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Methylenetetrahydrofolate reductase tagging polymorphisms are associated with risk of esophagogastric junction adenocarcinoma: a case-control study involving 2,740 Chinese Han subjects

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Oncotarget. 2017; 8:111482-111494. https://doi.org/10.18632/oncotarget.22845

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Guowen Ding, Yafeng Wang, Yu Chen, Jun Yin, Chao Liu, Yu Fan, Hao Qiu, Weifeng Tang and Shuchen Chen _

Abstract

Guowen Ding1,*, Yafeng Wang2,*, Yu Chen3,*, Jun Yin1, Chao Liu1, Yu Fan4, Hao Qiu5, Weifeng Tang6 and Shuchen Chen6

1Department of Cardiothoracic Surgery, Affiliated People’s Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, China

2Department of Cardiology, The People’s Hospital of Xishuangbanna Dai Autonomous Prefecture, Jinghong, Yunnan Province, China

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

4Department of Medical Oncology, Affiliated People’s Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, China

5Department of Immunology, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu Province, China

6Department of Thoracic Surgery, Fujian Medical University Union Hospital, Fuzhou, Fujian Province, China

*These authors have contributed equally to this work

Correspondence to:

Shuchen Chen, email: cscdoctor@163.com

Weifeng Tang, email: twf001001@126.com

Keywords: MTHFR; polymorphism; esophagogastric junction adenocarcinoma; risk

Received: May 16, 2017    Accepted: November 13, 2017    Published: December 01, 2017

ABSTRACT

In this study, we aimed to determine the potential association of MTHFR tagging single nucleotide polymorphisms (SNPs) with risk of developing esophagogastric junction adenocarcinoma (EGJA). MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms were genotyped in 1,677 healthy individuals and 1,063 patients with EGJA. We found that MTHFR rs1801133 G>A polymorphism was significantly associated with the risk of developing EGJA (AA vs. GG: adjusted P = 0.001; GA/AA vs. GG: adjusted P = 0.007 and AA vs. GA/GG: adjusted P = 0.001). However, for MTHFR rs4845882 G>A polymorphism, the decreased risk of EGJA was found in two genetic models (AA vs. GG: adjusted P = 0.002 and AA vs. GA/GG: adjusted P = 0.005). In addition, for MTHFR rs3753584 T>C and rs9651118 T>C polymorphisms, a tendency to decreased risk of EGJA was noted. In a subgroup analysis, a significantly decreased risk of EGJA in <64 years subgroup was identified. We found that MTHFR Grs1801133Trs3753584Grs4845882Ars4846048Crs9651118, Grs1801133Crs3753584Ars4845882Ars4846048Trs9651118 and Grs1801133Trs3753584Ars4845882Grs4846048Trs9651118 haplotypes significantly decreased the risk of EGJA (P = 0.002, P < 0.001 and P = 0.038, respectively). In conclusion, our study demonstrates that MTHFR rs1801133 G>A may be associated with the increased risk of EGJA. Meanwhile, MTHFR rs3753584 T>C, rs4845882 G>A and rs9651118 T>C polymorphisms and haplotypes may decrease the risk of EGJA in Eastern Chinese Han population. Further studies with large sample size and detailed gene-environmental data are needed to validate our conclusion.


INTRODUCTION

The increasing incidence of esophagogastric junction adenocarcinoma (EGJA) was observed worldwide [13] and was considered to have different etiology and risk factor compared with distal gastric carcinoma (GC) [4]. EGJA remains poor prognosis [5] and is a common public health problem. The vital risk factors contributing to the development of EGJA are obesity, gastro-esophageal reflux disease, smoking, foods preserved by salting and low intake of fruits and vegetables et al [6, 7]. However, these observed risk factors could not interpret the overall susceptibility to EGJA. Recently, more and more epidemiologic studies suggested that individual’s genetic factor might influence the pathogenesis of EGJA.

Accumulating evidences indicate that folate insufficiency may increase the susceptibility of multiple malignancies [8, 9]. In humans, the majority of methyl groups may be presented by folic acid for endocellular methylation reactions and DNA de novo deoxynucleoside synthesis. During DNA synthesis, lack of folate can cause uracil misincorporation and then affect the stability of DNA [10]. In folate metabolism and DNA synthesis, methylenetetrahydrofolate reductase (MTHFR) is an important enzyme which catalyzes the revivification of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. And 5-methyltetrahydrofolate is a main circulating and existing form of folate and is the methyl donor for DNA methylation and remethylation procedure of homocysteine to methionine. Based on the important role of participation in both DNA synthesis and methylation, any variant of MTHFR gene may involve in the carcinogenesis.

Human MTHFR is composed of 656 amino acids. MTHFR gene is located on the short arm of Chromosome 1. The human MTHFR gene is very polymorphic (http://www.ncbi.nlm.nih.gov/SNP) and a number of loci have been established, such as rs1537514, rs3753584, rs9651118, rs1537516, rs4845882, rs1801131, rs1801133, rs2066462, rs4846048 and rs3737967 polymorphisms, etc. Interestingly, many previous case-control studies demonstrated that MTHFR polymorphisms were correlated with the risk of multiple human malignancies [e.g., esophageal squamous cell carcinoma (ESCC) [11], gastric cardia adenocarcinoma (GCA) [12], cervicalcancer [13], breast cancer [14, 15] and childhood acute lymphoblastic leukemia [16] et al]. Thus, the single nucleotide polymorphisms (SNPs) in MTHFR genes on EGJA risk attracted our interest. Exploring the potential association of MTHFR SNPs with EGJA susceptibility may be conducive to the prevention and personalized diagnosis. In this study, we selected MTHFR tagging SNPs (rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C) and performed a case-control study to evaluate the effect of MTHFR genotypes for EGJA risk.

RESULTS

Baseline characteristics

A total of 1,063 sporadic patients with EGJA and 1,677 normal controls were recruited. Of the EJGA patients, 759 were male and 304 were female, with a mean age (± standard deviation) of 64.19 ±8.63 years. The normal controls comprised of 1,194 males and 483 females with a mean age of 63.91 ±10.22 years. The demographics (age and sex) was well matched (P = 0.165 and P = 0.909, respectively; Table 1). Of the smoking and alcohol consumption, a significant difference was observed between EGJA patients and controls (P < 0.001, Table 1). The frequency distribution of MTHFR genotypes was determined after genotyping the 2,740 study subjects. For MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms, success rates of genotyping were 99.01%, 99.09%, 99.05%, 99.09% and 98.98%, respectively (Table 2). In controls, the distribution of MTHFR genotype frequencies accorded with Hardy–Weinberg equilibrium (HWE), except for MTHFR rs4846048 A>G polymorphism (Table 2).

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

Variable

Overall Cases (n=1,063)

Overall Controls (n=1,677)

P a

n (%)

n (%)

Age (years)

64.19 ±8.63

63.91 ±10.22

0.451

Age (years)

0.165

 < 64

494 (46.47)

825 (49.19)

 ≥64

569 (53.53)

852 (50.81)

Sex

0.909

 Male

759 (71.40)

1194 (71.20)

 Female

304 (28.60)

483 (28.80)

Smoking status

<0.001

 Never

773 (72.72)

1323 (78.89)

 Ever

290 (27.28)

354 (21.11)

Alcohol use

<0.001

 Never

908 (85.42)

1507 (89.86)

 Ever

155 (14.58)

170 (10.14)

a Two-sided χ2 test and Student t test.

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

Genotyped SNPs

rs1801133 G>A

rs3753584 T>C

rs4845882 G>A

rs4846048 A>G

rs9651118 T>C

Chromosome

1

1

1

1

1

Function

Missense

NearGene-5

Intron

Intron

Intron

Chr Pos (Genome Build 36.3)

11778965

11787173

11765754

11768839

11784801

MAFa for Chinese in database

0.439

0.093

0.198

0.105

0.382

MAF in our controls (n = 1,677)

0.359

0.108

0.209

0.096

0.378

P value for HWEb test in our controls

0.679

0.691

0.972

0.014

0.270

Genotyping method

SNPscan

SNPscan

SNPscan

SNPscan

SNPscan

% Genotyping value

99.01%

99.09%

99.05%

99.09%

98.98%

aMAF: minor allele frequency.

bHWE: Hardy–Weinberg equilibrium.

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

The genotypes of MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms are presented in Table 3. For MTHFR rs1801133 G>A polymorphism, the risk of developing EGJA was significant in three genetic models [AA vs. GG: crude odds ratio (OR) = 1.50, 95% confidence interval (CI): 1.19–1.90, P = 0.001; GA/AA vs. GG: crude OR = 1.27, 95% CI: 1.08–1.49, P = 0.004 and AA vs. GA/GG: crude OR = 1.45, 95% CI: 1.17–1.80, P = 0.001; Table 3]. Adjustment for age, sex, smoking and drinking, the similar results were also found (AA vs. GG: adjusted OR = 1.47, 95% CI: 1.16–1.86, P = 0.001; GA/AA vs. GG: adjusted OR = 1.25, 95% CI: 1.06–1.47, P = 0.007 amd AA vs. GA/GG: adjusted OR = 1.43, 95% CI: 1.15–1.77, P = 0.001; Table 3). For MTHFR rs4845882 G>A polymorphism, the decreased risk of EGJA was found in two genetic models (AA vs. GG: crude OR = 0.47, 95% CI: 0.29–0.75, P = 0.002 and AA vs. GA/GG: crude OR = 0.50, 95% CI: 0.31–0.80, P = 0.004; Table 3). Adjustment for age, sex, smoking and drinking, the results were not materially changed (AA vs. GG: adjusted OR = 0.47, 95% CI: 0.29–0.76, P = 0.002 and AA vs. GA/GG: adjusted OR = 0.50, 95% CI: 0.31–0.81, P = 0.005; Table 3). In addition, these associations were still significant after a Bonferroni correction for multiple comparisons.

Table 3: Logistic regression analyses of associations between MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms and the risk of EGJA

Genotype

Cases
(n=1,063)

Controls
(n=1,677)

Crude OR
(95%CI)

P

Adjusted OR a
(95%CI)

P

n

%

n

%

MTHFR rs1801133 G>A

GG

367

35.29

683

40.82

1.00

1.00

GA

492

47.31

778

46.50

1.11 (0.94-1.32)

0.208

1.10 (0.93-1.30)

0.263

AA

181

17.40

212

12.67

1.50 (1.19-1.90)

0.001

1.47 (1.16-1.86)

0.001

GA + AA

673

64.71

990

59.18

1.27 (1.08-1.49)

0.004

1.25 (1.06-1.47)

0.007

GG+ GA

859

82.60

1,461

87.33

1.00

1.00

AA

181

17.40

212

12.67

1.45 (1.17-1.80)

0.001

1.43 (1.15-1.77)

0.001

A allele

854

41.06

1,202

35.92

MTHFR rs3753584 T>C

TT

855

82.13

1,330

79.45

1.00

1.00

CT

177

17.00

326

19.47

0.83 (0.67-1.01)

0.062

0.83 (0.68-1.01)

0.067

CC

9

0.86

18

1.08

0.76 (0.34-1.70)

0.504

0.73 (0.32-1.63)

0.440

CT+CC

186

17.87

344

20.55

0.84 (0.69-1.03)

0.087

0.84 (0.69-1.03)

0.091

TT+CT

1032

99.14

1,656

98.92

1.00

1.00

CC

9

0.86

18

1.08

0.80 (0.36-1.79)

0.591

0.77 (0.34-1.72)

0.516

C allele

195

9.37

362

10.81

MTHFR rs4845882 G>A

GG

687

66.06

1,049

62.66

1.00

1.00

GA

330

31.73

552

32.97

0.89 (0.75-1.05)

0.153

0.89 (0.75-1.05)

0.150

AA

23

2.21

73

4.36

0.47 (0.29-0.75)

0.002

0.47 (0.29-0.76)

0.002

GA+AA

353

33.94

625

37.34

0.86 (0.73-1.01)

0.074

0.86 (0.73-1.02)

0.075

GG+GA

1,017

97.79

1,601

95.64

1.00

1.00

AA

23

2.21

73

4.36

0.50 (0.31-0.80)

0.004

0.50 (0.31-0.81)

0.005

A allele

376

18.08

698

20.85

MTHFR rs4846048 A>G

AA

860

82.61

1,378

82.32

1.00

1.00

AG

171

16.43

272

16.25

0.98 (0.80-1.21)

0.883

0.99 (0.80-1.22)

0.921

GG

10

0.96

24

1.43

0.65 (0.31-1.37)

0.260

0.63 (0.30-1.34)

0.230

AG+GG

181

17.39

296

17.68

0.98 (0.80-1.20)

0.845

0.98 (0.80-1.21)

0.870

AA+AG

1,031

99.04

1,650

98.57

1.00

1.00

GG

10

0.96

24

1.43

0.67 (0.32-1.40)

0.284

0.65 (0.31-1.36)

0.250

G allele

191

9.17

320

9.56

MTHFR rs9651118 T>C

TT

423

40.75

638

38.11

1.00

1.00

TC

486

46.82

808

48.27

0.86 (0.73-1.02)

0.075

0.87 (0.74-1.03)

0.105

CC

129

12.43

228

13.62

0.81 (0.63-1.04)

0.094

0.83 (0.65-1.07)

0.150

TC+CC

615

59.25

1,036

61.89

0.90 (0.76-1.05)

0.171

0.91 (0.78-1.07)

0.260

TT+TC

909

87.57

1,446

86.38

1.00

1.00

CC

129

12.43

228

13.62

0.90 (0.71-1.13)

0.372

0.92 (0.73-1.16)

0.489

C allele

744

35.84

1,264

37.75

a Adjusted for age, sex, smoking and drinking status; Bold values are statistically significant (P <0.05).

Association of MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms with EGJA in Different Stratification Groups

In the stratified analyses by sex, age, drinking and smoking, the genotype frequencies of MTHFR rs1801133 G>A polymorphism are listed in Table 4. After adjustment by logistic regression analysis, the association of MTHFR rs1801133 G>A variants with EGJA risk was evident in some subgroups [male group: AA vs. GG: adjusted OR = 1.66, 95% CI 1.26–2.20, P < 0.001, GA/AA vs. GG: adjusted OR = 1.27, 95% CI 1.05–1.53, P = 0.015 and AA vs. GA/GG: adjusted OR = 1.61, 95% CI 1.24–2.08, P < 0.001; <64 years subgroup: aa vs. GG: adjusted OR = 1.51, 95% CI 1.06–2.14, P = 0.022, GA/AA vs. GG: adjusted OR = 1.38, 95% CI 1.09–1.74, P = 0.007 and AA vs. GA/GG: adjusted OR = 1.39, 95% CI 1.00–1.92, P = 0.049; ≥64 years subgroup: AA vs. GG: adjusted OR = 1.42, 95% CI 1.03–1.95, P = 0.032 and AA vs. GA/GG: adjusted OR = 1.47, 95% CI 1.10–1.96, P = 0.010; never smoking group: AA vs. GG: adjusted OR = 1.43, 95% CI 1.08–1.87, P = 0.012, GA/AA vs. GG: adjusted OR = 1.32, 95% CI 1.10–1.59, P = 0.004 and AA vs. GA/GG: adjusted OR = 1.33, 95% CI 1.04–1.72, P = 0.026; ever smoking group: AA vs. GG: adjusted OR = 1.62, 95% CI 1.01–2.61, P = 0.046 and AA vs. GA/GG: adjusted OR = 1.79, 95% CI 1.15–2.76, P = 0.009 and never drinking group: AA vs. GG: adjusted OR = 1.54, 95% CI 1.20–1.98, P = 0.001, GA/AA vs. GG: adjusted OR = 1.35, 95% CI 1.13–1.60, P = 0.001 and AA vs. GA/GG: adjusted OR = 1.45, 95% CI 1.15–1.82, P = 0.002; Table 4)].

Table 4: Stratified analyses between MTHFR rs1801133 G>A polymorphism and EGJA risk by sex, age, smoking status and alcohol consumption

Variable

MTHFR rs1801133 G>A (case/control)a

Adjusted ORb (95% CI); P

GG

GA

AA

GG

GA

AA

GA /AA

AA vs. (GA/GG)

Sex

Male

260/485

350/563

135/142

1.00

1.10 (0.90-1.34);
P: 0.360

1.66 (1.26-2.20);
P: < 0.001

1.27 (1.05-1.53);
P: 0.015

1.61 (1.24-2.08);
P: < 0.001

Female

107/198

142/215

46/70

1.00

1.10 (0.80-1.50);
P: 0.575

1.03 (0.66-1.60);
P: 0.909

1.17 (0.86-1.59);
P: 0.311

1.01 (0.67-1.53);
P: 0.954

Age

<64

173/362

233/363

76/98

1.00

1.24 (0.97-1.58);
P: 0.089

1.51 (1.06-2.14);
P: 0.022

1.38 (1.09-1.74);
P: 0.007

1.39 (1.00-1.92);
P: 0.049

≥64

194/321

259/415

105/114

1.00

0.97 (0.77-1.23);
P: 0.822

1.42 (1.03-1.95);
P: 0.032

1.12 (0.90-1.40);
P: 0.312

1.47 (1.10-1.96);
P: 0.010

Smoking status

Never

263/549

368/603

123/168

1.00

1.19 (0.98-1.44);
P: 0.085

1.43 (1.08-1.87);
P: 0.012

1.32 (1.10-1.59);
P: 0.004

1.33 (1.04-1.72);
P: 0.026

Ever

104/134

124/175

58/44

1.00

0.86 (0.61-1.22);
P: 0.396

1.62 (1.01-2.61);
P: 0.046

1.04 (0.75-1.45);
P: 0.805

1.79 (1.15-2.76);
P: 0.009

Alcohol consumption

Never

308/631

426/686

152/187

1.00

1.19 (0.99-1.42);
P: 0.065

1.54 (1.20-1.98);
P: 0.001

1.35 (1.13-1.60);
P: 0.001

1.45 (1.15-1.82);
P: 0.002

Ever

59/52

66/92

29/25

1.00

0.60 (0.36-1.00);
P: 0.051

1.10 (0.56-2.19);
P: 0.780

0.70 (0.43-1.13);
P: 0.138

1.47 (0.80-2.73);
P: 0.217

a The genotyping was successful in 1063 (97.84%) EGJA cases, and 1677 (99.76%) controls for MTHFR rs1801133 G>A;

b Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model;

Table 5 summarizes the results of association between MTHFR rs3753584 T>C polymorphism and EGJA risk in the stratified analysis. We found that MTHFR rs3753584 T>C polymorphism was associated with the decreased risk of EGJA in <64 years subgroup [tc vs. TT: adjusted OR = 0.70, 95% CI 0.52–0.93, P = 0.016 and TC/CC vs. TT: adjusted OR = 0.73, 95% CI 0.55–0.97, P = 0.032 (Table 5)].

Table 5: Stratified analyses between MTHFR rs3753584 T>C polymorphism and EGJA risk by sex, age, smoking status and alcohol consumption

Variable

MTHFR rs3753584 T>C (case/control)a

Adjusted ORb (95% CI); P

TT

TC

CC

TT

TC

CC

TC / CC

CC vs. (TC/TT)

Sex

Male

613/950

126/226

7/15

1.00

0.85 (0.67-1.08);
P: 0.177

0.69 (0.28-1.70);
P: 0.415

0.85 (0.67-1.08);
P: 0.184

0.72 (0.29-1.78);
P: 0.471

Female

242/380

51/100

2/3

1.00

0.80 (0.55-1.17);
P: 0.252

0.61 (0.08-4.43);
P: 0.622

0.83 (0.57-1.20);
P: 0.319

0.64 (0.09-4.72);
P: 0.663

Age

<64

398/640

79/177

5/7

1.00

0.70 (0.52-0.93);
P: 0.016

0.99 (0.31-3.19);
P: 0.987

0.73 (0.55-0.97);
P: 0.032

1.08 (0.34-3.47);
P: 0.899

≥64

457/691

98/149

4/11

1.00

0.98 (0.74-1.30);
P: 0.881

0.53 (0.17-1.68);
P: 0.282

0.97 (0.73-1.28);
P: 0.813

0.54 (0.17-1.71);
P: 0.296

Smoking status

Never

619/1,058

131/249

4/14

1.00

0.88 (0.70-1.11);
P: 0.283

0.47 (0.16-1.45);
P: 0.189

0.88 (0.70-1.11);
P: 0.288

0.49 (0.16-1.51);
P: 0.216

Ever

236/272

46/77

5/4

1.00

0.74 (0.49-1.11);
P: 0.144

1.22 (0.31-4.74);
P: 0.776

0.77 (0.52-1.15);
P: 0.202

1.29 (0.33-5.04);
P: 0.711

Alcohol consumption

Never

729/1,190

152/299

6/16

1.00

0.81 (0.65-1.00);
P: 0.052

0.59 (0.23-1.52);
P: 0.273

0.82 (0.66-1.01);
P: 0.064

0.63 (0.24-1.61);
P: 0.331

Ever

126/140

25/27

3/2

1.00

1.11 (0.60-2.06);
P: 0.748

1.52 (0.24-9.63);
P: 0.657

1.14 (0.63-2.07);
P: 0.668

1.49 (0.24-9.44);
P: 0.669

a The genotyping was successful in 1063 (97.93%) EGJA cases, and 1677 (99.82%) controls for MTHFR rs3753584 T>C;

b Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model;

The results of association between MTHFR rs4845882 G>A polymorphism and EGJA risk in the stratified analyses are summarized in Table 6. We found that MTHFR rs4845882 G>A polymorphism decreased the risk of EGJA in several subgroups [male group: AA vs. GG: adjusted OR = 0.47, 95% CI 0.27–0.83, P = 0.009 and AA vs. GA/GG: adjusted OR = 0.50, 95% CI 0.29–0.87, P = 0.014; <64 years subgroup: aa vs. GG: adjusted OR = 0.41, 95% CI 0.20–0.84, P = 0.015 and AA vs. GA/GG: adjusted OR = 0.45, 95% CI 0.22–0.91, P = 0.027; never smoking group: AA vs. GG: adjusted OR = 0.37, 95% CI 0.21–0.67, P = 0.001 and AA vs. GA/GG: adjusted OR = 0.39, 95% CI 0.22–0.70, P = 0.002 and never drinking group: AA vs. GG: adjusted OR = 0.44, 95% CI 0.26–0.74, P = 0.002, GA/AA vs. GG: adjusted OR = 0.83, 95% CI 0.69–0.98, P = 0.032 and AA vs. GA/GG: adjusted OR = 0.48, 95% CI 0.29–0.80, P = 0.005 (Table 6)].

Table 6: Stratified analyses between MTHFR rs4845882 G>A polymorphism and EGJA risk by sex, age, smoking status and alcohol consumption

Variable

MTHFR rs4845882 G>A (case/control)a

Adjusted ORb (95% CI); P

GG

GA

AA

GG

GA

AA

GA /AA

AA vs. (GA/GG)

Sex

Male

492/746

237/391

17/54

1.00

0.89 (0.73-1.09);
P: 0.268

0.47 (0.27-0.83);
P: 0.009

0.86 (0.71-1.05);
P: 0.113

0.50 (0.29-0.87);
P: 0.014

Female

195/303

93/161

6/19

1.00

0.86 (0.63-1.18);
P: 0.354

0.43 (0.16-1.12);
P: 0.084

0.86 (0.63-1.17);
P: 0.330

0.46 (0.18-1.21);
P: 0.117

Age

<64

320/507

152/279

10/37

1.00

0.83 (0.65-1.06);
P: 0.132

0.41 (0.20-0.84);
P: 0.015

0.81 (0.64-1.02);
P: 0.077

0.45 (0.22-0.91);
P: 0.027

≥64

367/542

178/273

13/36

1.00

0.94 (0.74-1.18);
P: 0.566

0.53 (0.28-1.01);
P: 0.052

0.91 (0.73-1.14);
P: 0.424

0.55 (0.29-1.04);
P: 0.068

Smoking status

Never

496/832

243/427

14/62

1.00

0.92 (0.76-1.12);
P: 0.416

0.37 (0.21-0.67);
P: 0.001

0.89 (0.73-1.07);
P: 0.207

0.39 (0.22-0.70);
P: 0.002

Ever

191/217

87/125

9/11

1.00

0.81 (0.58-1.14);
P: 0.220

0.96 (0.38-2.40);
P: 0.927

0.83 (0.60-1.16);
P: 0.269

1.04 (0.42-2.59);
P: 0.939

Alcohol consumption

Never

591/938

276/501

19/66

1.00

0.85 (0.71-1.01);
P: 0.065

0.44 (0.26-0.74);
P: 0.002

0.83 (0.69-0.98);
P: 0.032

0.48 (0.29-0.80);
P: 0.005

Ever

96/111

54/51

4/7

1.00

1.30 (0.80-2.12);
P: 0.293

0.85 (0.23-3.19);
P: 0.813

1.25 (0.78-2.02);
P: 0.355

0.78 (0.21-2.87);
P: 0.708

a The genotyping was successful in 1063 (97.84%) EGJA cases, and 1677 (99.82%) controls for MTHFR rs4845882 G>A;

b Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model;

Table 7 lists MTHFR rs4846048 A>G genotype frequencies in the stratified analysis. We found no significant difference in genotype distribution of MTHFR rs4846048 A>G polymorphism among EGJA cases and non-cancer controls.

Table 7: Stratified analyses between MTHFR rs4846048 A>G polymorphism and EGJA risk by sex, age, smoking status and alcohol consumption

Variable

MTHFR rs4846048 A>G (case/control)a

Adjusted ORb (95% CI); P

AA

AG

GG

AA

AG

GG

AG/GG

GG vs. (AG/AA)

Sex

Male

615/984

124/189

7/18

1.00

1.04 (0.81-1.33);
P: 0.772

0.58 (0.24-1.40);
P: 0.227

1.02 (0.80-1.29);
P: 0.904

0.58 (0.24-1.41);
P: 0.233

Female

245/394

47/83

3/6

1.00

0.88 (0.59-1.30);
P: 0.522

0.73 (0.18-2.96);
P: 0.659

0.90 (0.61-1.32);
P: 0.597

0.77 (0.19-3.11);
P: 0.711

Age

<64

398/677

78/134

6/12

1.00

0.99 (0.73-1.34);
P: 0.927

0.74 (0.27-2.02);
P: 0.562

0.99 (0.74-1.34);
P: 0.955

0.76 (0.28-2.07);
P: 0.590

≥64

462/701

93/138

4/12

1.00

1.00 (0.75-1.33);
P: 0.981

0.48 (0.15-1.51);
P: 0.210

0.97 (0.74-1.29);
P: 0.855

0.49 (0.16-1.53);
P: 0.221

Smoking status

Never

624/1,081

125/220

5/20

1.00

0.96 (0.75-1.22);
P: 0.725

0.43 (0.16-1.16);
P: 0.095

0.94 (0.74-1.19);
P: 0.611

0.45 (0.17-1.19);
P: 0.107

Ever

236/297

46/52

5/4

1.00

1.13 (0.73-1.75);
P: 0.582

1.49 (0.39-5.72);
P: 0.563

1.17 (0.77-1.78);
P: 0.473

1.47 (0.38-5.66);
P: 0.572

Alcohol consumption

Never

736/1,236

145/248

6/21

1.00

0.96 (0.77-1.20);
P: 0.709

0.47 (0.19-1.16);
P: 0.101

0.95 (0.76-1.18);
P: 0.613

0.48 (0.19-1.19);
P: 0.115

Ever

124/142

26/24

4/3

1.00

1.31 (0.70-2.46);
P: 0.406

1.77 (0.36-8.68);
P: 0.483

1.35 (0.74-2.46);
P: 0.323

1.70 (0.35-8.32);
P: 0.512

a The genotyping was successful in 1063 (97.93%) EGJA cases, and 1677 (99.82%) controls for MTHFR rs4846048 A>G;

b Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model;

The results of relationship between MTHFR rs9651118 T>C polymorphism and EGJA risk in the stratified analyses are summarized in Table 8. We found that MTHFR rs9651118 T>C polymorphism was associated with the decreased risk of EGJA in <64 years subgroup [tc vs. TT: adjusted OR = 0.78, 95% CI 0.61–0.99, P = 0.040 (Table 8)].

Table 8: Stratified analyses between MTHFR rs9651118 T>C polymorphism and EGJA risk by sex, age, smoking status and alcohol consumption

Variable

MTHFR rs9651118 T>C (case/control)a

Adjusted ORb (95% CI); P

TT

TC

CC

TT

TC

CC

TC / CC

CC vs. (TC/TT)

Sex

Male

309/447

339/574

95/170

1.00

0.82 (0.68-1.00);
P: 0.054

0.80 (0.60-1.07);
P: 0.134

0.86 (0.71-1.04);
P: 0.109

0.91 (0.69-1.19);
P: 0.492

Female

114/191

147/234

34/58

1.00

1.03 (0.76-1.40);
P: 0.857

0.99 (0.61-1.61);
P: 0.970

1.11 (0.82-1.50);
P: 0.501

1.01 (0.64-1.60);
P: 0.956

Age

<64

190/288

231/424

59/111

1.00

0.78 (0.61-0.99);
P: 0.040

0.80 (0.55-1.15);
P: 0.225

0.83 (0.66-1.05);
P: 0.128

0.95 (0.68-1.34);
P: 0.767

≥64

233/350

255/384

70/117

1.00

0.96 (0.76-1.21);
P: 0.728

0.89 (0.63-1.25);
P: 0.502

0.99 (0.80-1.23);
P: 0.914

0.93 (0.68-1.28);
P: 0.651

Smoking status

Never

299/499

357/636

95/186

1.00

0.88 (0.73-1.06);
P: 0.182

0.80 (0.60-1.06);
P: 0.126

0.92 (0.77-1.11);
P: 0.380

0.89 (0.68-1.16);
P: 0.374

Ever

124/140

129/173

34/43

1.00

0.83 (0.59-1.16);
P: 0.270

0.95 (0.56-1.59);
P: 0.835

0.87 (0.63-1.19);
P: 0.378

1.05 (0.64-1.72);
P: 0.843

Alcohol consumption

Never

357/564

415/728

113/213

1.00

0.86 (0.72-1.03);
P: 0.092

0.80 (0.62-1.04);
P: 0.096

0.90 (0.76-1.06);
P: 0.214

0.89 (0.70-1.14);
P: 0.369

Ever

66/74

71/80

16/15

1.00

0.93 (0.57-1.49);
P: 0.750

1.12 (0.50-2.51);
P: 0.783

0.97 (0.61-1.54);
P: 0.907

1.17 (0.54-2.53);
P: 0.686

a The genotyping was successful in 1063 (97.65%) EGJA cases, and 1677 (99.82%) controls for MTHFR rs9651118 T>C;

b Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model;

SNP haplotypes

We used a SHESIS software (http://analysis.bio-x.cn/myAnalysis.php) [17] to construct haplotypes of MTHFR gene (Table 9). Finally, five MTHFR haplotypes were identified. When MTHFR Ars1801133Trs3753584Grs4845882Ars4846048Trs9651118 haplotype was used as reference, we found that MTHFR Grs1801133Trs3753584Grs4845882Ars4846048Crs9651118, Grs1801133Crs3753584Ars4845882Ars4846048Trs9651118 and Grs1801133Trs3753584Ars4845882Grs4846048Trs9651118 haplotypes significantly decreased the risk of EGJA (P = 0.002, P < 0.001 and P = 0.038, respectively, Table 9).

Table 9: MTHFR haplotype frequencies (%) in patients and controls and risk of esophagogastric junction adenocarcinom

Haplotypes

Case (n=2,126)

Control (n=3,354)

Crude OR (95% CI)

P

n

%

n

%

Ars1801133Trs3753584Grs4845882Ars4846048Trs9651118

814

39.36

1169

34.92

1.00

Grs1801133Trs3753584Grs4845882Ars4846048Crs9651118

708

34.24

1244

37.16

0.82 (0.72-0.93)

0.002

Grs1801133Crs3753584Ars4845882Ars4846048Trs9651118

171

8.27

335

10.01

0.69 (0.56-0.85)

<0.001

Grs1801133Trs3753584Ars4845882Grs4846048Trs9651118

173

8.37

309

9.23

0.80 (0.65-0.99)

0.038

Grs1801133Trs3753584Grs4845882Ars4846048Trs9651118

130

6.29

206

6.15

0.91 (0.72-1.15)

0.416

Others

72

3.48

85

2.54

1.22 (0.88-1.69)

0.239

DISCUSSION

Incidence of EGJA has increased over the past two decades [18, 19]. Many studies demonstrated that the morbidity of EGJA was increased in Asian countries, such as China, Korea and Japan [1921]. However, the etiology of EGJA remains unknown. In this study, we explored the association between MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms and EGJA risk in Eastern Chinese Han population. We found that MTHFR rs1801133 G>A might be associated with the increased risk of EGJA. Meanwhile, MTHFR rs3753584 T>C, rs4845882 G>A and rs9651118 T>C polymorphisms decreased the risk of EGJA.

MTHFR gene lies in 1p36.3 and contains 11 exons with a length of about 1980 bp. In exon 4, a G to A variant at nucleotide 677 locus (rs1801133 G>A) directly leads to valine substitution for alanine, which is relevant to a reduction of MTHFR activity [22]. The individuals who carry heterozygous genetype (GA genetype) of MTHFR rs1801133 G>A polymorphism have 70% of normal enzyme activity, however, those who carry homozygous genotype (AA genetype) have only 30% of normal enzyme activity [23]. A case-control study reported that rs1801133 AA genotype was associated an increased risk of GCA [24]. Another case-control study also found that MTHFR rs1801133 AA and GA genotypes were associated the increased risk of GCA [25]. These results were in accordance with our conclusions. In the future, more replicated study should be conducted to verify these primary findings.

MTHFR rs3753584 T>C is situated in the intron region of MTHFR gene. There were only a few studies focusing on the association between MTHFR rs3753584 T>C and cancer risk. A previous study found that there was an increased lung cancer risk in carriers of MTHFR rs3753584 CC genotype compared with carriers of rs3753584 TT genotype [26]. However, no association was found between ESCC risk and MTHFR rs3753584 T>C polymorphism [11]. In addition, Wang et al. also reported that MTHFR rs3753584 T>C was not associated with GCA risk [12]. The present study concluded that rs3753584 TC and TC/CC genotypes were related to a decreased EGJA risk in <64 years subgroup. these apparent discrepancy findings may be due to the insufficient sample size. in future, more studies with large size and detailed environmental factors are indispensable explore relationship between MTHFR rs3753584 T>C and the risk of different cancers.

MTHFR rs4845882 G>A polymorphism lies in a intron region and is almost complete linkage disequilibrium (LD) with MTHFR rs1801131 A>C locus. Shen et al. found there was no significant relationship between MTHFR rs4845882 G>A polymorphism and gastric cancer risk [27]. Additionally, the association between MTHFR rs4845882 G>A and GCA risk was not concluded in a recent sutdy [12]. However, our study saw a decreased EGJA risk in the individuals carrying MTHFR rs4845882 AA genotype in male and <64 years subgroups. MTHFR rs9651118 T>C is situated in intron 2 and possesses low LD with rs1801133 G>A (r2 < 0.30). Fuctional annotation by HapReg demonstrated that MTHFR rs9651118 T>C coincides with MTHFR enhancers or promoters, which may correspond to the regions of open chromatin [28]. Several studies implicated that MTHFR rs9651118 C allele was associated with a reduced risk of lung cancer and prostate cancer [28, 29]. In addition, MTHFR rs9651118 C allele was associated with a decreased risk of breast cancer [30]. Our results suggested that MTHFR rs9651118 TC genotype may reduce EGJA susceptibility in <64 years subgroup, which were very similar to the findings of previous studies. In the future, these potential should be confirmed by functional studies.

In this case-control study, we constructed five MTHFR haplotypes to assess the potential inherited patterns of haplotype. We found that MTHFR Grs1801133Trs3753584Grs4845882Ars4846048Crs9651118, Grs1801133Crs3753584Ars4845882Ars4846048Trs9651118 and Grs1801133Trs3753584Ars4845882Grs4846048Trs9651118 haplotypes significantly decreased the risk of EGJA. To the best of our knowledge, we first explore the relationship of haplotypes in MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms with EGJA susceptibility. We also found that MTHFR rs1801133 G and rs4845882 A alleles might be protective factors for haplotype to EGJA.

However, several limitations in our study should be presented. First, for the controls were recruited from the local hospitals, the selection bias of the study population should not be ignored. Second, the data of plasma folate level were not available, which may affect the association between MTHFR SNPs and EGJA susceptibility. Thirdly, for lack of cancer stage, disease progression and overall survival data, we did not consider the influence of MTHFR SNPs on progress and prognosis of EGJA. Last but not least, other environmental and genetic factors were not considered. Further studies are necessary to explore the effect of interactions between environment and gene factors on EGJA risk.

In conclusion, our study demonstrates that MTHFR rs1801133 G>A may be associated with the increased risk of EGJA. Meanwhile, MTHFR rs3753584 T>C, rs4845882 G>A and rs9651118 T>C polymorphisms decrease the risk of EGJA in Eastern Chinese Han population. The further case-control studies are needed to confirm our findings.

MATERIALS AND METHODS

Subjects

Study conducted at the Affiliated Union Hospital of Fujian Medical University, Fujian Medical University Cancer Hospital and the Affiliated People’s Hospital of Jiangsu University was approved by the Ethics Committee of Fujian Medical University (Fuzhou, China) and Jiangsu University (Zhenjiang, China). Subjects were enrolled from three hospitals in Eastern China. Our study involved 2,740 study participants, comprising 1,063 histopathologically confirmed sporadic EGJA patients and 1,677 healthy normal controls. Among them, 280 EGJA patients and 840 controls were enrolled from Fujian Medical University Union Hospital and Cancer Hospital of Fujian Medical University from January 2014 to May 2016. In addition, 783 EGJA patients and 837 controls were enrolled from the Affiliated People’s Hospital of Jiangsu University between January 2008 and November 2016. All EJGA patients were Siewert type II. The control group involved normal individuals who visited these hospitals for health check. The healthy normal controls were unrelated to the EGJA patients and were cancer-free individuals. Data of demographic details and risk factors was obtained using a structured questionnaire. The definition of ‘ever smokers’ were subjects who smoked at least one cigarette per day over 1 year [11], and ‘ever drinkers’ were subjects who drank no less than three times a week for more than 6 months [11]. The corresponding data are listed in Table 1. The Ethical Committee of Fujian Medical University and Jiangsu University approved the study protocols (No. SQ2015-006-01 and No. 20150083, respectively).

Selection of SNPs

The MTHFR tagging SNPs (upstream and downstream of MTHFR gene extending 5 Kb, respectively) were selected from the database of CHB population using the HapMap Project (http://hapmap.ncbi.nlm.nih.gov/index.html.en) and Haploview 4.2 software. The major criterion were: (a) MAF ≥ 0.05 and call rate ≥ 95 %, (b) a HWE P ≥ 0.05, (c) a pairwise linkage disequilibrium (LD) r2 threshold of 0.8 between polymorphisms (r2 > 0.8) [11, 31, 32]. Finally, five MTHFR tagging SNPs (rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C) were eligible and included in this case-control study to evaluate the effect of MTHFR polymorphisms with EGJA risk. The primary information of MTHFR tagging SNPs is presented in Table 2.

DNA extraction and genotyping

Each participant donated 2ml blood sample which was stored in an EDTA-anticoagulated tube. We use the Promega Genomic DNA Purification Kit (Promega, Madison, USA) to extract the genomic DNA. SNPscanTM genotyping assay (Genesky Biotechologies Inc., Shanghai, China) [33, 34] was harnessed to determine the genotyping of MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms. Briefly, 150ng DNA sample was denatured at 98°C for 5min. The ligation reaction was done in an ABI 2720 thermal cycler. We used a 48-plex fluorescence PCR reaction for each ligation product amplification. In an ABI 3730XL sequencer, PCR products were analyzed by capillary electrophoresis. The obtained raw data were conducted by GeneMapper 4.1 software (Applied Biosystems, USA). One hundred and ten DNA samples (4%) were randomly selected to reanalyze the genotypes by different laboratory technicians and the reproducibility was 100%.

Statistical analysis

Age of EGJA patients and controls was expressed as mean ± standard deviation. And a Student’s t-test was harnessed to assess the difference for age. The Chi-square test (χ2) was used to compare age, sex, smoking, drinking and the genotypes distribution of MTHFR SNPs in patients and controls. We used multivariate logistic regression analysis to assess the risk of MTHFR rs1801133 G>A, rs3753584 T>C, rs4845882 G>A, rs4846048 A>G and rs9651118 T>C polymorphisms and considered the confounders such as sex, age, smoking and drinking status. The crude/adjusted ORs and 95% CIs were calculated using the SAS software (Version 9.4; SAS Institute Inc., Cary, NC, USA). A P < 0.05 (two sided) was considered as statistical significance. In this study, multiple comparisons were conducted by Bonferroni correction [35]. We used a SHESIS software (http://analysis.bio-x.cn/myAnalysis.php) [17] to construct MTHFR haplotypes.

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 conflicts of interest.

GRANT SUPPORT

This study was supported in part by Young and Middle-aged Talent Training Project of Health Development Planning Commission in Fujian Province (2016-ZQN-25 and 2014-ZQN-JC-11), Medical Innovation Project of Fujian Province (2014-CX-15 and 2014-CX-18), Nursery Garden Project of Fujian Medical University (2015MP020), Science and Technology Project of Fujian Province (2060203), Natural Science Foundation of Universities and Colleges of Jiangsu Province (Grant No. 16KJB310002), Senior Talents Scientific Research Foundation of Jiangsu University (Grant No. 16JDG066) and Jiangsu Provincial Key R & D Special Fund (BE2015666).

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