homozygote model

0.56 (0.37-0.84)

0.006

0.56 (0.37-0.84)

0.005

0.47(0.25-0.87)

0.017

0.46 (0.25-0.86)

0.015

0.64 (0.38-1.07)

0.089

0.64 (0.38-1.07)

0.090

Dominant model

0.83 (0.66-1.04)

0.109

0.83 (0.66-1.04)

0.106

0.74(0.54-1.02)

0.066

0.74(0.54-1.02)

0.063

0.91(0.68-1.21)

0.522

0.91(0.68-1.21)

0.512

Recessive model

0.60 (0.40-0.89)

0.011

0.60 (0.40-0.89)

0.011

0.53 (0.29-0.96)

0.037

0.52(0.28-0.95)

0.035

0.65 (0.40-1.07)

0.092

0.66 (0.40-1.08)

0.095

MTHFR rs4846048 A>G

additive model

1.12(0.83-1.49)

0.466

1.11(0.83-1.49)

0.474

1.08(0.71-1.64)

0.717

1.08 (0.71-1.63)

0.735

1.14 (0.79-1.65)

0.480

1.14(0.79-1.66)

0.482

homozygote model

1.01 (0.38-2.72)

0.982

0.98 (0.36-2.63)

0.961

0.46(0.06-3.43)

0.447

0.46 (0.06-3.48)

0.454

1.45 (0.49-4.30)

0.501

1.36 (0.45-4.07)

0.583

Dominant model

1.12(0.84-1.49)

0.439

1.11(0.84-1.48)

0.457

1.05(0.69-1.58)

0.826

1.04 (0.69-1.58)

0.839

1.18 (0.82-1.68)

0.373

1.17 (0.82-1.67)

0.393

Recessive model

1.00(0.37-2.68)

1.000

0.96(0.36-2.59)

0.938

0.46(0.06-3.42)

0.445

0.46(0.06-3.45)

0.450

1.43(0.48-4.22)

0.519

1.33 (0.45-3.98)

0.609

MTHFR rs4845882 G>A

additive model

1.04 (0.82-1.33)

0.741

1.05 (0.82-1.33)

0.720

0.96 (0.68-1.36)

0.832

0.97 (0.68-1.36)

0.843

1.11(0.82-1.50)

0.517

1.11(0.82-1.51)

0.508

homozygote model

1.65 (1.03-2.66)

0.039

1.64 (1.02-2.65)

0.043

1.41(0.70-2.82)

0.338

1.42 (0.71-2.85)

0.326

1.86(1.04-3.31)

0.035

1.79 (1.00-3.20)

0.050

Dominant model

1.13 (0.90-1.42)

0.308

1.13 (0.90-1.42)

0.300

1.03(0.74-1.43)

0.862

1.03(0.74-1.44)

0.845

1.21 (0.90-1.61)

0.204

1.20 (0.90-1.61)

0.211

Recessive model

1.65(1.03-2.63)

0.038

1.63 (1.01-2.60)

0.044

1.44 (0.73-2.86)

0.299

1.45 (0.73-2.88)

0.290

1.81(1.03-3.18)

0.041

1.73(0.98-3.07)

0.059

aAdjusted for age, sex, smoking status and alcohol use in a logistic regression model.

When compared with MTHFR rs1801133 GG genotype, MTHFR rs1801133 AA genotype was associated with a decreased risk of CRC (crude OR = 0.56, 95% CI: 0.37–0.84, P = 0.006). When compared with MTHFR rs1801133 GG/GA genotype, MTHFR rs1801133 AA genotype was also associated with a decreased risk of CRC (crude OR = 0.60, 95% CI: 0.40–0.89, P = 0.011). Adjustment for multiple factors (age, sex, smoking and drinking), the results were not essentially changed (AA vs. GG: adjusted OR = 0.56, 95% CI: 0.37–0.84, P = 0.005; AA vs. GG/GA: OR = 0.60, 95% CI: 0.40–0.89, P = 0.011; Table 4).

When compared with MTHFR rs4845882 GG genotype, a MTHFR rs4845882 AA genotype increased the risk of CRC (crude OR = 1.65, 95% CI: 1.03–2.66, P = 0.039). When compared with MTHFR rs4845882 GG/GA genotype, MTHFR rs4845882 AA genotype was associated with an increased risk of CRC (crude OR = 1.65, 95% CI: 1.03–2.63, P = 0.038). Adjustment for multiple factors (age, sex, smoking and drinking), the results were not essentially changed (AA vs. GG: adjusted OR =1.64 95% CI: 1.02–2.65, P = 0.043; AA vs. GG/GA: OR = 1.63, 95% CI: 1.01–2.60, P = 0.044; Table 4).

Association of MTHFR rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A polymorphisms with CRC in a stratification group

To determine whether the effect of MTHFR polymorphisms was influenced by CRC region, we performed a stratified analysis. For MTHFR rs3753584 T>C polymorphism, we found this polymorphism was associated with an increased risk of rectum cancer (CC+TC vs. TT: adjusted OR = 1.44, 95% CI = 1.04–2.00, P = 0.026; TC vs. TT: adjusted OR = 1.44, 95% CI = 1.04–2.00, P = 0.030) and of colon cancer (CC vs. TT+TC: adjusted OR = 3.74, 95% CI = 1.31–10.64, P = 0.014; CC vs. TT: adjusted OR = 3.63, 95% CI = 1.27–10.38, P = 0.016). For MTHFR rs9651118 T>C polymorphism, we found MTHFR rs9651118 CC genotypes might be associated with an increased risk of colon cancer (CC vs. TT+TC: adjusted OR = 1.53, 95% CI = 1.01–2.31, P = 0.044). However, we found MTHFR rs1801133 G>A polymorphism was associated with a decreased risk of colon cancer (AA vs. GG+GA: adjusted OR = 0.52, 95% CI = 0.28–0.95, P = 0.035; AA vs. GG: adjusted OR = 0.46, 95% CI = 0.25–0.86, P = 0.015). Other comparisons are presented in Table 4.

The power of the present study (α= 0.05)

For MTHFR rs3753584 T>C, the power value was 0.634 in homozygote model and 0.651 in recessive model among colon cancer group, and 0.586 in homozygote model and 0.614 in recessive model among rectum cancer group. In addition, for MTHFR rs9651118 T>C, the power value was 0.527 in recessive model among colon cancer group. Moreover, for MTHFR rs1801133 G>A, the power value was 0.815 in homozygote model and 0.748 in recessive model among overall CRC group, and 0.705 in homozygote model and 0.565 in recessive model among colon cancer group. For MTHFR rs4845882 G>A, the power value was 0.537 in homozygote model and 0.553 in recessive model among overall CRC group, and 0.494 in homozygote model and 0.540 in recessive model among rectum cancer group as well.

DISCUSSION

Recently, CRC incidence and related mortality are increasing rapidly worldwide. The individual's susceptibility to CRC may be influenced by some environmental exposure and genetic factors. Recently, many case-control studies have been directed towards the association between MTHFR polymorphisms and CRC risk. However, the sample size of most studies was relatively small. Here, we attempt to assess the association between MTHFR tagging SNPs (rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A) and susceptibility of CRC. Our results indicated several MTHFR tagging polymorphisms could affect the risk of CRC.

MTHFR rs1801133 G>A polymorphism leads to an amino acid transformation (alanine→valine at 226 position of MTHFR protein). The role of MTHFR enzyme is remethylation of homocysteine to methionine. Polymorphisms in MTHFR gene are corrected with the deficiency of MTHFR enzyme activity. Compared with rs1801133 GG homozygote, MTHFR rs1801133 AA homozygote decreases 70% of the enzyme activity and MTHFR rs1801133 GA heterozygote loss 35% of enzymatic function. This transformation may increase the plasma homocysteine (Hcy) concentration and decreases the plasma folic acid concentration [26]. Recently, several meta-analyses indicated that MTHFR rs1801133 G>A polymorphism decreased the risk of CRC in Asians [27, 28]. MTHFR rs1801133 G>A polymorphism locates on the NH2-terminal catalytic domain. In addition, MTHFR rs1801133 G>A polymorphism increases the availability of 5, 10-methylenetetrahydrofolate for DNA synthesis [29, 30], which may partially explain the protective factor of CRC. In combination with this case-control study, our results evidence that G→A mutation in MTHFR rs1801133 increases the availability of 5, 10-methylenetetrahydrofolate for DNA synthesis; thus, this SNP may be a protective factor of CRC.

To the best of our knowledge, it was the first epidemiological study to explore the relationship of MTHFR rs4845882 G>A polymorphism with CRC risk. MTHFR rs4845882 G>A is linkage disequilibrium (LD) with rs1801131 (1298 A>C). Several meta-analyses suggested that MTHFR rs1801131 A>C polymorphism affected risk of CRC in Asians [31, 32]. In the present study, we found MTHFR rs4845882 G>A polymorphism may be associated with the development of CRC. Since MTHFR rs4845882 G>A and rs1801131 A>C are in strong LD (r2 = 0.935, http://gvs.gs.washington.edu/GVS147/LDpairwiseR2.jsp?GET_TAYLORGRAM=1491876387794), the function of rs4845882 G>A may be affected by rs1801131 A>C.

A study reported that CC genotype of MTHFR rs9651118 conferred a reduced risk of breast cancer compared to TT genotype in a Chinese population [33]. Swartz et al. found MTHFR rs9651118 T>C polymorphism may be correlated with the decreased risk of lung cancer in Caucasians [34]. In our previous study, we found there was null association between MTHFR rs9651118 T>C polymorphism and esophageal squamous cell carcinoma in a Chinese Han population [21]. However, in this study, we found that rs9651118 CC genotype was relevant to increased risk of colon cancer. Rs9651118 T>C polymorphism is located on the intron of MTHFR gene. The function of this polymorphism is not well known. It was reported that MTHFR rs9651118 TT genotype elevated the level of Hcy compared with CC genotype [35]. In the future, more functional studies are required to identify the real biological effect of MTHFR rs9651118 T>C polymorphism on the etiology of CRC.

In a subgroup analysis by the region of CRC, MTHFR rs3753584 T>C polymorphism was associated with the risk of colon and rectum cancer. Although the function of MTHFR rs3753584 T>C polymorphism was not identified, a significantly increased risk of lung cancer was found for the variant allele carriers of this polymorphism, compared with individuals with wild homozygote [36]. In this study, we also found that C allele of MTHFR rs3753584 was probably correlated with an increased risk of colon and rectum cancer, which was consistent with the findings of those previous study.

Like all case-control studies, this study has some limitations. First, demographic variables and risk factor information only focused on age, sex, smoking and alcohol consumption. And other lifestyles were not collected, which might increase the possibility of confounding from environmental risk factors. Second, the source of non-cancer controls was hospital-based; which might not well represent the whole Chinese population. Third, in a stratified analysis by the region of CRC, the relatively small sample size may decrease the power of the results. Finally, these findings should be interpreted with very caution because the participants were only enrolled from Chinese Han population. Thus, the results may not permit extrapolation to other ethnicities.

In summary, the tagging polymorphisms in MTHFR gene (rs3753584 T>C, rs9651118 T>C and rs4845882 G>A) are associated with an increased risk of CRC. However, MTHFR rs1801133 G>A polymorphism confers a decreased risk to CRC. Our findings suggest that further validation studies are needed.

MATERIALS AND METHODS

Study population and patient selection

The study population consisted of 387 sporadic, consecutive CRC patients from the Department of General Surgery, Fujian Medical University Union Hospital (Fuzhou, China) between October 2014 and May 2016. Histological report confirmed the diagnosis of CRC. Non-cancer controls (n = 1,536) were randomly recruited from the Fujian Medical University Union Hospital (Fuzhou, China) and the Affiliated People's Hospital of Jiangsu University (Zhenjiang, China) between October 2014 and November 2016. The CRC patients and controls were matched for age, sex and residential area (Eastern China). Every participant was informed the aim of the present study and signed an informed consent. The protocol of the study was obtained by the institutional ethics committee of Fujian Medical University and Jiangsu University. Additionally, in this case-control study, we conformed to the principles of Declaration of Helsinki.

Data collection

Every participant was personally questioned and answered a questionnaire regarding age, sex, the status of cigarette smoking, and alcohol consumption. Participants who drink more than thrice/week for >6 months and smoke at least one cigarette/day over 1 year were considered positive. Clinical characteristics, such as pathological stage and tumor site, were obtained from the medical records (Table 1).

Selection of tagging SNPs

The tagging SNPs of MTHFR gene [30.4 Kbp spanning from 11780730 to 11811103 in chromosome 1 (upstream and downstream of the gene extending 5000 bases, respectively)] were analyzed and selected from the data of Chinese Han individuals in Beijing (CHB) via the HapMap Project (http://hapmap.ncbi.nlm.nih.gov/index.html.en) [20]. The detailed process and criterion were described previously [21]. The information of selected MTHFR tagging SNPs is presented in Table 2.

DNA extraction and genotyping

Ethylenediamine tetraacetic acid-anticoagulated intravenous blood was donated by every participant. Using the Promega DNA Blood Mini Kit (Promega, Madison, USA), genomic DNA was extracted from peripheral lymphocytes by the standard experimental protocol.

A custom-by-design 48-Plex SNPscan Kit (Genesky Biotechnologies Inc., Shanghai, China), double ligation and multiplex fluorescence PCR [22], was performed to identify the genotypes of MTHFR rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A polymorphisms as described in previous studies [23, 24]. A total of 77 samples were randomly selected and were tested again for quality control. Based on 4% of duplicated samples in this study, the accordance rates were 100%.

Statistical analysis

We used SAS statistical software, version 9.4 (SAS Institute, Cary, NC) for data analysis and a P < 0.05 (two–tailed) was considered to be a statistical significance. The quantitative variables were expressed as means ± standard deviation (SD). Student's t-test was harnessed to evaluate the difference of age between CRC patients and controls. Additionally, we used χ2 test to examine the differences in age, sex, smoking status, alcohol consumption and the frequencies of genotype between patients with CRC and controls. The Hardy-Weinberg equilibrium (HWE) equation was used to assess whether the proportion of MTHFR tagging SNPs genotypes obtained was in accordance with the expected value. An online calculator (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) was harnessed to calculate the P value of HWE [25]. The relationship of MTHFR rs3753584 T>C, rs9651118 T>C, rs1801133 G>A, rs4846048 A>G and rs4845882 G>A genotypes with CRC risk was estimated by crude/adjusted odds ratios (ORs) and 95% confidence intervals (CIs). The power of the present study (α= 0.05) was evaluated by the Power and Sample Size Calculator (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize).

ACKNOWLEDGMENTS

We appreciate all subjects who participated in this study. We wish to thank Dr. Yan Liu (Genesky Biotechnologies Inc., Shanghai, China) for technical support.

CONFLICTS OF INTEREST

The authors have no potential financial conflicts of interest.

GRANT SUPPORT

This study was supported by Natural Science Foundation of Universities and colleges of Jiangsu Province (Grant No. 16KJB310002), Senior Talents Scientific Research Foundation of Jiangsu University (Grant No. 16JDG066), Critical Patented Project of the Science and Technology Bureau of Fujian Province (Grant No. 2013YZ0002-2), the Special Program for the Development of Strategic Emerging Industries of Fujian Province (Grant No. 13YZ0201) and the Natural Science Foundation of Fujian Province (Grant No. 2015J01435).

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