Med Oncol (2014) 31:40 DOI 10.1007/s12032-014-0040-6
ORIGINAL PAPER
Association between OGG1 gene single nucleotide polymorphisms and risk of pancreatic cancer in Chinese Chengli Liu • Hui Huang • Cheng Wang • Yalin Kong • Hui Zhang • Hongyi Zhang
Received: 28 April 2014 / Accepted: 16 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Previous studies have suggested that the 8-oxoguanine DNA glycosylase gene (OGG1) has potentially influenced the risk of pancreatic cancer. The objective of this study was to assess the association between single nucleotide polymorphisms (SNPs) of OGG1 gene and risk of pancreatic cancer. A case–control study has been conducted in 370 pancreatic cancer patients and 395 healthy controls. Genotypes were determined using the polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing methods. The association analysis was evaluated by the unconditional logistic regression test. Our data suggested that the distributions of alleles and genotypes were statistically different between pancreatic cancer patients and healthy controls. The c.307G[C SNP was associated with the decreased risk of pancreatic cancer (C vs. G: OR 0.73, 95 % CI 0.59–0.91, P = 0.006). As for c.828A[G SNP, the significantly decreased risk of pancreatic cancer was detected (G vs. A: OR 0.74, 95 % CI 0.59–0.92, P = 0.006). The allele C of c.307G[C and allele G of c.828A[G SNPs might be associated with a protection from pancreatic cancer. Findings from this study indicate that OGG1 SNPs are associated with pancreatic cancer risk in Chinese Han population and could be useful molecular biomarkers for assessing the risk of pancreatic cancer.
C. Liu (&) C. Wang Y. Kong H. Zhang H. Zhang Department of Hepatobiliary Surgery, The Air Force General Hospital of People’s Liberation Army, No. 30 Fucheng Road, Haidian District, Beijing 100142, People’s Republic of China e-mail: [email protected] H. Huang Department of Hepatobiliary Surgery, The 309th Hospital of Chinese People’s Liberation Army, Beijing 100091, People’s Republic of China
Keywords Pancreatic cancer Cancer susceptibility OGG1 gene Single nucleotide polymorphisms Molecular markers
Introduction Pancreatic cancer is one of the most common cancerrelated deaths for both men and women worldwide [1–5]. It causes a constantly rising health burden in the world, with a 5-year survival rate of less than 5 % [1–3]. It has been proposed that the possible risk factors for pancreatic cancer include gender, age, alcohol consumption, smoking status, body mass index, overweight, diabetes mellitus, genetic variants and family history of pancreatic cancer [3, 6–9]. Up to date, the exact mechanism of pancreatic cancer still remains uncertain. Previous studies have shown that the 8-oxoguanine DNA glycosylase gene (OGG1) is an important candidate gene for influencing the development of pancreatic cancer [3–5, 10–14]. The OGG1 gene, located on chromosome 3p26, is one of the component of DNA base excision repair (BER) pathway, which play an important role in repairing damaged DNA [14]. OGG1 is polygenetic gene, and the OGG1 genetic variants may affect the expression and function of OGG1 proteins, which contributing to the risk of pancreatic cancer and influencing the prognosis of patients. Currently, several single nucleotide polymorphisms (SNPs), such as proline (Pro) 90 glutamine (Gln), Serine (Ser) 209Ser, Arginine (Arg) 299 Glutamine (Gln) and Ser326 Cysteine (Cys), have been considered to be associated with the risk of pancreatic cancer [3–5, 11–13]. However, no related studies have reported the effects of OGG1c.307G[C and c.828A[G SNPs on influencing the risk of pancreatic cancer. Therefore, considering the importance of the role of
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Table 1 Characteristics of the pancreatic cancer cases and controls
Med Oncol (2014) 31:40
Characteristics
Pancreatic cancer cases (n)
Controls (n)
Number
370 (%)
395 (%)
Male
242 (65.41)
253 (64.05)
Female
128 (34.59)
142 (35.95)
v2 values
P values*
0.1535
0.6952
0.0025
0.9598
0.9548
0.3285
0.0720
0.7885
0.0060
0.9381
3.1439
0.0762
2.8092
0.0937
Gender (n)
Age (years) Mean ± SD
57.31 ± 15.45
58.26 ± 16.37
\55
167 (45.14)
179 (45.32)
C55
203 (54.86)
216 (54.68)
211 (57.03)
239 (60.51)
159 (42.97)
156 (39.49)
Never
226 (61.08)
245 (62.03)
Ever
144 (38.92)
150 (37.97)
\23
176 (47.57)
189 (47.85)
C23
194 (52.43)
206 (52.15)
Smoking status Never Ever Alcohol consumption
Body mass index
Diabetes mellitus (n) Yes
107 (28.92)
92 (23.29)
No
263 (71.08)
303 (76.71)
Family history of pancreatic cancer (n) SD standard deviation
Yes
68 (18.38)
55 (13.92)
* P values calculated by Chisquare (v2) test
No
302 (81.62)
340 (86.08)
OGG1 genetic variants in the development of pancreatic cancer, the purpose of this study is to evaluate the potential association between these two SNPs and the risk of pancreatic cancer.
of the Air Force General Hospital of People’s Liberation Army (Beijing, China). Written informed consent was obtained from each study individual. DNA extraction and genotyping
Materials and methods Studied subjects The study included 370 patients diagnosed and pathologically confirmed with pancreatic cancer and 395 healthy unrelated individuals as controls. All subjects were enrolled from the Air Force General Hospital of People’s Liberation Army (Beijing, China) between January 2010 and October 2013. The healthy controls were frequency-matched to pancreatic cancer patients on gender and age, excluding those with a history of cancer and other medical diseases. All individuals were of Chinese Han ethnicity. Table 1 summarizes the general characteristics of the pancreatic cancer patients and healthy controls, including gender, age, alcohol consumption, smoking status, diabetes mellitus body mass index and family history of pancreatic cancer. The study was approved by the Ethics Committees
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The peripheral venous blood was collected from each enrolled pancreatic cancer patients and healthy controls. Genomic DNA was extracted from the peripheral venous blood using the proteinase K digestion and phenol–chloroform extraction. According to the reference sequences (GenBank IDs: NG_012106.1 and NM_002542.5) of human OGG1 gene, the specific polymerase chain reaction (PCR) primers were designed by Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). The primers sequences, annealing temperature, amplification fragment size and region are given in Table 2. The PCR amplification was carried out in a total volume of 20 lL mixture which containing 50 ng template DNA, 19 buffer (100 mmol Tris–HCl, pH 8.3; 500 mmol KCl), 0.25 lmol primers, 2.0 mmol MgCl2, 0.25 mmol dNTPs (Bioteke Corporation, Beijing, China) and 0.5 U Taq DNA polymerase (Promega, Madison, WI, USA). The PCR was stared at 94 °C for 5 min, followed by 32 cycles at 94 °C
Med Oncol (2014) 31:40
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Table 2 The PCR primers and PCR–RFLP method used for investigating OGG1 SNPs SNPs
Primer sequences
c.307G[C
50 -CCACACCAGACGAGCTGGAG-30 0
5 -TAATCCCCATTTTACAGGTGGC-3
Annealing temperature (°C)
Amplification fragment (bp)
Region
Restriction enzyme
Genotype (bp)
61.0
212
Exon2
MaeI
GG:170,42
0
GC:212,170,42 CC:212,
c.828A[G
50 -CAACAGTAACCCCAGAGTGAAGG-30
61.7
216
Exon5
MaeII
50 -TGGTAGGGTGCCAGCTGTAGTC-30
AA:191,25 AG:216,191,25 GG:216
SNPs single nucleotide polymorphisms, PCR polymerase chain reaction, PCR–RFLP PCR–restriction fragment length polymorphism
for 32 s, at the corresponding temperature (presented in Table 2) for 32 s, at 72 °C for 32 s, and then an extension at 72 °C for 5 min. The OGG1 SNPs were genotyped by the PCR–restriction fragment length polymorphism (PCR– RFLP) method. According to the manufacturer’s instructions, each PCR amplified products was digested with 5 units selected restriction enzyme (MBI Fermentas, St. Leon-Rot, Germany, Table 2) at 37 °C for 10 h and then verified by agarose gel electrophoresis containing ethidium bromide and observed under UV light. For quality control, 10 % subjects that present different genotypes were random examined using DNA sequencing method (ABI3730xl DNA Analyzer, Applied Biosystems, Foster City, CA, USA) to confirm the genotyping results from PCR–RFLP method. Statistical analysis The Hardy–Weinberg equilibrium (HWE) test was performed for the distributions of OGG1 SNPs in the case and control groups. The Chi-square (v2) test was utilized to compare the distributions of OGG1 SNPs, and the differences of general characteristics between pancreatic cancer patients and healthy controls. To evaluate the potential associations between the OGG1 SNPs and the susceptibility to pancreatic cancer, the odds ratios (ORs) with their 95 % confidence intervals (CIs) were estimated by unconditional logistic regression analysis. The statistically significant was settled at P value less than 0.05. All statistical analyses were analyzed by the STATA 14.0 (Stata Corp., College Station, TX, USA) and SPSS 15.0 (SPSS Inc., Chicago, IL, USA) software programs.
Results Subject characteristics The subject characteristics are summarized in Table 1. A total of 765 subjects were enrolled in this case–control study,
which containing 370 pancreatic cancer patients (male: 242, female: 128, mean age ± standard deviation (SD): 57.31 ± 15.45) and 395 healthy controls (male: 253, female: 142, mean age ± SD: 58.26 ± 16.37). The healthy controls were comparable with the pancreatic cancer patients in regard to the distributions of gender, age, alcohol consumption, smoking status, diabetes mellitus, body mass index and family history of pancreatic cancer (all p values [0.05, Table 1). Genotyping and distribution of OGG1 SNPs Through the PCR–RFLP and DNA sequencing methods, two novel OGG1 SNPs (c.307G[C and c.828A[G) were detected in the Chinese Han populations. As for c.307G[C SNP, sequence analysis indicates that this SNP is a nonsynonymous mutation. It causes from G to C mutations in exon2 of human OGG1 gene and leads to aspartic acid (Asp) to histidine (His) amino acid replacement (p.Asp103His, reference sequences GenBank IDs: NG_012106.1, NM_002542.5 and NP_002533.1). The PCR amplified products of this SNP were digested with MaeI restriction enzyme and divided into three genotypes: GG (170 and 42 bp), GC (212, 170 and 42 bp) and CC (212 bp, Table 2). As for c.828A[G SNP, sequence analysis reveals that this SNP is a synonymous mutation. It causes from A to G mutations in exon5 of human OGG1 gene (p.Gln276Gln). The MaeII restriction enzyme has been utilized to digest the PCR amplified products of c.828A[G SNP. All three possible genotypes have been observed: AA (191 and 25 bp), AG (216, 191 and 25 bp) and GG (216 bp, Table 2). Table 3 summarizes the genotypic and allelic frequencies of these two SNPs in cases and controls. As for c.307G[C SNP, the frequencies of allele G and genotype GG in pancreatic cancer patients were higher than healthy controls (Table 3). The allelic frequencies of pancreatic cancer patients (G, 73.78 %; C, 26.22 %) were not consistent with healthy controls (G, 67.34 %; C, 32.66 %), the differences being statistically significant (v2 = 7.6180, P = 0.0058, Table 3). The genotypic frequencies of pancreatic cancer patients
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0.1196 30.65 469 69.35 1061 84 39.35 301 49.67
Association of OGG1 SNPs with pancreatic cancer risk Table 4 shows the potential association of OGG1 SNPs with pancreatic cancer risk. As for c.307G[C SNP, there were significantly decreased risk of pancreatic cancer in the heterozygote comparison (GC versus (vs.) GG: OR 0.66, 95 % CI 0.49–0.90, v2 = 7.02, P = 0.008), dominant model (CC/GC vs. GG: OR 0.66, 95 % CI 0.50–0.88, v2 = 8.28, P = 0.004) and allele contrast (C vs. G: OR 0.73, 95 % CI 0.59–0.91, v2 = 7.61, P = 0.006). As for c.828A[G SNP, the significantly decreased risk of pancreatic cancer were found in the homozygote comparison (GG vs. AA: OR 0.58, 95 % CI 0.36–0.93, v2 = 5.09, P = 0.024), heterozygote comparison (AG vs. AA: OR 0.73, 95 % CI 0.54–0.99, v2 = 3.99, P = 0.046), dominant model (GG/AG vs. AA: OR 0.76, 95 % CI 0.52–0.93, v2 = 6.19, P = 0.013) and allele contrast (G vs. A: OR 0.74, 95 % CI 0.59–0.92, v2 = 7.59, P = 0.006).
Discussion
* P values calculated by Chi-square (v2) test
70.46
452
29.54
3.8077
0.1490
380
v2 = 7.1155, P = 0.0285*
10.98
v2 = 7.5947, P = 0.0059*
4.2476
(GG, 56.49 %; GC, 34.59 %; CC, 8.92 %) were significantly different from those in healthy controls (GG, 46.08 %; GC, 42.53 %; CC, 11.39 %; v2 = 8.3079, P = 0.0157, Table 3). As for c.828A[G SNP, the allele frequencies of pancreatic cancer patients were significantly different from healthy controls (for pancreatic cancer patients: A, 72.70 %; G, 27.30 %; for healthy controls: A, 66.20 %; G, 33.80 %, v2 = 7.5947, P = 0.0059). The genotype frequencies of pancreatic cancer patients were not consistent with healthy controls, the differences being statistically significant (for pancreatic cancer patients: AA, 54.32 %; AG, 36.76 %; GG, 8.92 %; for healthy controls: AA, 45.32 %; AG, 41.77 %; GG, 12.91 %, v2 = 7.1155, P = 0.0285, Table 3). The genotype distribution of these two SNPs in cases and healthy controls fitted with HWE (all p values[0.05).
v2 = 7.6180, P = 0.0058*
1078 10.20 78 38.69 296 51.11 391 Total (n = 765)
v2 = 8.3079, P = 0.0157*
1.7486
2.0231 27.30
33.80 267
202 72.70
66.20 523
538 8.92 33
51
36.76
41.77
136
165
54.32
45.32
201
179 0.8060
0.1262 4.1396
0.4314 32.66
26.22 194
258 67.34
73.78 546
532
8.92
11.39
33
45
34.59
42.53 168
128 56.49 209
182
Pancreatic cancer patients (n = 370)
Healthy subjects (n = 395)
% C % G % CC % GC % GG
Allele frequencies (%) Genotype frequencies (%)
46.08
G % A AA % AG % AA
Genotype frequencies (%)
%
Allele frequencies (%)
%
v2 c.828A[G P v2 c.307G[C Groups
Table 3 The genotype and allele frequencies of c.307G[C and c.828A[G SNPs of OGG1 gene in pancreatic cancer patients and healthy controls
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12.91
0.3637
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Page 4 of 6
P
40
Pancreatic cancer is nowadays a common malignancy worldwide and has a high incidence. It is well known that the development of pancreatic cancer is a complex and multifactorial process from environmental and genetic factors [15–17]. There is no doubt that the genetic factors play key functions in the pathogenesis of pancreatic cancer [10, 15–22]. In recent years, OGG1 gene has been selected as one of the most potentially candidate genes for affecting pancreatic cancer risk [3–5, 10–14], and these observations indicated that OGG1 genetic variants might be associated with the risk of pancreatic cancer [3, 4, 11–13]. Zhang and his colleagues detected a statistically significantly increased risk for the variant allele (326Cys) of OGG1 Ser326Cys genetic variant compared with the wild-
Med Oncol (2014) 31:40
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Table 4 The association between OGG1 c.307G[C and c.828A[G SNPs and pancreatic cancer risk SNPs
Comparisons
OR (95 % CI)
v2 value
P values*
c.307G[C
CC vs. GG (homozygote comparison)
0.64 (0.39–1.04)
3.23
0.072
GC vs. GG (heterozygote comparison)
0.66 (0.49–0.90)
7.02
0.008
CC/GC vs. GG (dominant model)
0.66 (0.50–0.88)
8.28
0.004
CC vs. GC/GG (recessive model)
0.76 (0.47–1.22)
1.27
0.259
C vs. G (allele contrast)
0.73 (0.59–0.91)
7.61
0.006
GG vs. AA (homozygote comparison)
0.58 (0.36–0.93)
5.09
0.024
c.828A[G
AG vs. AA (heterozygote comparison)
0.73 (0.54–0.99)
3.99
0.046
GG/AG vs. AA (dominant model)
0.70 (0.52–0.93)
6.19
0.013
GG vs. AG/AA (recessive model)
0.66 (0.42–1.05)
3.11
0.078
G vs. A (allele contrast)
0.74 (0.59–0.92)
7.59
0.006
SNPs single nucleotide polymorphisms, OR odds ratio, CI confidence interval, vs. versus * P values calculated by Chi-square (v2) test
type allele (326Ser) (Ser/Cys or Cys/Cys vs. Ser/Ser: OR 1.57, 95 % CI 1.04–2.39). Results from Zhang’s study suggested that OGG1 genetic variants could influence the risk of pancreatic cancer [13]. Li et al. indicated that the homozygous variants of OGG1 G2657A showed a weak but significant effect on overall survival of patients with pancreatic cancer. The observations indicated that OGG1 genetic variants significantly influenced the clinical outcome of pancreatic cancer patients [11]. Chen et al. [5] observed that that the OGG1 Pro90Gln and Ser209Ser SNPs were statistically associated with the decreased risk of pancreatic cancer compared with wild genotype (for Pro90Gln, AA vs. CC: OR 0.44, 95 % CI 0.27–0.73, P = 0.001; for Ser209Ser, CC vs. TT: OR 0.57, 95 % CI 0.35–0.94, P = 0.028). Li et al. [4] found that there was a weak interaction of OGG1 Ser326Cys genetic polymorphism CC/CG genotype with diabetes in increased risk of pancreatic cancer. Nakao et al. [3] found that there was no significant association with pancreatic cancer risk for the OGG1 Ser326Cys genetic variant. McWilliams et al. [12] suggested that no significant differences in the risk of pancreatic cancer were detected for the OGG1 Arg299Gln and Ser326Cys SNPs. However, results from these studies are still inconsistent rather than conclusive. In the current study, the influencing of OGG1 c.307G[C and c.828A[G genetic variants on the risk of pancreatic cancer was determined by association analysis in 370 pancreatic cancer patients and 395 healthy controls. We detected that these two SNPs were statistically associated with pancreatic cancer and have significant impact on the risk of pancreatic cancer in Chinese Han population (Table 4). Our data suggested that the frequencies of allele and genotype in pancreatic cancer patients were significantly different from those of controls for these two SNPs (All P \ 0.05, Table 3). As for c.307G[C SNP, the GC
genotype and CC/GC carriers were statistically associated with the decreased susceptibility to pancreatic cancer compared to wild GG genotype (P = 0.008 and 0.004, Table 4). As for c.828A[G SNP, the GG genotype, AG genotype and GG/AG carriers were statistically associated with the decreased susceptibility to pancreatic cancer compared with wild AA genotype (P = 0.024, 0.046 and 0.013, Table 4). Results from this study indicated that the allele C of c.307G[C and allele G of c.828A[G SNPs may contribute to be associated with a protection from pancreatic cancer (P = 0.006, Table 4). The OGG1 genetic variants could impact on the expression and function of OGG1 proteins, which influencing the risk of pancreatic cancer. Our sequences analysis suggested that c.307G[C genetic variant is a nonsynonymous mutation and causes Asp to His amino acid replacement (p.Asp103His), and it might be after the function of OGG1 protein. Although the c.828A[G genetic variant is a synonymous coding variant in OGG1 gene (p.Gln276Gln), this genetic variant may linked to other known nonsynonymous genetic variants, such as Pro90Gln, Ser209Ser, Arg299Gln and Ser326Cys, which have been approved to influence the function of OGG1 proteins and significantly associated with the risk of pancreatic cancer [4, 11, 13]. To the best of our knowledge, this is the first investigation regarding the potential influence of OGG1 c.307G[C and c.828A[G SNPs with the risk of pancreatic cancer. Our findings indicate that these two SNPs are significantly associated with the decreased risk of pancreatic cancer in Chinese Han population and could be useful molecular biomarkers for evaluating the risk of pancreatic cancer. In summary, our data provide evidence for the association of OGG1 c.307G[C and c.828A[G SNPs with the risk of pancreatic cancer. Our finding is suggestive but need to be confirmed in larger different ethnic populations,
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and the underlying molecular mechanisms for pancreatic cancer carcinogenesis should be fully elucidated. Acknowledgments
None.
Conflict of interest
The authors declare no conflict of interests.
11.
12.
References 13. 1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin. 2007;57:43–66. 2. Tanaka M, Okazaki T, Suzuki H, Abbruzzese JL, Li D. Association of multi-drug resistance gene polymorphisms with pancreatic cancer outcome. Cancer. 2011;117:744–51. 3. Nakao M, Hosono S, Ito H, Watanabe M, Mizuno N, Sato S, Yatabe Y, Yamao K, Ueda R, Tajima K, Tanaka H, Matsuo K. Selected polymorphisms of base excision repair genes and pancreatic cancer risk in Japanese. J Epidemiol. 2012;22:477–83. 4. Li D, Suzuki H, Liu B, Morris J, Liu J, Okazaki T, Li Y, Chang P, Abbruzzese JL. DNA repair gene polymorphisms and risk of pancreatic cancer. Clin Cancer Res. 2009;15:740–6. 5. Chen H, Zhou B, Lan X, Wei D, Yuan T, Chen P. Association between single-nucleotide polymorphisms of OGG1 gene and pancreatic cancer risk in Chinese Han population. Tumour Biol. 2014;35:809–13. 6. Li D, Frazier M, Evans DB, Hess KR, Crane CH, Jiao L, Abbruzzese JL. Single nucleotide polymorphisms of RecQ1, RAD54L, and ATM genes are associated with reduced survival of pancreatic cancer. J Clin Oncol. 2006;24:1720–8. 7. Lowenfels AB, Maisonneuve P. Epidemiology and risk factors for pancreatic cancer. Best Pract Res Clin Gastroenterol. 2006;20:197–209. 8. Larsson SC, Orsini N, Wolk A. Body mass index and pancreatic cancer risk: a meta-analysis of prospective studies. Int J Cancer. 2007;120:1993–8. 9. Luo J, Iwasaki M, Inoue M, Sasazuki S, Otani T, Ye W, Tsugane S. Body mass index, physical activity and the risk of pancreatic cancer in relation to smoking status and history of diabetes: a large-scale population-based cohort study in Japan—the JPHC study. Cancer Causes Control. 2007;18:603–12. 10. Duell EJ, Bracci PM, Moore JH, Burk RD, Kelsey KT, Holly EA. Detecting pathway-based gene–gene and gene–environment
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ORIGINAL PAPER
Association between OGG1 gene single nucleotide polymorphisms and risk of pancreatic cancer in Chinese Chengli Liu • Hui Huang • Cheng Wang • Yalin Kong • Hui Zhang • Hongyi Zhang
Received: 28 April 2014 / Accepted: 16 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Previous studies have suggested that the 8-oxoguanine DNA glycosylase gene (OGG1) has potentially influenced the risk of pancreatic cancer. The objective of this study was to assess the association between single nucleotide polymorphisms (SNPs) of OGG1 gene and risk of pancreatic cancer. A case–control study has been conducted in 370 pancreatic cancer patients and 395 healthy controls. Genotypes were determined using the polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing methods. The association analysis was evaluated by the unconditional logistic regression test. Our data suggested that the distributions of alleles and genotypes were statistically different between pancreatic cancer patients and healthy controls. The c.307G[C SNP was associated with the decreased risk of pancreatic cancer (C vs. G: OR 0.73, 95 % CI 0.59–0.91, P = 0.006). As for c.828A[G SNP, the significantly decreased risk of pancreatic cancer was detected (G vs. A: OR 0.74, 95 % CI 0.59–0.92, P = 0.006). The allele C of c.307G[C and allele G of c.828A[G SNPs might be associated with a protection from pancreatic cancer. Findings from this study indicate that OGG1 SNPs are associated with pancreatic cancer risk in Chinese Han population and could be useful molecular biomarkers for assessing the risk of pancreatic cancer.
C. Liu (&) C. Wang Y. Kong H. Zhang H. Zhang Department of Hepatobiliary Surgery, The Air Force General Hospital of People’s Liberation Army, No. 30 Fucheng Road, Haidian District, Beijing 100142, People’s Republic of China e-mail: [email protected] H. Huang Department of Hepatobiliary Surgery, The 309th Hospital of Chinese People’s Liberation Army, Beijing 100091, People’s Republic of China
Keywords Pancreatic cancer Cancer susceptibility OGG1 gene Single nucleotide polymorphisms Molecular markers
Introduction Pancreatic cancer is one of the most common cancerrelated deaths for both men and women worldwide [1–5]. It causes a constantly rising health burden in the world, with a 5-year survival rate of less than 5 % [1–3]. It has been proposed that the possible risk factors for pancreatic cancer include gender, age, alcohol consumption, smoking status, body mass index, overweight, diabetes mellitus, genetic variants and family history of pancreatic cancer [3, 6–9]. Up to date, the exact mechanism of pancreatic cancer still remains uncertain. Previous studies have shown that the 8-oxoguanine DNA glycosylase gene (OGG1) is an important candidate gene for influencing the development of pancreatic cancer [3–5, 10–14]. The OGG1 gene, located on chromosome 3p26, is one of the component of DNA base excision repair (BER) pathway, which play an important role in repairing damaged DNA [14]. OGG1 is polygenetic gene, and the OGG1 genetic variants may affect the expression and function of OGG1 proteins, which contributing to the risk of pancreatic cancer and influencing the prognosis of patients. Currently, several single nucleotide polymorphisms (SNPs), such as proline (Pro) 90 glutamine (Gln), Serine (Ser) 209Ser, Arginine (Arg) 299 Glutamine (Gln) and Ser326 Cysteine (Cys), have been considered to be associated with the risk of pancreatic cancer [3–5, 11–13]. However, no related studies have reported the effects of OGG1c.307G[C and c.828A[G SNPs on influencing the risk of pancreatic cancer. Therefore, considering the importance of the role of
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Table 1 Characteristics of the pancreatic cancer cases and controls
Med Oncol (2014) 31:40
Characteristics
Pancreatic cancer cases (n)
Controls (n)
Number
370 (%)
395 (%)
Male
242 (65.41)
253 (64.05)
Female
128 (34.59)
142 (35.95)
v2 values
P values*
0.1535
0.6952
0.0025
0.9598
0.9548
0.3285
0.0720
0.7885
0.0060
0.9381
3.1439
0.0762
2.8092
0.0937
Gender (n)
Age (years) Mean ± SD
57.31 ± 15.45
58.26 ± 16.37
\55
167 (45.14)
179 (45.32)
C55
203 (54.86)
216 (54.68)
211 (57.03)
239 (60.51)
159 (42.97)
156 (39.49)
Never
226 (61.08)
245 (62.03)
Ever
144 (38.92)
150 (37.97)
\23
176 (47.57)
189 (47.85)
C23
194 (52.43)
206 (52.15)
Smoking status Never Ever Alcohol consumption
Body mass index
Diabetes mellitus (n) Yes
107 (28.92)
92 (23.29)
No
263 (71.08)
303 (76.71)
Family history of pancreatic cancer (n) SD standard deviation
Yes
68 (18.38)
55 (13.92)
* P values calculated by Chisquare (v2) test
No
302 (81.62)
340 (86.08)
OGG1 genetic variants in the development of pancreatic cancer, the purpose of this study is to evaluate the potential association between these two SNPs and the risk of pancreatic cancer.
of the Air Force General Hospital of People’s Liberation Army (Beijing, China). Written informed consent was obtained from each study individual. DNA extraction and genotyping
Materials and methods Studied subjects The study included 370 patients diagnosed and pathologically confirmed with pancreatic cancer and 395 healthy unrelated individuals as controls. All subjects were enrolled from the Air Force General Hospital of People’s Liberation Army (Beijing, China) between January 2010 and October 2013. The healthy controls were frequency-matched to pancreatic cancer patients on gender and age, excluding those with a history of cancer and other medical diseases. All individuals were of Chinese Han ethnicity. Table 1 summarizes the general characteristics of the pancreatic cancer patients and healthy controls, including gender, age, alcohol consumption, smoking status, diabetes mellitus body mass index and family history of pancreatic cancer. The study was approved by the Ethics Committees
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The peripheral venous blood was collected from each enrolled pancreatic cancer patients and healthy controls. Genomic DNA was extracted from the peripheral venous blood using the proteinase K digestion and phenol–chloroform extraction. According to the reference sequences (GenBank IDs: NG_012106.1 and NM_002542.5) of human OGG1 gene, the specific polymerase chain reaction (PCR) primers were designed by Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). The primers sequences, annealing temperature, amplification fragment size and region are given in Table 2. The PCR amplification was carried out in a total volume of 20 lL mixture which containing 50 ng template DNA, 19 buffer (100 mmol Tris–HCl, pH 8.3; 500 mmol KCl), 0.25 lmol primers, 2.0 mmol MgCl2, 0.25 mmol dNTPs (Bioteke Corporation, Beijing, China) and 0.5 U Taq DNA polymerase (Promega, Madison, WI, USA). The PCR was stared at 94 °C for 5 min, followed by 32 cycles at 94 °C
Med Oncol (2014) 31:40
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Table 2 The PCR primers and PCR–RFLP method used for investigating OGG1 SNPs SNPs
Primer sequences
c.307G[C
50 -CCACACCAGACGAGCTGGAG-30 0
5 -TAATCCCCATTTTACAGGTGGC-3
Annealing temperature (°C)
Amplification fragment (bp)
Region
Restriction enzyme
Genotype (bp)
61.0
212
Exon2
MaeI
GG:170,42
0
GC:212,170,42 CC:212,
c.828A[G
50 -CAACAGTAACCCCAGAGTGAAGG-30
61.7
216
Exon5
MaeII
50 -TGGTAGGGTGCCAGCTGTAGTC-30
AA:191,25 AG:216,191,25 GG:216
SNPs single nucleotide polymorphisms, PCR polymerase chain reaction, PCR–RFLP PCR–restriction fragment length polymorphism
for 32 s, at the corresponding temperature (presented in Table 2) for 32 s, at 72 °C for 32 s, and then an extension at 72 °C for 5 min. The OGG1 SNPs were genotyped by the PCR–restriction fragment length polymorphism (PCR– RFLP) method. According to the manufacturer’s instructions, each PCR amplified products was digested with 5 units selected restriction enzyme (MBI Fermentas, St. Leon-Rot, Germany, Table 2) at 37 °C for 10 h and then verified by agarose gel electrophoresis containing ethidium bromide and observed under UV light. For quality control, 10 % subjects that present different genotypes were random examined using DNA sequencing method (ABI3730xl DNA Analyzer, Applied Biosystems, Foster City, CA, USA) to confirm the genotyping results from PCR–RFLP method. Statistical analysis The Hardy–Weinberg equilibrium (HWE) test was performed for the distributions of OGG1 SNPs in the case and control groups. The Chi-square (v2) test was utilized to compare the distributions of OGG1 SNPs, and the differences of general characteristics between pancreatic cancer patients and healthy controls. To evaluate the potential associations between the OGG1 SNPs and the susceptibility to pancreatic cancer, the odds ratios (ORs) with their 95 % confidence intervals (CIs) were estimated by unconditional logistic regression analysis. The statistically significant was settled at P value less than 0.05. All statistical analyses were analyzed by the STATA 14.0 (Stata Corp., College Station, TX, USA) and SPSS 15.0 (SPSS Inc., Chicago, IL, USA) software programs.
Results Subject characteristics The subject characteristics are summarized in Table 1. A total of 765 subjects were enrolled in this case–control study,
which containing 370 pancreatic cancer patients (male: 242, female: 128, mean age ± standard deviation (SD): 57.31 ± 15.45) and 395 healthy controls (male: 253, female: 142, mean age ± SD: 58.26 ± 16.37). The healthy controls were comparable with the pancreatic cancer patients in regard to the distributions of gender, age, alcohol consumption, smoking status, diabetes mellitus, body mass index and family history of pancreatic cancer (all p values [0.05, Table 1). Genotyping and distribution of OGG1 SNPs Through the PCR–RFLP and DNA sequencing methods, two novel OGG1 SNPs (c.307G[C and c.828A[G) were detected in the Chinese Han populations. As for c.307G[C SNP, sequence analysis indicates that this SNP is a nonsynonymous mutation. It causes from G to C mutations in exon2 of human OGG1 gene and leads to aspartic acid (Asp) to histidine (His) amino acid replacement (p.Asp103His, reference sequences GenBank IDs: NG_012106.1, NM_002542.5 and NP_002533.1). The PCR amplified products of this SNP were digested with MaeI restriction enzyme and divided into three genotypes: GG (170 and 42 bp), GC (212, 170 and 42 bp) and CC (212 bp, Table 2). As for c.828A[G SNP, sequence analysis reveals that this SNP is a synonymous mutation. It causes from A to G mutations in exon5 of human OGG1 gene (p.Gln276Gln). The MaeII restriction enzyme has been utilized to digest the PCR amplified products of c.828A[G SNP. All three possible genotypes have been observed: AA (191 and 25 bp), AG (216, 191 and 25 bp) and GG (216 bp, Table 2). Table 3 summarizes the genotypic and allelic frequencies of these two SNPs in cases and controls. As for c.307G[C SNP, the frequencies of allele G and genotype GG in pancreatic cancer patients were higher than healthy controls (Table 3). The allelic frequencies of pancreatic cancer patients (G, 73.78 %; C, 26.22 %) were not consistent with healthy controls (G, 67.34 %; C, 32.66 %), the differences being statistically significant (v2 = 7.6180, P = 0.0058, Table 3). The genotypic frequencies of pancreatic cancer patients
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0.1196 30.65 469 69.35 1061 84 39.35 301 49.67
Association of OGG1 SNPs with pancreatic cancer risk Table 4 shows the potential association of OGG1 SNPs with pancreatic cancer risk. As for c.307G[C SNP, there were significantly decreased risk of pancreatic cancer in the heterozygote comparison (GC versus (vs.) GG: OR 0.66, 95 % CI 0.49–0.90, v2 = 7.02, P = 0.008), dominant model (CC/GC vs. GG: OR 0.66, 95 % CI 0.50–0.88, v2 = 8.28, P = 0.004) and allele contrast (C vs. G: OR 0.73, 95 % CI 0.59–0.91, v2 = 7.61, P = 0.006). As for c.828A[G SNP, the significantly decreased risk of pancreatic cancer were found in the homozygote comparison (GG vs. AA: OR 0.58, 95 % CI 0.36–0.93, v2 = 5.09, P = 0.024), heterozygote comparison (AG vs. AA: OR 0.73, 95 % CI 0.54–0.99, v2 = 3.99, P = 0.046), dominant model (GG/AG vs. AA: OR 0.76, 95 % CI 0.52–0.93, v2 = 6.19, P = 0.013) and allele contrast (G vs. A: OR 0.74, 95 % CI 0.59–0.92, v2 = 7.59, P = 0.006).
Discussion
* P values calculated by Chi-square (v2) test
70.46
452
29.54
3.8077
0.1490
380
v2 = 7.1155, P = 0.0285*
10.98
v2 = 7.5947, P = 0.0059*
4.2476
(GG, 56.49 %; GC, 34.59 %; CC, 8.92 %) were significantly different from those in healthy controls (GG, 46.08 %; GC, 42.53 %; CC, 11.39 %; v2 = 8.3079, P = 0.0157, Table 3). As for c.828A[G SNP, the allele frequencies of pancreatic cancer patients were significantly different from healthy controls (for pancreatic cancer patients: A, 72.70 %; G, 27.30 %; for healthy controls: A, 66.20 %; G, 33.80 %, v2 = 7.5947, P = 0.0059). The genotype frequencies of pancreatic cancer patients were not consistent with healthy controls, the differences being statistically significant (for pancreatic cancer patients: AA, 54.32 %; AG, 36.76 %; GG, 8.92 %; for healthy controls: AA, 45.32 %; AG, 41.77 %; GG, 12.91 %, v2 = 7.1155, P = 0.0285, Table 3). The genotype distribution of these two SNPs in cases and healthy controls fitted with HWE (all p values[0.05).
v2 = 7.6180, P = 0.0058*
1078 10.20 78 38.69 296 51.11 391 Total (n = 765)
v2 = 8.3079, P = 0.0157*
1.7486
2.0231 27.30
33.80 267
202 72.70
66.20 523
538 8.92 33
51
36.76
41.77
136
165
54.32
45.32
201
179 0.8060
0.1262 4.1396
0.4314 32.66
26.22 194
258 67.34
73.78 546
532
8.92
11.39
33
45
34.59
42.53 168
128 56.49 209
182
Pancreatic cancer patients (n = 370)
Healthy subjects (n = 395)
% C % G % CC % GC % GG
Allele frequencies (%) Genotype frequencies (%)
46.08
G % A AA % AG % AA
Genotype frequencies (%)
%
Allele frequencies (%)
%
v2 c.828A[G P v2 c.307G[C Groups
Table 3 The genotype and allele frequencies of c.307G[C and c.828A[G SNPs of OGG1 gene in pancreatic cancer patients and healthy controls
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12.91
0.3637
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P
40
Pancreatic cancer is nowadays a common malignancy worldwide and has a high incidence. It is well known that the development of pancreatic cancer is a complex and multifactorial process from environmental and genetic factors [15–17]. There is no doubt that the genetic factors play key functions in the pathogenesis of pancreatic cancer [10, 15–22]. In recent years, OGG1 gene has been selected as one of the most potentially candidate genes for affecting pancreatic cancer risk [3–5, 10–14], and these observations indicated that OGG1 genetic variants might be associated with the risk of pancreatic cancer [3, 4, 11–13]. Zhang and his colleagues detected a statistically significantly increased risk for the variant allele (326Cys) of OGG1 Ser326Cys genetic variant compared with the wild-
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Table 4 The association between OGG1 c.307G[C and c.828A[G SNPs and pancreatic cancer risk SNPs
Comparisons
OR (95 % CI)
v2 value
P values*
c.307G[C
CC vs. GG (homozygote comparison)
0.64 (0.39–1.04)
3.23
0.072
GC vs. GG (heterozygote comparison)
0.66 (0.49–0.90)
7.02
0.008
CC/GC vs. GG (dominant model)
0.66 (0.50–0.88)
8.28
0.004
CC vs. GC/GG (recessive model)
0.76 (0.47–1.22)
1.27
0.259
C vs. G (allele contrast)
0.73 (0.59–0.91)
7.61
0.006
GG vs. AA (homozygote comparison)
0.58 (0.36–0.93)
5.09
0.024
c.828A[G
AG vs. AA (heterozygote comparison)
0.73 (0.54–0.99)
3.99
0.046
GG/AG vs. AA (dominant model)
0.70 (0.52–0.93)
6.19
0.013
GG vs. AG/AA (recessive model)
0.66 (0.42–1.05)
3.11
0.078
G vs. A (allele contrast)
0.74 (0.59–0.92)
7.59
0.006
SNPs single nucleotide polymorphisms, OR odds ratio, CI confidence interval, vs. versus * P values calculated by Chi-square (v2) test
type allele (326Ser) (Ser/Cys or Cys/Cys vs. Ser/Ser: OR 1.57, 95 % CI 1.04–2.39). Results from Zhang’s study suggested that OGG1 genetic variants could influence the risk of pancreatic cancer [13]. Li et al. indicated that the homozygous variants of OGG1 G2657A showed a weak but significant effect on overall survival of patients with pancreatic cancer. The observations indicated that OGG1 genetic variants significantly influenced the clinical outcome of pancreatic cancer patients [11]. Chen et al. [5] observed that that the OGG1 Pro90Gln and Ser209Ser SNPs were statistically associated with the decreased risk of pancreatic cancer compared with wild genotype (for Pro90Gln, AA vs. CC: OR 0.44, 95 % CI 0.27–0.73, P = 0.001; for Ser209Ser, CC vs. TT: OR 0.57, 95 % CI 0.35–0.94, P = 0.028). Li et al. [4] found that there was a weak interaction of OGG1 Ser326Cys genetic polymorphism CC/CG genotype with diabetes in increased risk of pancreatic cancer. Nakao et al. [3] found that there was no significant association with pancreatic cancer risk for the OGG1 Ser326Cys genetic variant. McWilliams et al. [12] suggested that no significant differences in the risk of pancreatic cancer were detected for the OGG1 Arg299Gln and Ser326Cys SNPs. However, results from these studies are still inconsistent rather than conclusive. In the current study, the influencing of OGG1 c.307G[C and c.828A[G genetic variants on the risk of pancreatic cancer was determined by association analysis in 370 pancreatic cancer patients and 395 healthy controls. We detected that these two SNPs were statistically associated with pancreatic cancer and have significant impact on the risk of pancreatic cancer in Chinese Han population (Table 4). Our data suggested that the frequencies of allele and genotype in pancreatic cancer patients were significantly different from those of controls for these two SNPs (All P \ 0.05, Table 3). As for c.307G[C SNP, the GC
genotype and CC/GC carriers were statistically associated with the decreased susceptibility to pancreatic cancer compared to wild GG genotype (P = 0.008 and 0.004, Table 4). As for c.828A[G SNP, the GG genotype, AG genotype and GG/AG carriers were statistically associated with the decreased susceptibility to pancreatic cancer compared with wild AA genotype (P = 0.024, 0.046 and 0.013, Table 4). Results from this study indicated that the allele C of c.307G[C and allele G of c.828A[G SNPs may contribute to be associated with a protection from pancreatic cancer (P = 0.006, Table 4). The OGG1 genetic variants could impact on the expression and function of OGG1 proteins, which influencing the risk of pancreatic cancer. Our sequences analysis suggested that c.307G[C genetic variant is a nonsynonymous mutation and causes Asp to His amino acid replacement (p.Asp103His), and it might be after the function of OGG1 protein. Although the c.828A[G genetic variant is a synonymous coding variant in OGG1 gene (p.Gln276Gln), this genetic variant may linked to other known nonsynonymous genetic variants, such as Pro90Gln, Ser209Ser, Arg299Gln and Ser326Cys, which have been approved to influence the function of OGG1 proteins and significantly associated with the risk of pancreatic cancer [4, 11, 13]. To the best of our knowledge, this is the first investigation regarding the potential influence of OGG1 c.307G[C and c.828A[G SNPs with the risk of pancreatic cancer. Our findings indicate that these two SNPs are significantly associated with the decreased risk of pancreatic cancer in Chinese Han population and could be useful molecular biomarkers for evaluating the risk of pancreatic cancer. In summary, our data provide evidence for the association of OGG1 c.307G[C and c.828A[G SNPs with the risk of pancreatic cancer. Our finding is suggestive but need to be confirmed in larger different ethnic populations,
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and the underlying molecular mechanisms for pancreatic cancer carcinogenesis should be fully elucidated. Acknowledgments
None.
Conflict of interest
The authors declare no conflict of interests.
11.
12.
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