Med Oncol (2014) 31:272 DOI 10.1007/s12032-014-0272-5
ORIGINAL PAPER
Uracil-DNA glycosylase (UNG) rs246079 G/A polymorphism is associated with decreased risk of esophageal cancer in a Chinese population Jun Yin • Yonghua Sang • Liang Zheng • Liming Wang • Luorongxin Yuan Chao Liu • Xu Wang • Yijun Shi • Aizhong Shao • Guowen Ding • Suocheng Chen • Weifeng Tang • Haiyong Gu
•
Received: 31 August 2014 / Accepted: 26 September 2014 / Published online: 10 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Esophageal cancer is the sixth leading cause of cancer-associated death worldwide. In addition to environmental risk factors, genetic factors might play an important role in esophageal cancer carcinogenesis. We conducted a hospital-based case–control study to evaluate the association between functional single nucleotide polymorphisms (SNPs) in uracil-DNA glycosylase (UNG) and the development of esophageal cancer. A total of 380 esophageal squamous cell carcinoma (ESCC) cases and 380 controls were recruited for this study. The UNG rs3219218 A/G and UNG rs246079 G/A genotypes were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). When the UNG rs246079 GG homozygote genotype was used as the reference group, the GA genotype was associated with a significantly decreased risk for ESCC (GA vs. GG: adjusted OR 0.67, 95 % CI 0.49–0.91, P = 0.011); the AA genotype was not associated with the risk of ESCC.
Jun Yin and Yonghua Sang have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s12032-014-0272-5) contains supplementary material, which is available to authorized users. J. Yin L. Yuan C. Liu X. Wang Y. Shi A. Shao G. Ding S. Chen W. Tang (&) H. Gu (&) Department of Cardiothoracic Surgery, Affiliated People’s Hospital of Jiangsu University, 8 Dianli Rd, Zhenjiang 212000, China e-mail: [email protected] H. Gu e-mail: [email protected]
In stratification analyses, a significantly decreased risk of ESCC associated with the UNG rs246079 G/A polymorphism was evident among women, younger patients and never-smokers and never-drinkers. The UNG rs3219218 A/G polymorphism was not associated with the risk for ESCC. These findings indicated that UNG rs246079 G/A might contribute to a decreased risk of ESCC in specific populations. Because of the limited sample size, further studies including a larger and more diverse population, as well as tissue-specific biological characterization, are required to confirm the current findings. Keywords UNG Polymorphisms Esophageal cancer Molecular epidemiology Abbreviations UNG Uracil-DNA glycosylase LD Linkage disequilibrium OR Odds ratio CI Confidential interval SNPs Single nucleotide polymorphisms ESCC Esophageal squamous cell carcinoma
L. Zheng Department of Cardiothoracic Surgery, The First People’s Hospital of Changzhou and The Third Affiliated Hospital of Suzhou University, Changzhou 213003, China L. Wang Department of Chemotherapy, Cancer Institute, People’s Hospital Affiliated to Jiangsu University, Zhenjiang 212002, Jiangsu, China
Y. Sang Department of Cardiothoracic Surgery, The Second Affiliated Hospital of Soochow University, Suzhou 215002, Jiangsu, China
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Introduction Esophageal cancer is the sixth leading cause of cancerassociated death worldwide [1]. For esophageal cancer patients, the 5-year survival rate of patients with esophageal cancer is very poor and has been reported to be approximately 12.3 % in Europe [2]. Esophageal squamous cell carcinoma (ESCC) accounts for more than 90 % of esophageal cancers [3]. ESCC carcinogenesis is multifactorial, and environmental factors such as smoking and heavy drinking [4] and genetic factors are well-known risk factors. Single nucleotide polymorphisms (SNPs) might play an important role in the etiology of ESCC. Many DNA-damaging agents cause genome instability, which represents a serious problem for cells and organisms if the damaged DNA cannot be repaired [5]. Such DNAdamaging agents [6] and deficiencies of the DNA-repair system [7] have been implicated in the development of cancers. During DNA replication, uracil-DNA glycosylase (UNG) excises misincorporated uracil from DNA,
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preventing U/G mispairs and may result in C/G to T/A transition mutations [8]. UNG-deficient mice have an increased incidence of B cell lymphoma [9], as well as aberrant somatic hypermutation and class-switch recombination in B cells [10]. Uracil-processing gene polymorphisms may result in altered enzyme activity, thus affecting uracil concentrations and increasing uracil misincorporation, leading to human diseases [11]. Four UNG genes have been identified in humans, namely UNG, SMUG1, MBD4 and TDG [8, 12, 13]. Chanson et al. [14] showed that two SNPs in SMUG1 and one SNP in UNG were associated with increased blood uracil-DNA concentrations, whereas one SNP in the DUT gene was associated with decreased uracil-DNA concentrations. A study in a Taiwan Han Chinese population detected UNG rs3219218 A/G and UNG rs246079 G/A in 183 rheumatoid arthritis (RA) cases and 192 controls and confirmed that the UNG locus was associated with RA [15]. The G allele of the UNG rs246079 SNP has been identified as a high-risk factor for the development of RA.
Fig. 1 Genotyping of UNG rs3219218 A/G and UNG rs246079 G/A by MALDI-TOF–MS
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However, few studies have investigated the role of the UNG rs3219218 A/G and UNG rs246079 G/A polymorphisms in the susceptibility to cancer. Functional genetic variations in the UNG gene may contribute to the development of ESCC. The objective of the present study was to evaluate the association between the UNG rs3219218 A/G and UNG rs246079 G/A genotypes and the risk of ESCC in a hospital-based case–control study. Genotyping analyses of the two UNG SNPs were performed in 380 ESCC cases and 380 controls in a Chinese population.
Materials and methods Ethical approval of the study protocol This hospital-based case–control study was approved by the Review Board of Jiangsu University (Zhenjiang, China). The study was conducted in compliance with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals. All subjects provided written informed consent to be included in the study.
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Isolation of DNA and genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) Blood samples were collected from patients using vacutainers and transferred to tubes lined with ethylenediamine tetra-acetic acid (EDTA). Genomic DNA was isolated from whole blood with the QIAamp DNA Blood Mini Kit (Qiagen, Berlin, Germany). Sample DNA (10 ng) was amplified by PCR according to the manufacturer’s recommendations. For UNG rs3219218 A/G and UNG rs246079 G/A, genotyping was undertaken by MALDITOF MS as previously described [16]. SNP genotyping was carried out using the MassARRAY system (Sequenom, San Diego, California) by MALDI-TOF MS according to the manufacturer’s instructions. Completed genotyping reactions were spotted onto a 384-well spectroCHIP (Sequenom) using a MassARRAY Nanodispenser (Sequenom). Genotype calling was performed in real time with MassARRAY RT software version 3.1 (Sequenom, San Table 1 Distribution of selected demographic variables and risk factors in ESCC cases and controls Variable
Patients and controls A total of 380 patients with esophageal cancer were consecutively recruited from the Affiliated People’s Hospital of Jiangsu University and Affiliated Hospital of Jiangsu University (Zhenjiang, China) between October 2008 and November 2009. Diagnosis was done by biopsy and all cases of esophageal cancer were ESCC. The exclusion criteria were patients who had previously been diagnosed with cancer; any metastasized cancer; and patients who had received radiotherapy or chemotherapy. The controls were patients without cancer who were frequency-matched to the cases with regard to age (±5 years) and sex recruited from the two hospitals mentioned above during the same time period. Most of the controls were admitted to the hospitals for the treatment of trauma and included 306 trauma patients, 45 infectious disease patients and 29 hypertension patients. Each subject was personally questioned by trained interviewers using a pre-tested questionnaire to obtain information on demographic data (e.g., age, sex) and related risk factors (including tobacco smoking and alcohol consumption). After the interview, 2-mL samples of venous blood were collected from each subject. Individuals who smoked one cigarette per day for [1 year were defined as ‘‘smokers.’’ Subjects who consumed C3 alcoholic drinks a week for [6 months were considered to be ‘‘alcohol drinkers.’’
Cases (n = 380)
Controls (n = 380)
N
N
%
Pa
%
Age (years)
0.056
\60
142
37.4
117
30.8
C60
238
62.6
263
69.2
Age [(years), mean ± SD]
62.84 (± 8.50)
63.44 (± 7.19)
Men
269
70.8
257
67.6
Women
111
29.2
123
32.4
Never
220
57.9
253
66.6
Ever
160
42.1
127
33.4
253
66.6
270
71.1
127
33.4
110
28.9
LN meta (?)
85
23.9
LN meta (-)
270
76.1
I
51
15.6
II
202
62.0
III
60
18.4
IV
13
4.0
Sex
0.346
Tobacco use
Alcohol use Never Ever
0.296b
0.014
0.183
Lymph node metastasis
TNM stages
Bold values are statistically significant (P \ 0.05) a
Two-sided v2 test
b
Student t test
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Table 2 Logistic regression analyses of associations between UNG rs3219218 A/G and UNG rs246079 G/A polymorphisms and risk of ESCC Genotype
Cases (n = 380)
Controls (n = 380)
Crude OR (95 % CI)
N
(%)
N
(%)
AA
208
55.61
209
55.73
1.00 (reference value)
AG GG
135 31
36.10 8.29
144 22
38.40 5.87
0.94 (0.70–1.28) 1.42 (0.79–2.53)
P
Adjusted ORa (95 % CI)
P
UNG rs3219218 A/G
GG versus AG versus AA
1.00 (reference value) 0.699 0.239
0.95 (0.70–1.30) 1.49 (0.83–2.67)
0.764 0.180
1.02 (0.77–1.37)
0.876
0.403
AG/GG
166
44.39
166
44.27
1.01 (0.75–1.34)
AA/AG
343
91.71
353
94.13
1.00 (reference value)
GG
31
8.29
22
5.87
1.45 (0.82–2.56)
A allele
551
73.66
562
74.93
1.00 (reference value)
G allele
197
26.34
188
25.07
1.07 (0.85–1.35)
GG
217
57.87
186
49.21
1.00 (reference value)
GA
125
33.33
157
41.53
AA
33
8.80
35
9.26
0.974
1.00 (reference value) 0.198
1.52 (0.86–2.69)
0.151
– 0.574
–
–
0.68 (0.50–0.93)
0.014
0.67 (0.49–0.91)
0.011
0.81 (0.48–1.35)
0.417
0.79 (0.47–1.32)
0.360
0.69 (0.52–0.93)
0.013
UNG rs246079 G/A
AA versus GA versus GG
1.00 (reference value)
0.048
GA/AA
158
42.13
GG/GA
342
91.20
AA G allele
33 559
8.80 74.53
A allele
191
25.47
192
50.79
0.71 (0.53–0.94)
343
90.74
1.00 (reference value)
35 529
9.26 69.97
0.95 (0.57–1.56) 1.00 (reference value)
0.826
0.93 (0.56–1.53) –
0.762
227
30.03
0.80 (0.64–1.00)
0.048
–
–
0.017
1.00 (reference value)
UNG rs3219218 A/G and UNG rs246079 G/A combinations No-mutation variant genotypeb
94
25.34
76
20.27
1.00 (reference value)
1-mutation variant genotype
174
46.90
187
49.87
0.74 (0.51–1.06)
0.102
0.73 (0.51–1.06)
0.098
2-mutation variant genotype
100
26.95
111
29.60
0.72 (0.48–1.07)
0.103
0.72 (0.48–1.07)
0.102
3-mutation variant genotype
3
0.81
1
0.27
2.89 (0.30–27.95)
0.360
3.08 (0.32–30.00)
0.333
4-mutation variant genotype
0
0
0
0
–
–
–
–
1.00 (reference value)
a
Adjusted for age, sex, smoking and drinking status; Bold values are statistically significant (P \ 0.05)
b
Variant genotype means UNG rs3219218 AG or GG, UNG rs246079 GA or AA
Diego, California) and analyzed using the MassARRAY Typer software version 4.0 (Sequenom, San Diego, California) (Fig. 1). For quality control, repeated analyses were performed for 10 % of randomly selected samples by MALDI-TOF MS. Statistical analyses Differences in the distributions of demographic characteristics, selected variables, and genotypes of the UNG rs3219218 A/G and UNG rs246079 G/A variants between the cases and controls were evaluated using Student’s t test and the v2 test. The associations between the five SNPs and risk of ESCC were estimated by computing the odds ratios (ORs) and their 95 % confidence intervals (CIs) using
123
logistic regression analyses for crude ORs and adjusted ORs when adjusting for age, sex, smoking and drinking status. The Hardy–Weinberg equilibrium (HWE) was tested by a goodness-of-fit v2 test to compare the observed genotype frequencies to the expected ones among the control subjects. All statistical analyses were performed with SAS 9.1.3 (SAS Institute, Cary, NC, USA).
Results Characteristics of the study population Characteristics of cases and controls included in the study are summarized in Table 1. The cases and controls
68/59
114/95
103/91
Female
\63
C63
85/63
Ever
c
b
48/45
77/112
62/50
63/107
64/69
61/88
28/54
97/103
11/10
22/25
14/14
19/21
15/17
18/18
12/8
21/27
59/55
99/137
76/64
82/128
79/86
79/106
40/62
118/130
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
0.88 (0.34–2.31); P: 0.796; Ph:0.835
P: 0.844; Ph:0.318
P: 0.353; Ph:0.835
P: 0.011; Ph:0.318 0.95 (0.54–1.65);
0.74 (0.39–1.40);
P: 0.469; Ph:0.810
P: 0.869; Ph:0.111 0.61 (0.42–0.89);
0.74 (0.32–1.69);
P: 0.749; Ph:0.810
P: 0.008; Ph:0.111 0.96 (0.58–1.59);
0.90 (0.45–1.77);
P: 0.476; Ph:0.899
P: 0.359; Ph:0.264 0.58 (0.39–0.87);
0.76 (0.36–1.62);
P: 0.673; Ph:0.899
P: 0.009; Ph:0.264 0.81 (0.52–1.27);
0.86 (0.41–1.77);
0.56 (0.36–0.86);
1.34 (0.51–3.53); P: 0.560; Ph:0.247
P: 0.159; Ph:0.247
0.45 (0.25–0.80); P: 0.006; Ph:0.095
0.64 (0.34–1.19);
0.79 (0.55–1.14);
AA
P: 0.201; Ph:0.095
GA
Ph for heterogeneity; Bold values are statistically significant (P or Ph \ 0.05)
Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model
The genotyping was successful in 375 (98.7 %) ESCC cases, and 378 (99.5 %) controls for UNG rs246079 G/A
69/55
Ever
a
148/131
Never
Alcohol consumption
132/123
Never
Smoking status
Age
149/127
GA/AA
GG
AA
GG
GA
Adjusted ORb (95 % CI); P; Pch
UNG rs246079 G [ A (case/control)a
Male
Sex
Variable
Table 3 Stratified analyses between UNG rs246079 G/A polymorphism and ESCC risk by sex, age, smoking status and alcohol consumption
P: 0.797; Ph:0.358
0.93 (0.55–1.58);
P: 0.012; Ph:0.358
0.63 (0.44–0.91);
P: 0.692; Ph:0.201
0.91 (0.56–1.46);
P: 0.017; Ph:0.201
0.63 (0.43–0.92);
P: 0.302; Ph:0.363
0.80 (0.53–1.22);
P: 0.017; Ph:0.363
0.61 (0.40–0.92);
0.56 (0.33–0.95); P: 0.032; Ph:0.315
P: 0.114; Ph:0.315
0.76 (0.53–1.07);
GA/AA
P: 0.829; Ph:0.831
0.90 (0.36–2.29);
P: 0.740; Ph:0.831
0.90 (0.49–1.67);
P: 0.481; Ph:0.526
0.75 (0.34–1.67);
P: 0.755; Ph:0.526
1.11 (0.57–2.15);
P: 0.616; Ph:0.677
0.83 (0.40–1.73);
P: 0.812; Ph:0.677
1.09 (0.54–2.20);
1.81 (0.70–4.67); P: 0.221; Ph:0.117
P: 0.260; Ph:0.117
0.71 (0.39–1.29);
AA versus (GA/GG)
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272 Page 6 of 8 Table 4 UNG haplotype frequencies (%) in cases and controls and risk of ESCC
Bold values are statistically significant (P \ 0.05)
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Haplotypes
Cases (n = 754)
Controls (n = 756)
N
%
N
%
UNG Ars3219218Grs246079
367
48.67
341
45.11
1.00 (reference value)
UNG Ars3219218Ars246079
189
25.07
226
29.89
0.78 (0.61–0.99)
0.042
UNG Grs3219218Grs246079
195
25.86
188
24.87
0.96 (0.75–1.24)
0.771
UNG Grs3219218Ars246079
3
0.40
1
0.13
2.78 (0.29–26.83)
0.376
appeared to be adequately matched on age and sex as suggested by the v2 tests (P = 0.056 and P = 0.346, respectively). As shown in Table 1, no significant difference was detected on drinking status between the cases and the controls (P = 0.183), but smoking rate was higher in ESCC patients than in control subjects (P = 0.014). Lymph node metastasis data were available in 355 (93.4 %) of 380 ESCC patients; regional lymph node metastasis was present in 85 (23.9 %) cases. Tumor, nodal and metastatic (TNM) stage data were available in 326 (85.8 %) of 380 patients (stage I: 51; stage II: 202; stage III: 60, stage IV: 13). The primary information for UNG rs3219218 A/G and UNG rs246079 G/A SNPs was in Table S1. The genotyping success rate was 98.55 % for UNG rs3219218 A/G and 99.08 % for UNG rs246079 G/A in all 760 samples. The concordance rates of repeated analyses were 100 % for all two SNPs. Minor allele frequency (MAF) in our controls was similar to MAF for Chinese in database for all two SNPs (Table S1). The observed genotype frequencies for UNG rs3219218 A/G and UNG rs246079 G/A polymorphisms in the controls were in HWE (P = 0.667 and P = 0.822) (Table S1). Associations between UNG rs3219218 A/G and UNG rs246079 G/A polymorphisms and risk of ESCC The genotype distributions of UNG rs3219218 A/G and UNG rs246079 G/A in the cases and the controls are shown in Table 2. In the single locus analyses, the genotype frequencies of UNG rs3219218 A/G were 55.61 % (AA), 36.10 % (AG) and 8.29 % (GG) in the case patients and 55.73 % (AA), 38.40 % (AG) and 5.87 % (GG) in the control subjects, and the difference was not statistically significant (P = 0.403). In the recessive model, when the UNG rs3219218 AA/AG genotypes were used as the reference group, the GG homozygote genotype was not associated with the risk for ESCC (adjusted OR 1.52, 95 % CI 0.86–2.69, P = 0.151). When the UNG rs3219218 AA homozygote genotype was used as the reference group, the AG genotype was not associated with the risk for ESCC (AG vs. AA: adjusted OR 0.95, 95 % CI 0.70–1.30, P = 0.764); the GG genotype was not associated with the risk for ESCC (GG vs. AA: adjusted OR 1.49, 95 % CI
123
Crude OR (95 % CI)
P
0.83–2.67, P = 0.180). In the dominant model, the UNG rs3219218 AG/GG variants were not associated with the risk for ESCC, compared with the UNG rs3219218 AA genotype (adjusted OR 1.02, 95 % CI 0.77–1.37, P = 0.876) (Table 2). The genotype frequencies of UNG rs246079 G/A were 57.87 % (GG), 33.33 % (GA) and 8.80 % (AA) in the case patients and 49.21 % (GG), 41.53 % (GA) and 9.26 % (AA) in the control subjects, and the difference was statistically significant (P = 0.048). When the UNG rs246079 GG homozygote genotype was used as the reference group, the GA genotype was associated with a significantly decreased risk for ESCC (GA vs. GG: adjusted OR 0.67, 95 % CI 0.49–0.91, P = 0.011); the AA genotype was not associated with the risk of ESCC. In the dominant model, the UNG rs246079 GA/AA variants were associated with a significantly decreased risk for ESCC, compared with the UNG rs246079 GG genotype (adjusted OR 0.69, 95 % CI 0.52–0.93, P = 0.013). In the recessive model, when the UNG rs246079 GG/GA genotypes were used as the reference group, the AA homozygote genotype was not associated with the risk for ESCC (adjusted OR 0.93, 95 % CI 0.56–1.53, P = 0.762) (Table 2). Stratification analyses of UNG rs246079 G/A polymorphism and risk of ESCC To evaluate the effects of UNG rs246079 G/A genotypes on ESCC risk according to different age, sex, smoking and alcohol drinking status, we performed the stratification analyses (Table 3). A significantly decreased risk of ESCC associated with the UNG rs246079 G/A polymorphism was evident among women patients, younger patients and patients who never smoking or drinking (Table 3). Haplotype analysis of UNG polymorphisms and susceptibility of ESCC As shown in Table 4, haplotype analysis was performed and haplotypes were derived from the observed genotypes of these two UNG polymorphisms. Compared with the most common haplotype UNG Ars3219218Grs246079, the UNG Ars3219218Ars246079 haplotype was associated with
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significantly decreased risk of ESCC (OR 0.78, 95 % CI 0.61–0.99, P = 0.042). The UNG Grs3219218Grs246079 and UNG Grs3219218Ars246079 haplotypes were not associated with the risk of ESCC (Table 4).
Discussion Four UNGs have been identified in humans and encoded by the UNG, SMUG1, MBD4 and TDG genes [8, 12, 13]. The current study determined the association between the UNG rs3219218 A/G and UNG rs246079 G/A polymorphisms and the risks of ESCC in a Chinese population and provided the first positive evidence for a relationship between UNG rs246079 G/A polymorphism and ESCC. The UNG rs246079 GA allele appeared to decrease the risk of ESCC. However, no positive associations were found between the UNG rs3219218 A/G and ESCC risk. An association between autoantibodies and DNA-repair proteins has previously been reported in connective tissue diseases [17]. Among these, UNG excises misincorporated uracil from DNA, preventing the formation of U/G mispairs that could cause C/G to T/A transition mutations during DNA replication [8]. Many DNA-damaging agents cause genome instability, which could represent a serious problem for cells and organisms if the damaged DNA cannot be repaired [5]. Such DNA-damaging agents [6] and deficiencies of the DNA-repair system [7] have been implicated in the development of autoimmune disorders and cancers. Genetic alterations of UNG may play a role in the development of a subset of primary glioblastomas [18]. UNG rs246079 G/A was associated with lung cancer risk at the gene level [19]. UNG gene polymorphisms may affect repair capability or immunological function, thus conferring a risk of developing ESCC. Based on UNG rs246079 G/A mutant alleles in the control group, case samples and control samples, the power of our study (a = 0.05) was 0.640 in 375 ESCC cases and 378 controls with an OR of 0.68. The frequencies of genetic polymorphisms often vary between ethnic groups. In the present Chinese study, the allele frequency of UNG rs246079 A was 0.300 in 380 control subjects, which is consistent with the values reported in the SNP database for the Chinese Han (0.314), lower than that of European population (0.617) and higher than that of Sub-Saharan African population (0.200) (http:// www.ncbi.nlm.nih.gov/SNP). Several limitations of the present study need to be addressed. First, this was a hospital-based case–control study, and the subjects were not fully representative of the general population; therefore, selection bias was unavoidable. Second, the polymorphisms investigated, based on their functional considerations, may not offer a
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comprehensive view of the genetic variability of UNG. Third, because of the relatively small number of patients evaluated, a single case–control study is not sufficient to fully interpret the relationship between UNG polymorphisms and susceptibility to ESCC. Studies with larger numbers of subjects are necessary to confirm our findings. Finally, environmental factors differ between Chinese populations and others; because ESCC risk is likely to be influenced by gene–gene and gene–environment interactions, the UNG gene may be associated with different degrees of genetic risk in different ethnic groups and under different environmental exposures. In conclusion, the present study provided strong evidence that the UNG rs246079 G/A functional polymorphism may contribute to the risk of ESCC, whereas UNG rs3219218 A/G polymorphisms may not be associated with the risk of ESCC. However, further studies are needed to confirm the results of this preliminary study. Acknowledgments We appreciate all patients who participated in this study. This study was supported in part by National Natural Science Foundation of China (81472332, 81370001, 81300037, 81101889, 81000028), Jiangsu Province Natural Science Foundation (BK2010333, BK2011481), Social Development Foundation of Zhenjiang (SH2010017) and Changzhou Young Talents and ScienceTechnology Foundation of Health Bureau (QN201102). Conflict of interest
None.
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ORIGINAL PAPER
Uracil-DNA glycosylase (UNG) rs246079 G/A polymorphism is associated with decreased risk of esophageal cancer in a Chinese population Jun Yin • Yonghua Sang • Liang Zheng • Liming Wang • Luorongxin Yuan Chao Liu • Xu Wang • Yijun Shi • Aizhong Shao • Guowen Ding • Suocheng Chen • Weifeng Tang • Haiyong Gu
•
Received: 31 August 2014 / Accepted: 26 September 2014 / Published online: 10 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Esophageal cancer is the sixth leading cause of cancer-associated death worldwide. In addition to environmental risk factors, genetic factors might play an important role in esophageal cancer carcinogenesis. We conducted a hospital-based case–control study to evaluate the association between functional single nucleotide polymorphisms (SNPs) in uracil-DNA glycosylase (UNG) and the development of esophageal cancer. A total of 380 esophageal squamous cell carcinoma (ESCC) cases and 380 controls were recruited for this study. The UNG rs3219218 A/G and UNG rs246079 G/A genotypes were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). When the UNG rs246079 GG homozygote genotype was used as the reference group, the GA genotype was associated with a significantly decreased risk for ESCC (GA vs. GG: adjusted OR 0.67, 95 % CI 0.49–0.91, P = 0.011); the AA genotype was not associated with the risk of ESCC.
Jun Yin and Yonghua Sang have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s12032-014-0272-5) contains supplementary material, which is available to authorized users. J. Yin L. Yuan C. Liu X. Wang Y. Shi A. Shao G. Ding S. Chen W. Tang (&) H. Gu (&) Department of Cardiothoracic Surgery, Affiliated People’s Hospital of Jiangsu University, 8 Dianli Rd, Zhenjiang 212000, China e-mail: [email protected] H. Gu e-mail: [email protected]
In stratification analyses, a significantly decreased risk of ESCC associated with the UNG rs246079 G/A polymorphism was evident among women, younger patients and never-smokers and never-drinkers. The UNG rs3219218 A/G polymorphism was not associated with the risk for ESCC. These findings indicated that UNG rs246079 G/A might contribute to a decreased risk of ESCC in specific populations. Because of the limited sample size, further studies including a larger and more diverse population, as well as tissue-specific biological characterization, are required to confirm the current findings. Keywords UNG Polymorphisms Esophageal cancer Molecular epidemiology Abbreviations UNG Uracil-DNA glycosylase LD Linkage disequilibrium OR Odds ratio CI Confidential interval SNPs Single nucleotide polymorphisms ESCC Esophageal squamous cell carcinoma
L. Zheng Department of Cardiothoracic Surgery, The First People’s Hospital of Changzhou and The Third Affiliated Hospital of Suzhou University, Changzhou 213003, China L. Wang Department of Chemotherapy, Cancer Institute, People’s Hospital Affiliated to Jiangsu University, Zhenjiang 212002, Jiangsu, China
Y. Sang Department of Cardiothoracic Surgery, The Second Affiliated Hospital of Soochow University, Suzhou 215002, Jiangsu, China
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Introduction Esophageal cancer is the sixth leading cause of cancerassociated death worldwide [1]. For esophageal cancer patients, the 5-year survival rate of patients with esophageal cancer is very poor and has been reported to be approximately 12.3 % in Europe [2]. Esophageal squamous cell carcinoma (ESCC) accounts for more than 90 % of esophageal cancers [3]. ESCC carcinogenesis is multifactorial, and environmental factors such as smoking and heavy drinking [4] and genetic factors are well-known risk factors. Single nucleotide polymorphisms (SNPs) might play an important role in the etiology of ESCC. Many DNA-damaging agents cause genome instability, which represents a serious problem for cells and organisms if the damaged DNA cannot be repaired [5]. Such DNAdamaging agents [6] and deficiencies of the DNA-repair system [7] have been implicated in the development of cancers. During DNA replication, uracil-DNA glycosylase (UNG) excises misincorporated uracil from DNA,
Med Oncol (2014) 31:272
preventing U/G mispairs and may result in C/G to T/A transition mutations [8]. UNG-deficient mice have an increased incidence of B cell lymphoma [9], as well as aberrant somatic hypermutation and class-switch recombination in B cells [10]. Uracil-processing gene polymorphisms may result in altered enzyme activity, thus affecting uracil concentrations and increasing uracil misincorporation, leading to human diseases [11]. Four UNG genes have been identified in humans, namely UNG, SMUG1, MBD4 and TDG [8, 12, 13]. Chanson et al. [14] showed that two SNPs in SMUG1 and one SNP in UNG were associated with increased blood uracil-DNA concentrations, whereas one SNP in the DUT gene was associated with decreased uracil-DNA concentrations. A study in a Taiwan Han Chinese population detected UNG rs3219218 A/G and UNG rs246079 G/A in 183 rheumatoid arthritis (RA) cases and 192 controls and confirmed that the UNG locus was associated with RA [15]. The G allele of the UNG rs246079 SNP has been identified as a high-risk factor for the development of RA.
Fig. 1 Genotyping of UNG rs3219218 A/G and UNG rs246079 G/A by MALDI-TOF–MS
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However, few studies have investigated the role of the UNG rs3219218 A/G and UNG rs246079 G/A polymorphisms in the susceptibility to cancer. Functional genetic variations in the UNG gene may contribute to the development of ESCC. The objective of the present study was to evaluate the association between the UNG rs3219218 A/G and UNG rs246079 G/A genotypes and the risk of ESCC in a hospital-based case–control study. Genotyping analyses of the two UNG SNPs were performed in 380 ESCC cases and 380 controls in a Chinese population.
Materials and methods Ethical approval of the study protocol This hospital-based case–control study was approved by the Review Board of Jiangsu University (Zhenjiang, China). The study was conducted in compliance with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals. All subjects provided written informed consent to be included in the study.
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Isolation of DNA and genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) Blood samples were collected from patients using vacutainers and transferred to tubes lined with ethylenediamine tetra-acetic acid (EDTA). Genomic DNA was isolated from whole blood with the QIAamp DNA Blood Mini Kit (Qiagen, Berlin, Germany). Sample DNA (10 ng) was amplified by PCR according to the manufacturer’s recommendations. For UNG rs3219218 A/G and UNG rs246079 G/A, genotyping was undertaken by MALDITOF MS as previously described [16]. SNP genotyping was carried out using the MassARRAY system (Sequenom, San Diego, California) by MALDI-TOF MS according to the manufacturer’s instructions. Completed genotyping reactions were spotted onto a 384-well spectroCHIP (Sequenom) using a MassARRAY Nanodispenser (Sequenom). Genotype calling was performed in real time with MassARRAY RT software version 3.1 (Sequenom, San Table 1 Distribution of selected demographic variables and risk factors in ESCC cases and controls Variable
Patients and controls A total of 380 patients with esophageal cancer were consecutively recruited from the Affiliated People’s Hospital of Jiangsu University and Affiliated Hospital of Jiangsu University (Zhenjiang, China) between October 2008 and November 2009. Diagnosis was done by biopsy and all cases of esophageal cancer were ESCC. The exclusion criteria were patients who had previously been diagnosed with cancer; any metastasized cancer; and patients who had received radiotherapy or chemotherapy. The controls were patients without cancer who were frequency-matched to the cases with regard to age (±5 years) and sex recruited from the two hospitals mentioned above during the same time period. Most of the controls were admitted to the hospitals for the treatment of trauma and included 306 trauma patients, 45 infectious disease patients and 29 hypertension patients. Each subject was personally questioned by trained interviewers using a pre-tested questionnaire to obtain information on demographic data (e.g., age, sex) and related risk factors (including tobacco smoking and alcohol consumption). After the interview, 2-mL samples of venous blood were collected from each subject. Individuals who smoked one cigarette per day for [1 year were defined as ‘‘smokers.’’ Subjects who consumed C3 alcoholic drinks a week for [6 months were considered to be ‘‘alcohol drinkers.’’
Cases (n = 380)
Controls (n = 380)
N
N
%
Pa
%
Age (years)
0.056
\60
142
37.4
117
30.8
C60
238
62.6
263
69.2
Age [(years), mean ± SD]
62.84 (± 8.50)
63.44 (± 7.19)
Men
269
70.8
257
67.6
Women
111
29.2
123
32.4
Never
220
57.9
253
66.6
Ever
160
42.1
127
33.4
253
66.6
270
71.1
127
33.4
110
28.9
LN meta (?)
85
23.9
LN meta (-)
270
76.1
I
51
15.6
II
202
62.0
III
60
18.4
IV
13
4.0
Sex
0.346
Tobacco use
Alcohol use Never Ever
0.296b
0.014
0.183
Lymph node metastasis
TNM stages
Bold values are statistically significant (P \ 0.05) a
Two-sided v2 test
b
Student t test
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Table 2 Logistic regression analyses of associations between UNG rs3219218 A/G and UNG rs246079 G/A polymorphisms and risk of ESCC Genotype
Cases (n = 380)
Controls (n = 380)
Crude OR (95 % CI)
N
(%)
N
(%)
AA
208
55.61
209
55.73
1.00 (reference value)
AG GG
135 31
36.10 8.29
144 22
38.40 5.87
0.94 (0.70–1.28) 1.42 (0.79–2.53)
P
Adjusted ORa (95 % CI)
P
UNG rs3219218 A/G
GG versus AG versus AA
1.00 (reference value) 0.699 0.239
0.95 (0.70–1.30) 1.49 (0.83–2.67)
0.764 0.180
1.02 (0.77–1.37)
0.876
0.403
AG/GG
166
44.39
166
44.27
1.01 (0.75–1.34)
AA/AG
343
91.71
353
94.13
1.00 (reference value)
GG
31
8.29
22
5.87
1.45 (0.82–2.56)
A allele
551
73.66
562
74.93
1.00 (reference value)
G allele
197
26.34
188
25.07
1.07 (0.85–1.35)
GG
217
57.87
186
49.21
1.00 (reference value)
GA
125
33.33
157
41.53
AA
33
8.80
35
9.26
0.974
1.00 (reference value) 0.198
1.52 (0.86–2.69)
0.151
– 0.574
–
–
0.68 (0.50–0.93)
0.014
0.67 (0.49–0.91)
0.011
0.81 (0.48–1.35)
0.417
0.79 (0.47–1.32)
0.360
0.69 (0.52–0.93)
0.013
UNG rs246079 G/A
AA versus GA versus GG
1.00 (reference value)
0.048
GA/AA
158
42.13
GG/GA
342
91.20
AA G allele
33 559
8.80 74.53
A allele
191
25.47
192
50.79
0.71 (0.53–0.94)
343
90.74
1.00 (reference value)
35 529
9.26 69.97
0.95 (0.57–1.56) 1.00 (reference value)
0.826
0.93 (0.56–1.53) –
0.762
227
30.03
0.80 (0.64–1.00)
0.048
–
–
0.017
1.00 (reference value)
UNG rs3219218 A/G and UNG rs246079 G/A combinations No-mutation variant genotypeb
94
25.34
76
20.27
1.00 (reference value)
1-mutation variant genotype
174
46.90
187
49.87
0.74 (0.51–1.06)
0.102
0.73 (0.51–1.06)
0.098
2-mutation variant genotype
100
26.95
111
29.60
0.72 (0.48–1.07)
0.103
0.72 (0.48–1.07)
0.102
3-mutation variant genotype
3
0.81
1
0.27
2.89 (0.30–27.95)
0.360
3.08 (0.32–30.00)
0.333
4-mutation variant genotype
0
0
0
0
–
–
–
–
1.00 (reference value)
a
Adjusted for age, sex, smoking and drinking status; Bold values are statistically significant (P \ 0.05)
b
Variant genotype means UNG rs3219218 AG or GG, UNG rs246079 GA or AA
Diego, California) and analyzed using the MassARRAY Typer software version 4.0 (Sequenom, San Diego, California) (Fig. 1). For quality control, repeated analyses were performed for 10 % of randomly selected samples by MALDI-TOF MS. Statistical analyses Differences in the distributions of demographic characteristics, selected variables, and genotypes of the UNG rs3219218 A/G and UNG rs246079 G/A variants between the cases and controls were evaluated using Student’s t test and the v2 test. The associations between the five SNPs and risk of ESCC were estimated by computing the odds ratios (ORs) and their 95 % confidence intervals (CIs) using
123
logistic regression analyses for crude ORs and adjusted ORs when adjusting for age, sex, smoking and drinking status. The Hardy–Weinberg equilibrium (HWE) was tested by a goodness-of-fit v2 test to compare the observed genotype frequencies to the expected ones among the control subjects. All statistical analyses were performed with SAS 9.1.3 (SAS Institute, Cary, NC, USA).
Results Characteristics of the study population Characteristics of cases and controls included in the study are summarized in Table 1. The cases and controls
68/59
114/95
103/91
Female
\63
C63
85/63
Ever
c
b
48/45
77/112
62/50
63/107
64/69
61/88
28/54
97/103
11/10
22/25
14/14
19/21
15/17
18/18
12/8
21/27
59/55
99/137
76/64
82/128
79/86
79/106
40/62
118/130
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
1.00 (reference value)
0.88 (0.34–2.31); P: 0.796; Ph:0.835
P: 0.844; Ph:0.318
P: 0.353; Ph:0.835
P: 0.011; Ph:0.318 0.95 (0.54–1.65);
0.74 (0.39–1.40);
P: 0.469; Ph:0.810
P: 0.869; Ph:0.111 0.61 (0.42–0.89);
0.74 (0.32–1.69);
P: 0.749; Ph:0.810
P: 0.008; Ph:0.111 0.96 (0.58–1.59);
0.90 (0.45–1.77);
P: 0.476; Ph:0.899
P: 0.359; Ph:0.264 0.58 (0.39–0.87);
0.76 (0.36–1.62);
P: 0.673; Ph:0.899
P: 0.009; Ph:0.264 0.81 (0.52–1.27);
0.86 (0.41–1.77);
0.56 (0.36–0.86);
1.34 (0.51–3.53); P: 0.560; Ph:0.247
P: 0.159; Ph:0.247
0.45 (0.25–0.80); P: 0.006; Ph:0.095
0.64 (0.34–1.19);
0.79 (0.55–1.14);
AA
P: 0.201; Ph:0.095
GA
Ph for heterogeneity; Bold values are statistically significant (P or Ph \ 0.05)
Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model
The genotyping was successful in 375 (98.7 %) ESCC cases, and 378 (99.5 %) controls for UNG rs246079 G/A
69/55
Ever
a
148/131
Never
Alcohol consumption
132/123
Never
Smoking status
Age
149/127
GA/AA
GG
AA
GG
GA
Adjusted ORb (95 % CI); P; Pch
UNG rs246079 G [ A (case/control)a
Male
Sex
Variable
Table 3 Stratified analyses between UNG rs246079 G/A polymorphism and ESCC risk by sex, age, smoking status and alcohol consumption
P: 0.797; Ph:0.358
0.93 (0.55–1.58);
P: 0.012; Ph:0.358
0.63 (0.44–0.91);
P: 0.692; Ph:0.201
0.91 (0.56–1.46);
P: 0.017; Ph:0.201
0.63 (0.43–0.92);
P: 0.302; Ph:0.363
0.80 (0.53–1.22);
P: 0.017; Ph:0.363
0.61 (0.40–0.92);
0.56 (0.33–0.95); P: 0.032; Ph:0.315
P: 0.114; Ph:0.315
0.76 (0.53–1.07);
GA/AA
P: 0.829; Ph:0.831
0.90 (0.36–2.29);
P: 0.740; Ph:0.831
0.90 (0.49–1.67);
P: 0.481; Ph:0.526
0.75 (0.34–1.67);
P: 0.755; Ph:0.526
1.11 (0.57–2.15);
P: 0.616; Ph:0.677
0.83 (0.40–1.73);
P: 0.812; Ph:0.677
1.09 (0.54–2.20);
1.81 (0.70–4.67); P: 0.221; Ph:0.117
P: 0.260; Ph:0.117
0.71 (0.39–1.29);
AA versus (GA/GG)
Med Oncol (2014) 31:272 Page 5 of 8 272
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272 Page 6 of 8 Table 4 UNG haplotype frequencies (%) in cases and controls and risk of ESCC
Bold values are statistically significant (P \ 0.05)
Med Oncol (2014) 31:272
Haplotypes
Cases (n = 754)
Controls (n = 756)
N
%
N
%
UNG Ars3219218Grs246079
367
48.67
341
45.11
1.00 (reference value)
UNG Ars3219218Ars246079
189
25.07
226
29.89
0.78 (0.61–0.99)
0.042
UNG Grs3219218Grs246079
195
25.86
188
24.87
0.96 (0.75–1.24)
0.771
UNG Grs3219218Ars246079
3
0.40
1
0.13
2.78 (0.29–26.83)
0.376
appeared to be adequately matched on age and sex as suggested by the v2 tests (P = 0.056 and P = 0.346, respectively). As shown in Table 1, no significant difference was detected on drinking status between the cases and the controls (P = 0.183), but smoking rate was higher in ESCC patients than in control subjects (P = 0.014). Lymph node metastasis data were available in 355 (93.4 %) of 380 ESCC patients; regional lymph node metastasis was present in 85 (23.9 %) cases. Tumor, nodal and metastatic (TNM) stage data were available in 326 (85.8 %) of 380 patients (stage I: 51; stage II: 202; stage III: 60, stage IV: 13). The primary information for UNG rs3219218 A/G and UNG rs246079 G/A SNPs was in Table S1. The genotyping success rate was 98.55 % for UNG rs3219218 A/G and 99.08 % for UNG rs246079 G/A in all 760 samples. The concordance rates of repeated analyses were 100 % for all two SNPs. Minor allele frequency (MAF) in our controls was similar to MAF for Chinese in database for all two SNPs (Table S1). The observed genotype frequencies for UNG rs3219218 A/G and UNG rs246079 G/A polymorphisms in the controls were in HWE (P = 0.667 and P = 0.822) (Table S1). Associations between UNG rs3219218 A/G and UNG rs246079 G/A polymorphisms and risk of ESCC The genotype distributions of UNG rs3219218 A/G and UNG rs246079 G/A in the cases and the controls are shown in Table 2. In the single locus analyses, the genotype frequencies of UNG rs3219218 A/G were 55.61 % (AA), 36.10 % (AG) and 8.29 % (GG) in the case patients and 55.73 % (AA), 38.40 % (AG) and 5.87 % (GG) in the control subjects, and the difference was not statistically significant (P = 0.403). In the recessive model, when the UNG rs3219218 AA/AG genotypes were used as the reference group, the GG homozygote genotype was not associated with the risk for ESCC (adjusted OR 1.52, 95 % CI 0.86–2.69, P = 0.151). When the UNG rs3219218 AA homozygote genotype was used as the reference group, the AG genotype was not associated with the risk for ESCC (AG vs. AA: adjusted OR 0.95, 95 % CI 0.70–1.30, P = 0.764); the GG genotype was not associated with the risk for ESCC (GG vs. AA: adjusted OR 1.49, 95 % CI
123
Crude OR (95 % CI)
P
0.83–2.67, P = 0.180). In the dominant model, the UNG rs3219218 AG/GG variants were not associated with the risk for ESCC, compared with the UNG rs3219218 AA genotype (adjusted OR 1.02, 95 % CI 0.77–1.37, P = 0.876) (Table 2). The genotype frequencies of UNG rs246079 G/A were 57.87 % (GG), 33.33 % (GA) and 8.80 % (AA) in the case patients and 49.21 % (GG), 41.53 % (GA) and 9.26 % (AA) in the control subjects, and the difference was statistically significant (P = 0.048). When the UNG rs246079 GG homozygote genotype was used as the reference group, the GA genotype was associated with a significantly decreased risk for ESCC (GA vs. GG: adjusted OR 0.67, 95 % CI 0.49–0.91, P = 0.011); the AA genotype was not associated with the risk of ESCC. In the dominant model, the UNG rs246079 GA/AA variants were associated with a significantly decreased risk for ESCC, compared with the UNG rs246079 GG genotype (adjusted OR 0.69, 95 % CI 0.52–0.93, P = 0.013). In the recessive model, when the UNG rs246079 GG/GA genotypes were used as the reference group, the AA homozygote genotype was not associated with the risk for ESCC (adjusted OR 0.93, 95 % CI 0.56–1.53, P = 0.762) (Table 2). Stratification analyses of UNG rs246079 G/A polymorphism and risk of ESCC To evaluate the effects of UNG rs246079 G/A genotypes on ESCC risk according to different age, sex, smoking and alcohol drinking status, we performed the stratification analyses (Table 3). A significantly decreased risk of ESCC associated with the UNG rs246079 G/A polymorphism was evident among women patients, younger patients and patients who never smoking or drinking (Table 3). Haplotype analysis of UNG polymorphisms and susceptibility of ESCC As shown in Table 4, haplotype analysis was performed and haplotypes were derived from the observed genotypes of these two UNG polymorphisms. Compared with the most common haplotype UNG Ars3219218Grs246079, the UNG Ars3219218Ars246079 haplotype was associated with
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significantly decreased risk of ESCC (OR 0.78, 95 % CI 0.61–0.99, P = 0.042). The UNG Grs3219218Grs246079 and UNG Grs3219218Ars246079 haplotypes were not associated with the risk of ESCC (Table 4).
Discussion Four UNGs have been identified in humans and encoded by the UNG, SMUG1, MBD4 and TDG genes [8, 12, 13]. The current study determined the association between the UNG rs3219218 A/G and UNG rs246079 G/A polymorphisms and the risks of ESCC in a Chinese population and provided the first positive evidence for a relationship between UNG rs246079 G/A polymorphism and ESCC. The UNG rs246079 GA allele appeared to decrease the risk of ESCC. However, no positive associations were found between the UNG rs3219218 A/G and ESCC risk. An association between autoantibodies and DNA-repair proteins has previously been reported in connective tissue diseases [17]. Among these, UNG excises misincorporated uracil from DNA, preventing the formation of U/G mispairs that could cause C/G to T/A transition mutations during DNA replication [8]. Many DNA-damaging agents cause genome instability, which could represent a serious problem for cells and organisms if the damaged DNA cannot be repaired [5]. Such DNA-damaging agents [6] and deficiencies of the DNA-repair system [7] have been implicated in the development of autoimmune disorders and cancers. Genetic alterations of UNG may play a role in the development of a subset of primary glioblastomas [18]. UNG rs246079 G/A was associated with lung cancer risk at the gene level [19]. UNG gene polymorphisms may affect repair capability or immunological function, thus conferring a risk of developing ESCC. Based on UNG rs246079 G/A mutant alleles in the control group, case samples and control samples, the power of our study (a = 0.05) was 0.640 in 375 ESCC cases and 378 controls with an OR of 0.68. The frequencies of genetic polymorphisms often vary between ethnic groups. In the present Chinese study, the allele frequency of UNG rs246079 A was 0.300 in 380 control subjects, which is consistent with the values reported in the SNP database for the Chinese Han (0.314), lower than that of European population (0.617) and higher than that of Sub-Saharan African population (0.200) (http:// www.ncbi.nlm.nih.gov/SNP). Several limitations of the present study need to be addressed. First, this was a hospital-based case–control study, and the subjects were not fully representative of the general population; therefore, selection bias was unavoidable. Second, the polymorphisms investigated, based on their functional considerations, may not offer a
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comprehensive view of the genetic variability of UNG. Third, because of the relatively small number of patients evaluated, a single case–control study is not sufficient to fully interpret the relationship between UNG polymorphisms and susceptibility to ESCC. Studies with larger numbers of subjects are necessary to confirm our findings. Finally, environmental factors differ between Chinese populations and others; because ESCC risk is likely to be influenced by gene–gene and gene–environment interactions, the UNG gene may be associated with different degrees of genetic risk in different ethnic groups and under different environmental exposures. In conclusion, the present study provided strong evidence that the UNG rs246079 G/A functional polymorphism may contribute to the risk of ESCC, whereas UNG rs3219218 A/G polymorphisms may not be associated with the risk of ESCC. However, further studies are needed to confirm the results of this preliminary study. Acknowledgments We appreciate all patients who participated in this study. This study was supported in part by National Natural Science Foundation of China (81472332, 81370001, 81300037, 81101889, 81000028), Jiangsu Province Natural Science Foundation (BK2010333, BK2011481), Social Development Foundation of Zhenjiang (SH2010017) and Changzhou Young Talents and ScienceTechnology Foundation of Health Bureau (QN201102). Conflict of interest
None.
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