Tumor Biol. DOI 10.1007/s13277-015-3703-9
RESEARCH ARTICLE
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 1,2 & Li-ping Sun 1,2 & Qian Xu 1,2 & Yuan Yuan 1,2
Received: 17 April 2015 / Accepted: 22 June 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
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
* Yuan Yuan [email protected] 1
Tumor Etiology and Screening Department of Cancer Institute and General Surgery, The First Affiliated Hospital of China Medical University, Heping District, Nanjing North Street 155#, Shenyang City 110001, China
2
Key Laboratory of Cancer Etiology and Prevention, Liaoning Provincial Education Department, China Medical University, Shenyang 110001, China
(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 riskmodifier 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].
Tumor Biol.
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 [11–14]. 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 [18–20]. 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 5ml 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. 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
Tumor Biol. Table 1 Baseline characteristics of the study subjects
P value
Gastric cancer
P value
Healthy control
Atrophic gastritis
Total Age Mean±SD Sex Male Female H. pylori-IgG
394
394
56.33±8.684
56.16±8.533
0.788
56.36±8.745
0.967
270 (68.5 %) 124 (31.5 %)
270 (68.5 %) 124 (31.5 %)
1.000
270 (68.5 %) 124 (31.5 %)
1.000
Positive Negative Missing Smoking Yes No Missing Alcohol Yes No Missing
78 (19.8 %) 313 (79.4 %) 3 (0.8 %)
244 (61.9 %) 147 (37.3 %) 3 (0.8 %)
0.000
110 (27.9 %) 100 (25.4 %) 184 (46.7 %)
0.000
110 (27.9 %) 133 (33.8 %) 151 (38.3 %)
107 (27.2 %) 179 (45.4 %) 108 (27.4 %)
0.076
105 (26.6 %) 113 (28.7 %) 176 (44.7 %)
0.575
76 (19.3 %) 167 (42.4 %) 151 (38.3 %)
80 (20.3 %) 206 (52.3 %) 108 (27.4 %)
0.444
65 (16.5 %) 114 (28.9 %) 215 (54.6 %)
0.297
– –
– –
130 (44.2 %) 164 (55.8 %)
394
Lauren’s classificationa Intestinal type Diffuse type a
– –
Some of the gastric cancer cases failed to be classified into either group of intestinal type or diffuse type
disequilibrium (LD) information to maximally represent (r2 > 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 χ2 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
Tumor Biol.
SHEsis analysis platform to calculate LD index (D′ and r2) 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. 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). 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. 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). 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
Table 2
Information of ERCC8 tagSNPs
SNP_ID
Alleles captured
Position
Predicted functional effects
rs158572
rs158570, rs158572, rs976080, rs158914, rs4647102, rs158935, rs11744756, rs4235483, rs929780 rs2306350, rs158916, rs4647078, rs2306351, rs4647108, rs158928 rs12520314 rs7722373 rs12657309
Intron
Intronic enhancer, TF binding site
5′ upstream
Upstream with no known function
Intron Intron Intron
Intronic with no known function Intronic enhancer Intronic with no known function
rs158916 rs12520314 rs7722373 rs12657309
Tumor Biol.
Fig. 1 Plot of information of linkage disequilibrium (r2) among common ERCC8 polymorphisms based on CHB HapMap population
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).
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 agematched 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,
HWE Hardy–Weinberg equilibrium in control population, CI confidence interval, OR odds ratio
OR was obtained in the logistic regression models. Analyses of results with P50
≤50
rs158916 Male
Female
>50
≤50
Healthy control vs gastric cancer
OR (95 % CI)
P value
1.497 (0.879–2.551) 2.104 (0.189–23.359) 1.519 (0.901–2.563)
0.138 0.545 0.117
0.900 (0.450–1.799) NA 1.000 (0.508–1.968)
0.766 NA 1.000
1.107 (0.685–1.788) 2.047 (0.184–22.721) 1.131 (0.706–1.814)
0.678 0.560 0.608
1.789 (0.742–4.310) NA 2.027 (0.856–4.801)
0.195 NA 0.108
0.749 (0.498–1.124) 1.320 (0.412–4.226) 0.788 (0.532–1.165)
0.163 0.641 0.232
0.969 (0.525–1.789) 0.242 (0.027–2.208)
0.920
AA GA GG GG+GA AA GA GG GG+GA AA GA GG GG+GA AA
243/231 26/37 1/2 27/39 104/104 20/18 0/2 20/20 259/253 37/40 1/2 38/42 88/82
GA GG GG+GA
9/15 0/2 9/17
TT CT CC CC+CT TT CT CC
197/209 68/54 5/7 73/61 94/97 26/26 4/1
CC+CT TT CT CC CC+CT TT CT
30/27 216/238 76/50 5/7 81/57 75/68 18/30
0.872 (0.482–1.577)
0.209 0.651
0.597 (0.400–0.892) 1.271 (0.397–4.063) 0.639 (0.434–0.939)
0.012 0.686 0.023
1.838 (0.940–3.593)
CC CC+CT
4/1 22/31
0.276 (0.030–2.528) 1.554 (0.822–2.940)
OR (95 % CI)
P value
AA GA GG GG+GA AA GA GG GG+GA AA GA GG GG+GA AA
243/219 26/45 1/6 27/51 104/103 20/20 0/1 20/21 259/240 37/49 1/7 38/56 88/82
1.920 (1.146–3.218) 6.658 (0.795–55.735) 2.096 (1.270–3.459)
0.012 0.044 0.004
1.010 (0.513–1.987) NA 1.060 (0.542–2.072)
0.978 NA 0.864
1.429 (0.901–2.267) 7.554 (0.923–61.851) 1.590 (1.016–2.489)
0.129 0.059 0.042
GA GG GG+GA
9/16 0 9/16
1.908 (0.799–4.555) NA 1.908 (0.799–4.555)
0.146 NA 0.146
TT CT CC CC+CT TT CT
197/208 68/55 5/7 73/62 94/99 26/22
0.766 (0.511–1.149) 1.326 (0.414–4.247) 0.804 (0.544–1.189)
0.198 0.635 0.275
0.803 (0.426–1.515)
0.499
4/3 30/25 216/230 76/58 5/8 81/66 75/77 18/19
0.712 (0.155–3.267) 0.791 (0.434–1.443)
0.662 0.445
0.717 (0.486–1.057) 1.503 (0.484–4.664) 0.765 (0.526–1.113)
0.093 0.481 0.161
0.075
CC CC+CT TT CT CC CC+CT TT CT
1.028 (0.501–2.110)
0.940
0.254 0.175
CC CC+CT
4/2 22/21
0.487 (0.087–2.739) 0.930 (0.472–1.830)
0.414 0.833
OR was obtained in the logistic regression models. Analyses of results with P
RESEARCH ARTICLE
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 1,2 & Li-ping Sun 1,2 & Qian Xu 1,2 & Yuan Yuan 1,2
Received: 17 April 2015 / Accepted: 22 June 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
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
* Yuan Yuan [email protected] 1
Tumor Etiology and Screening Department of Cancer Institute and General Surgery, The First Affiliated Hospital of China Medical University, Heping District, Nanjing North Street 155#, Shenyang City 110001, China
2
Key Laboratory of Cancer Etiology and Prevention, Liaoning Provincial Education Department, China Medical University, Shenyang 110001, China
(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 riskmodifier 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].
Tumor Biol.
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 [11–14]. 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 [18–20]. 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 5ml 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. 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
Tumor Biol. Table 1 Baseline characteristics of the study subjects
P value
Gastric cancer
P value
Healthy control
Atrophic gastritis
Total Age Mean±SD Sex Male Female H. pylori-IgG
394
394
56.33±8.684
56.16±8.533
0.788
56.36±8.745
0.967
270 (68.5 %) 124 (31.5 %)
270 (68.5 %) 124 (31.5 %)
1.000
270 (68.5 %) 124 (31.5 %)
1.000
Positive Negative Missing Smoking Yes No Missing Alcohol Yes No Missing
78 (19.8 %) 313 (79.4 %) 3 (0.8 %)
244 (61.9 %) 147 (37.3 %) 3 (0.8 %)
0.000
110 (27.9 %) 100 (25.4 %) 184 (46.7 %)
0.000
110 (27.9 %) 133 (33.8 %) 151 (38.3 %)
107 (27.2 %) 179 (45.4 %) 108 (27.4 %)
0.076
105 (26.6 %) 113 (28.7 %) 176 (44.7 %)
0.575
76 (19.3 %) 167 (42.4 %) 151 (38.3 %)
80 (20.3 %) 206 (52.3 %) 108 (27.4 %)
0.444
65 (16.5 %) 114 (28.9 %) 215 (54.6 %)
0.297
– –
– –
130 (44.2 %) 164 (55.8 %)
394
Lauren’s classificationa Intestinal type Diffuse type a
– –
Some of the gastric cancer cases failed to be classified into either group of intestinal type or diffuse type
disequilibrium (LD) information to maximally represent (r2 > 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 χ2 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
Tumor Biol.
SHEsis analysis platform to calculate LD index (D′ and r2) 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. 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). 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. 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). 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
Table 2
Information of ERCC8 tagSNPs
SNP_ID
Alleles captured
Position
Predicted functional effects
rs158572
rs158570, rs158572, rs976080, rs158914, rs4647102, rs158935, rs11744756, rs4235483, rs929780 rs2306350, rs158916, rs4647078, rs2306351, rs4647108, rs158928 rs12520314 rs7722373 rs12657309
Intron
Intronic enhancer, TF binding site
5′ upstream
Upstream with no known function
Intron Intron Intron
Intronic with no known function Intronic enhancer Intronic with no known function
rs158916 rs12520314 rs7722373 rs12657309
Tumor Biol.
Fig. 1 Plot of information of linkage disequilibrium (r2) among common ERCC8 polymorphisms based on CHB HapMap population
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).
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 agematched 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,
HWE Hardy–Weinberg equilibrium in control population, CI confidence interval, OR odds ratio
OR was obtained in the logistic regression models. Analyses of results with P50
≤50
rs158916 Male
Female
>50
≤50
Healthy control vs gastric cancer
OR (95 % CI)
P value
1.497 (0.879–2.551) 2.104 (0.189–23.359) 1.519 (0.901–2.563)
0.138 0.545 0.117
0.900 (0.450–1.799) NA 1.000 (0.508–1.968)
0.766 NA 1.000
1.107 (0.685–1.788) 2.047 (0.184–22.721) 1.131 (0.706–1.814)
0.678 0.560 0.608
1.789 (0.742–4.310) NA 2.027 (0.856–4.801)
0.195 NA 0.108
0.749 (0.498–1.124) 1.320 (0.412–4.226) 0.788 (0.532–1.165)
0.163 0.641 0.232
0.969 (0.525–1.789) 0.242 (0.027–2.208)
0.920
AA GA GG GG+GA AA GA GG GG+GA AA GA GG GG+GA AA
243/231 26/37 1/2 27/39 104/104 20/18 0/2 20/20 259/253 37/40 1/2 38/42 88/82
GA GG GG+GA
9/15 0/2 9/17
TT CT CC CC+CT TT CT CC
197/209 68/54 5/7 73/61 94/97 26/26 4/1
CC+CT TT CT CC CC+CT TT CT
30/27 216/238 76/50 5/7 81/57 75/68 18/30
0.872 (0.482–1.577)
0.209 0.651
0.597 (0.400–0.892) 1.271 (0.397–4.063) 0.639 (0.434–0.939)
0.012 0.686 0.023
1.838 (0.940–3.593)
CC CC+CT
4/1 22/31
0.276 (0.030–2.528) 1.554 (0.822–2.940)
OR (95 % CI)
P value
AA GA GG GG+GA AA GA GG GG+GA AA GA GG GG+GA AA
243/219 26/45 1/6 27/51 104/103 20/20 0/1 20/21 259/240 37/49 1/7 38/56 88/82
1.920 (1.146–3.218) 6.658 (0.795–55.735) 2.096 (1.270–3.459)
0.012 0.044 0.004
1.010 (0.513–1.987) NA 1.060 (0.542–2.072)
0.978 NA 0.864
1.429 (0.901–2.267) 7.554 (0.923–61.851) 1.590 (1.016–2.489)
0.129 0.059 0.042
GA GG GG+GA
9/16 0 9/16
1.908 (0.799–4.555) NA 1.908 (0.799–4.555)
0.146 NA 0.146
TT CT CC CC+CT TT CT
197/208 68/55 5/7 73/62 94/99 26/22
0.766 (0.511–1.149) 1.326 (0.414–4.247) 0.804 (0.544–1.189)
0.198 0.635 0.275
0.803 (0.426–1.515)
0.499
4/3 30/25 216/230 76/58 5/8 81/66 75/77 18/19
0.712 (0.155–3.267) 0.791 (0.434–1.443)
0.662 0.445
0.717 (0.486–1.057) 1.503 (0.484–4.664) 0.765 (0.526–1.113)
0.093 0.481 0.161
0.075
CC CC+CT TT CT CC CC+CT TT CT
1.028 (0.501–2.110)
0.940
0.254 0.175
CC CC+CT
4/2 22/21
0.487 (0.087–2.739) 0.930 (0.472–1.830)
0.414 0.833
OR was obtained in the logistic regression models. Analyses of results with P
