Gene 542 (2014) 64–68
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Laryngeal cancer risk and common single nucleotide polymorphisms in nucleotide excision repair pathway genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5 and XPA Baocai Lu a, Jing Li a, Qingzu Gao b, Wenfa Yu a,⁎, Qinghui Yang c, Xiaoyu Li d a
Department of Otorhinolaryngology, The First AffiliatedHospital of Xinxiang Medical University, Weihui, China Department of Pathology, The First Affiliated Hospital of Xinxiang Medical University, Weihui, China c Oncology Department, The First Affiliated Hospital of Xinxiang Medical University, Weihui, China d Department of Otolaryngology Head and Neck Surgery, The Affiliated Hospital of Jining Medical University, Jining, China b
a r t i c l e
i n f o
Article history: Accepted 24 February 2014 Available online 26 February 2014 Keywords: Single nucleotide polymorphism Laryngeal cancer DNA repair gene
a b s t r a c t Because the molecular mechanisms underlying the development of laryngeal cancer are not well understood, we conducted a case–control study to determine the association between eight common SNPs in NER pathway genes and risk of laryngeal cancer, and the association between genetic polymorphisms and environmental factors. A 1:1 matched case–control study of 176 cases and 176 controls was conducted. Laryngeal cancer cases were more likely to smoke and drink (all P values b 0.05). Subjects with the ERCC1 rs11615 CC genotype and C allele had an increased risk of laryngeal cancer. Similarly, individuals with the ERCC5 rs17655 GG genotype and G allele had an increased risk of laryngeal cancer. Gene–gene interaction analysis showed that subjects carrying ERCC1 rs11615 C allele and XPG/ERCC5 rs17655 G allele had a greatly increased risk of breast cancer. Stratified analysis revealed that the interaction between polymorphisms of ERCC1 rs11615 and ERCC5 rs17655 and smoking on cancer risk was statistically significant, and ERCC1 rs11615 polymorphisms also had a significant interaction with drinking habit. In conclusion, our study suggests that ERCC1 rs11615 and ERCC5 rs17655 polymorphisms are associated with increased risk of laryngeal cancer, and that they confer more risk among smokers and drinkers. © 2014 Published by Elsevier B.V.
1. Introduction Laryngeal carcinoma is the most common malignancy of the upper respiratory tract. It is believed to result from complex interactions among many genetic and environmental factors. The estimated incidence, mortality and 5-year prevalence of laryngeal carcinoma worldwide are 156,877, 83,376 and 441,675, respectively (GLOBOCAN, 2012). An increasing number of epidemiological studies suggest that
Abbreviations: ERCC1, excision repair cross-complementation group 1; ERCC2, excision repair cross-complementation group 2; ERCC3, excision repair crosscomplementation group 3; ERCC4, excision repair cross-complementation group 4; ERCC5, excision repair cross-complementation group 5; XPD, xeroderma pigmentosum D; XPB, xeroderma pigmentosum B; XPF, xeroderma pigmentosum F; XPG, xeroderma pigmentosum G; XPA, xeroderma pigmentosum A; SNP, single nucleotide polymorphism; NER, nuclear excision repair; BER, base excision repair; SSBR, double-strand break repair; HRR, homologous recombination repair; PCR, polymerase chain reaction; CI, confidence interval; 95% OR, odds ratio; HWE, Hardy–Weinberg equilibrium; SPSS, Statistical Package for Social Science; DNA, deoxyribose nucleic acid; TCR, transcription coupled repair; GGR, global genomic nucleotide excision repair. ⁎ Corresponding author at: Department of Otorhinolaryngology, The First Affiliated Hospital of Xinxiang Medical University, Weihui,China. E-mail address: [email protected] (W. Yu).
http://dx.doi.org/10.1016/j.gene.2014.02.043 0378-1119/© 2014 Published by Elsevier B.V.
genetics, environmental exposures, human papilloma virus (HPV) infection, smoking and long-term smoking and high alcohol consumption contribute to the development of laryngeal cancer during specific life phases (Bosetti et al., 2006; Kupisz et al., 2010; Parkin et al., 2002; Sadri et al., 2006; Zhuo et al., 2009). The molecular mechanisms underlying development of laryngeal cancer are not well understood, but it is widely recognized that environmental carcinogens induce DNA damage, which could in turn induce genomic instability. The bulky DNA adducts generated by tobacco carcinogens are mainly repaired by nucleotide excision repair (NER). Other cellular DNA repair pathways, such as base excision repair (BER), double-strand break repair (SSBR), and homologous recombination repair (HRR) also play important roles in the carcinogenesis of laryngeal cancer by repairing single strand and double strand DNA breaks induced by smoking, ionizing radiation, and other DNAdamaging agents (Bentley et al., 2004; Joenje, 1989; Stern et al., 2001). NER is the most common pathway for repairing bulky DNA lesions and maintaining genomic stability. SNPs in genes involved in NER could alter the efficacy of DNA repair. However, only a few studies have examined the contribution of SNPs in NER pathway genes to risk of laryngeal cancer (Abbasi et al., 2009; Cui et al., 2006). These demonstrated that variants in the xeroderma pigmentosum (XP)
B. Lu et al. / Gene 542 (2014) 64–68
complementation family of proteins can play an important role in laryngeal cancer risk. We conducted a case–control study to determine the association between eight common SNPs in NER pathway genes and risk of laryngeal cancer, and the association between genetic polymorphisms and environmental factors. 2. Materials and methods 2.1. Study subjects Laryngeal cancer patients were recruited at the The First Affiliated Hospital of Xinxiang Medical University between May 2008 and May 2011. After patients provided informed consent, 5 mL whole blood was collected at the time of enrollment in the study. All cases were newly diagnosed and confirmed by histopathological examination. Patients with a history of cancer were excluded. At the same time, controls were selected from inpatients in the hospital's departments of orthopedics, dermatology, and digestive diseases, and were frequency matched to cases by age (within five years) and sex. When we selected one case, we selected one control subject matched to case by sex and age. Controls had to have no history of head and neck, esophageal, or lung cancer. A structured questionnaire was taken to collect the demographic characteristics of all cases and controls by doctors or nurses, including sex, age, tobacco smoking and alcohol drinking as well as family history of cancer. Current and former drinkers were defined as individuals who drank more than 200 g of alcohol per month and continued for one year. Current and former smokers were defined as individuals who smoked more than one packet of cigarettes per month and continued for one year. Clinical characteristics were collected from medical records. The study design was approved by the ethics committee of The First Affiliated Hospital of Xinxiang Medical University. 2.2. Genotype analysis For genotype determination, DNA was extracted from a peripheral blood sample using a TIANamp blood DNA kit (Tiangen Biotech, Beijing, China). The ten SNP genotyping was conducted in a 384-well plate format on the Sequenom MassARRAY® Analyzer 4 system (Sequenom, San Diego, CA, USA), which combines polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technologies. Primers and probes of the ten SNPs for polymerase chain reaction (PCR) amplification were designed by Sequenom® Assay Design 3.1 software (Sequenom®). Each PCR reaction (20 μL)
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contained 200 ng of DNA template, 200 μM of dNTP, 1 unit of Taq DNA polymerase, and 200 μM of primers, as well as 1.5 mM of MgCl2. The PCR reaction was conducted at 95 °C for 10 min to activate the Taq polymerase, followed by 45 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 60 s. The MassARRAY Analyzer Compact with ACQUAIRE Module (Sequenom) acquired the spectra from the SpectroCHIP, and spectral data were saved into the MassARRAY database. A random subgroup of 10% of cases and controls was subjected to repeat analysis to confirm consistency. The consistency rate was 100%.
2.3. Statistical analyses The χ2 test was used to assess the differences between cases and controls with regard to demographic characteristics. The distribution of genotypes in controls was analyzed for deviation from Hardy– Weinberg equilibriums (HWE) in controls using a goodness-of-fit χ2 test. The two groups' genotype distributions were compared using a χ2 test. Conditional logistic regression was performed to identify the effects of the ten SNPs on the risk of laryngeal cancer, expressed in odds ratios (ORs) and 95% confidence intervals (CIs). All comparisons were two-sided, and P b 0.05 was considered as statistically significant. All statistical analyses were conducted using the SPSS® statistical package, version 16.0 for Windows.
3. Results Of the 197 laryngeal cancer patients who were screened, 176 (89.34%) were eligible and were included in the study (47 females and 129 males) (Table 1). For the control group, 176 controls were selected and matched by sex and age at the same time the cases were recruited. The average ages of included cases and controls were 58.3 ± 7.5 and 58.8 ± 9.1 years old, respectively. Laryngeal cancer cases were more likely to have smoking and drinking habits (all P values b 0.05). Of the laryngeal cancer cases, 63.64% were at stages III–IV and 52.27% had well-differentiated tumors (92 cases). Genotype distributions of ERCC2 rs13181, ERCC2 rs1799793, ERCC4 rs6498486, ERCC4 rs2276465, ERCC5 rs17655, and ERCC5 rs1047768 were within the parameters of the Hardy–Weinberg equilibrium (HWE) for the control population, but distributions of ERCC1 rs11615, ERCC1 rs3212986, ERCC3 rs4150441, and XPA rs1800975 deviated from HWE (Table 2) (both P values b 0.05). The minor allele frequencies of the eight SNPs among the controls were similar to the general
Table 1 Characteristics of included laryngeal cancer patients and controls. %
t or χ2 test
P value
0.56
0.29
0.1
0.75
26.70 73.30
–
–
126 50
71.59 28.41
31.31
b0.001
3.47 (2.18–5.55)
117 59
66.48 33.52
5.74
0.02
1.69 (1.07–2.66)
Characteristics
Cases N = 176
Controls N = 176
%
Age, years ≤55 N55 Sex Female Male Smoking status Never Current and former Alcohol consumption Never Current and former Stage I–II III–IV Degree of tumor differentiation Well Moderate Poor
57.3 ± 7.5 80 96
45.7 54.3
56.8 ± 9.1 83 93
46.9 53.1
47 129
26.70 73.30
47 129
74 102
42.05 57.95
95 81
53.98 46.02
64 112
36.36 63.64
92 58 26
52.27 32.96 14.77
OR (95% CI)
1.07 (0.69–1.66)
1.0 (0.61–1.65)
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B. Lu et al. / Gene 542 (2014) 64–68
Table 2 Genotype characteristics of the ten single nucleotide polymorphisms (SNPs). Gene
SNP
ERCC1 ERCC1 XPD/ERCC2 XPB/ERCC3 XPF/ERCC4 XPF/ERCC4 XPG/ERCC5 XPA a b
MAFa
Alleles
rs11615 rs3212986 rs13181 rs4150441 rs6498486 rs2276465 rs17655 rs1800975
C/T G/T G/T A/G A/C A/G C/G A/G
HWEb (P value) in controls
Control group
From dbSNP
0.403 0.335 0.307 0.349 0.301 0.329 0.403 0.386
0.363 0.29 0.2369 0.435 0.2635 0.2649 0.3774 0.3466
0.001 b0.05 0.09 b0.05 0.06 0.15 0.07 b0.05
Minor allele frequency. Hardy–Weinberg equilibrium.
population in the NCBI dbSNP databases. The minor allele frequencies of all SNPs were more than 10%. There were significant differences in the genotype distributions of ERCC1 rs11615 and ERCC5 rs17655 between cases and controls (All P values b 0.05) (Table 3). Subjects carrying the ERCC1 rs11615 CC genotype (adjusted OR = 1.95, 95% CI = 1.09–3.63, P value = 0.03) had an increased risk of laryngeal cancer when compared with the TT genotype, and those carrying a C allele showed a marginally enhanced risk (adjusted OR = 1.53, 95% CI = 0.98–2.36, P value = 0.06). Similarly, individuals with the ERCC5 rs17655 GG genotype and G allele
showed a significantly increased risk relative to the CC genotype, with adjusted ORs (95% CIs) of 2.10 (1.17–3.75) and 1.76 (1.23–3.14), respectively. However, no associations were found between polymorphisms in ERCC1 rs3212986, ERCC2 rs13181, ERCC2 rs1799793, ERCC3 rs4150441, ERCC4 rs6498486, ERCC4 rs2276465, ERCC5 rs1047768, or XPA rs1800975 and risk of laryngeal cancer. We further analyze the gene–gene interaction on the risk of laryngeal cancer (Table 4). Individuals carrying ERCC1 rs11615 C allele and XPG/ERCC5 rs17655 G allele had a greatly increased risk of breast cancer compared to ERCC1 rs11615 T allele and XPG/ERCC5 rs17655 C allele
Table 3 Associations between NER repair gene polymorphisms and risk of laryngeal cancer. SNP ERCC1 rs11615 Allele ERCC1 rs3212986 Allele XPD/ERCC2 rs13181 Allele XPB/ERCC3 rs4150441 Allele XPF/ERCC4 rs6498486 Allele XPF/ERCC4 rs2276465 Allele XPG/ERCC5 rs17655 Allele XPA rs1800975 Allele
TT TC CC T C TT TG GG T G GG GT TT G T AA AG GG A G AA AC CC A C AA AG GG A G CC CG GG C G GG GA AA G A
Cases N = 176
%
Controls N = 176
%
P value
OR (95% CI)a
P value
OR (95% CI)a
61 66 49 188 164 83 57 36 223 129 85 60 31 230 122 78 68 30 224 128 83 69 24 235 117 78 64 34 220 132 53 69 54 178 176 78 55 43 211 141
34.66 37.5 27.84 53.41 46.59 47.16 32.39 20.45 63.35 36.65 48.3 34.09 17.61 65.345 34.655 44.32 38.64 17.05 63.64 36.36 47.16 39.2 13.64 66.76 33.24 44.32 36.36 19.32 62.5 37.5 30.11 39.20 30.68 49.72 50.28 44.32 31.25 24.43 59.945 40.055
79 62 35 220 132 90 54 32 234 118 94 56 26 244 108 82 65 29 229 123 90 66 20 246 106 87 62 27 236 116 78 62 36 216 136 82 54 40 218 136
44.89 35.23 19.89 62.50 37.50 51.14 30.68 18.18 66.48 33.52 53.41 31.82 14.77 69.32 30.68 46.59 36.93 16.48 65.055 34.945 51.14 37.5 11.36 69.89 30.11 49.43 35.23 15.34 67.045 32.955 44.32 35.23 20.45 61.93 38.07 46.59 30.68 22.73 61.93 38.07
– 0.19 0.03 – 0.01 – 0.58 0.49 – 0.39 – 0.48 0.36 – 0.26 – 0.69 0.78 – 0.69 – 0.59 0.44 – 0.37 – 0.55 0.26 – 0.21 – 0.04 0.004 – 0.003 – 0.78 0.65 – 0.65
1.0 (Ref.) 1.38 (0.83–2.30) 1.81 (1.01–3.26)⁎ 1.0 (Ref.) 1.45 (1.06–1.98)⁎ 1.0 (Ref.) 1.14 (0.69–1.90) 1.22 (0.67–2.23) 1.0 (Ref.) 1.15 (0.83–1.59) 1.0 (Ref.) 1.18 (0.72–1.94) 1.32 (0.69–2.51) 1.0 (Ref.) 1.20 (0.86–1.66) 1.0 (Ref.) 1.10 (0.68–1.79) 1.09 (0.57–2.07) 1.0 (Ref.) 1.06 (0.77–1.47) 1.0 (Ref.) 1.13 (0.70–1.82) 1.30 (0.64–2.68) 1.0 (Ref.) 1.15 (0.83–1.61) 1.0 (Ref.) 1.15 (0.70–1.88) 1.40 (0.75–2.65) 1.0 (Ref.) 1.22 (0.88–1.68) 1.0 (Ref.) 1.64 (0.98–2.75) 2.21 (1.23–3.96)⁎ 1.0 (Ref.) 1.57 (1.15–2.14)⁎ 1.0 (Ref.) 1.07 (0.64–1.79) 1.13 (0.64–1.99) 1.0 (Ref.) 1.07 (0.78–1.47)
– 0.22 0.03 – 0.006 – 0.53 0.36 – 0.34 – 0.42 0.32 – 0.14 – 0.62 0.54 – 0.63 – 0.53 0.62 – 0.25 – 0.42 0.22 – 0.11 – 0.1 0.003 – 0.001 – 0.62 0.56 – 0.5
1.0 (Ref.) 1.35 (0.79–2.39) 1.95 (1.09–3.63)⁎ 1.0 (Ref.) 1.56 (1.12–2.16)⁎ 1.0 (Ref.) 1.20 (0.73–1.97) 1.28 (0.71–2.34) 1.0 (Ref.) 1.21 (0.87–1.67) 1.0 (Ref.) 1.21 (0.76–2.04) 1.37 (0.79–2.62) 1.0 (Ref.) 1.32 (0.91–1.73) 1.0 (Ref.) 1.15 (0.74–1.82) 0.89 (0.47–1.71) 1.0 (Ref.) 1.15 (0.82–1.59) 1.0 (Ref.) 1.18 (0.70–1.82) 1.24 (0.61–2.66) 1.0 (Ref.) 1.24 (0.91–1.74) 1.0 (Ref.) 1.27 (0.79–2.03) 1.46 (0.76–2.71) 1.0 (Ref.) 1.41 (0.95–1.82) 1.0 (Ref.) 1.52 (0.87–2.57) 2.10 (1.17–3.75)⁎ 1.0 (Ref.) 1.64 (1.20–2.27)⁎ 1.0 (Ref.) 1.15 (0.67–1.82) 1.22 (0.70–2.04) 1.0 (Ref.) 1.20 (0.86–1.62)
a Adjusted for sex, age, and smoking and drinking status. ⁎ P value b 0.05.
B. Lu et al. / Gene 542 (2014) 64–68 Table 4 Interaction between ERCC1 rs11615 and ERCC5 rs17655 polymorphisms in laryngeal cancer risk. ERCC1 rs11615
XPG/ERCC5 rs17655
Cases
Controls P value
T allele C allele T allele C allele
C allele C allele G allele G allele
103 75 85 89
122 94 98 38
a
– 0.78 0.89 b0.001
OR (95% CI)a 1.0 (Ref.) 0.95 (0.62–1.44) 1.03 (0.68–1.55) 2.77 (1.71–4.53)
Adjusted for sex, age, and smoking and drinking status.
(adjusted OR = 1.53, 95% CI = 1.71–4.53, P value b 0.001). However, the test for interaction between ERCC1 rs11615 and XPG/ERCC5 rs17655 genetic variants was not significant (P for interaction = 0.15). We analyzed the association between ERCC1 rs11615 and ERCC5 rs17655 polymorphisms and laryngeal cancer risk stratified by smoking and drinking status (Tables 5 and 6). The ERCC1 rs11615 CC and ERCC5 rs17655 GG genotypes carried a greatly increased risk of laryngeal cancer for ever smokers (for rs11615 CC vs. TT genotype, adjusted OR = 3.24, 95% CI = 1.03–6.73; for rs17655 GG vs. CC genotype, adjusted OR = 2.84, 95% CI = 1.06–7.27). Similarly, the ERCC5 rs17655 G allele was associated with a moderately increased risk of this cancer in ever smokers compared with the C allele (adjusted OR = 1.84, 95% CI = 1.10–3.11). The interactions between ERCC1 rs11615 and ERCC5 rs17655 polymorphisms and smoking on cancer risk were statistically significant (P for interactions = 0.36 and 0.15, respectively). Ever drinkers carrying ERCC5 rs17655 GG and a G allele had a greatly increased risk of laryngeal cancer (for rs17655 GG vs. CC, adjusted OR = 3.79, 95% CI = 1.48–10.43; for G allele vs. C allele, adjusted OR = 2.18, 95% CI = 1.30–3.66). ERCC1 rs11615 CC and C allele carried a marginally increased risk of laryngeal cancer in drinkers (for CC vs. TT genotype, adjusted OR = 2.36, 95% CI = 0.97–6.05; for C allele vs T allele, adjusted OR = 1.64, 95% CI = 0.99–2.74). A significant interaction was found between ERCC5 rs17655 polymorphism and drinking habit (P = 0.008). 4. Discussion We aimed to identify SNPs that contribute to the risk of laryngeal cancer in order to better predict individual risk and understand the pathogenesis of the disease. Only two other recent studies have examined the association between NER genes and laryngeal cancer risk (Abbasi et al., 2009; Cui et al., 2006). Cui reports that the XPG Asp1104Asp genotype may be associated with decreased susceptibility to laryngeal cancer (Cui et al., 2006). Abbasi et al. found that the ERCC6 Arg1230Pro polymorphism is associated with an increased risk for laryngeal cancer, and that the ERCC5 Asp1104His and RAD23B Ala249Val polymorphisms carry an increased cancer risk among heavy smokers and heavy drinkers (Abbasi et al., 2009). However, the results remained confusing. Therefore, we investigated ten SNPs in six NER genes (ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, and XPA) in a case–control study of laryngeal cancer patients and matched controls.
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The allele frequencies we observed were similar to frequencies given for Asian populations in the NCBI reference assembly (http://www.ncbi. nlm.nih.gov/snp/). Multivariate logistic regression analysis revealed that two of the ten investigated SNPs, ERCC1 rs11615 and ERCC5 rs17655, showed a significant association with laryngeal cancer risk in the overall population and in individuals who had ever smoked or habitually consumed alcohol. For the other SNPs, no significant associations were found. ERCC1 is located on 19q 13.32, comprises 10 exons and encodes a 297 acetaldehyde ammonia product, which comprises a subunit of the NER complex required for the incision step. Since the ERCC1 protein plays an important role in the NER and influences genomic instability, polymorphisms in ERCC1 may be involved in carcinogenesis (Jones et al., 2012; Shannon et al., 1999). ERCC1 rs11615 variants are reportedly associated with susceptibility to non-small cell lung cancer, breast cancer, and glioma as well as gastric cancer (He et al., 2012; Yang et al., 2013; Yin et al., 2012; Zhang et al., 2012). Likewise, the ERCC1 rs11615 C allele has been found to be associated with an elevated cancer risk in Asian populations (He et al., 2012; Yang et al., 2013; Yin et al., 2012; Zhang et al., 2012). Only one published study found no association between ERCC1 rs11615 variants and susceptibility to laryngeal cancer (Albino et al., 2004). In contrast, our study found that the ERCC1 rs11615 CC genotype significantly increases susceptibility to laryngeal cancer. The precise mechanism for the association of the ERCC1 rs11615 polymorphism with cancer remains unclear, and the inconsistency of findings on that association may be explained by differences in population background, source of control subjects, and sample size. ERCC5 has been mapped to chromosome 13q33 and consists of 15 exons spanning ~30 kb of genomic DNA. It encodes the 3′-endonuclease XPG, which is involved in the excision step of NER and is responsible for a 1186 amino acid structure-specific endonuclease. XPG is essential for the two incision steps in NER, namely transcription coupled repair (TCR), which specifically targets and repairs DNA damage on the transcribed strand of actively expressed genes (Le Page et al., 2000), and global genomic nucleotide excision repair (GGR), which repairs DNA damage in the remaining genome (Kiyohara and Yoshimasu, 2007). Mutations in XPG can lead to changes in the function of DNA damage repair, which may lead to carcinogenesis. A meta-analysis of 44 published case–control studies indicated that ERCC5 rs17655 polymorphism was associated with individual susceptibility to cancer (Zhu et al., 2012). Another recent study indicates that ERCC5 rs17655 GG is associated with increased risk of laryngeal cancer (Abbasi et al., 2009), which is consistent with our results. In our subgroup analysis, we further stratified for smoking and drinking status and analyzed the risk modifying effect of genotype in both groups. These analyses revealed that the ERCC1 rs11615 and ERCC5 rs17655 polymorphisms enhanced risk of laryngeal cancer in individuals who were ever smokers or drinkers. Overall, this finding seems to strengthen the role of ERCC1 rs11615 and ERCC5 rs17655 in the etiology of this cancer. Since our results show that ever smokers and drinkers have an increased susceptibility to laryngeal cancer, the
Table 5 Interactions of ERCC1 rs11615 and ERCC5 rs17655 polymorphisms with smoking in laryngeal cancer risk. SNP
ERCC1 rs11615
XPG/ERCC5 rs17655
a
Never smokers (N)
TT CC T allele C allele CC GG C allele G allele
Cases
Controls
28 19 83 65 25 20 79 69
56 25 157 95 54 27 153 99
Adjusted for sex, age, and drinking status.
P value
OR (95% CI)a
– 0.21 – 0.22 – 0.22 – 0.15
1.0 (Ref.) 1.62 (0.72–3.56) 1.0 (Ref.) 1.29 (0.84–2.0) Ref. 1.72 (0.75–3.83) 1.0 (Ref.) 1.35 (0.88–2.08)
Ever smokers (N) Cases
Controls
33 30 105 99 30 34 98 106
23 10 63 37 23 10 63 37
P value
OR (95% CI)a
– 0.04 – 0.06 – 0.02 – 0.02
1.0 (Ref.) 3.24 (1.03–6.73) 1.0 (Ref.) 1.61 (0.96–2.71) Ref. 2.84 (1.06–7.27) 1.0 (Ref.) 1.84 (1.10–3.11)
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Table 6 Interactions of ERCC1 rs11615 and ERCC5 rs17655 polymorphisms with drinking in laryngeal cancer risk. SNP
ERCC1 rs11615
XPG/ERCC5 rs17655
a
Never drinkers (N)
TT CC T allele C allele CC GG T allele C allele
Cases
Controls
37 23 109 81 34 24 105 85
53 22 148 86 50 26 141 93
P value
– 0.23 – 0.22 – 0.33 0.30
OR (95% CI)a
1.0 (Ref.) 1.55 (0.73–3.41) 1.0 (Ref.) 1.27 (0.85–1.93) 1.0 (Ref.) 1.31 (0.61–2.86) 1.0 (Ref.) 1.23 (0.82–1.84)
Current and former drinkers (N) Cases
Controls
24 26 79 83 21 30 72 90
26 13 72 46 27 11 75 43
P value
OR (95% CI)a
– 0.05 – 0.04 – 0.005 – 0.0016
1.0 (Ref.) 2.36 (0.97–6.05) 1.0 (Ref.) 1.64 (0.99–2.74) 1.0 (Ref.) 3.79 (1.48–10.43) 1.0 (Ref.) 2.18 (1.30–3.66)
Adjusted for sex, age, and smoking status.
ERCC1 rs11615 CC and ERCC5 rs17655 GG genotypes may have an additive effect on the risk of this cancer in these higher-risk subgroups. Several limitations should be considered in this study. First, all the patients and control subjects were selected from one hospital, and some level of selection bias could not be avoided. But the allele frequencies in controls were similar to minor allele frequencies in Chinese population from NCBI reference assembly, which indicated that the control subjects could represent the Chinese population. Second, we only find that the ERCC1 polymorphisms are marginally associated with risk of laryngeal cancer, the marginal significance may be induced by the small numbers of cases and controls. Therefore, further large sample studies are greatly needed to confirm their association. In conclusion, our study suggests that ERCC1 rs11615 and ERCC5 rs17655 polymorphisms are associated with increased risk of laryngeal cancer, and that they confer more risk among smokers and drinkers. Our findings may be helpful in identifying individuals at increased risk for developing laryngeal cancer. As the biological impact of these polymorphisms is unclear, further large population-based studies are needed to evaluate the function of NER genes in the development of laryngeal cancer. Conflict of interest There is no conflict of interest in this study. Acknowledgments This work was supported by the Funding Scheme for Young Backbone Teachers of Colleges and Universities of Henan Province (2010GGJS-120) and the Project of Health Science and technology Innovation Talents of Henan Province (2010141). References Abbasi, R., et al., 2009. Laryngeal cancer risk associated with smoking and alcohol consumption is modified by genetic polymorphisms in ERCC5, ERCC6 and RAD23B but not by polymorphisms in five other nucleotide excision repair genes. International Journal of Cancer 125, 1431–1439.
Albino, A.P., et al., 2004. Induction of H2AX phosphorylation in pulmonary cells by tobacco smoke: a new assay for carcinogens. Cell Cycle 3, 1062–1068. Bentley, J., et al., 2004. DNA double strand break repair in human bladder cancer is error prone and involves microhomology-associated end-joining. Nucleic Acids Research 32, 5249–5259. Bosetti, C., et al., 2006. Effects of smoking cessation on the risk of laryngeal cancer: an overview of published studies. Oral Oncology 42, 866–872. Cui, Y., et al., 2006. Polymorphism of xeroderma pigmentosum group G and the risk of lung cancer and squamous cell carcinomas of the oropharynx, larynx and esophagus. International Journal of Cancer 118, 714–720. GLOBOCAN, 2012. International Agency for Research on Cancer (Online). Available at http://globocan.iarc.fr/factsheets/populations/factsheet (Accessed on 30th October 2013). He, J., et al., 2012. Polymorphisms in ERCC1 and XPF genes and risk of gastric cancer in an eastern Chinese population. PLoS One 7, e49308. Joenje, H., 1989. Genetic toxicology of oxygen. Mutation Research 219, 193–208. Jones, A.R., et al., 2012. Novel oxidatively activated agents modify DNA and are enhanced by ERCC1 silencing. Chemical Research in Toxicology 25, 2542–2552. Kiyohara, C., Yoshimasu, K., 2007. Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: a meta-analysis. International Journal of Medical Sciences 4, 59–71. Kupisz, K., et al., 2010. Preliminary results of prognostic significance of proliferating cell nuclear antigen expression in advanced primary larynx carcinomas and lymph node metastases. Archives of Medical Science 6, 65–70. Le Page, F., et al., 2000. Transcription-coupled repair of 8-oxoguanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome. Cell 101, 159–171. Parkin, D.M., et al., 2002. Cancer Incidence in Five Continents. Vol. VIII. IARC Scientific Publication No. 155. International Agency for Research on Cancer, Lyon (France). Sadri, M., et al., 2006. Laryngeal dysplasia: aetiology and molecular biology. Journal of Laryngology and Otology 120, 170–177. Shannon, M., et al., 1999. Characterization of the mouse XPF DNA repair gene and differential expression during spermatogenesis. Genomics 62, 427–435. Stern, M.C., et al., 2001. DNA repair gene XRCC1 polymorphisms, smoking, and bladder cancer risk. Cancer Epidemiology, Biomarkers & Prevention 10, 125–131. Yang, Z., et al., 2013. Polymorphisms in the ERCC1 and XPF genes and risk of breast cancer in a Chinese population. Genetic Testing and Molecular Biomarkers 17, 700–706. Yin, J., et al., 2012. A specific diplotype defined by PPP1R13L rs1970764, CD3EAP rs967591 and ERCC1 rs11615 and lung cancer risk in a Chinese population. Lung Cancer 76, 286–291. Zhang, N., et al., 2012. ERCC1 polymorphisms and risk of adult glioma in a Chinese population: a hospital-based case–control study. Cancer Investigation 30, 199–202. Zhu, M.L., et al., 2012. Association between the ERCC5 Asp1104His polymorphism and cancer risk: a meta-analysis. PLoS One 7, e36293. Zhuo, W.L., et al., 2009. Polymorphisms of CYP1A1 and GSTM1 and laryngeal cancer risk: evidence-based meta-analyses. Journal of Cancer Research and Clinical Oncology 135, 1081–1090.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Laryngeal cancer risk and common single nucleotide polymorphisms in nucleotide excision repair pathway genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5 and XPA Baocai Lu a, Jing Li a, Qingzu Gao b, Wenfa Yu a,⁎, Qinghui Yang c, Xiaoyu Li d a
Department of Otorhinolaryngology, The First AffiliatedHospital of Xinxiang Medical University, Weihui, China Department of Pathology, The First Affiliated Hospital of Xinxiang Medical University, Weihui, China c Oncology Department, The First Affiliated Hospital of Xinxiang Medical University, Weihui, China d Department of Otolaryngology Head and Neck Surgery, The Affiliated Hospital of Jining Medical University, Jining, China b
a r t i c l e
i n f o
Article history: Accepted 24 February 2014 Available online 26 February 2014 Keywords: Single nucleotide polymorphism Laryngeal cancer DNA repair gene
a b s t r a c t Because the molecular mechanisms underlying the development of laryngeal cancer are not well understood, we conducted a case–control study to determine the association between eight common SNPs in NER pathway genes and risk of laryngeal cancer, and the association between genetic polymorphisms and environmental factors. A 1:1 matched case–control study of 176 cases and 176 controls was conducted. Laryngeal cancer cases were more likely to smoke and drink (all P values b 0.05). Subjects with the ERCC1 rs11615 CC genotype and C allele had an increased risk of laryngeal cancer. Similarly, individuals with the ERCC5 rs17655 GG genotype and G allele had an increased risk of laryngeal cancer. Gene–gene interaction analysis showed that subjects carrying ERCC1 rs11615 C allele and XPG/ERCC5 rs17655 G allele had a greatly increased risk of breast cancer. Stratified analysis revealed that the interaction between polymorphisms of ERCC1 rs11615 and ERCC5 rs17655 and smoking on cancer risk was statistically significant, and ERCC1 rs11615 polymorphisms also had a significant interaction with drinking habit. In conclusion, our study suggests that ERCC1 rs11615 and ERCC5 rs17655 polymorphisms are associated with increased risk of laryngeal cancer, and that they confer more risk among smokers and drinkers. © 2014 Published by Elsevier B.V.
1. Introduction Laryngeal carcinoma is the most common malignancy of the upper respiratory tract. It is believed to result from complex interactions among many genetic and environmental factors. The estimated incidence, mortality and 5-year prevalence of laryngeal carcinoma worldwide are 156,877, 83,376 and 441,675, respectively (GLOBOCAN, 2012). An increasing number of epidemiological studies suggest that
Abbreviations: ERCC1, excision repair cross-complementation group 1; ERCC2, excision repair cross-complementation group 2; ERCC3, excision repair crosscomplementation group 3; ERCC4, excision repair cross-complementation group 4; ERCC5, excision repair cross-complementation group 5; XPD, xeroderma pigmentosum D; XPB, xeroderma pigmentosum B; XPF, xeroderma pigmentosum F; XPG, xeroderma pigmentosum G; XPA, xeroderma pigmentosum A; SNP, single nucleotide polymorphism; NER, nuclear excision repair; BER, base excision repair; SSBR, double-strand break repair; HRR, homologous recombination repair; PCR, polymerase chain reaction; CI, confidence interval; 95% OR, odds ratio; HWE, Hardy–Weinberg equilibrium; SPSS, Statistical Package for Social Science; DNA, deoxyribose nucleic acid; TCR, transcription coupled repair; GGR, global genomic nucleotide excision repair. ⁎ Corresponding author at: Department of Otorhinolaryngology, The First Affiliated Hospital of Xinxiang Medical University, Weihui,China. E-mail address: [email protected] (W. Yu).
http://dx.doi.org/10.1016/j.gene.2014.02.043 0378-1119/© 2014 Published by Elsevier B.V.
genetics, environmental exposures, human papilloma virus (HPV) infection, smoking and long-term smoking and high alcohol consumption contribute to the development of laryngeal cancer during specific life phases (Bosetti et al., 2006; Kupisz et al., 2010; Parkin et al., 2002; Sadri et al., 2006; Zhuo et al., 2009). The molecular mechanisms underlying development of laryngeal cancer are not well understood, but it is widely recognized that environmental carcinogens induce DNA damage, which could in turn induce genomic instability. The bulky DNA adducts generated by tobacco carcinogens are mainly repaired by nucleotide excision repair (NER). Other cellular DNA repair pathways, such as base excision repair (BER), double-strand break repair (SSBR), and homologous recombination repair (HRR) also play important roles in the carcinogenesis of laryngeal cancer by repairing single strand and double strand DNA breaks induced by smoking, ionizing radiation, and other DNAdamaging agents (Bentley et al., 2004; Joenje, 1989; Stern et al., 2001). NER is the most common pathway for repairing bulky DNA lesions and maintaining genomic stability. SNPs in genes involved in NER could alter the efficacy of DNA repair. However, only a few studies have examined the contribution of SNPs in NER pathway genes to risk of laryngeal cancer (Abbasi et al., 2009; Cui et al., 2006). These demonstrated that variants in the xeroderma pigmentosum (XP)
B. Lu et al. / Gene 542 (2014) 64–68
complementation family of proteins can play an important role in laryngeal cancer risk. We conducted a case–control study to determine the association between eight common SNPs in NER pathway genes and risk of laryngeal cancer, and the association between genetic polymorphisms and environmental factors. 2. Materials and methods 2.1. Study subjects Laryngeal cancer patients were recruited at the The First Affiliated Hospital of Xinxiang Medical University between May 2008 and May 2011. After patients provided informed consent, 5 mL whole blood was collected at the time of enrollment in the study. All cases were newly diagnosed and confirmed by histopathological examination. Patients with a history of cancer were excluded. At the same time, controls were selected from inpatients in the hospital's departments of orthopedics, dermatology, and digestive diseases, and were frequency matched to cases by age (within five years) and sex. When we selected one case, we selected one control subject matched to case by sex and age. Controls had to have no history of head and neck, esophageal, or lung cancer. A structured questionnaire was taken to collect the demographic characteristics of all cases and controls by doctors or nurses, including sex, age, tobacco smoking and alcohol drinking as well as family history of cancer. Current and former drinkers were defined as individuals who drank more than 200 g of alcohol per month and continued for one year. Current and former smokers were defined as individuals who smoked more than one packet of cigarettes per month and continued for one year. Clinical characteristics were collected from medical records. The study design was approved by the ethics committee of The First Affiliated Hospital of Xinxiang Medical University. 2.2. Genotype analysis For genotype determination, DNA was extracted from a peripheral blood sample using a TIANamp blood DNA kit (Tiangen Biotech, Beijing, China). The ten SNP genotyping was conducted in a 384-well plate format on the Sequenom MassARRAY® Analyzer 4 system (Sequenom, San Diego, CA, USA), which combines polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technologies. Primers and probes of the ten SNPs for polymerase chain reaction (PCR) amplification were designed by Sequenom® Assay Design 3.1 software (Sequenom®). Each PCR reaction (20 μL)
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contained 200 ng of DNA template, 200 μM of dNTP, 1 unit of Taq DNA polymerase, and 200 μM of primers, as well as 1.5 mM of MgCl2. The PCR reaction was conducted at 95 °C for 10 min to activate the Taq polymerase, followed by 45 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 60 s. The MassARRAY Analyzer Compact with ACQUAIRE Module (Sequenom) acquired the spectra from the SpectroCHIP, and spectral data were saved into the MassARRAY database. A random subgroup of 10% of cases and controls was subjected to repeat analysis to confirm consistency. The consistency rate was 100%.
2.3. Statistical analyses The χ2 test was used to assess the differences between cases and controls with regard to demographic characteristics. The distribution of genotypes in controls was analyzed for deviation from Hardy– Weinberg equilibriums (HWE) in controls using a goodness-of-fit χ2 test. The two groups' genotype distributions were compared using a χ2 test. Conditional logistic regression was performed to identify the effects of the ten SNPs on the risk of laryngeal cancer, expressed in odds ratios (ORs) and 95% confidence intervals (CIs). All comparisons were two-sided, and P b 0.05 was considered as statistically significant. All statistical analyses were conducted using the SPSS® statistical package, version 16.0 for Windows.
3. Results Of the 197 laryngeal cancer patients who were screened, 176 (89.34%) were eligible and were included in the study (47 females and 129 males) (Table 1). For the control group, 176 controls were selected and matched by sex and age at the same time the cases were recruited. The average ages of included cases and controls were 58.3 ± 7.5 and 58.8 ± 9.1 years old, respectively. Laryngeal cancer cases were more likely to have smoking and drinking habits (all P values b 0.05). Of the laryngeal cancer cases, 63.64% were at stages III–IV and 52.27% had well-differentiated tumors (92 cases). Genotype distributions of ERCC2 rs13181, ERCC2 rs1799793, ERCC4 rs6498486, ERCC4 rs2276465, ERCC5 rs17655, and ERCC5 rs1047768 were within the parameters of the Hardy–Weinberg equilibrium (HWE) for the control population, but distributions of ERCC1 rs11615, ERCC1 rs3212986, ERCC3 rs4150441, and XPA rs1800975 deviated from HWE (Table 2) (both P values b 0.05). The minor allele frequencies of the eight SNPs among the controls were similar to the general
Table 1 Characteristics of included laryngeal cancer patients and controls. %
t or χ2 test
P value
0.56
0.29
0.1
0.75
26.70 73.30
–
–
126 50
71.59 28.41
31.31
b0.001
3.47 (2.18–5.55)
117 59
66.48 33.52
5.74
0.02
1.69 (1.07–2.66)
Characteristics
Cases N = 176
Controls N = 176
%
Age, years ≤55 N55 Sex Female Male Smoking status Never Current and former Alcohol consumption Never Current and former Stage I–II III–IV Degree of tumor differentiation Well Moderate Poor
57.3 ± 7.5 80 96
45.7 54.3
56.8 ± 9.1 83 93
46.9 53.1
47 129
26.70 73.30
47 129
74 102
42.05 57.95
95 81
53.98 46.02
64 112
36.36 63.64
92 58 26
52.27 32.96 14.77
OR (95% CI)
1.07 (0.69–1.66)
1.0 (0.61–1.65)
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B. Lu et al. / Gene 542 (2014) 64–68
Table 2 Genotype characteristics of the ten single nucleotide polymorphisms (SNPs). Gene
SNP
ERCC1 ERCC1 XPD/ERCC2 XPB/ERCC3 XPF/ERCC4 XPF/ERCC4 XPG/ERCC5 XPA a b
MAFa
Alleles
rs11615 rs3212986 rs13181 rs4150441 rs6498486 rs2276465 rs17655 rs1800975
C/T G/T G/T A/G A/C A/G C/G A/G
HWEb (P value) in controls
Control group
From dbSNP
0.403 0.335 0.307 0.349 0.301 0.329 0.403 0.386
0.363 0.29 0.2369 0.435 0.2635 0.2649 0.3774 0.3466
0.001 b0.05 0.09 b0.05 0.06 0.15 0.07 b0.05
Minor allele frequency. Hardy–Weinberg equilibrium.
population in the NCBI dbSNP databases. The minor allele frequencies of all SNPs were more than 10%. There were significant differences in the genotype distributions of ERCC1 rs11615 and ERCC5 rs17655 between cases and controls (All P values b 0.05) (Table 3). Subjects carrying the ERCC1 rs11615 CC genotype (adjusted OR = 1.95, 95% CI = 1.09–3.63, P value = 0.03) had an increased risk of laryngeal cancer when compared with the TT genotype, and those carrying a C allele showed a marginally enhanced risk (adjusted OR = 1.53, 95% CI = 0.98–2.36, P value = 0.06). Similarly, individuals with the ERCC5 rs17655 GG genotype and G allele
showed a significantly increased risk relative to the CC genotype, with adjusted ORs (95% CIs) of 2.10 (1.17–3.75) and 1.76 (1.23–3.14), respectively. However, no associations were found between polymorphisms in ERCC1 rs3212986, ERCC2 rs13181, ERCC2 rs1799793, ERCC3 rs4150441, ERCC4 rs6498486, ERCC4 rs2276465, ERCC5 rs1047768, or XPA rs1800975 and risk of laryngeal cancer. We further analyze the gene–gene interaction on the risk of laryngeal cancer (Table 4). Individuals carrying ERCC1 rs11615 C allele and XPG/ERCC5 rs17655 G allele had a greatly increased risk of breast cancer compared to ERCC1 rs11615 T allele and XPG/ERCC5 rs17655 C allele
Table 3 Associations between NER repair gene polymorphisms and risk of laryngeal cancer. SNP ERCC1 rs11615 Allele ERCC1 rs3212986 Allele XPD/ERCC2 rs13181 Allele XPB/ERCC3 rs4150441 Allele XPF/ERCC4 rs6498486 Allele XPF/ERCC4 rs2276465 Allele XPG/ERCC5 rs17655 Allele XPA rs1800975 Allele
TT TC CC T C TT TG GG T G GG GT TT G T AA AG GG A G AA AC CC A C AA AG GG A G CC CG GG C G GG GA AA G A
Cases N = 176
%
Controls N = 176
%
P value
OR (95% CI)a
P value
OR (95% CI)a
61 66 49 188 164 83 57 36 223 129 85 60 31 230 122 78 68 30 224 128 83 69 24 235 117 78 64 34 220 132 53 69 54 178 176 78 55 43 211 141
34.66 37.5 27.84 53.41 46.59 47.16 32.39 20.45 63.35 36.65 48.3 34.09 17.61 65.345 34.655 44.32 38.64 17.05 63.64 36.36 47.16 39.2 13.64 66.76 33.24 44.32 36.36 19.32 62.5 37.5 30.11 39.20 30.68 49.72 50.28 44.32 31.25 24.43 59.945 40.055
79 62 35 220 132 90 54 32 234 118 94 56 26 244 108 82 65 29 229 123 90 66 20 246 106 87 62 27 236 116 78 62 36 216 136 82 54 40 218 136
44.89 35.23 19.89 62.50 37.50 51.14 30.68 18.18 66.48 33.52 53.41 31.82 14.77 69.32 30.68 46.59 36.93 16.48 65.055 34.945 51.14 37.5 11.36 69.89 30.11 49.43 35.23 15.34 67.045 32.955 44.32 35.23 20.45 61.93 38.07 46.59 30.68 22.73 61.93 38.07
– 0.19 0.03 – 0.01 – 0.58 0.49 – 0.39 – 0.48 0.36 – 0.26 – 0.69 0.78 – 0.69 – 0.59 0.44 – 0.37 – 0.55 0.26 – 0.21 – 0.04 0.004 – 0.003 – 0.78 0.65 – 0.65
1.0 (Ref.) 1.38 (0.83–2.30) 1.81 (1.01–3.26)⁎ 1.0 (Ref.) 1.45 (1.06–1.98)⁎ 1.0 (Ref.) 1.14 (0.69–1.90) 1.22 (0.67–2.23) 1.0 (Ref.) 1.15 (0.83–1.59) 1.0 (Ref.) 1.18 (0.72–1.94) 1.32 (0.69–2.51) 1.0 (Ref.) 1.20 (0.86–1.66) 1.0 (Ref.) 1.10 (0.68–1.79) 1.09 (0.57–2.07) 1.0 (Ref.) 1.06 (0.77–1.47) 1.0 (Ref.) 1.13 (0.70–1.82) 1.30 (0.64–2.68) 1.0 (Ref.) 1.15 (0.83–1.61) 1.0 (Ref.) 1.15 (0.70–1.88) 1.40 (0.75–2.65) 1.0 (Ref.) 1.22 (0.88–1.68) 1.0 (Ref.) 1.64 (0.98–2.75) 2.21 (1.23–3.96)⁎ 1.0 (Ref.) 1.57 (1.15–2.14)⁎ 1.0 (Ref.) 1.07 (0.64–1.79) 1.13 (0.64–1.99) 1.0 (Ref.) 1.07 (0.78–1.47)
– 0.22 0.03 – 0.006 – 0.53 0.36 – 0.34 – 0.42 0.32 – 0.14 – 0.62 0.54 – 0.63 – 0.53 0.62 – 0.25 – 0.42 0.22 – 0.11 – 0.1 0.003 – 0.001 – 0.62 0.56 – 0.5
1.0 (Ref.) 1.35 (0.79–2.39) 1.95 (1.09–3.63)⁎ 1.0 (Ref.) 1.56 (1.12–2.16)⁎ 1.0 (Ref.) 1.20 (0.73–1.97) 1.28 (0.71–2.34) 1.0 (Ref.) 1.21 (0.87–1.67) 1.0 (Ref.) 1.21 (0.76–2.04) 1.37 (0.79–2.62) 1.0 (Ref.) 1.32 (0.91–1.73) 1.0 (Ref.) 1.15 (0.74–1.82) 0.89 (0.47–1.71) 1.0 (Ref.) 1.15 (0.82–1.59) 1.0 (Ref.) 1.18 (0.70–1.82) 1.24 (0.61–2.66) 1.0 (Ref.) 1.24 (0.91–1.74) 1.0 (Ref.) 1.27 (0.79–2.03) 1.46 (0.76–2.71) 1.0 (Ref.) 1.41 (0.95–1.82) 1.0 (Ref.) 1.52 (0.87–2.57) 2.10 (1.17–3.75)⁎ 1.0 (Ref.) 1.64 (1.20–2.27)⁎ 1.0 (Ref.) 1.15 (0.67–1.82) 1.22 (0.70–2.04) 1.0 (Ref.) 1.20 (0.86–1.62)
a Adjusted for sex, age, and smoking and drinking status. ⁎ P value b 0.05.
B. Lu et al. / Gene 542 (2014) 64–68 Table 4 Interaction between ERCC1 rs11615 and ERCC5 rs17655 polymorphisms in laryngeal cancer risk. ERCC1 rs11615
XPG/ERCC5 rs17655
Cases
Controls P value
T allele C allele T allele C allele
C allele C allele G allele G allele
103 75 85 89
122 94 98 38
a
– 0.78 0.89 b0.001
OR (95% CI)a 1.0 (Ref.) 0.95 (0.62–1.44) 1.03 (0.68–1.55) 2.77 (1.71–4.53)
Adjusted for sex, age, and smoking and drinking status.
(adjusted OR = 1.53, 95% CI = 1.71–4.53, P value b 0.001). However, the test for interaction between ERCC1 rs11615 and XPG/ERCC5 rs17655 genetic variants was not significant (P for interaction = 0.15). We analyzed the association between ERCC1 rs11615 and ERCC5 rs17655 polymorphisms and laryngeal cancer risk stratified by smoking and drinking status (Tables 5 and 6). The ERCC1 rs11615 CC and ERCC5 rs17655 GG genotypes carried a greatly increased risk of laryngeal cancer for ever smokers (for rs11615 CC vs. TT genotype, adjusted OR = 3.24, 95% CI = 1.03–6.73; for rs17655 GG vs. CC genotype, adjusted OR = 2.84, 95% CI = 1.06–7.27). Similarly, the ERCC5 rs17655 G allele was associated with a moderately increased risk of this cancer in ever smokers compared with the C allele (adjusted OR = 1.84, 95% CI = 1.10–3.11). The interactions between ERCC1 rs11615 and ERCC5 rs17655 polymorphisms and smoking on cancer risk were statistically significant (P for interactions = 0.36 and 0.15, respectively). Ever drinkers carrying ERCC5 rs17655 GG and a G allele had a greatly increased risk of laryngeal cancer (for rs17655 GG vs. CC, adjusted OR = 3.79, 95% CI = 1.48–10.43; for G allele vs. C allele, adjusted OR = 2.18, 95% CI = 1.30–3.66). ERCC1 rs11615 CC and C allele carried a marginally increased risk of laryngeal cancer in drinkers (for CC vs. TT genotype, adjusted OR = 2.36, 95% CI = 0.97–6.05; for C allele vs T allele, adjusted OR = 1.64, 95% CI = 0.99–2.74). A significant interaction was found between ERCC5 rs17655 polymorphism and drinking habit (P = 0.008). 4. Discussion We aimed to identify SNPs that contribute to the risk of laryngeal cancer in order to better predict individual risk and understand the pathogenesis of the disease. Only two other recent studies have examined the association between NER genes and laryngeal cancer risk (Abbasi et al., 2009; Cui et al., 2006). Cui reports that the XPG Asp1104Asp genotype may be associated with decreased susceptibility to laryngeal cancer (Cui et al., 2006). Abbasi et al. found that the ERCC6 Arg1230Pro polymorphism is associated with an increased risk for laryngeal cancer, and that the ERCC5 Asp1104His and RAD23B Ala249Val polymorphisms carry an increased cancer risk among heavy smokers and heavy drinkers (Abbasi et al., 2009). However, the results remained confusing. Therefore, we investigated ten SNPs in six NER genes (ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, and XPA) in a case–control study of laryngeal cancer patients and matched controls.
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The allele frequencies we observed were similar to frequencies given for Asian populations in the NCBI reference assembly (http://www.ncbi. nlm.nih.gov/snp/). Multivariate logistic regression analysis revealed that two of the ten investigated SNPs, ERCC1 rs11615 and ERCC5 rs17655, showed a significant association with laryngeal cancer risk in the overall population and in individuals who had ever smoked or habitually consumed alcohol. For the other SNPs, no significant associations were found. ERCC1 is located on 19q 13.32, comprises 10 exons and encodes a 297 acetaldehyde ammonia product, which comprises a subunit of the NER complex required for the incision step. Since the ERCC1 protein plays an important role in the NER and influences genomic instability, polymorphisms in ERCC1 may be involved in carcinogenesis (Jones et al., 2012; Shannon et al., 1999). ERCC1 rs11615 variants are reportedly associated with susceptibility to non-small cell lung cancer, breast cancer, and glioma as well as gastric cancer (He et al., 2012; Yang et al., 2013; Yin et al., 2012; Zhang et al., 2012). Likewise, the ERCC1 rs11615 C allele has been found to be associated with an elevated cancer risk in Asian populations (He et al., 2012; Yang et al., 2013; Yin et al., 2012; Zhang et al., 2012). Only one published study found no association between ERCC1 rs11615 variants and susceptibility to laryngeal cancer (Albino et al., 2004). In contrast, our study found that the ERCC1 rs11615 CC genotype significantly increases susceptibility to laryngeal cancer. The precise mechanism for the association of the ERCC1 rs11615 polymorphism with cancer remains unclear, and the inconsistency of findings on that association may be explained by differences in population background, source of control subjects, and sample size. ERCC5 has been mapped to chromosome 13q33 and consists of 15 exons spanning ~30 kb of genomic DNA. It encodes the 3′-endonuclease XPG, which is involved in the excision step of NER and is responsible for a 1186 amino acid structure-specific endonuclease. XPG is essential for the two incision steps in NER, namely transcription coupled repair (TCR), which specifically targets and repairs DNA damage on the transcribed strand of actively expressed genes (Le Page et al., 2000), and global genomic nucleotide excision repair (GGR), which repairs DNA damage in the remaining genome (Kiyohara and Yoshimasu, 2007). Mutations in XPG can lead to changes in the function of DNA damage repair, which may lead to carcinogenesis. A meta-analysis of 44 published case–control studies indicated that ERCC5 rs17655 polymorphism was associated with individual susceptibility to cancer (Zhu et al., 2012). Another recent study indicates that ERCC5 rs17655 GG is associated with increased risk of laryngeal cancer (Abbasi et al., 2009), which is consistent with our results. In our subgroup analysis, we further stratified for smoking and drinking status and analyzed the risk modifying effect of genotype in both groups. These analyses revealed that the ERCC1 rs11615 and ERCC5 rs17655 polymorphisms enhanced risk of laryngeal cancer in individuals who were ever smokers or drinkers. Overall, this finding seems to strengthen the role of ERCC1 rs11615 and ERCC5 rs17655 in the etiology of this cancer. Since our results show that ever smokers and drinkers have an increased susceptibility to laryngeal cancer, the
Table 5 Interactions of ERCC1 rs11615 and ERCC5 rs17655 polymorphisms with smoking in laryngeal cancer risk. SNP
ERCC1 rs11615
XPG/ERCC5 rs17655
a
Never smokers (N)
TT CC T allele C allele CC GG C allele G allele
Cases
Controls
28 19 83 65 25 20 79 69
56 25 157 95 54 27 153 99
Adjusted for sex, age, and drinking status.
P value
OR (95% CI)a
– 0.21 – 0.22 – 0.22 – 0.15
1.0 (Ref.) 1.62 (0.72–3.56) 1.0 (Ref.) 1.29 (0.84–2.0) Ref. 1.72 (0.75–3.83) 1.0 (Ref.) 1.35 (0.88–2.08)
Ever smokers (N) Cases
Controls
33 30 105 99 30 34 98 106
23 10 63 37 23 10 63 37
P value
OR (95% CI)a
– 0.04 – 0.06 – 0.02 – 0.02
1.0 (Ref.) 3.24 (1.03–6.73) 1.0 (Ref.) 1.61 (0.96–2.71) Ref. 2.84 (1.06–7.27) 1.0 (Ref.) 1.84 (1.10–3.11)
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B. Lu et al. / Gene 542 (2014) 64–68
Table 6 Interactions of ERCC1 rs11615 and ERCC5 rs17655 polymorphisms with drinking in laryngeal cancer risk. SNP
ERCC1 rs11615
XPG/ERCC5 rs17655
a
Never drinkers (N)
TT CC T allele C allele CC GG T allele C allele
Cases
Controls
37 23 109 81 34 24 105 85
53 22 148 86 50 26 141 93
P value
– 0.23 – 0.22 – 0.33 0.30
OR (95% CI)a
1.0 (Ref.) 1.55 (0.73–3.41) 1.0 (Ref.) 1.27 (0.85–1.93) 1.0 (Ref.) 1.31 (0.61–2.86) 1.0 (Ref.) 1.23 (0.82–1.84)
Current and former drinkers (N) Cases
Controls
24 26 79 83 21 30 72 90
26 13 72 46 27 11 75 43
P value
OR (95% CI)a
– 0.05 – 0.04 – 0.005 – 0.0016
1.0 (Ref.) 2.36 (0.97–6.05) 1.0 (Ref.) 1.64 (0.99–2.74) 1.0 (Ref.) 3.79 (1.48–10.43) 1.0 (Ref.) 2.18 (1.30–3.66)
Adjusted for sex, age, and smoking status.
ERCC1 rs11615 CC and ERCC5 rs17655 GG genotypes may have an additive effect on the risk of this cancer in these higher-risk subgroups. Several limitations should be considered in this study. First, all the patients and control subjects were selected from one hospital, and some level of selection bias could not be avoided. But the allele frequencies in controls were similar to minor allele frequencies in Chinese population from NCBI reference assembly, which indicated that the control subjects could represent the Chinese population. Second, we only find that the ERCC1 polymorphisms are marginally associated with risk of laryngeal cancer, the marginal significance may be induced by the small numbers of cases and controls. Therefore, further large sample studies are greatly needed to confirm their association. In conclusion, our study suggests that ERCC1 rs11615 and ERCC5 rs17655 polymorphisms are associated with increased risk of laryngeal cancer, and that they confer more risk among smokers and drinkers. Our findings may be helpful in identifying individuals at increased risk for developing laryngeal cancer. As the biological impact of these polymorphisms is unclear, further large population-based studies are needed to evaluate the function of NER genes in the development of laryngeal cancer. Conflict of interest There is no conflict of interest in this study. Acknowledgments This work was supported by the Funding Scheme for Young Backbone Teachers of Colleges and Universities of Henan Province (2010GGJS-120) and the Project of Health Science and technology Innovation Talents of Henan Province (2010141). References Abbasi, R., et al., 2009. Laryngeal cancer risk associated with smoking and alcohol consumption is modified by genetic polymorphisms in ERCC5, ERCC6 and RAD23B but not by polymorphisms in five other nucleotide excision repair genes. International Journal of Cancer 125, 1431–1439.
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