Molecular Biology Reports

, Volume 36, Issue 6, pp 1461–1467

PARP-1 Val762Ala polymorphism, CagA+H. pylori infection and risk for gastric cancer in Han Chinese population

Authors

  • Quanbao Zhang
    • Department of General SurgeryThe First Hospital of Lanzhou University
    • Department of General SurgeryThe First Hospital of Lanzhou University
  • Xun Li
    • Department of General SurgeryThe First Hospital of Lanzhou University
  • Wence Zhou
    • Department of General SurgeryThe First Hospital of Lanzhou University
  • Bin Shi
    • Department of ICUThe First Hospital of Lanzhou University
  • Hao Chen
    • Department of General SurgeryThe First Hospital of Lanzhou University
  • Wenzhen Yuan
    • Department of Surgery OncologyThe First Hospital of Lanzhou University
Article

DOI: 10.1007/s11033-008-9336-y

Cite this article as:
Zhang, Q., Li, Y., Li, X. et al. Mol Biol Rep (2009) 36: 1461. doi:10.1007/s11033-008-9336-y

Abstract

Introduction PARP-1 plays important role in the BER (base excision repair) and maintenance of genomic integrity. Previous study found the Val762Ala genetic variant in the PARP-1 gene contributed to susceptibility of some cancers and decreased PARP-1 enzyme activity in response to oxidative damage. Helicobacter pylori (H. pylori) infection was thought to be one of the major causes of gastric cancer. In this study, we investigated the association between the PARP-1 Val762Ala polymorphism, CagA+H. pylori infection, and the risk for gastric cancer. Methods This hospital-based, case–control study was performed involving 556 individuals (236 cases with gastric cancer and 320 controls without evidence of neoplasm and gastrointestinal disease) using a PCR-RFLP method. Chi-square test and logistic regression analysis were used to count OR and 95% CI. Results 762Ala/Ala genotype was overrepresented in the cases (16.9%) compared with controls (10.3%), (OR, 1.942; 95% CI, 1.157–3.257, P = 0.011). Multivariate analysis showed that two factors were significantly associated with risk of gastric cancer, including CagA+H. pylori infection (OR, 2.562; 95% CI, 1.174–5.240, P = 0.037), PARP-1 762AA genotype (OR, 1.772; 95% CI, 1.065–3.867; P = 0.042). Stratification analysis indicated that among Cag+H. pylori positive subjects, 762Ala/Ala carriers had higher risk for developing gastric cancer compared with 762Val/Val carrier (OR, 2.337; 95% CI, 1.148–4.758; P = 0.017). Conclusion PARP-1 762Ala/Ala could be a risk factor for gastric cancer in Han Chinese population; PARP-1 762Val/Ala polymorphism and Cag+H. pylori infection jointly contribute to higher risk for gastric cancer.

Keywords

Poly(ADP ribose) polymerase-1PolymorphismHelicobacter pyloriCagAGastric cancerRisk

Introduction

Gastric cancer is one of the leading causes of death from malignant tumors in the whole world. Especially, in Gansu province of China, mortality of gastric cancer is highest among all tumors [1]. In the process of blocking gene mutation and preventing initiation of tumor, the DNA repair genes have played critical role [2], of which Poly(ADP-ribose) polymerase (PARP-1) exerts important effect in the BER (base excision repair) and maintenance of genomic integrity [35]. PARP-1 is an abundant nuclear protein and composed of three domains: DNA-binding, automodification and NAD-binding domains [6]. PARP-1 specifically recognizes and binds DNA strand breaks via two tandem-arrayed N-terminal zinc fingers, and recruits other DNA repair proteins to jointly perform the DNA damage repair. PARP-1 is capable of catalyzing polyADP-ribosylation of various proteins, including histones, X-ray repair cross-complementing factor-1 (XRCC-1), NF-B, p53 [7] and PARP-1 itself, using nicotinamide adenine dinucleotide (NAD) as a substrate [8, 9]. Research found that PARP-1−/− mice show a higher susceptibility to carcinogenesis induced by alkylating agents [9]. There are many identified single nucleotide polymorphisms in the PARP-1 gene, and some of which were reported to be associated with risk for a few malignant tumors, such as germ cell [9], prostate [10], lung [11] and esophagus [12].

Helicobacter pylori (H. pylori) infection is closely related to initiation of gastric cancer, and thought to be one of the major causes of stomach cancer [13, 14]. The International Agency of Research on Cancer, sponsored by the World Health Organization, has categorized Helicobacter pylori infection as a class I carcinogen and a definite cause of human gastric cancer [15]. Previous Studies have showed that H. pylori infection results in DNA oxidative damage by its main virulence CagA and VacA [16, 17]. Many studies confirmed that carriage of CagA strains increases the risk for the development of gastric cancer [1821].

According to the previous studies [912], we hypothesized that PARP-1 762Ala/Ala may be associated with risk of gastric cancer. In addition, many previous reports suggested that interaction of H. pylori infection with the genetic variation of host jointly contributes to the gastric carcinogenesis [18, 22, 23]. Therefore, in the study, we specially analyzed the interaction of CagA+H. pylori infection and PARP-1 Val762Ala polymorphism for risk of gastric cancer.

Materials and methods

Study population

The data were collected from the First Hospital of Lanzhou University (Gansu, China). A hospital-based, case–control study consists of 236 cases and 320 controls. The recruiting criteria for cases include newly diagnosed, histopathologically confirmed and previously untreated patients in the oncology, gastroenterology and general surgery department from January 2006 to May 2008. Controls composed of 320 individuals were recruited from health examinees in the clinic department and exclusive of tumors and gastrointestinal diseases. According to principle of case–control study, the controls were selected to match to the cases by inhabiting region, sex and age (±5 years). At recruitment, each subject was informed of the detailed study protocol, and signed informed consent. A questionnaire collected information on (a) demographic factors, such as age, sex; (b) medical history and medication use; (c) smoking and drinking history; and (d) family history of cancer. Subject with at least one first-degree relative (include parent, brother or sister) with gastric cancer were considered to have a positive FH. Meanwhile, each subject donated 5 ml peripheral vein blood, and all specimens were kept frozen at −20°C. The study was approved by the institutional review board of the First Hospital of Lanzhou University.

Sample DNA extraction

Blood samples were collected with a standard venipuncture technique using EDTA containing tubes. Genomic DNA was extracted from peripheral blood leukocytes using EZ Spin Column Genetic DNA Isolation Kit (BBI, Canada) following the manufacturer’s instruction.

CagA+H. pylori strain infection test

CagA+H. pylori infection examination was tested with H. pylori IgG enzyme-linked immunosorbent assay (ELISA) kit (Bio-Check, USA) and CagA–Ig G ELISA kit (Shanghai Jingyin Biotech, China) following the manufacturers’ instructions respectively. The subject whose serology results were positive to both tests was identified as CagA+H. pylori infected.

Genotyping of PARP-1 Val762Ala polymorphism

The analysis of the Val762Ala polymorphism was performed by PCR-based restriction fragment length polymorphism (PCR-RFLP) as previously described [11]. The primers used in the amplification were: PARPF 5′-TTTGCTCCTCCAGGCCALAALACG-3′,and PARPR 5′-CATCGATGGGATCCTTGCTGCT-3′, which produce a 110-bp fragment. Each PCR contained 10× Buffer 5 μl, 4× dNTPs 10 mmol/l 5 μl, 25 mmol/l MgCl2 6 μl, each primer (50 μmol/l) 1 μl, Taq polymerase 1.0 unit (Sangon, Shanghai, China),template DNA 3 μl, ddH2O 28 μl, and a total volume of 50 μl. Heating was performed in Alpha UnitTM Block Assembly for PTC DNA EngineTM. The amplification conditions were 94°C during 5 min for the initial denaturation step, followed by 35 cycles of denaturation at 94°C (30 s), annealing at 62°C (30 s) and extension at 72°C (45 s). The final extension step consisted 10 min at 72°C. As a negative control PCR mix without DNA sample was used to ensure contamination free PCR product.

Detection of the polymorphism in PCR products by digestion with 10 units of AccII restriction endonuclease (TaKaRa, Japan) during 8 h at 37°C, then electrophoresed on 2.5% agarose gel stained with ethidium bromide. Fragment sizes of 110 bp indicated a wild-type homozygous 762Val/Val genotype, and fragment of 90 bp and 20-bp bands indicated the homozygous 762Ala/Ala genotype. The presence of all the three bands (110, 90, and 20 bp) indicated a heterozygous 762Val/Ala genotype. The 20 bp fragment can not be distinguished from the primer–dimmer band in the agarose gel (see Fig. 1). Analysis of genotypes was independently performed by two of the authors. Cases with discordant results between the two persons or with the absence of a PCR product were rejected. Also a second PCR-RFLP analysis was performed in 15% of all samples to confirm the genotype.
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-008-9336-y/MediaObjects/11033_2008_9336_Fig1_HTML.gif
Fig. 1

PCR-RFLP analysis of PARP-1 Val762Ala polymorphism. M, DNA ladder; Lanes 1–3, Homozygous 762VV genotype; lanes 4 and 5, Heterozygous 762VA genotype; lanes 6–8, Homozygous 762AA genotype

Statistical analysis

Student and Chi-square test analysis was used to compare categorical variables and genotype frequencies between cases and controls, using a 5% level of significance. Odds ratio (OR) and its 95% confidence interval (CI) were used to estimate the association between genotype and the risk of gastric cancer. Multivariate logistic regression was used to analyze all risk factors with gastric cancer. Allele frequency distribution was stratified by age, sex, CagA+H. pylori infection and FH, and assessment for interaction was considered in the model. All data analysis was performed using the computer software Statistical Package for Social Sciences-SPSS for Windows (version 11.5).

Results

Subject characteristics

The demographic information on study subjects is shown in Table 1. There were no statistically significant differences between the cases and controls in terms of age, sex, smoking and drinking distributions. However, CagA+H. pylori positive subjects were overrepresented in the cases compared with the controls (58.5% versus 48.1%; P < 0.05), and more cases had FH than controls (19.5% vs. 11.3%; P < 0.05).
Table 1

Characteristics of study subjects

Characteristics and category

Controls; n = 320 (%)

Cancer cases; n = 236 (%)

P value

Age

    Mean + SD

59.26 ± 11.08

58.32 ± 10.53

0.786

Age

    ≤50

35 (10.9)

24 (10.2)

0.992

    51–60

128 (40.0)

96 (40.7)

    61–70

132 (41.3)

98 (41.5)

    ≥71

25 (7.8)

18 (7.6)

Sex

    Male

214 (66.9)

155 (65.1)

0.666

    Female

106 (33.1)

83 (34.9)

Smokinga

    No

196 (61.3)

129 (54.7)

0.119

    Yes

124 (38.8)

107 (45.3)

Drinkingb

    No

215 (67.2)

140 (69.0)

0.133

    Yes

105 (32.8)

96 (31.0)

CagA+H. pylori

    Negative

166 (51.9)

98 (41.5)

0.016

    Positive

154 (48.1)

138 (58.5)

Family historyc

    No

284 (88.8)

190 (80.5)

0.007

    Yes

36 (11.3)

46 (19.5)

P values from Student’s t test, Chi-square test

aSmoking history, at least one per day for 6 months or longer

bDrinking history, at least once a week for 6 months or longer

cFamily history, first-degree relatives with gastric cancer (parents, brother or sister)

Allelic distribution of PARP-1 Val762Ala polymorphism

The distribution of Val762Ala PARP-1 Val762Ala genotypes is shown in Table 2. The frequency of the 762VV, VA and AA genotypes were 56.6%, 33.1% and 10.3%, respectively in controls, 47.9%, 35.2% and 16.9% in cases. All genotypic distributions are in Hardy-Weinberg equilibrium (P > 0.05). 762AA carriers were more frequently found among the cases than the controls (OR, 1.942; 95% CI, 1.157–3.257, P = 0.011); and carrying 762Ala allele carriers had a increased risk for gastric cancer compared with those carrying 762Val allele (OR, 1.449; 95% CI, 1.006–2.086, P = 0.046).
Table 2

PARP-1 Val762Ala genotype distribution in case and control group

 

Genotype n (%)

Allele frequency

VV

VA

AA

V (%)

A

Control

181 (56.6)

106 (33.1)

33 (10.3)

73.1

26.9%

Case

113 (47.9)

83 (35.2)

40 (16.9)

65.5

34.5%

OR

1 (ref)

1.254

1.942

 

1.449

95% CI

0.865–1.818

1.157–3.257

 

1.006–2.086

P value

 

0.231

0.011

 

0.046

P values from χ2 test

Multivariate logistic regress of risk factors, including age, sex, smoking, drinking, CagA+H. pylori infection and family history and Val762Ala genotype

In order to assess significant risk factors with gastric cancer, we put age, sex, smoking, drinking, CagA+H. pylori infection, family history and Val762Ala genotype into multivariate logistic regression analysis. Result is showed in Table 3, at last step, there were two factors significantly associated with risk of gastric cancer, including CagA+H. pylori infection (OR, 2.562; 95% CI, 1.174–5.240, P = 0.037) and PARP-1 762AA genotype (OR, 1.772; 95% CI, 1.065–3.867; P = 0.042) (OR, 1.435; 95% CI, 0.818–3.826, P = 0.085). Age, sex, smoking and drinking and family history were excluded from risk factors for gastric cancer (P > 0.05).
Table 3

Multivariate logistic regress analysis of risk factors with gastric cancer

Variable

B

SE

Wald χ2

P value

OR

95% CI

CagA+Hp

0.862

0.411

4.321

0.037

2.562

1.174–5.240

Genotype AA

0.474

0.263

3.324

0.042

1.772

1.065–3.867

Constant

−2.015

0.496

16.828

0.001

0.112

 

Adopted method: Backward LR, α = 0.05

Distribution of PARP-1 Val762Ala genotype stratified by age, sex, CagA+H. pylori infection and family history

We stratified the distribution of PARP-1 Val762Ala genotype by age, sex, CagA+H. pylori infection and family history. As show in Table 4, among subjects <65 years of age, 762AA carriers had 2-fold increased risk for developing gastric cancer compared with 762VV carriers (OR, 2.008; 95% CI, 1.025–3.935; P = 0.040); among CagA+H. pylori IgG positive subjects, 762AA carriers had 2.3-fold increased risk for developing gastric cancer compared with 762VV carriers (OR, 2.337; 95% CI, 1.148–4.758; P = 0.017). Among subjects having family history, 762AA carriers’ risk for gastric cancer is slightly but not significantly higher than 762VV carriers’ (OR, 1.742; 95% CI: 0.981–3.094, P = 0.057). There was no statistically significant difference was observed in genotype distribution by sex (P > 0.05).
Table 4

Distribution of PARP-1 genotype by age, sex, CagA+H. pylori infection and family history

Characteristic

Controls

Cases

VA vs VV

AA vs VV

VV

VA

AA

VV

VA

AA

OR (95% CI)

P

OR (95% CI)

P

Age

    <65 years

122

76

18

81

63

24

1.249 (0.807−1.932)

0.318

2.008 (1.025–3.935)

0.040

    ≥65 years

59

30

15

32

20

16

1.229 (0.604–2.502)

0.569

1.967 (0.861–4.490)

0.105

Sex

    Male

118

74

22

73

57

25

1.245 (0.792–1.957)

0.342

1.837 (0.966–3.494)

0.062

    Female

63

32

11

40

26

15

1.280 (0.667–2.456)

0.458

2.148 (0.897–5.142)

0.082

CagA+Hp

    Negative

92

56

18

47

37

14

1.293 (0.751–2.228)

0.354

1.522 (0.697–3.327)

0.290

    Positive

89

50

15

66

46

26

1.241 (0.744–2.069)

0.409

2.337 (1.148–4.758)

0.017

Family history

    No

162

93

29

93

68

29

1.096 (0.414–2.899)

0.853

2.613 (0.708–9.637)

0.220

    Yes

19

13

4

20

15

11

1.274 (0.851–1.907)

0.240

1.742 (0.981–3.094)

0.057

P values from χ2 test or Fisher’s exact test, comparing characteristics, adjusted for case/control status

Furthermore, we compared PARP-1 Val762Ala genotype distribution of control Chinese in previous study with present one [11]. As is showed in Table 5, we found Val762Ala genotype distribution of control Chinese was significantly different between two studies (P < 0.01).
Table 5

Comparison of PARP-1 Val762Ala genotype distribution in Chinese control group

 

n

Genotype n (%)

P value

VV

VA

AA

Previous study

1000

359 (35.9)

504 (50.4)

137 (13.7)

<0.01

Present study

310

181 (56.6)

106 (33.1)

33 (10.3)

 

P values from χ2 test

Discussion

The gastric carcinogenesis is a multifactor and multistep process in which not only various carcinogens in environment, but the genetic variation is involved. Through repairing DNA damage and maintaining genetic stability, PARP-1 has played important role in prevention of carcinogenesis. PARP-1 participates in DNA repair as DNA damage activates PARP-1 to catalyze extensive polymerization of ADP-ribose from its substrate NAD+ to nuclear proteins, most notably PARP-1 itself. Previous study monitored PARP activity through conversion of [32P] NAD+ to PAR [poly (ADP-ribose)] in PARP-1 wide type and PARP-1 −/− mice. That research found in the stomach PARIS (PAR in situ) were most intense in the mucosa and relatively absent in submucosa and muscular wall, moreover, PARIS activity is largely depleted in liver, colon, small intestine, brain, testis, pancreas, bladder, and stomach of PARP-1 −/− mice [24]. In addition, Inhibition of PARP or its genetic inactivation significantly reduced ischemia reperfusion injury in the bowel as evaluated by histological examination and functional measurements [25]. Both of the above studies showed that PARP-1 expresses in gastro-intestinal tract and plays certain role in pathogenesis of gut.

Previous studies suggested that genetic variation in DNA repair genes were associated with risk of various malignant tumors. In this study, we found that the subjects carrying 762Ala/Ala genotype had a 2-fold increased risk for developing gastric cancer compared with those carrying Val/Val genotype (OR, 1.942; 95% CI, 1.157–3.257, P = 0.011), and subjects carrying Ala/Ala allele had increased risk for gastric cancer compared with Val/Val allele (OR, 1.449; 95% CI, 1.006–2.086, P = 0.046). Moreover, result from multivariate logistic regression also supports that 762Ala/Ala genotype is a significant risk factor for gastric cancer (OR, 1.772; 95% CI, 1.065–3.867; P = 0.042). The finding is consistent with previous reports [1012]. From statistic analysis, we drew the conclusion that PARP-1 Val762Ala polymorphism is related to the risk for gastric cancer among Han Chinese population.

It is a well established concept that SNPs altering the conserved amino acids are more likely to be associated with cancer susceptibility [26]. Previous studies found that in the exon 17 of PARP-1, T-to-C transition at the position 2,444 leads to an amino acid exchange of valine to alanine at residue 762 which is located in the catalytic domain of PARP-1 and is highly conserved among species [6]. Recently, Lockett showed that the Ala/Ala genotype of the Val762Ala genetic variant in the PARP-1 gene contributes to susceptibility of prostate cancer and decreased PARP-1 enzyme activity in response to H2O2 in an allele dosage-dependent manner [10]. Zhang found that polymorphism of PARP-1 Val762Ala is associated with an increased risk for smoking-related lung cancer in Han Chinese, which showed the gene–environment interaction has played important role in carcinogenesis [11]. Many previous Studies have showed that infection with CagA+H. pylori were associated with increased risk of developing gastric cancer compared with CagA [1921]. In the study, we also found that CagA+H. pylori infection was a definite risk factor for gastric cancer (OR, 2.562; 95% CI, 1.174–5.240, P = 0.037), and by stratified analysis, we found CagA+H. pylori positive subjects with 762Ala/Ala genotype have stronger association with risk for gastric cancer. The results confirmed that the interaction of gene–environment has played a great role in the gastric carcinogenesis. Previous study showed H. pylori impairs DNA mismatch repair in gastric epithelial cells and increase the accumulation of genetic mutation in epithelial cells and risk for gastric cancer [27]. CagA, the main virulence of H. pylori could result in DNA oxidative damage, and PARP-1 Val762Ala decreases the enzyme activity of repairing DNA damage. They jointly contribute to the increased risk for gastric cancer. Previously many studies have found that interaction of H. pylori infection with the genetic variation of host jointly contributes to the gastric carcinogenesis [18, 22, 23]. Meanwhile, we found the subjects <65 years of age with 762Ala/Ala genotype have higher risk for gastric cancer. The reasonable explanation is subjects with 762Ala/Ala genotype are susceptible to gastric cancer, so, at relatively younger age, they have higher possibility to suffer from gastric cancer than those with 762Val/Val genotype.

In addition, we found Val762Ala genotype distribution was significantly different in control Chinese between previous study and ours (P < 0.01) [11]. There are some factors could result in the discrepancy: the genetic and environmental backgrounds are different between two studied regions, Beijing is near northeast China, Gansu province in northwest China where is one of highest mortality regions of gastric cancer in China [1]; Control was selected from health examinees without tumors and gastrointestinal disease in our study, and the previous just selected cancer-free subjects from a nutritional survey. Control sample size is relatively smaller in our study than it in previous (320 vs. 1,000).

In summary, PARP-1 762Ala/Ala could be a risk factor for gastric cancer in Han Chinese population; PARP-1 762Val/Ala polymorphism and CagA+H. pylori infection jointly contribute to higher risk for gastric cancer. However, the genetic and environmental factors involved in carcinogenesis are interacted and complicated. Therefore, the present conclusion remains to be further confirmed by large sample sizes investigation among different races and regions, finally, to determine whether the PARP-1 Val762Ala polymorphism would be a susceptibility biomarker for gastric cancer.

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