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Tumor Biology

, Volume 36, Issue 12, pp 9525–9535 | Cite as

Effect of ERCC8 tagSNPs and their association with H. pylori infection, smoking, and alcohol consumption on gastric cancer and atrophic gastritis risk

  • Jing-jing Jing
  • Li-ping Sun
  • Qian Xu
  • Yuan Yuan
Research Article

Abstract

Excision repair cross-complementing group 8 (ERCC8) plays a critical role in DNA repair. Genetic polymorphisms in ERCC8 may contribute to the risk of cancer development. We selected tag single nucleotide polymorphisms (tagSNPs) in Chinese patients from the HapMap database to investigate associations with gastric cancer and its precursors. Genomic DNA was extracted from 394 controls, 394 atrophic gastritis, and 394 gastric cancer cases in northern Chinese patients, and genotypes were identified using the Sequenom MassARRAY system. We found that the ERCC8 rs158572 GG+GA genotype showed a 1.651-fold (95 % confidence interval (CI) = 1.109–2.457, P = 0.013) increased risk of gastric cancer compared with the AA genotype, especially in diffuse type. Stratified analysis comparing the common genotype revealed significantly increased gastric cancer risk in males and individuals older than 50 years with rs158572 GA/GG/GG+GA genotypes, while individuals older than 50 years with rs158916 CT/CC+CT genotypes were less susceptible to atrophic gastritis. Haplotype analysis showed that the G-T haplotype was associated with increased risk of gastric cancer. Statistically significant interactions between the two ERCC8 tagSNPs and Helicobacter pylori infection were observed for gastric cancer and atrophic gastritis risk (P < 0.05). Smokers and drinkers with ERCC8 rs158572 GG+GA genotype were more susceptible to gastric cancer compared with non-smokers and non-drinkers homozygous for AA. Our findings suggested that ERCC8 rs158572 and rs158916, alone or together with environmental factors, might be associated with gastric cancer and atrophic gastritis susceptibility. Further validation of our results in larger populations along with additional studies evaluating the underlying molecular function is required.

Keywords

ERCC8 TagSNP Gastric cancer Atrophic gastritis Susceptibility 

Introduction

Gastric cancer is one of the most common cancers worldwide, accounting for 10 % (738,000) of all cancer-related deaths in 2008 [1]. However, its etiology and pathogenesis are not yet clearly defined. Gastric cancer development generally involves at least three key steps, including superficial gastritis, precancerous conditions (i.e., atrophy, intestinal metaplasia and dysplasia), and carcinoma [2]. Several factors including bacterial, genetic, and environmental factors may contribute to gastric cancer etiology. Together, this indicates that gastric carcinogenesis is a complex multistep process with the involvement of various cancer susceptibility genes and multiple factors [3, 4].

Inherited cancer susceptibility genes, known as risk-modifier genes, could affect an individual’s risk of developing gastric cancer [5]. More than 80 % of cancer cases may be caused by gene–environment interactions [6], in which environmental factors often cause damage to DNA. Mutations in human DNA repair genes have been implicated in the genetic background of gastric cancer [7].

Excision repair cross-complementing group 8 (ERCC8) spans approximately 80 kb at 5q12.1 and is organized into 12 exons. ERCC8 was cloned by functional complementation of the UV sensitivity of the Cockayne syndrome complementation group A (CSA) cell line [8]; thus, the gene is alternatively known as CSA. Defects in ERCC8 were first found as the cause of Cockayne syndrome [9], and an increasing number of novel mutations in ERCC8 have been described [10].

ERCC8 (CSA) protein is involved in DNA repair, oxidative injury repair, and ubiquitination, and scaffolding for protein–protein interactions. ERCC8 plays a specific function as an essential factor of the transcription-couple repair (TCR) pathway of the nucleotide excision repair (NER) DNA repair pathway [11, 12, 13, 14]. As part of an E3 ubiquitin ligase complex [15], ERCC8 is required for the recruitment of other ancillary NER factors to repair sites [16] and likely triggers the degradation of CSB (ERCC6) at a late stage of the TCR process [17]. ERCC8 is also involved in the response to oxidative stress and helps prevent the accumulation of various oxidized DNA bases in vivo [18, 19, 20]. Thus, ERCC8 is a crucial gene of the TCR pathway.

Despite the potential importance of ERCC8, information of the association between its genetic polymorphisms and disease remains limited. Only two published studies of ERCC8 single nucleotide polymorphisms (SNPs) are available [21, 22], and no significant results have been reported to date. ERCC8 has many SNP sites, which may influence its gene transcription and protein function to further influence DNA repair capacity. Tag single nucleotide polymorphism (tagSNP) is the representative polymorphism site among several linked disequilibrium SNP sites, and studies on the association of tagSNPs and cancer risk are generally accepted as a first-line screening strategy.

In the current study, two tagSNPs rs158572 and rs158916 were selected from the HapMap database to investigate the potential effect of genetic variations in ERCC8 and their interaction with non-genetic factors on gastric cancer and atrophic gastritis risk in a Chinese population.

Materials and methods

Study population

The subjects in the present study were mainly recruited from a population-based, combined serologic/endoscopic screening program for gastric cancer in the Zhuanghe area of Liaoning Province from 1997 to 2011. The screening population selection and recruitment process were reported previously [23]. A small fraction of gastric cancer cases were from patients who were histologically certificated at the First Affiliated Hospital of China Medical University.

Gastric tissue specimens were collected using a standard endoscopic technique, and venous blood was drawn from each subject simultaneously. The biopsy specimens were paraffin embedded and stained for histological diagnosis by two histologists. Histopathological findings were assessed according to the visual analog scale of the updated Sydney System for gastritis [24] and the World Health Organization (WHO) criteria for gastric cancer [25]. Gastric adenocarcinoma patients were classified into two subgroups based on the histopathology according to the Lauren’s classification [26]. A 5-ml fasting venous blood sample was obtained for DNA isolation and measurement for Helicobacter pylori serology. The segregated blood clots and serum were immediately frozen and stored until analysis. Meanwhile, data regarding sex, age, smoking, and alcohol consumption were also taken by questionnaire and the records were computerized. Individuals who smoked at least once a day for more than 1 year were defined as ever smokers and included current smokers and former smokers who had quit smoking for more than 1 year; the remainder were defined as never smokers. Individuals who consumed one or more alcoholic drinks per week for at least 1 year were considered drinkers, and the rest were defined as non-drinkers. Participants with other malignancies were excluded from this study.

All the enrolled subjects were histologically classified into three groups: gastric cancer, atrophic gastritis, and healthy control subjects. The eligible controls were confirmed to have relative normal mucosa or only mild superficial gastritis. Healthy control cases were frequency matched to cases of atrophic gastritis and gastric cancer by gender (1:1) and age (±5 years) for individual association analysis. A total of 1182 individuals were adopted for association analysis of ERCC8 genotypic effects on atrophic gastritis and gastric cancer risk, including 394 gastric cancer, 394 atrophic gastritis, and 394 healthy control cases. The frequency distributions of demographic and other selected characteristics of the participants are summarized in Table 1.
Table 1

Baseline characteristics of the study subjects

 

Healthy control

Atrophic gastritis

P value

Gastric cancer

P value

Total

394

394

 

394

 

Age

 Mean ± SD

56.33 ± 8.684

56.16 ± 8.533

0.788

56.36 ± 8.745

0.967

Sex

 Male

270 (68.5 %)

270 (68.5 %)

1.000

270 (68.5 %)

1.000

 Female

124 (31.5 %)

124 (31.5 %)

124 (31.5 %)

H. pylori-IgG

 Positive

78 (19.8 %)

244 (61.9 %)

0.000

110 (27.9 %)

0.000

 Negative

313 (79.4 %)

147 (37.3 %)

100 (25.4 %)

 Missing

3 (0.8 %)

3 (0.8 %)

184 (46.7 %)

Smoking

 Yes

110 (27.9 %)

107 (27.2 %)

0.076

105 (26.6 %)

0.575

 No

133 (33.8 %)

179 (45.4 %)

113 (28.7 %)

 Missing

151 (38.3 %)

108 (27.4 %)

176 (44.7 %)

Alcohol

 Yes

76 (19.3 %)

80 (20.3 %)

0.444

65 (16.5 %)

0.297

 No

167 (42.4 %)

206 (52.3 %)

114 (28.9 %)

 Missing

151 (38.3 %)

108 (27.4 %)

215 (54.6 %)

Lauren’s classificationa

 Intestinal type

130 (44.2 %)

 

 Diffuse type

164 (55.8 %)

 

aSome of the gastric cancer cases failed to be classified into either group of intestinal type or diffuse type

Ethics statement

This research was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University (Shenyang, China). During epidemiological interviews, written informed consent was obtained from each participant.

TagSNP selection criteria

Genotypic data of a HapMap Chinese Han Beijing (CHB) population (release 27, phase I + II + III, http://www.HapMap.org) were extracted within extended gene regions of ERCC8 encompassing 10 kb of upstream and downstream flanking sequence. Using Haploview 4.2 [27], tagSNPs were selected based on pairwise linkage disequilibrium (LD) information to maximally represent (r 2 > 0.8) the common SNPs (minor allele frequency >0.05). Further, the potentially functional SNPs of interest were predicted by FASTSNP software (http://fastsnp.ibms.sinica.edu.tw/pages/input_CandidateGeneSearch.jsp). SNPs located at the two ends of the ERCC8 gene (i.e., the 5′ near gene, 5′UTR, 3′UTR, and 3′ near gene) or affecting transcription factor binding site (TFBS) activity were preferred.

SNP genotyping

Whole blood from individuals was collected, and blood clots were allowed to form by incubating clot-activating tubes at room temperature for 1 h. Each clot was transferred to a 2-ml centrifuge tube and stored at −80 °C until DNA extraction. Genomic DNA was extracted from blood samples using a routine phenol–chloroform method and then diluted to a working concentration (50 ng/μl) for ERCC8 genotyping. All samples were placed randomly into 384-well plates and blinded for disease status. The genotyping assay was performed by CapitalBio (Beijing, China) using the Sequenom MassARRAY platform (Sequenom, San Diego, CA, USA). A total of 50 samples were repeatedly genotyped and the concordance rate was 100 %, demonstrating that the genotyping was correct.

H. pylori serology examination

H. pylori serology testing was performed to check the status of H. pylori infection using enzyme-linked immunosorbent assay (ELISA; H. pylori-immunoglobulin (Ig)G ELISA kit; BIOHIT Plc, Helsinki, Finland), as described previously [28]. Briefly, approximately 5 ml fasting venous blood was obtained from each individual and the serum sample was collected after 10 min centrifugation at 3500×g, and a serum aliquot was immediately frozen and stored until analysis. H. pylori-IgG concentrations of the serum sample were detected using the ELISA kit according to the manufacturer’s protocol. A numerical reading exceeding 34 enzyme immune units was considered to be H. pylori infection positive.

Statistical analysis

SNP genotypes were first tested for Hardy–Weinberg equilibrium against the controls. Continuous variables were presented as mean ± SD and compared by Student’s t test. Discrete variables were represented as frequencies and percentages and evaluated by Pearson’s χ 2 test. The associations between ERCC8 genotypes and gastric cancer risk were estimated by multivariate logistic regression and were expressed as odds ratios (ORs) with 95 % confidence intervals (CIs). On the basis of the observed frequencies of two SNPs, we used the SHEsis analysis platform to calculate LD index (D′ and r 2) and infer haplotype frequencies [29, 30]. Interaction effects were assessed from the likelihood ratio test, comparing the fit of the logistic model that included the main effects of sex, age, environmental risk factor, and genotype with a fully parameterized model containing the multiplicative interaction terms of genotype and environmental risk factor. Joint effects between genotypes and environmental risk factors were evaluated using the full regression model. The abovementioned analyses were performed with SPSS 13.0 software (SPSS, Chicago, IL, USA). A two-side P value of less than 0.05 was considered statistically significant.

Results

ERCC8 tagSNP selection results

According to the tagSNP selection criteria, five tagSNPs were identified (rs158572, rs158916, rs12520314, rs7722373, and rs12657309) by Haploview, and two (rs158572, rs158916) of them captured the majority of SNPs in this region. Furthermore, rs158916 was located at 5′ upstream region, and rs158572 was predicted to be a transcription factor binding site as well as an intron enhancer by FASTSNP software (Table 2). Thus, rs158572 and rs158916 were finally selected for genotyping. The locations and characterization of the selected SNPs are listed in Fig. 1.
Table 2

Information of ERCC8 tagSNPs

SNP_ID

Alleles captured

Position

Predicted functional effects

rs158572

rs158570, rs158572, rs976080, rs158914, rs4647102, rs158935, rs11744756, rs4235483, rs929780

Intron

Intronic enhancer, TF binding site

rs158916

rs2306350, rs158916, rs4647078, rs2306351, rs4647108, rs158928

5′ upstream

Upstream with no known function

rs12520314

rs12520314

Intron

Intronic with no known function

rs7722373

rs7722373

Intron

Intronic enhancer

rs12657309

rs12657309

Intron

Intronic with no known function

Fig. 1

Plot of information of linkage disequilibrium (r 2) among common ERCC8 polymorphisms based on CHB HapMap population

ERCC8 polymorphisms and risk of atrophic gastritis and gastric cancer

The genotypic frequencies of the two tagSNPs rs158572 and rs158916 were in agreement with the Hardy–Weinberg equilibrium in the controls (both P > 0.05). To examine whether the risks of gastric cancer and its precancerous conditions were related to ERCC8 genotype, we analyzed the association between ERCC8 tagSNPs and the risk of atrophic gastritis and gastric cancer in the total population and in subpopulations according to sex and age. For gastric cancer susceptibility, compared with the common AA genotype, rs158572 GA genotype and GG+GA genotypes were associated with increased risk of gastric cancer in the total population, with corresponding ORs of 1.523 (95 % CI 1.014–2.288, P = 0.043) and 1.651 (95 % CI 1.109–2.457, P = 0.013), respectively (Table 3). In a stratified analysis, GA, GG, and GG+GA genotypes were also observed to be associated with an elevated gastric cancer risk in the subgroups of males and aged >50 years (Table 4).
Table 3

Association of ERCC8 polymorphisms with the risks of atrophic gastritis and gastric cancer

 

Healthy control vs atrophic gastritis

Healthy control vs gastric cancer

P HWE

Healthy control

Atrophic gastritis

OR (95 % CI)

P value

Healthy control

Gastric cancer

OR (95 % CI)

P value

rs158572

 AA

347

335

1.0 (ref)

 

347

322

1.0 (ref)

 

0.684

 GA

46

55

1.238 (0.814–1.884)

0.318

46

65

1.523 (1.014–2.288)

0.043

 GG

1

4

4.118 (0.458–37.047)

0.207

1

7

7. 543 (0.923–61.649)

0.059

 GG+GA

47

59

1.300 (0.862–1.962)

0.211

47

72

1.651 (1.109–2.457)

0.013

rs158916

 TT

291

306

1.0 (ref)

 

291

307

1.0 (ref)

 

0.667

 CT

94

80

0.809 (0.577–1.135)

0.221

94

77

0.776 (0.552–1.092)

0.146

 CC

9

8

0.845 (0.322–2.221)

0.733

9

10

1. 053 (0.422–2.629)

0.912

 CC+CT

103

88

0.812 (0.586–1.126)

0.213

103

87

0.801 (0.577–1.111)

0.183

OR was obtained in the logistic regression models. Analyses of results with P < 0.05 were highlighted in italic characters

HWE Hardy–Weinberg equilibrium in control population, CI confidence interval, OR odds ratio

Table 4

Association of ERCC8 polymorphisms with the risks of atrophic gastritis and gastric cancer stratified by sex and age

 

Healthy control vs atrophic gastritis

Healthy control vs gastric cancer

  

OR (95 % CI)

P value

  

OR (95 % CI)

P value

rs158572

 Male

AA

243/231

  

AA

243/219

  

GA

26/37

1.497 (0.879–2.551)

0.138

GA

26/45

1.920 (1.146–3.218)

0.012

GG

1/2

2.104 (0.189–23.359)

0.545

GG

1/6

6.658 (0.795–55.735)

0.044

GG+GA

27/39

1.519 (0.901–2.563)

0.117

GG+GA

27/51

2.096 (1.270–3.459)

0.004

 Female

AA

104/104

  

AA

104/103

  

GA

20/18

0.900 (0.450–1.799)

0.766

GA

20/20

1.010 (0.513–1.987)

0.978

GG

0/2

NA

NA

GG

0/1

NA

NA

GG+GA

20/20

1.000 (0.508–1.968)

1.000

GG+GA

20/21

1.060 (0.542–2.072)

0.864

 >50

AA

259/253

  

AA

259/240

  

GA

37/40

1.107 (0.685–1.788)

0.678

GA

37/49

1.429 (0.901–2.267)

0.129

GG

1/2

2.047 (0.184–22.721)

0.560

GG

1/7

7.554 (0.923–61.851)

0.059

GG+GA

38/42

1.131 (0.706–1.814)

0.608

GG+GA

38/56

1.590 (1.016–2.489)

0.042

 ≤50

AA

88/82

  

AA

88/82

  

GA

9/15

1.789 (0.742–4.310)

0.195

GA

9/16

1.908 (0.799–4.555)

0.146

GG

0/2

NA

NA

GG

0

NA

NA

GG+GA

9/17

2.027 (0.856–4.801)

0.108

GG+GA

9/16

1.908 (0.799–4.555)

0.146

rs158916

 Male

TT

197/209

  

TT

197/208

  

CT

68/54

0.749 (0.498–1.124)

0.163

CT

68/55

0.766 (0.511–1.149)

0.198

CC

5/7

1.320 (0.412–4.226)

0.641

CC

5/7

1.326 (0.414–4.247)

0.635

CC+CT

73/61

0.788 (0.532–1.165)

0.232

CC+CT

73/62

0.804 (0.544–1.189)

0.275

 Female

TT

94/97

  

TT

94/99

  

CT

26/26

0.969 (0.525–1.789)

0.920

CT

26/22

0.803 (0.426–1.515)

0.499

CC

4/1

0.242 (0.027–2.208)

0.209

CC

4/3

0.712 (0.155–3.267)

0.662

CC+CT

30/27

0.872 (0.482–1.577)

0.651

CC+CT

30/25

0.791 (0.434–1.443)

0.445

 >50

TT

216/238

  

TT

216/230

  

CT

76/50

0.597 (0.400–0.892)

0.012

CT

76/58

0.717 (0.486–1.057)

0.093

CC

5/7

1.271 (0.397–4.063)

0.686

CC

5/8

1.503 (0.484–4.664)

0.481

CC+CT

81/57

0.639 (0.434–0.939)

0.023

CC+CT

81/66

0.765 (0.526–1.113)

0.161

 ≤50

TT

75/68

  

TT

75/77

  

CT

18/30

1.838 (0.940–3.593)

0.075

CT

18/19

1.028 (0.501–2.110)

0.940

CC

4/1

0.276 (0.030–2.528)

0.254

CC

4/2

0.487 (0.087–2.739)

0.414

CC+CT

22/31

1.554 (0.822–2.940)

0.175

CC+CT

22/21

0.930 (0.472–1.830)

0.833

OR was obtained in the logistic regression models. Analyses of results with P < 0.05 were highlighted in italic characters

CI confidence interval, OR odds ratio, NA not available

With regard to the rs158916 polymorphism, no overall or stratified effect of this polymorphism on gastric cancer risk was observed. As to atrophic gastritis susceptibility, no positive association was observed between both SNPs and atrophic gastritis risk in the total population. Only rs158916 CT and CC+CT genotypes were observed to be associated with a reduced atrophic gastritis risk in the subgroup of aged >50 years (OR = 0.597, 95 % CI 0.400–0.892, P = 0.012; OR = 0.639, 95 % CI 0.434–0.939, P = 0.023, respectively) (Table 4).

Genetic effect of ERCC8 polymorphisms on the risk of gastric cancer was further assessed in subpopulations according to histological subtype for gastric cancer case based on the Lauran’s classification. In the stratified analysis of histology (Table 5), compared with the common AA genotype, rs158572 GA genotype and GG+GA genotypes were associated with increased risk of diffuse-type gastric cancer, with corresponding ORs of 1.915 (95 % CI 1.173–3.127, P = 0.009) and 2.045 (95 % CI 1.267–3.299, P = 0.003), respectively. There was no difference between rs158916 polymorphism and intestinal- or diffuse-type gastric cancer.
Table 5

Association of ERCC8 polymorphisms with different histological-type gastric cancer risk

 

Healthy control

Intestinal-type gastric cancer

OR (95 % CI)

P value

Diffuse-type gastric cancer

OR (95 % CI)

P value

rs158572

 AA

347

108

1.0 (ref)

 

130

1.0 (ref)

 

 GA

46

18

1.257 (0.700–2.259)

0.444

33

1.915 (1.173–3.127)

0.009

 GG

1

2

6.426 (0.577–71.557)

0.130

3

8.008 (0.826–77.657)

0.073

 GG+GA

47

20

1.367 (0.776–2.408)

0.279

36

2.045 (1.267–3.299)

0.003

rs158916

 TT

291

98

1.0 (ref)

 

131

1.0 (ref)

 

 CT

94

27

0.853 (0.525–1.386)

0.521

31

0.733 (0.465–1.155)

0.180

 CC

9

5

1.650 (0.540–5.040)

0.380

2

0.494 (0.105–2.316)

0.371

 CC+CT

103

32

0.923 (0.584–1.458)

0.730

33

0.712 (0.457–1.108)

0.132

OR was obtained in the logistic regression models. Analyses of results with P < 0.05 were highlighted in italic characters

CI confidence interval, OR odds ratio

In addition, the combined effects of these two ERCC8 polymorphisms were assessed by haplotype analyses. The G-T haplotype was associated with increased risk of gastric cancer (OR = 1.793, 95 % CI 1.228–2.618, P = 0.002) (Table 6).
Table 6

Association of haplotype of the two ERCC8 SNPs (rs158572-rs158916) with atrophic gastritis and gastric cancer risks

Haplotype

Healthy control (%)

(N = 394)

Atrophic gastritis (%)

(N = 394)

χ 2

P

OR (95 % CI)

Healthy control (%)

(N = 394)

Gastric cancer (%)

(N = 394)

χ 2

P

OR (95 % CI)

A-C

109.84 (13.9)

95.98 (12.2)

1.118

0.290

0.854 (0.636–1.145)

109.84 (13.9)

96.75 (12.3)

0.992

0.319

0.862 (0.643–1.155)

A-T

630.16 (80.0)

629.02 (79.8)

0.032

0.858

0.978 (0.764–1.252)

630.16 (80.0)

612.25 (77.7)

1.441

0.230

0.862 (0.676–1.099)

G-Ca

2.16 (0.3)

0.02 (0.0)

/

/

/

2.16 (0.3)

2.16 (0.003)

0.25 (0.0)

/

/

G-T

45.84 (5.8)

62.98 (8.0)

2.850

0.091

1.402 (0.946–2.080)

45.84 (5.8)

78.75 (10.0)

9.353

0.002

1.793 (1.228–2.618)

Analyses of results with P < 0.05 were highlighted in italic characters

aAll those frequencies P < 0.03 were ignored in the analyses

Interaction analysis between ERCC8 genotypes and non-genetic factors on the risk of atrophic gastritis and gastric cancer

We also examined the effects of H. pylori infection, smoking, and alcohol consumption on the association between ERCC8 genetic variants and the risk of gastric cancer in subjects with available information. The heterozygous genotype and rare homozygous genotype were combined to evaluate the interactive effects. The common genotype carriers without H. pylori infection or consumption of smoking and drinking were regarded as references for each interactive analysis. For the rs158572 polymorphism, the risk of gastric cancer was significantly enhanced by H. pylori positive, ever-smoking, and alcohol drinking for individuals carrying GG+GA genotypes, with corresponding ORs of 5.248 (95 % CI 2.361–11.663, P = 0.000), 3.722 (95 % CI 1.563–8.864, P = 0.015), and 3.885 (95 % CI 1.461–10.332, P = 0.014), respectively. The risks of atrophic gastritis was elevated 7.778-fold (95 % CI 3.857–15.686, P = 0.000) in individuals carrying GG+GA genotypes and H. pylori infection (Table 7). For the rs158916 polymorphism, H. pylori positivity also led to a 5.777-fold increase (95 % CI 3.224–10.352, P = 0.000) in gastric cancer risk and an 8.731-fold increase (95 % CI 5.520–14.519, P = 0.000) in atrophic gastritis risk for subjects carrying CC+CT genotypes (Table 8).
Table 7

Interaction between ERCC8 rs158572 and environment factors

rs158572

Healthy control vs atrophic gastritis

Healthy control vs gastric cancer

AA

GG+GA

AA

GG+GA

H. pylori (−)

No. of controls/cases

278/127

35/20

278/81

35/19

OR (95 % CI)

1.0 (ref)

1.253 (0.695–2.261)

1.0 (ref)

1.829 (0.990–3.380)

H. pylori (+)

No. of controls/cases

67/205

11/39

67/93

11/17

OR (95 % CI)

6.691 (4.731–9.464)

7.778 (3.857–15.686)

4.757 (3.184–7.107)

5.248 (2.361–11.663)

P for interaction

 

0.000

0.000

Smoking (−)

No. of controls/cases

117/148

16/31

117/94

16/19

OR (95 % CI)

1.0 (ref)

1.505 (0.780–2.902)

1.0 (ref)

1.423 (0.677–2.991)

Smoking (+)

No. of controls/cases

101/93

9/14

101/86

9/19

OR (95 % CI)

0.743 (0.493–1.119)

1.198 (0.489–2.938)

1.508 (0.961–2.367)

3.722 (1.563–8.864)

P for interaction

 

0.178

0.015

Alcohol (−)

No. of controls/cases

149/172

18/34

149/95

18/19

OR (95 % CI)

1.0 (ref)

1.562 (0.857–2.958)

1.0 (ref)

1.585 (0.772–3.255)

Alcohol (+)

No. of controls/cases

69/69

7/11

69/52

7/13

OR (95 % CI)

0.967 (0.633–1.476)

1.427 (0.530–3.836)

1.654 (1.022–2.676)

3.885 (1.461–10.332)

P for interaction

 

0.415

0.014

Analyses of results with P < 0.05 were highlighted in italic characters

OR and 95 % CI were calculated by logistic regression analysis; interaction effects of ERCC8 genotypes and environmental factors on gastric cancer and atrophic gastritis risk were evaluated by the likelihood ratio test using a full model

Table 8

Interaction between ERCC8 rs158916 and environmental factors

rs158916

Healthy control vs atrophic gastritis

Healthy control vs gastric cancer

AA

CC+CT

AA

CC+CT

H. pylori (−)

No. of controls/cases

84/30

229/117

84/21

229/79

OR (95 % CI)

1.0 (ref)

1.432 (0.891–2.300)

1.0 (ref)

1.382 (0.802–2.382)

H. pylori (+)

No. of controls/cases

18/57

60/187

18/24

60/86

OR (95 % CI)

8.852 (4.500–17.414)

8.731 (5.250–14.519)

5.211 (2.385–11.386)

5.777 (3.224–10.352)

P for interaction

 

0.000

0.000

Smoking (−)

No. of controls/cases

38/37

95/142

38/23

95/90

OR (95 % CI)

1.0 (ref)

1.565 (0.923–2.654)

1.0 (ref)

1.536 (0.834–2.834)

Smoking (+)

No. of controls/cases

29/26

81/81

29/31

81/74

OR (95 % CI)

0.945 (0.458–1.950)

1.071 (0.601–1.910)

2.528 (1.172–5.455)

2.131 (1.107–4.103)

P for interaction

 

0.159

0.066

Alcohol (−)

No. of controls/cases

49/44

118/162

49/28

118/86

OR (95 % CI)

1.0 (ref)

1.572 (0.975–2.535)

1.0 (ref)

1.261 (0.722–2.204)

Alcohol (+)

No. of controls/cases

18/19

58/61

18/16

58/49

OR (95 % CI)

1.366 (0.625–2.986)

1.335 (0.758–2.350)

2.249 (0.960–5.267)

2.039 (1.079–3.855)

P for interaction

 

0.320

0.075

Analyses of results with P < 0.05 were highlighted in italic characters

OR and 95 % CI were calculated by logistic regression analysis; interaction effects of ERCC8 genotypes and environmental factors on gastric cancer and atrophic gastritis risk were evaluated by the likelihood ratio test using a full model

Discussion

Despite the crucial role of ERCC8 in TCR, to the best of our knowledge, there has been no report focusing on the association of ERCC8 and related disease risk. This is the first study to evaluate the genetic effects of ERCC8 polymorphisms on the risk of gastric cancer and their interactions with environmental factors. First, the findings of this gender- and age-matched study demonstrated an association of rs158572 polymorphism in ERCC8 with risk of gastric cancer, especially for males and people older than 50 years. Regarding rs158916, variant genotypes had a protective effect on atrophic gastritis in those older than 50 years. Haplotype analysis showed that rs158572G-rs158916T was associated with increased risk of gastric cancer. Additionally, the rs158572 genetic variant of ERCC8 may interact with H. pylori infection, smoking, and alcohol drinking to increase the risk of gastric cancer and atrophic gastritis. ERCC8 rs158916 SNP was associated with H. pylori infection in the development of atrophic gastritis and gastric cancer.

On reviewing the literature, we found that concerns regarding ERCC8 polymorphisms are not taken seriously enough, with only two studies to date that separately concentrated on the prognosis and treatment of advanced cancer [21, 22]. However, associations between ERCC8 gene polymorphisms and cancer susceptibility have remained unclear. Our findings shed light on the relationship between polymorphisms in ERCC8 and risk of developing gastric cancer, and suggest a potential value of ERCC8 polymorphisms in the early diagnosis of gastric cancer. Sequence variants, especially SNP sites that alter the binding capacity of transcription factors to promoter/regulatory regions, hold great promise in altering gene transcription and thereby modulation of cancer risk [31]. Driven by such an assumption, rs158572 variant G genotype was predicted as a transcription factor binding site of P54/c-ets-1 [32] (http://www.uniprot.org/help/uniprotkb), but the wild-type A genotype likely does not show this binding ability. Based on the predicted functional effects, the switch from A to G allele would gain ability to bind to P54, which may repress transcription to decrease the expression and function of ERCC8. Regarding rs158916, compared with the wild-type genotype, the secondary structure of variant C genotype was predicted to be more unstable (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi), and thus promote RNA cleavage and improve ERCC8 expression levels and repair ability. This may to some extent explain the protective effect of the rs158916 polymorphism. In addition, as tagSNPs, these two polymorphisms were in LD with several other functional sites, which may support the associations of these two tagSNPs on gastric cancer and atrophic gastritis susceptibility. Thus, genetic variants of ERCC8 may change its transcription and expression, and thus affecting the function of ERCC8 protein in the TCR pathway. This may partially explain the observed association of ERCC8 gene polymorphisms with increased cancer risk. Nevertheless, further studies are still required to confirm this predicted biological significance.

Stratification analysis of sex and age demonstrated an association of rs158572 polymorphism in ERCC8 with risk of gastric cancer in males and people older than 50 years, while rs158916 CC/CT genotypes had a protective effect on atrophic gastritis in subjects older than 50 years. In general, men are more vulnerable to gastric cancer and their mortality rate of gastric cancer exceeds that in women [33]. Higher exposure rates to environmental risk factors such as smoking, alcohol drinking, and other unhealthy lifestyle habits by males may also contribute to the additional risk of gastric cancer development [34]. These factors together may, at least in part, explain why a more perceptible association of rs158572 polymorphism with gastric cancer risk in males was observed. DNA injury accumulates with increased age, and the cumulative mutations are required much more for DNA repair capacity; thus, the protective or adverse effects of genetic polymorphisms would be further highlighted.

Different histological type of gastric cancer is thought to bear its distinct pathogenic mechanism that involves distinguishing genetic components [35]. To investigate the association of ERCC8 polymorphisms and different types of gastric cancer, we compared the frequency distribution between intestinal- and diffuse-type gastric cancer. In rs158572 GA and GG+GA genotype subjects, we observed a 1.915-fold and 2.045-fold increased risk for diffuse-type gastric cancer, which indicated a tendency of association between this polymorphism with diffuse gastric cancer risk. However, large-scale study in the future could draw a more reliable conclusion on this difference.

Moreover, different statuses of environmental exposure and immune function among people of different ages may partially affect the association between polymorphisms and disease risk in different age groups. Haplotype analyses demonstrated a joint effect of rs158572G and rs159616T on the development of gastric cancer. These data support the hypothesis that the action of genetic variation at the ERCC8 locus is influenced by other genetic influences. The two polymorphisms may be closely linked and their effects are difficult to separate. However, it is just a statistical estimation and further studies are required to confirm its biological validity.

The stomach is constantly under stimulation from endogenous and exogenous factors, leading to a dynamic balance between damage and repair [36]. Several non-genetic factors, including H. pylori infection, smoking, and alcohol consumption, have been identified or suspected to increase cancer risk [37, 38]. These risk factors may aggravate the burden of DNA repair, thereby contributing additional risk for gastric cancer development, especially in subjects with insufficient DNA repair capacity. In the present study, obvious interactive effects between the two ERCC8 SNPs and H. pylori infection were found to influence the risk of atrophic gastritis and gastric cancer. Furthermore, rs158572 interacted with smoking and alcohol drinking to increase gastric cancer risk. These environmental risk factors act through different mechanisms of cancer induction, and persistent exposure to H. pylori infection and tobacco and alcohol consumption may result in accumulating DNA lesions by modifying DNA bases, breaking DNA strands, and/or altering DNA structures, thereby triggering cellular carcinogenesis [39, 40, 41, 42].

This study had some limitations. One major limitation is that the sample size may still be relatively inadequate for stratification and interaction analyses, particularly for rare alleles. Larger samples and even multicenter studies should be used in the future. In addition, we cannot obtain the detailed information to discriminate between the kinds of drinkers as heavy/mild drinkers. We expect to improve the questionnaire to obtain more detailed information in the future follow-up visit. Moreover, because most of the patients were from gastroscopic screening population rather than clinical cases, thus, the data analysis about clinical pathology parameters were limited. Furthermore, molecular evidence regarding the modulation of DNA transcription and mRNA expression are warranted to support the results of our study.

In conclusion, this study reports for the first time that ERCC8 rs158572 GG+GA genotypes can increase gastric cancer risk, especially in diffuse type, males, and people older than 50 years, and ERCC8 rs158916 CC+CT genotypes have a protective effect on atrophic gastritis in those older than 50 years. The G-T haplotype was associated with increased risk of gastric cancer. Moreover, genetic variants of ERCC8 rs158572 may interact with H. pylori infection, smoking, and alcohol drinking to increase the risk of gastric cancer and atrophic gastritis. ERCC8 rs158916 SNP had an interaction with H. pylori infection in the development of atrophic gastritis and gastric cancer. While the functional interpretation remains elusive, additional large-scale studies, particularly in other ethnic populations, are still needed to validate our findings.

Notes

Acknowledgments

This work is supported by grants from the National Key Basic Research Program of China (973 Program Ref No.2010CB529304) and the National Natural Science Foundation of China (Ref No.31200968).

Authors’ contributions

YY conceived and designed the experiments and revised the manuscript. JJ and QX performed the experiments. JJ and LS analyzed the data. LS and QX collected the serum/biopsy samples. YY contributed the reagents/materials/analysis tools. JJ and YY wrote the paper.

Conflicts of interest

None

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Jing-jing Jing
    • 1
    • 2
  • Li-ping Sun
    • 1
    • 2
  • Qian Xu
    • 1
    • 2
  • Yuan Yuan
    • 1
    • 2
  1. 1.Tumor Etiology and Screening Department of Cancer Institute and General SurgeryThe First Affiliated Hospital of China Medical UniversityShenyang CityChina
  2. 2.Key Laboratory of Cancer Etiology and Prevention, Liaoning Provincial Education DepartmentChina Medical UniversityShenyangChina

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