224 Research paper
Single nucleotide polymorphisms in the DNA repair genes in HPV-positive cervical cancer Deepti Bajpaia, Ayan Banerjeea, Sujata Pathaka, Bhaskar Thakurb, Sunesh K. Jainc and Neeta Singha Genetic variation in DNA repair genes can modulate DNA repair capacity and may be related to the risk of cancer. The human papillomavirus is considered to be a necessary but not sufficient cause for cervical cancer and, therefore, other factors contribute to the carcinogenesis. A hereditary component for this neoplasia has been reported. Evaluation of the association of six polymorphisms was carried out in the following DNA repair genes: XRCC1 (Arg194Trp, Arg280His, and Arg399Gln), ERCC1 (Asp118Asp), ERCC2 (Lys751Gln), and ERCC4 (Arg415Gln). The cases (n = 110) included 65 squamous cell carcinomas (SCCs) and 45 squamous intraepithelial lesions (SIL). Controls (n = 68) were recruited from among women without cervical abnormalities. Genotypes were determined by PCRrestriction fragment length polymorphism and DNA sequencing. A positive association was observed between the polymorphisms of XRCC1 genes, that is, in codons 194 [P = 0.001, odds ratio (OR) = 20.1, 95% confidence interval (CI) = 5.9–68.8], 280 (P = 0.001, OR = 5.4, 95% CI = 2.3–12.6), and 399 (P = 0.008, OR = 4.2, 95% CI = 1.5–12.1) and cervical cancer. SIL patients also showed a significant association with codon 194 (P = 0.012, OR = 3.8, 95% CI = 1.3–10.6), but not with 280 (P = 0.35) and 399 (P = 0.81). A positive correlation was also found in ERCC4 Gln415Gln in both SCCs and SILs (P = 0.001,
OR = 21.3, 95% CI = 7.1–64.0 and P = 0.001, OR = 7.8, 95% CI = 2.9–20.9, respectively). For ERCC2 Gln751Gln, the association was significant for both SCCs (P = 0.001, OR = 10.1, 95% CI = 2.6–37.9) and SILs (P = 0.001, OR = 8.9, 95% CI = 2.8–28.3). However, the risk of SCC did not appear to differ significantly among individuals with the ERCC1 Asp118Asp genotype (P = 0.404). For SILs, it appeared to be a protective genotype (95% CI = 0.1–0.7). This study indicates that variant types of DNA repair genes play an important role in modifying individual susceptibility to SCC. European Journal of Cancer Prevention 25:224–231 Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
Introduction
critically involved in protecting against mutations that lead to cancer and/or inherited genetic disease (Radman et al., 1995). We have selected four DNA repair genes, representing two different repair pathways, for this study. Three of the genes, ERCC2, ERCC4, and ERCC1, belong to the nucleotide excision repair (NER) pathway and are members of a complex of 13–15 proteins that removes bulky adducts and thymidine dimers from DNA (Grossman and Wei, 1994). The fourth gene, XRCC1, was originally isolated as a radiation-sensitive mutant and assigned to the double-strand break/recombination pathway of DNA repair (Lindhal et al., 1997).
Although it is well known that human papillomaviruses (HPVs) are the causal agents for cervical cancer, HPV infections are very common relative to rare incidences of cancer, indicating that many infections resolve spontaneously (Schiffman et al., 2007) or persist without cancer progression. Host genetic factors may play a role in cervical carcinogenesis and are believed to influence persistence of HPV infection and perhaps progression to cervical cancer (Hemminki et al., 1999; Czene et al., 2002; Hildesheim and Wang, 2002; Carrington et al., 2005; Hemminki and Chen, 2006; Hussain et al., 2008). The role of nongenetic cofactors in persistence and progression has been well studied, but there are very few studies on the role of host genetic factors in the pathogenesis of cervical cancer. Many studies have documented that the genes involved in DNA repair and maintenance of genome integrity are All supplementary digital content is available directly from the corresponding author. 0959-8278 Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
European Journal of Cancer Prevention 2016, 25:224–231 Keywords: cervical cancer, ERCC1, ERCC2, ERCC4, genetic susceptibility, polymorphism (genetics), XRCC1 Departments of aBiochemistry, bBiostatistics and cObstetrics and Gynaecology, All India Institute of Medical Sciences, New Delhi, India Correspondence to Neeta Singh, PhD, Department of Biochemistry, Room No 3027-A, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India Tel: + 91 11 26594945; fax: + 91 11 2685663; e-mail: [email protected] Received 25 September 2014 Accepted 18 February 2015
The associations of single nucleotide polymorphisms (SNPs) in DNA repair genes and various types of cancer have been described extensively, with differing findings. For example, previous studies have reported that XRCC1 Arg194Trp C > T (TT) increases the risk of esophageal (Xing et al., 2002) and bile duct cancer (Huang et al., 2008), but decreases the risk of gastric carcinoma (Shen et al., 2000). XRCC1 Arg280His G > A has been reported DOI: 10.1097/CEJ.0000000000000159
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DNA repair genes in cervical cancer Bajpai et al. 225
to be a risk factor for breast cancer (Loizidou et al., 2008) and a protective factor for bile duct cancer (Huang et al., 2008). Niwa et al. (2005) first reported that the XRCC1 Arg399Gln G > A polymorphism is related to the increased susceptibility to cervical cancer in a Japanese population. For the Lys751Gln polymorphism in ERCC2, the homozygous variant genotype was associated with a higher risk of head and neck cancer (Sturgis et al., 2000), skin cancer (Dong et al., 2013), and lung cancer (Simone Benhamou and Sarasin, 2005). The ERCC1 C118T polymorphism presented an increased risk of cervical cancer in a Chinese population (Kwon et al., 2007; Zhang et al., 2011). In a study by Jorgensen et al. (2009), the ERCC4 variant allele was statistically significantly associated with benign breast disease. To date, no published data have examined the association of the polymorphism of the above DNA repair genes with cervical cancer in an Indian population. We genotyped six variants of the four DNA repair genes XRCC1 (Arg194Trp, Arg399Gln, Arg280His), ERCC1 (Asp118Asp), ERCC2 (Lys751Gln), and ERCC4 (Arg415Gln), and assessed their association with squamous intraepithelial lesions (SIL) and cervical carcinoma and with other epidemiological risk factors.
Materials and methods Study participants
The study protocol was approved by the institutional ethics committee. All participants were genetically unrelated women from the Indian subcontinent. Patients histopathologically confirmed with cervical cancer or SIL were consecutively recruited at AIIMS; they were 18–65 years of age. The invasive cervical cancer cases were histologically proven squamous cell carcinoma (SCC). The exclusion criteria included self-reported cancer history, previous radiotherapy or chemotherapy, and a family history of cancer. The controls were recruited during the same period from the Department of Obstetrics and Gynecology. The controls were women who were matched to the cases in terms of age, and who were histologically confirmed to have a normal cervix, with no personal or familial history of cancer or any other genetic disease. Questionnaire
A total of 110 eligible patients (65 cervical carcinoma patients and 45 SIL patients) and 68 eligible control women completed the questionnaires and consented to provide blood as well as cervical tissue samples for genotyping and HPV detection. Detailed information on demographic factors was obtained. Following enrollment, ∼ 4 ml of venous blood as well as cervical tissue was collected from each participant and maintained at − 70°C. Genotyping assays
DNA was extracted from both whole blood and cervical tissue using a DNA extraction kit (Qiagen, Germantown,
Maryland, USA). Genotyping was performed using PCRrestriction fragment length polymorphism methods with appropriate primer sets (Supplementary table 1). The PCR conditions were as follows: (i) activation of Fast Taq polymerase (Chromus Biotech, Bangalore, India) at 94°C for 2 min, (ii) 35 cycles of denaturation at 94°C for 10 s; annealing (as per primer) for 20 s; elongation at 72°C for 5 s, and (iii) extension at 72°C for 5 min. Each PCR product was digested with an appropriate restriction enzyme (Fermentas Life Sciences, New Delhi, India) (Supplementary table 1) for 2 h at 37°C. The products were then resolved on a 3.5% agarose gel. Restriction fragment length polymorphism results were confirmed by DNA sequencing (Techno Concept Ind Pvt Ltd, New Delhi, India) of a few PCR products. HPV analysis
Genomic DNA was extracted from the above samples. HPV DNA testing was performed as described previously (Singh et al., 2009). Statistical analysis
The sample size for both patients and controls was calculated using QUANTO software, version 1.0 (http://hydra.usc. edu/gxe), and was found to be adequate. The sample size achieved less than 80% statistical power. χ2 analysis was used to assess deviation from Hardy–Weinberg equilibrium and to compare the genotype/allele frequency between the patients and the controls. Odds ratios (ORs) were obtained by unconditional logistic regression analysis and adjusted for age, age at first live birth, and smoking as a continuous variable. All statistical analyses were carried out using the SPSS software, version 15 (SPSS Inc., Chicago, Illinois, USA). The OR was calculated using unconditional logistic regression for risk genotypes with the wild-type genotype as a reference. Haplotype analysis was carried out using the PLINK software (http://pngu.mgh.harvard.edu/∼purcell/plink/).
Results Characteristics of the study population
A total of 110 patients (65 carcinomas and 45 SILs) and 68 controls were enrolled in this study. The characteristics of the study participants are shown in Table 1. There was no significant difference in age between the controls and the patients. The mean age of the cancer patients, SIL patients, and control women was 43, 42, and 43 years, respectively. The reproductive history, including age at menarche and age at first child birth, in all the groups was not significantly different, but the parities and use of oral contraceptives (OCP) differed significantly (Table 1). There were 13 (20%), 5 (11.1%), and 5 (7.3%) smokers in the carcinoma, SIL, and control groups, respectively. HPV infections in carcinoma and SIL patients were 94 and 90%, respectively, whereas in control women, the infection rate was only 17% (P < 0.001).
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226 European Journal of Cancer Prevention 2016, Vol 25 No 3
Table 1
Clinicopathological characteristics of SCC, SILs, and controls SILs
Variables Age (years) (mean ± SD) Age at menarche (years) (mean ± SD) Age at first live birth (years) (mean ± SD) Parity, (mean ± SD) Smoking status Nonsmoker Smoker Oral contraceptives Nonuser User HPV status Positive Negative
SCC
Controls n = 68 [n (%)]
n = 45 [n (%)]
P value
n = 65 [n (%)]
Pa value
43.2 ± 10.3 14.1 ± 0.4 23.5 ± 1.3 3.4 ± 1.2
41.2 ± 9.4 14.1 ± 1.2 22.8 ± 1.7 3.7 ± 1.0
0.298 0.972 0.027 0.113
43.9 ± 9.4 13.9 ± 1.4 22.9 ± 1.1 3.9 ± 1.3
0.651 0.324 0.013 0.014
63 (92.6) 5 (7.3)
40 (88.8) 5 (11.1)
0.491
52 (80) 13 (20)
0.033
50 (73.5) 18 (26.4)
29 (64.4) 6 (13.3)
0.303
28 (43.1) 37 (56.9)
0.303
12 (17.6) 56 (82.3)
40 (8.8) 5 (11.1)
0.001
61 (93.8) 4 (6.1)
0.001
a
HPV, human papillomavirus; SCC, squamous cell carcinoma; SILs, squamous intraepithelial lesions. Bold values indicates statistical significance. χ -test.
a 2
Genotype and allelic frequency distribution of XRCC1 gene polymorphisms and risk associated with SCC
Table 2
Controls (N = 68) [n (%)]
SCC (N = 65) [n (%)]
ORa (95% CI)
Controls (N = 68) [n (%)]
P value
SILs (N = 45) [n (%)]
ORa (95% CI)
P value
b
b
XRCC1-194 (Exon 6) (rs1799782) CC 44 (64.7) CT 11 (16.1) TT 13 (19.1) CT + TT 24 (35.2) Allele C 99 (72.7) Allele T 37 (54.4) XRCC1-280b (Exon 9) (rs25486) GG 48 (92.3) GA 7 (10.7) AA 3 (1.5) GA + AA 20 (12.3) Allele G 103 (6.6) Allele A 33 (93.3) XRCC1-399b (Exon 10) (rs25487) GG 23 (33.8) GA 33 (48.5) AA 12 (17.6) GA + AA 45 (66.1) Allele G 93 (71.5) Allele A 43 (66.1)
Genotype and allelic frequency distribution of XRCC1 gene polymorphisms and risk associated with SILs
Table 3
11 16 38 54 38 92
(16.9) (24.6) (58.4) (83.1) (29.2) (70.7)
Reference 11.5 (3.1–41.9) 20.1 (5.9–68.8) 16.0 (5.1–49.9) Reference 6.5 (3.7–11.5)
20 6 39 45 46 84
(30.7) (9.2) (60) (69.2) (35.3) (64.6)
Reference 2.0 (0.6–8.3) 7.4 (2.9–18.7) 5.4 (2.3–12.6) Reference 5.7 (3.2–10.1)
12 22 31 53 46 84
(18.4) (33.8) (47.6) (81.5) (35.3) (64.6)
Reference 0.9 (0.3–2.6) 4.2 (1.5–12.1) 1.9 (0.8–4.6) Reference 3.9 (2.3–6.8)
0.001 0.001 0.001 0.001
0.241 0.001 0.001 0.001
0.886 0.008 0.169 0.001
XRCC1-194 (Exon 6) (rs1799782) CC 44 (64.7) CT 11 (16.1) TT 13 (19.1) CT + TT 24 (35.2) Allele C 99 (72.7) Allele T 37 (54.4) XRCC1-280b (Exon 9) (rs25486) GG 48 (92.3) GA 7 (10.7) AA 13 (1.5) GA + AA 20 (12.3) Allele G 103(6.6) Allele A 33 (93.3) XRCC1-399b (Exon 10) (rs25487) GG 23 (33.8) GA 33 (48.5) AA 12 (17.6) GA + AA 45 (66.1) Allele G 93 (71.5) Allele A 43 (66.2)
13 19 13 32 45 45
(28.8) (42.2) (28.8) (71.1) (50) (50)
Reference 6.8 (2.4–18.6) 3.8 (1.3–10.6) 5.1 (2.2–12.1) Reference 2.7 (1.5–4.9)
30 11 4 15 71 19
(66.6) (24.4) (8.8) (33.3) (78.8) (42.2)
Reference 2.6 (0.9–7.7) 0.6 (0.2–1.9) 1.3 (0.6–3.0) Reference 0.8 (0.4–1.6)
14 23 8 31 51 39
(31.1) (51.1) (17.7) (68.8) (56.6) (43.3)
Reference 1.2 (0.5–3.0) 1.1 (0.4–3.6) 1.2 (0.5–2.8) Reference 1.6 (0.9–3.0)
0.001 0.012 0.001 0.001
0.082 0.351 0.552 0.581
0.617 0.813 0.635 0.073
CI, confidence interval; OR, odds ratio; SCC, squamous cell carcinoma. Bold values indicates statistical significance. a Adjusted for age at first live birth, parity, smoking status, and use of oral contraceptives. b Genotypic frequencies [N (%)].
CI, confidence interval; OR, odds ratio; SILs, squamous intraepithelial lesions. Bold values indicates statistical significance. a Adjusted for age at first live birth, parity, smoking status, and use of oral contraceptives. b Genotypic frequencies [N (%)].
Association of the BER gene polymorphism with SCC and SIL susceptibility
genotype (CT + TT) also showed an increased risk of SILs (P = 0.001, OR = 5.1) (Table 3).
The genotypic frequency distribution between SCC and controls is shown in Table 2 and that between SILs and controls is shown in Table 3. Our results indicated that the homozygous TT genotype of XRCC1 codon 194 and the AA genotype of XRCC1 codon 280 presented 20- and 7.4-fold, respectively, higher risk of cervical cancer. The variant genotype of XRCC1 codon 399 was associated with a 4.2-fold higher risk of SCC. The heterozygous genotype of only XRCC1 C194T presented a significant risk (Table 2). In SIL cases, the homozygous variant TT of exon 6 presented a 3.8-fold higher risk. The combined variant
Association of the NER gene polymorphism with SCC and SIL susceptibility
For ERCC1, the polymorphic variation occurs at codon 118, converting a common codon usage (AAC) into an infrequent one (AAT), both coding asparagine. We found no significant association between the ERCC1 118 polymorphism at both genotypic and allelic levels (Table 4). However, we found that in SIL cases, there was a significant difference between the variant homozygous TT genotype among SILs and controls [P = 0.010, OR = 0.3, 95% confidence interval (CI) = 0.1–0.7] (Table 5).
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DNA repair genes in cervical cancer Bajpai et al. 227
Genotype and allelic frequency distribution of ERCC1, ERCC2, and ERCC4 gene polymorphisms and risk associated with SCC
Table 4
Controls (N = 68) [n (%)]
SCC (N = 65) [n (%)]
ORa (95% CI)
P value
b
ERCC1-118 (Exon 4) (rs3212986) CC 14(20.5) 10 CT 15 (22.1) 6 TT 39 (57.3) 49 CT + TT 54 (79.4) 55 Allele C 43 (31.6) 26 Allele T 93 (68.3) 104 ERCC2-751b (Exon 23) (rs1052559) AA 52 (76.4) 27 AC 9 (13.2) 24 CC 7 (10.2) 14 AC + CC 16 (23.5) 38 Allele A 113 (83.1) 78 Allele C 23 (16.9) 52 b ERCC4-415 (Exon 8) (rs1800067) GG 45 (66.1) 8 GA 12 (17.6) 15 AA 11 (16.1) 42 GA + AA 23 (33.8) 57 Allele G 102 (75) 31 Allele A 34 (25) 99
(15.3) (9.2) (75.3) (84.6) (19.1) (76.4)
Reference 0.4 (0.1–1.6) 1.5 (0.5–4.4) 1.2 (0.4–3.4) Reference 1.8 (1.0–3.4)
(41.5) (36.9) (21.5) (58.4) (60) (40)
Reference 6.5 (2.3–18.1) 10.1 (2.6–37.9) 7.5 (3.0–18.9) Reference 3.3 (1.8–6.1)
(12.3) (23.1) (64.6) (87.6) (23.8) (76.1)
Reference 8.1 (2.4–27.2) 21.3 (7.1–64.0) 15.0 (5.6–40.5) Reference 9.6 (5.3–17.4)
0.191 0.404 0.688 0.031
0.001 0.001 0.001 0.001
0.001 0.001 0.001 0.001
CI, confidence interval; OR, odds ratio; SCC, squamous cell carcinoma. Bold values indicates statistical significance. a Adjusted for age at first live birth, parity, smoking status, and use of oral contraceptives. b Genotypic frequencies [N (%)].
In ERCC2, the polymorphism occurs at codon 751, exon 23. The homozygous variant genotype (CC) was associated significantly with risk of SCC. The heterozygous (AC) and the combined heterozygous and variant genotype (AC + CC) also presented a significantly increased risk of SCC. At the allelic level, the variant allele also posed a 3.3-fold higher risk of SCC (Table 4). In SILs, Genotype and allelic frequency distribution of ERCC1, ERCC2, and ERCC4 gene polymorphisms and risk associated with SILs
Table 5
Controls (N = 68) [n (%)] ERCC1-118b (Exon 4) (rs3212986) CC 14 (20.5) CT 15 (22.0) TT 39 (57.3) CT + TT 54 (79.4) Allele C 43 (31.6) Allele T 93 (68.3) b ERCC2-751 (Exon 23) (rs1052559) AA 52 (76.4) AC 9 (13.2) CC 7 (10.2) AC + CC 16 (23.5) Allele A 113 (83.1) Allele C 23 (16.1) ERCC4-415b (Exon 8) rs1800067 GG 45 (66.17) GA 12 (17.64) AA 11 (16.17) GA + AA 23 (33.8) Allele G 102 (75) Allele A 34 (25)
SILs (N = 45) [n (%)]
ORa (95% CI)
14 21 10 31 49 41
(31.1) (46.6) (22.2) (68.8) (54.4) (45.5)
Reference 1.2 (0.4–3.4) 0.3 (0.1–0.7) 0.5 (0.2–1.3) Reference 0.4 (0.2–0.7)
12 20 13 33 44 46
(26.6) (44.4) (28.8) (73.3) (48.8) (51.1)
Reference 9.6 (3.4–27.1) 8.9 (2.8–28.3) 9.3 (3.8–22.9) Reference 5.1 (2.7–9.9)
11 13 21 34 43 55
(24.4) (28.8) (46.6) (75.5) (47.7) (61.1)
Reference 4.4 (1.6–12.3) 7.8 (2.9–20.9) 6.9 (2.8–16.8) Reference 3.8 (2.1–7.0)
P value
0.674 0.010 0.171 0.001
0.001 0.001 0.001
In ERCC4, the polymorphic site is at codon 415, exon 8. We observed that the polymorphism in ERCC4 presented the overall highest risk of SCC. The homozygous variant genotype (AA) was associated significantly with the risk of SCC (P = 0.001 OR = 21.3, 95% CI = 7.1–64.0). The heterozygous (GA) and the combined heterozygous and variant genotype (GA + AA) also presented a significantly increased risk of SCC. At the allelic level, the variant allele (A) also posed a 9.6-fold higher risk of SCC (Table 4). In SILs, similar results to those for SCC were obtained (Table 5). Association of XRCC1, ERCC1, and ERCC2 haplotypes with SCC and SIL
To elucidate the combined influence of the polymorphisms in these three genes, we constructed XRCC1 Exon 6/9/10, ERCC1 Exon 4, and ERCC2 Exon 8 haplotypes (Table 6). The haplotype containing the wildtype allele of all the five polymorphisms was considered as a reference. The haplotype TAATC was most frequent in SCC (40%) and showed a four-fold increased risk, whereas in the SILs haplotype, TTGAC was predominantly present (21%). Haplotype TTAAC was present in all three study groups. Haplotype TTGGA was present only in patients with invasive cancer and in the controls. In logistic regression analysis, haplotypes TCAAA and TCGAA, being significantly lower in patients, appeared to be protective haplotypes. The Table 6 Haplotype analysis of XRCC1 (− 26304C/T, − 27466G/A, − 28152G/A), ERCC1 − 19007C/T, and ERCC2 − 35931A/C polymorphism, and cervical cancer and SIL risk Haplotype
Cancer
Control
Odds ratio (95% CI)
CCGGA TTAAC TTGGA TCGGA TTAAA TCAAA TTGAA TCGAA
20.01 40.02 7.36 7.97 17.38 0.26 6.04 0.93
31.86 14.08 1.95 23.95 5.75 3.85 4.15 14.4
Reference 4.08 (2.04–8.15) 3.99 (0.80–19.8) 0.27 (0.11–0.64) 3.44 (1.28–9.26) 0.06 (0.00–3.48) 1.48 (0.41–5.34) 0.05 (0.00–0.48)
Haplotype
SIL
Control
Odds ratio (95% CI)
CCGGA TTAAC TTGAC CTGGC TTAAA TCAAA TTGAA TCGAA TCGGA
49.14 23.75 21.64 4.6 0 0 0 0.85 0
33.07 13.36 1.37 0.77 5.54 3.52 5.73 12.06 24.55
Reference 2.01 (0.96–4.22) 19.88 (3.44–114.6) 6.21 (0.54–70.5) – – – 0.06 (0.00–0.57) –
P value ** * *
P value
0.001
0.004 0.001 0.001 0.001
CI, confidence interval; OR, odds ratio; SILs, squamous intraepithelial lesions. Bold values indicates statistical significance. Adjusted for age at first live birth, parity, smoking status, and use of oral contraceptives. b Genotypic frequencies [N (%)]. a
the homozygous variant genotype (CC) was associated significantly with the risk of SILs (P = 0.001; OR = 8.9; 95% CI = 2.8–28.3). The heterozygous (AC), the combined heterozygous and variant genotype (AC + CC), and also the variant allele (C) posed a higher risk of SIL (Table 5).
CI, confidence interval; SILs, squamous intraepithelial lesions. *Significant value. **Highly significant value.
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**
228 European Journal of Cancer Prevention 2016, Vol 25 No 3
haplotypes TTAAC, TTGAC, and TTAAA, with relatively higher frequencies in patients, appeared to be susceptible haplotypes. Analysis of the XRCC1, ERCC1, ERCC2, and ERCC4 gene polymorphism with HPV infection
We evaluated the gene–HPV interaction to study the modulation of SCC and the risk of SIL. Polymorphisms in XRCC1, ERCC1, ERCC2, and ERCC4 were associated significantly with HPV infection in SCC, SILs, and control samples as well (Table 7). There was more than a 95% association (Table 7) of HPV infection in the homozygous variant genotype in all three groups (i.e. SCC, SIL, and controls). In control samples, there was a uniform increase in individuals with HPV infection compared with among wild-type, heterozygous, and polymorphic homozygous genotypes of all the genes (Table 7). The maximum association was observed in individuals who carried a polymorphism in XRCC1 codon 194 and 280. In future, this group of the control population can be followed up to determine whether eventually they develop cervical precancer or frank cancer. Combined analysis of polymorphisms in all the genes across groups
For those individuals with a variant in more than one gene, there was a 9.8-fold increased risk of SCC (OR = 9.8, 95% CI = 3.0–31.8). In SIL cases, the frequency of those carrying a polymorphism in more than gene was comparatively low and, hence, the associated risk was not significant (P = 0.954) (data not shown).
Discussion Cervical cancer remains a major cause of cancer deaths in women worldwide (Waggoner, 2003). Currently, no Table 7
biological or genetic markers are available to predict which precancerous lesion will progress to invasive cervical cancer. Although infection with high-risk HPV is recognized to be an essential initiating event in cervical tumorigenesis, this alone is not sufficient for progression to invasive cancer (Zur Hausen, 2002). Despite the recent progress in understanding the molecular aspects of cervical cancer, the genetic basis of progression of precursor SILs to invasive cancer in the multistep progression of cervical cancer remains poorly understood (Gius et al., 2007). Therefore, identification of other ‘genetic hits’ in cervical cancer is important to understand its biology. Studies carried out over the past few years have identified variant alleles for a number of DNA repair genes, some of which may modify DNA repair capacity. With limited sample size, our current data suggest that amino acid substitution variants of DNA repair genes involved in base excision repair and NER pathways may contribute to cervical cancer pathogenesis. In the present study, we genotyped four common polymorphisms of the BER gene XRCC1, and NER genes ERCC1, ERCC2, and ERCC4, and tested the association between the distributions of their genotypes with cervical precancer and invasive cancer. These polymorphisms have been shown to have functional significance and may in be part responsible for the interindividual difference in the capacity of DNA repair in the general population and for low DNA repair efficacy in cancer patients (Kohno et al., 2006). Infection with the oncogenic types of HPV has been established as a main cause of cervical cancer (Singh et al., 2009) and SILs (Cuzick et al., 1994). We also found a significant association of HPV infection both in SIL and in SCC cases. An association between smoking and cervical cancer has been reported (Winkelstein, 1990), as
Association of HPV positivity in DNA repair genes in controls, SILs, and SCC Heterozygous (% HPV positive)
Polymorphic homozygous (% HPV positive)
P valuea
0.0 2.1 4.4 0.0 8.2 8.9
27.3 28.6 21.2 6.7 21.2 41.7
92.3 92.3 58.3 35.9 38.6 54.6
0.001 0.001 0.001 0.005 0.029 0.001
92.3 90.0 92.9 100.0 91.7 90.9
100.0 90.9 86.0 85.7 100.0 100.0
69.2 75.0 87.5 80.0 69.2 80.9
0.017 0.570 1.000 0.220 0.014 0.238
90.9 90.0 91.7 70.0 85.2 62.5
81.3 83.3 90.9 100.0 100.0 93.3
100.0 97.4 96.8 97.0 100.0 100.0
0.016 0.188 0.850 0.013 0.068 0.001
Controls Wild type (% HPV positive) XRCC1 codon 194 XRCC1 codon 280 XRCC1 codon 399 ERCC1 codon 118 ERCC2 codon 751 ERCC4 codon 415 SILs XRCC1 codon 194 XRCC1 codon 280 XRCC1 codon 399 ERCC1 codon 118 ERCC2 codon 751 ERCC4 codon 415 SCC XRCC1 codon 194 XRCC1 codon 280 XRCC1 codon 399 ERCC1 codon 118 ERCC2 codon 751 ERCC4 codon 415
HPV, human papillomavirus; SCC, squamous cell carcinoma; SILs, squamous intraepithelial lesions. Bold values indicates statistical significance. χ (comparison between genotypes).
a 2
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DNA repair genes in cervical cancer Bajpai et al. 229
well as smoking-related DNA damage in the cervical epithelium (Simons et al., 1993). In our study, we found that smoking posed a significant risk (P = 0.033) in SCC, whereas no significant association was found in SILs. Long-term use of OCP could be a cofactor that increases the risk of cervical carcinoma by up to four-fold in women who are positive for cervical HPV DNA (Moreno et al., 2002). We found a significant number of OCP users in SCC (57%) compared with only 26% among the controls. However, in SILs, OCP use was not associated significantly with the disease (P = 0.303). High parity, as evident in studies by Jensen et al. (2013), seems to increase the risk of SCC of the cervix among HPVpositive women. In our study, high parity was also associated significantly with SCC cases (P = 0.013), but we found no significant difference in SIL cases. Our study also assessed the difference in age at first live birth in SCC, SILs, and controls. There was a significant difference between SCC cases and controls (P = 0.013), whereas the difference in age at first live birth was not statistically significant between SILs and controls. XRCC1 is one of the > 20 genes that participate in the base excision repair pathway. A large number of molecular epidemiologic studies have been carried out to evaluate the role of the Arg399Gln polymorphism in the risk of cancer; however, the results remain conflicting rather than conclusive. The present study indicated that the XRCC1 Exon 6 (C > T) polymorphism was associated with both cervical precancer and cancer. Detailed metaanalysis by Shuai et al. (2012) showed that the variant 194Trp allele was associated significantly with an increased risk of cervical cancer. They also reported similar findings in a subgroup of an Asian population. To the best of our knowledge, this is the first study that has investigated the association of XRCC1 polymorphisms and cervical precancer and cancer risk. No association was observed between the XRCC1 Exon 10 (G > A) polymorphism and cervical precancer risk. In the case of a polymorphism at Exon 6 (C > T) and Exon 9 (G > A), a significantly increased risk for homozygous genotypes TT and AA, respectively, to have cervical cancer and precancer was observed compared with the controls by logistic regression analysis. To date, relatively few studies have been carried out to examine the association between the Arg280His variant and the risk of cancer and altered DNA adducts. Our observation is in agreement with the findings of Kang et al. (2007), in which the Arg280His polymorphism was associated significantly with SCC of the skin in a Korean population. A previous study showed that the polymorphism of ERCC1 was associated with the prognosis of patients receiving chemotherapy in human gastric, cervical, colorectal, non-small cell lung cancer, and bone cancer (Kwon et al., 2007). In our study, we did not observe any association between the ERCC1 118C/T polymorphism and the risk of cervical precancer and cancer. Han et al.
(2012) also reported that the C19007T polymorphism in ERCC1 might not be associated with the risk and invasiveness of cervical cancer in Korean women. The polymorphism in ERCC2 codon Lys751Gln is reported to be a risk allele and patients with the Gln allele genotype have a significantly increased risk of developing lung cancer (Zhan et al., 2010). The Gln/Gln variant of the ERCC2 correlated with a higher risk of skin, glioma, and breast cancer. To date, no studies have addressed the association between alterations in this region of the ERCC2 gene and SIL. Our data support the previous results that indicated that homozygous individuals with the Gln/Gln genotype had an increased risk compared with carriers of the Lys/Lys genotype. Several studies have investigated the associations between the ERCC4 polymorphisms and the risk of cancers, including cancer of the breast, lung, head and neck, skin, pancreas, and bladder, but the results are not consistent. An association was found between the Arg415Gln polymorphism and the risk of lung cancer (adjusted OR = 2.11, 95% CI = 0.9–2.5; Arg/Gln vs. Arg/ Arg) in Koreans (Zhan et al., 2010). We observed that both polymorphic homozygous and heterozygous genotypes of ERCC4 were associated significantly with increased cervical precancer and cancer risk. In a multistep, multigenic process such as carcinogenesis, SNP in a single gene is unlikely to alter the expression or the function of specific proteins to the extent of producing a pathological phenotype. Various studies have shown groups of SNPs within an individual gene to be associated with cervical cancer (Shao et al., 2008). Therefore, haplotype analysis of XRCC1, ERCC1, and ERCC2 was carried out. Two of the haplotypes, TTAAC and TTAAA, showed a higher risk for cervical cancer. Similarly, one haplotype, TTGAC, showed a statistically increased risk for SILs. These findings indicated that the combined genotype was associated with cervical cancer as well as precancer risk. Our findings on an association between XRCC1, ERCC1, and ERCC2 haplotypes and the risk of cervical cancer and precancer further strengthen our observation that polymorphisms in XRCC1, ERCC1, and ERCC2 may aid understanding of the development of the disease. On comparing the prevalence of the different polymorphisms in the study population, it became evident that more than one gene variant occurred in a considerable number of individuals, but because of the limited sample size, we cannot comment whether the risk associated with combined polymorphism was because of linkage among the DNA repair genes. Furthermore, we observed that in the control population, HPV infection increased from the wild-type genotype to the heterozygous and was maximum in the polymorphic homozygous genotype. As such individuals were exposed to major risk factors, a follow-up of the same could show whether they progress to cervical cancer. However, the
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230 European Journal of Cancer Prevention 2016, Vol 25 No 3
association of HPV and genotypes in the patient population did not show any trend. This could be because of already high baseline HPV positivity in the patient samples. Our study also showed that both blood and tumor samples can act as sources to detect polymorphism in DNA repair genes as findings of both blood and tissue samples matched 100%. Therefore, this interesting finding of a complete correlation between tissue and blood samples suggests that gene polymorphism in blood may be used as a noninvasive method to evaluate the risk of cervical cancer. Additional work is required to determine whether polymorphisms do indeed lead to a reduced DNA repair capacity in vivo and to identify the effects of these phenotypes in different environmental conditions. The identification of polymorphisms in many genes and the determination of their functional importance in cervical cancer will enable the design of susceptibility–risk models for de-novo and therapy-related disease. In summary, the present study suggested that XRCC1, ERCC2, and ERCC4 SNPs act as predisposing factors in cervical precancer and increased risk of invasive cervical cancer. This perhaps indicates that reduced DNA repair capacity of these gene polymorphisms modulated the SCC and SILs. Given that the sample size of our study was comparatively small, modest effects cannot be ruled out. However, like most association studies, larger studies involving more SNPs in one gene or genes in the same biologic pathway are warranted to replicate the study in different populations.
Acknowledgements The authors are grateful to all the study participants, without whom this study would not have been possible. Deepti Bajpai is grateful to the Indian Council of Medical Research for providing the Senior Research Fellowship. Conflicts of interest
There are no conflicts of interest.
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Sturgis EM, Zheng R, Li L, Castillo EJ, Eicher SA, Chen M, et al. (2000). XPD/ERCC2 polymorphisms and risk of head and neck cancer: a case–control analysis. Carcinogenesis 21:2219–2223. Waggoner SE (2003). Cervical cancer. Lancet 361:2217–2225. Winkelstein W Jr (1990). Smoking and cervical cancer – current status: a review. Am J Epidemiol 131:945–957. discussion 958–960. Xing D, Qi J, Miao X, Lu W, Tan W, Lin D (2002). Polymorphisms of DNA repair genes XRCC1 and XPD and their associations with risk of esophageal squamous cell carcinoma in a Chinese population. Int J Cancer 100:600–605.
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Single nucleotide polymorphisms in the DNA repair genes in HPV-positive cervical cancer Deepti Bajpaia, Ayan Banerjeea, Sujata Pathaka, Bhaskar Thakurb, Sunesh K. Jainc and Neeta Singha Genetic variation in DNA repair genes can modulate DNA repair capacity and may be related to the risk of cancer. The human papillomavirus is considered to be a necessary but not sufficient cause for cervical cancer and, therefore, other factors contribute to the carcinogenesis. A hereditary component for this neoplasia has been reported. Evaluation of the association of six polymorphisms was carried out in the following DNA repair genes: XRCC1 (Arg194Trp, Arg280His, and Arg399Gln), ERCC1 (Asp118Asp), ERCC2 (Lys751Gln), and ERCC4 (Arg415Gln). The cases (n = 110) included 65 squamous cell carcinomas (SCCs) and 45 squamous intraepithelial lesions (SIL). Controls (n = 68) were recruited from among women without cervical abnormalities. Genotypes were determined by PCRrestriction fragment length polymorphism and DNA sequencing. A positive association was observed between the polymorphisms of XRCC1 genes, that is, in codons 194 [P = 0.001, odds ratio (OR) = 20.1, 95% confidence interval (CI) = 5.9–68.8], 280 (P = 0.001, OR = 5.4, 95% CI = 2.3–12.6), and 399 (P = 0.008, OR = 4.2, 95% CI = 1.5–12.1) and cervical cancer. SIL patients also showed a significant association with codon 194 (P = 0.012, OR = 3.8, 95% CI = 1.3–10.6), but not with 280 (P = 0.35) and 399 (P = 0.81). A positive correlation was also found in ERCC4 Gln415Gln in both SCCs and SILs (P = 0.001,
OR = 21.3, 95% CI = 7.1–64.0 and P = 0.001, OR = 7.8, 95% CI = 2.9–20.9, respectively). For ERCC2 Gln751Gln, the association was significant for both SCCs (P = 0.001, OR = 10.1, 95% CI = 2.6–37.9) and SILs (P = 0.001, OR = 8.9, 95% CI = 2.8–28.3). However, the risk of SCC did not appear to differ significantly among individuals with the ERCC1 Asp118Asp genotype (P = 0.404). For SILs, it appeared to be a protective genotype (95% CI = 0.1–0.7). This study indicates that variant types of DNA repair genes play an important role in modifying individual susceptibility to SCC. European Journal of Cancer Prevention 25:224–231 Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
Introduction
critically involved in protecting against mutations that lead to cancer and/or inherited genetic disease (Radman et al., 1995). We have selected four DNA repair genes, representing two different repair pathways, for this study. Three of the genes, ERCC2, ERCC4, and ERCC1, belong to the nucleotide excision repair (NER) pathway and are members of a complex of 13–15 proteins that removes bulky adducts and thymidine dimers from DNA (Grossman and Wei, 1994). The fourth gene, XRCC1, was originally isolated as a radiation-sensitive mutant and assigned to the double-strand break/recombination pathway of DNA repair (Lindhal et al., 1997).
Although it is well known that human papillomaviruses (HPVs) are the causal agents for cervical cancer, HPV infections are very common relative to rare incidences of cancer, indicating that many infections resolve spontaneously (Schiffman et al., 2007) or persist without cancer progression. Host genetic factors may play a role in cervical carcinogenesis and are believed to influence persistence of HPV infection and perhaps progression to cervical cancer (Hemminki et al., 1999; Czene et al., 2002; Hildesheim and Wang, 2002; Carrington et al., 2005; Hemminki and Chen, 2006; Hussain et al., 2008). The role of nongenetic cofactors in persistence and progression has been well studied, but there are very few studies on the role of host genetic factors in the pathogenesis of cervical cancer. Many studies have documented that the genes involved in DNA repair and maintenance of genome integrity are All supplementary digital content is available directly from the corresponding author. 0959-8278 Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
European Journal of Cancer Prevention 2016, 25:224–231 Keywords: cervical cancer, ERCC1, ERCC2, ERCC4, genetic susceptibility, polymorphism (genetics), XRCC1 Departments of aBiochemistry, bBiostatistics and cObstetrics and Gynaecology, All India Institute of Medical Sciences, New Delhi, India Correspondence to Neeta Singh, PhD, Department of Biochemistry, Room No 3027-A, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India Tel: + 91 11 26594945; fax: + 91 11 2685663; e-mail: [email protected] Received 25 September 2014 Accepted 18 February 2015
The associations of single nucleotide polymorphisms (SNPs) in DNA repair genes and various types of cancer have been described extensively, with differing findings. For example, previous studies have reported that XRCC1 Arg194Trp C > T (TT) increases the risk of esophageal (Xing et al., 2002) and bile duct cancer (Huang et al., 2008), but decreases the risk of gastric carcinoma (Shen et al., 2000). XRCC1 Arg280His G > A has been reported DOI: 10.1097/CEJ.0000000000000159
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DNA repair genes in cervical cancer Bajpai et al. 225
to be a risk factor for breast cancer (Loizidou et al., 2008) and a protective factor for bile duct cancer (Huang et al., 2008). Niwa et al. (2005) first reported that the XRCC1 Arg399Gln G > A polymorphism is related to the increased susceptibility to cervical cancer in a Japanese population. For the Lys751Gln polymorphism in ERCC2, the homozygous variant genotype was associated with a higher risk of head and neck cancer (Sturgis et al., 2000), skin cancer (Dong et al., 2013), and lung cancer (Simone Benhamou and Sarasin, 2005). The ERCC1 C118T polymorphism presented an increased risk of cervical cancer in a Chinese population (Kwon et al., 2007; Zhang et al., 2011). In a study by Jorgensen et al. (2009), the ERCC4 variant allele was statistically significantly associated with benign breast disease. To date, no published data have examined the association of the polymorphism of the above DNA repair genes with cervical cancer in an Indian population. We genotyped six variants of the four DNA repair genes XRCC1 (Arg194Trp, Arg399Gln, Arg280His), ERCC1 (Asp118Asp), ERCC2 (Lys751Gln), and ERCC4 (Arg415Gln), and assessed their association with squamous intraepithelial lesions (SIL) and cervical carcinoma and with other epidemiological risk factors.
Materials and methods Study participants
The study protocol was approved by the institutional ethics committee. All participants were genetically unrelated women from the Indian subcontinent. Patients histopathologically confirmed with cervical cancer or SIL were consecutively recruited at AIIMS; they were 18–65 years of age. The invasive cervical cancer cases were histologically proven squamous cell carcinoma (SCC). The exclusion criteria included self-reported cancer history, previous radiotherapy or chemotherapy, and a family history of cancer. The controls were recruited during the same period from the Department of Obstetrics and Gynecology. The controls were women who were matched to the cases in terms of age, and who were histologically confirmed to have a normal cervix, with no personal or familial history of cancer or any other genetic disease. Questionnaire
A total of 110 eligible patients (65 cervical carcinoma patients and 45 SIL patients) and 68 eligible control women completed the questionnaires and consented to provide blood as well as cervical tissue samples for genotyping and HPV detection. Detailed information on demographic factors was obtained. Following enrollment, ∼ 4 ml of venous blood as well as cervical tissue was collected from each participant and maintained at − 70°C. Genotyping assays
DNA was extracted from both whole blood and cervical tissue using a DNA extraction kit (Qiagen, Germantown,
Maryland, USA). Genotyping was performed using PCRrestriction fragment length polymorphism methods with appropriate primer sets (Supplementary table 1). The PCR conditions were as follows: (i) activation of Fast Taq polymerase (Chromus Biotech, Bangalore, India) at 94°C for 2 min, (ii) 35 cycles of denaturation at 94°C for 10 s; annealing (as per primer) for 20 s; elongation at 72°C for 5 s, and (iii) extension at 72°C for 5 min. Each PCR product was digested with an appropriate restriction enzyme (Fermentas Life Sciences, New Delhi, India) (Supplementary table 1) for 2 h at 37°C. The products were then resolved on a 3.5% agarose gel. Restriction fragment length polymorphism results were confirmed by DNA sequencing (Techno Concept Ind Pvt Ltd, New Delhi, India) of a few PCR products. HPV analysis
Genomic DNA was extracted from the above samples. HPV DNA testing was performed as described previously (Singh et al., 2009). Statistical analysis
The sample size for both patients and controls was calculated using QUANTO software, version 1.0 (http://hydra.usc. edu/gxe), and was found to be adequate. The sample size achieved less than 80% statistical power. χ2 analysis was used to assess deviation from Hardy–Weinberg equilibrium and to compare the genotype/allele frequency between the patients and the controls. Odds ratios (ORs) were obtained by unconditional logistic regression analysis and adjusted for age, age at first live birth, and smoking as a continuous variable. All statistical analyses were carried out using the SPSS software, version 15 (SPSS Inc., Chicago, Illinois, USA). The OR was calculated using unconditional logistic regression for risk genotypes with the wild-type genotype as a reference. Haplotype analysis was carried out using the PLINK software (http://pngu.mgh.harvard.edu/∼purcell/plink/).
Results Characteristics of the study population
A total of 110 patients (65 carcinomas and 45 SILs) and 68 controls were enrolled in this study. The characteristics of the study participants are shown in Table 1. There was no significant difference in age between the controls and the patients. The mean age of the cancer patients, SIL patients, and control women was 43, 42, and 43 years, respectively. The reproductive history, including age at menarche and age at first child birth, in all the groups was not significantly different, but the parities and use of oral contraceptives (OCP) differed significantly (Table 1). There were 13 (20%), 5 (11.1%), and 5 (7.3%) smokers in the carcinoma, SIL, and control groups, respectively. HPV infections in carcinoma and SIL patients were 94 and 90%, respectively, whereas in control women, the infection rate was only 17% (P < 0.001).
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226 European Journal of Cancer Prevention 2016, Vol 25 No 3
Table 1
Clinicopathological characteristics of SCC, SILs, and controls SILs
Variables Age (years) (mean ± SD) Age at menarche (years) (mean ± SD) Age at first live birth (years) (mean ± SD) Parity, (mean ± SD) Smoking status Nonsmoker Smoker Oral contraceptives Nonuser User HPV status Positive Negative
SCC
Controls n = 68 [n (%)]
n = 45 [n (%)]
P value
n = 65 [n (%)]
Pa value
43.2 ± 10.3 14.1 ± 0.4 23.5 ± 1.3 3.4 ± 1.2
41.2 ± 9.4 14.1 ± 1.2 22.8 ± 1.7 3.7 ± 1.0
0.298 0.972 0.027 0.113
43.9 ± 9.4 13.9 ± 1.4 22.9 ± 1.1 3.9 ± 1.3
0.651 0.324 0.013 0.014
63 (92.6) 5 (7.3)
40 (88.8) 5 (11.1)
0.491
52 (80) 13 (20)
0.033
50 (73.5) 18 (26.4)
29 (64.4) 6 (13.3)
0.303
28 (43.1) 37 (56.9)
0.303
12 (17.6) 56 (82.3)
40 (8.8) 5 (11.1)
0.001
61 (93.8) 4 (6.1)
0.001
a
HPV, human papillomavirus; SCC, squamous cell carcinoma; SILs, squamous intraepithelial lesions. Bold values indicates statistical significance. χ -test.
a 2
Genotype and allelic frequency distribution of XRCC1 gene polymorphisms and risk associated with SCC
Table 2
Controls (N = 68) [n (%)]
SCC (N = 65) [n (%)]
ORa (95% CI)
Controls (N = 68) [n (%)]
P value
SILs (N = 45) [n (%)]
ORa (95% CI)
P value
b
b
XRCC1-194 (Exon 6) (rs1799782) CC 44 (64.7) CT 11 (16.1) TT 13 (19.1) CT + TT 24 (35.2) Allele C 99 (72.7) Allele T 37 (54.4) XRCC1-280b (Exon 9) (rs25486) GG 48 (92.3) GA 7 (10.7) AA 3 (1.5) GA + AA 20 (12.3) Allele G 103 (6.6) Allele A 33 (93.3) XRCC1-399b (Exon 10) (rs25487) GG 23 (33.8) GA 33 (48.5) AA 12 (17.6) GA + AA 45 (66.1) Allele G 93 (71.5) Allele A 43 (66.1)
Genotype and allelic frequency distribution of XRCC1 gene polymorphisms and risk associated with SILs
Table 3
11 16 38 54 38 92
(16.9) (24.6) (58.4) (83.1) (29.2) (70.7)
Reference 11.5 (3.1–41.9) 20.1 (5.9–68.8) 16.0 (5.1–49.9) Reference 6.5 (3.7–11.5)
20 6 39 45 46 84
(30.7) (9.2) (60) (69.2) (35.3) (64.6)
Reference 2.0 (0.6–8.3) 7.4 (2.9–18.7) 5.4 (2.3–12.6) Reference 5.7 (3.2–10.1)
12 22 31 53 46 84
(18.4) (33.8) (47.6) (81.5) (35.3) (64.6)
Reference 0.9 (0.3–2.6) 4.2 (1.5–12.1) 1.9 (0.8–4.6) Reference 3.9 (2.3–6.8)
0.001 0.001 0.001 0.001
0.241 0.001 0.001 0.001
0.886 0.008 0.169 0.001
XRCC1-194 (Exon 6) (rs1799782) CC 44 (64.7) CT 11 (16.1) TT 13 (19.1) CT + TT 24 (35.2) Allele C 99 (72.7) Allele T 37 (54.4) XRCC1-280b (Exon 9) (rs25486) GG 48 (92.3) GA 7 (10.7) AA 13 (1.5) GA + AA 20 (12.3) Allele G 103(6.6) Allele A 33 (93.3) XRCC1-399b (Exon 10) (rs25487) GG 23 (33.8) GA 33 (48.5) AA 12 (17.6) GA + AA 45 (66.1) Allele G 93 (71.5) Allele A 43 (66.2)
13 19 13 32 45 45
(28.8) (42.2) (28.8) (71.1) (50) (50)
Reference 6.8 (2.4–18.6) 3.8 (1.3–10.6) 5.1 (2.2–12.1) Reference 2.7 (1.5–4.9)
30 11 4 15 71 19
(66.6) (24.4) (8.8) (33.3) (78.8) (42.2)
Reference 2.6 (0.9–7.7) 0.6 (0.2–1.9) 1.3 (0.6–3.0) Reference 0.8 (0.4–1.6)
14 23 8 31 51 39
(31.1) (51.1) (17.7) (68.8) (56.6) (43.3)
Reference 1.2 (0.5–3.0) 1.1 (0.4–3.6) 1.2 (0.5–2.8) Reference 1.6 (0.9–3.0)
0.001 0.012 0.001 0.001
0.082 0.351 0.552 0.581
0.617 0.813 0.635 0.073
CI, confidence interval; OR, odds ratio; SCC, squamous cell carcinoma. Bold values indicates statistical significance. a Adjusted for age at first live birth, parity, smoking status, and use of oral contraceptives. b Genotypic frequencies [N (%)].
CI, confidence interval; OR, odds ratio; SILs, squamous intraepithelial lesions. Bold values indicates statistical significance. a Adjusted for age at first live birth, parity, smoking status, and use of oral contraceptives. b Genotypic frequencies [N (%)].
Association of the BER gene polymorphism with SCC and SIL susceptibility
genotype (CT + TT) also showed an increased risk of SILs (P = 0.001, OR = 5.1) (Table 3).
The genotypic frequency distribution between SCC and controls is shown in Table 2 and that between SILs and controls is shown in Table 3. Our results indicated that the homozygous TT genotype of XRCC1 codon 194 and the AA genotype of XRCC1 codon 280 presented 20- and 7.4-fold, respectively, higher risk of cervical cancer. The variant genotype of XRCC1 codon 399 was associated with a 4.2-fold higher risk of SCC. The heterozygous genotype of only XRCC1 C194T presented a significant risk (Table 2). In SIL cases, the homozygous variant TT of exon 6 presented a 3.8-fold higher risk. The combined variant
Association of the NER gene polymorphism with SCC and SIL susceptibility
For ERCC1, the polymorphic variation occurs at codon 118, converting a common codon usage (AAC) into an infrequent one (AAT), both coding asparagine. We found no significant association between the ERCC1 118 polymorphism at both genotypic and allelic levels (Table 4). However, we found that in SIL cases, there was a significant difference between the variant homozygous TT genotype among SILs and controls [P = 0.010, OR = 0.3, 95% confidence interval (CI) = 0.1–0.7] (Table 5).
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DNA repair genes in cervical cancer Bajpai et al. 227
Genotype and allelic frequency distribution of ERCC1, ERCC2, and ERCC4 gene polymorphisms and risk associated with SCC
Table 4
Controls (N = 68) [n (%)]
SCC (N = 65) [n (%)]
ORa (95% CI)
P value
b
ERCC1-118 (Exon 4) (rs3212986) CC 14(20.5) 10 CT 15 (22.1) 6 TT 39 (57.3) 49 CT + TT 54 (79.4) 55 Allele C 43 (31.6) 26 Allele T 93 (68.3) 104 ERCC2-751b (Exon 23) (rs1052559) AA 52 (76.4) 27 AC 9 (13.2) 24 CC 7 (10.2) 14 AC + CC 16 (23.5) 38 Allele A 113 (83.1) 78 Allele C 23 (16.9) 52 b ERCC4-415 (Exon 8) (rs1800067) GG 45 (66.1) 8 GA 12 (17.6) 15 AA 11 (16.1) 42 GA + AA 23 (33.8) 57 Allele G 102 (75) 31 Allele A 34 (25) 99
(15.3) (9.2) (75.3) (84.6) (19.1) (76.4)
Reference 0.4 (0.1–1.6) 1.5 (0.5–4.4) 1.2 (0.4–3.4) Reference 1.8 (1.0–3.4)
(41.5) (36.9) (21.5) (58.4) (60) (40)
Reference 6.5 (2.3–18.1) 10.1 (2.6–37.9) 7.5 (3.0–18.9) Reference 3.3 (1.8–6.1)
(12.3) (23.1) (64.6) (87.6) (23.8) (76.1)
Reference 8.1 (2.4–27.2) 21.3 (7.1–64.0) 15.0 (5.6–40.5) Reference 9.6 (5.3–17.4)
0.191 0.404 0.688 0.031
0.001 0.001 0.001 0.001
0.001 0.001 0.001 0.001
CI, confidence interval; OR, odds ratio; SCC, squamous cell carcinoma. Bold values indicates statistical significance. a Adjusted for age at first live birth, parity, smoking status, and use of oral contraceptives. b Genotypic frequencies [N (%)].
In ERCC2, the polymorphism occurs at codon 751, exon 23. The homozygous variant genotype (CC) was associated significantly with risk of SCC. The heterozygous (AC) and the combined heterozygous and variant genotype (AC + CC) also presented a significantly increased risk of SCC. At the allelic level, the variant allele also posed a 3.3-fold higher risk of SCC (Table 4). In SILs, Genotype and allelic frequency distribution of ERCC1, ERCC2, and ERCC4 gene polymorphisms and risk associated with SILs
Table 5
Controls (N = 68) [n (%)] ERCC1-118b (Exon 4) (rs3212986) CC 14 (20.5) CT 15 (22.0) TT 39 (57.3) CT + TT 54 (79.4) Allele C 43 (31.6) Allele T 93 (68.3) b ERCC2-751 (Exon 23) (rs1052559) AA 52 (76.4) AC 9 (13.2) CC 7 (10.2) AC + CC 16 (23.5) Allele A 113 (83.1) Allele C 23 (16.1) ERCC4-415b (Exon 8) rs1800067 GG 45 (66.17) GA 12 (17.64) AA 11 (16.17) GA + AA 23 (33.8) Allele G 102 (75) Allele A 34 (25)
SILs (N = 45) [n (%)]
ORa (95% CI)
14 21 10 31 49 41
(31.1) (46.6) (22.2) (68.8) (54.4) (45.5)
Reference 1.2 (0.4–3.4) 0.3 (0.1–0.7) 0.5 (0.2–1.3) Reference 0.4 (0.2–0.7)
12 20 13 33 44 46
(26.6) (44.4) (28.8) (73.3) (48.8) (51.1)
Reference 9.6 (3.4–27.1) 8.9 (2.8–28.3) 9.3 (3.8–22.9) Reference 5.1 (2.7–9.9)
11 13 21 34 43 55
(24.4) (28.8) (46.6) (75.5) (47.7) (61.1)
Reference 4.4 (1.6–12.3) 7.8 (2.9–20.9) 6.9 (2.8–16.8) Reference 3.8 (2.1–7.0)
P value
0.674 0.010 0.171 0.001
0.001 0.001 0.001
In ERCC4, the polymorphic site is at codon 415, exon 8. We observed that the polymorphism in ERCC4 presented the overall highest risk of SCC. The homozygous variant genotype (AA) was associated significantly with the risk of SCC (P = 0.001 OR = 21.3, 95% CI = 7.1–64.0). The heterozygous (GA) and the combined heterozygous and variant genotype (GA + AA) also presented a significantly increased risk of SCC. At the allelic level, the variant allele (A) also posed a 9.6-fold higher risk of SCC (Table 4). In SILs, similar results to those for SCC were obtained (Table 5). Association of XRCC1, ERCC1, and ERCC2 haplotypes with SCC and SIL
To elucidate the combined influence of the polymorphisms in these three genes, we constructed XRCC1 Exon 6/9/10, ERCC1 Exon 4, and ERCC2 Exon 8 haplotypes (Table 6). The haplotype containing the wildtype allele of all the five polymorphisms was considered as a reference. The haplotype TAATC was most frequent in SCC (40%) and showed a four-fold increased risk, whereas in the SILs haplotype, TTGAC was predominantly present (21%). Haplotype TTAAC was present in all three study groups. Haplotype TTGGA was present only in patients with invasive cancer and in the controls. In logistic regression analysis, haplotypes TCAAA and TCGAA, being significantly lower in patients, appeared to be protective haplotypes. The Table 6 Haplotype analysis of XRCC1 (− 26304C/T, − 27466G/A, − 28152G/A), ERCC1 − 19007C/T, and ERCC2 − 35931A/C polymorphism, and cervical cancer and SIL risk Haplotype
Cancer
Control
Odds ratio (95% CI)
CCGGA TTAAC TTGGA TCGGA TTAAA TCAAA TTGAA TCGAA
20.01 40.02 7.36 7.97 17.38 0.26 6.04 0.93
31.86 14.08 1.95 23.95 5.75 3.85 4.15 14.4
Reference 4.08 (2.04–8.15) 3.99 (0.80–19.8) 0.27 (0.11–0.64) 3.44 (1.28–9.26) 0.06 (0.00–3.48) 1.48 (0.41–5.34) 0.05 (0.00–0.48)
Haplotype
SIL
Control
Odds ratio (95% CI)
CCGGA TTAAC TTGAC CTGGC TTAAA TCAAA TTGAA TCGAA TCGGA
49.14 23.75 21.64 4.6 0 0 0 0.85 0
33.07 13.36 1.37 0.77 5.54 3.52 5.73 12.06 24.55
Reference 2.01 (0.96–4.22) 19.88 (3.44–114.6) 6.21 (0.54–70.5) – – – 0.06 (0.00–0.57) –
P value ** * *
P value
0.001
0.004 0.001 0.001 0.001
CI, confidence interval; OR, odds ratio; SILs, squamous intraepithelial lesions. Bold values indicates statistical significance. Adjusted for age at first live birth, parity, smoking status, and use of oral contraceptives. b Genotypic frequencies [N (%)]. a
the homozygous variant genotype (CC) was associated significantly with the risk of SILs (P = 0.001; OR = 8.9; 95% CI = 2.8–28.3). The heterozygous (AC), the combined heterozygous and variant genotype (AC + CC), and also the variant allele (C) posed a higher risk of SIL (Table 5).
CI, confidence interval; SILs, squamous intraepithelial lesions. *Significant value. **Highly significant value.
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**
228 European Journal of Cancer Prevention 2016, Vol 25 No 3
haplotypes TTAAC, TTGAC, and TTAAA, with relatively higher frequencies in patients, appeared to be susceptible haplotypes. Analysis of the XRCC1, ERCC1, ERCC2, and ERCC4 gene polymorphism with HPV infection
We evaluated the gene–HPV interaction to study the modulation of SCC and the risk of SIL. Polymorphisms in XRCC1, ERCC1, ERCC2, and ERCC4 were associated significantly with HPV infection in SCC, SILs, and control samples as well (Table 7). There was more than a 95% association (Table 7) of HPV infection in the homozygous variant genotype in all three groups (i.e. SCC, SIL, and controls). In control samples, there was a uniform increase in individuals with HPV infection compared with among wild-type, heterozygous, and polymorphic homozygous genotypes of all the genes (Table 7). The maximum association was observed in individuals who carried a polymorphism in XRCC1 codon 194 and 280. In future, this group of the control population can be followed up to determine whether eventually they develop cervical precancer or frank cancer. Combined analysis of polymorphisms in all the genes across groups
For those individuals with a variant in more than one gene, there was a 9.8-fold increased risk of SCC (OR = 9.8, 95% CI = 3.0–31.8). In SIL cases, the frequency of those carrying a polymorphism in more than gene was comparatively low and, hence, the associated risk was not significant (P = 0.954) (data not shown).
Discussion Cervical cancer remains a major cause of cancer deaths in women worldwide (Waggoner, 2003). Currently, no Table 7
biological or genetic markers are available to predict which precancerous lesion will progress to invasive cervical cancer. Although infection with high-risk HPV is recognized to be an essential initiating event in cervical tumorigenesis, this alone is not sufficient for progression to invasive cancer (Zur Hausen, 2002). Despite the recent progress in understanding the molecular aspects of cervical cancer, the genetic basis of progression of precursor SILs to invasive cancer in the multistep progression of cervical cancer remains poorly understood (Gius et al., 2007). Therefore, identification of other ‘genetic hits’ in cervical cancer is important to understand its biology. Studies carried out over the past few years have identified variant alleles for a number of DNA repair genes, some of which may modify DNA repair capacity. With limited sample size, our current data suggest that amino acid substitution variants of DNA repair genes involved in base excision repair and NER pathways may contribute to cervical cancer pathogenesis. In the present study, we genotyped four common polymorphisms of the BER gene XRCC1, and NER genes ERCC1, ERCC2, and ERCC4, and tested the association between the distributions of their genotypes with cervical precancer and invasive cancer. These polymorphisms have been shown to have functional significance and may in be part responsible for the interindividual difference in the capacity of DNA repair in the general population and for low DNA repair efficacy in cancer patients (Kohno et al., 2006). Infection with the oncogenic types of HPV has been established as a main cause of cervical cancer (Singh et al., 2009) and SILs (Cuzick et al., 1994). We also found a significant association of HPV infection both in SIL and in SCC cases. An association between smoking and cervical cancer has been reported (Winkelstein, 1990), as
Association of HPV positivity in DNA repair genes in controls, SILs, and SCC Heterozygous (% HPV positive)
Polymorphic homozygous (% HPV positive)
P valuea
0.0 2.1 4.4 0.0 8.2 8.9
27.3 28.6 21.2 6.7 21.2 41.7
92.3 92.3 58.3 35.9 38.6 54.6
0.001 0.001 0.001 0.005 0.029 0.001
92.3 90.0 92.9 100.0 91.7 90.9
100.0 90.9 86.0 85.7 100.0 100.0
69.2 75.0 87.5 80.0 69.2 80.9
0.017 0.570 1.000 0.220 0.014 0.238
90.9 90.0 91.7 70.0 85.2 62.5
81.3 83.3 90.9 100.0 100.0 93.3
100.0 97.4 96.8 97.0 100.0 100.0
0.016 0.188 0.850 0.013 0.068 0.001
Controls Wild type (% HPV positive) XRCC1 codon 194 XRCC1 codon 280 XRCC1 codon 399 ERCC1 codon 118 ERCC2 codon 751 ERCC4 codon 415 SILs XRCC1 codon 194 XRCC1 codon 280 XRCC1 codon 399 ERCC1 codon 118 ERCC2 codon 751 ERCC4 codon 415 SCC XRCC1 codon 194 XRCC1 codon 280 XRCC1 codon 399 ERCC1 codon 118 ERCC2 codon 751 ERCC4 codon 415
HPV, human papillomavirus; SCC, squamous cell carcinoma; SILs, squamous intraepithelial lesions. Bold values indicates statistical significance. χ (comparison between genotypes).
a 2
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DNA repair genes in cervical cancer Bajpai et al. 229
well as smoking-related DNA damage in the cervical epithelium (Simons et al., 1993). In our study, we found that smoking posed a significant risk (P = 0.033) in SCC, whereas no significant association was found in SILs. Long-term use of OCP could be a cofactor that increases the risk of cervical carcinoma by up to four-fold in women who are positive for cervical HPV DNA (Moreno et al., 2002). We found a significant number of OCP users in SCC (57%) compared with only 26% among the controls. However, in SILs, OCP use was not associated significantly with the disease (P = 0.303). High parity, as evident in studies by Jensen et al. (2013), seems to increase the risk of SCC of the cervix among HPVpositive women. In our study, high parity was also associated significantly with SCC cases (P = 0.013), but we found no significant difference in SIL cases. Our study also assessed the difference in age at first live birth in SCC, SILs, and controls. There was a significant difference between SCC cases and controls (P = 0.013), whereas the difference in age at first live birth was not statistically significant between SILs and controls. XRCC1 is one of the > 20 genes that participate in the base excision repair pathway. A large number of molecular epidemiologic studies have been carried out to evaluate the role of the Arg399Gln polymorphism in the risk of cancer; however, the results remain conflicting rather than conclusive. The present study indicated that the XRCC1 Exon 6 (C > T) polymorphism was associated with both cervical precancer and cancer. Detailed metaanalysis by Shuai et al. (2012) showed that the variant 194Trp allele was associated significantly with an increased risk of cervical cancer. They also reported similar findings in a subgroup of an Asian population. To the best of our knowledge, this is the first study that has investigated the association of XRCC1 polymorphisms and cervical precancer and cancer risk. No association was observed between the XRCC1 Exon 10 (G > A) polymorphism and cervical precancer risk. In the case of a polymorphism at Exon 6 (C > T) and Exon 9 (G > A), a significantly increased risk for homozygous genotypes TT and AA, respectively, to have cervical cancer and precancer was observed compared with the controls by logistic regression analysis. To date, relatively few studies have been carried out to examine the association between the Arg280His variant and the risk of cancer and altered DNA adducts. Our observation is in agreement with the findings of Kang et al. (2007), in which the Arg280His polymorphism was associated significantly with SCC of the skin in a Korean population. A previous study showed that the polymorphism of ERCC1 was associated with the prognosis of patients receiving chemotherapy in human gastric, cervical, colorectal, non-small cell lung cancer, and bone cancer (Kwon et al., 2007). In our study, we did not observe any association between the ERCC1 118C/T polymorphism and the risk of cervical precancer and cancer. Han et al.
(2012) also reported that the C19007T polymorphism in ERCC1 might not be associated with the risk and invasiveness of cervical cancer in Korean women. The polymorphism in ERCC2 codon Lys751Gln is reported to be a risk allele and patients with the Gln allele genotype have a significantly increased risk of developing lung cancer (Zhan et al., 2010). The Gln/Gln variant of the ERCC2 correlated with a higher risk of skin, glioma, and breast cancer. To date, no studies have addressed the association between alterations in this region of the ERCC2 gene and SIL. Our data support the previous results that indicated that homozygous individuals with the Gln/Gln genotype had an increased risk compared with carriers of the Lys/Lys genotype. Several studies have investigated the associations between the ERCC4 polymorphisms and the risk of cancers, including cancer of the breast, lung, head and neck, skin, pancreas, and bladder, but the results are not consistent. An association was found between the Arg415Gln polymorphism and the risk of lung cancer (adjusted OR = 2.11, 95% CI = 0.9–2.5; Arg/Gln vs. Arg/ Arg) in Koreans (Zhan et al., 2010). We observed that both polymorphic homozygous and heterozygous genotypes of ERCC4 were associated significantly with increased cervical precancer and cancer risk. In a multistep, multigenic process such as carcinogenesis, SNP in a single gene is unlikely to alter the expression or the function of specific proteins to the extent of producing a pathological phenotype. Various studies have shown groups of SNPs within an individual gene to be associated with cervical cancer (Shao et al., 2008). Therefore, haplotype analysis of XRCC1, ERCC1, and ERCC2 was carried out. Two of the haplotypes, TTAAC and TTAAA, showed a higher risk for cervical cancer. Similarly, one haplotype, TTGAC, showed a statistically increased risk for SILs. These findings indicated that the combined genotype was associated with cervical cancer as well as precancer risk. Our findings on an association between XRCC1, ERCC1, and ERCC2 haplotypes and the risk of cervical cancer and precancer further strengthen our observation that polymorphisms in XRCC1, ERCC1, and ERCC2 may aid understanding of the development of the disease. On comparing the prevalence of the different polymorphisms in the study population, it became evident that more than one gene variant occurred in a considerable number of individuals, but because of the limited sample size, we cannot comment whether the risk associated with combined polymorphism was because of linkage among the DNA repair genes. Furthermore, we observed that in the control population, HPV infection increased from the wild-type genotype to the heterozygous and was maximum in the polymorphic homozygous genotype. As such individuals were exposed to major risk factors, a follow-up of the same could show whether they progress to cervical cancer. However, the
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230 European Journal of Cancer Prevention 2016, Vol 25 No 3
association of HPV and genotypes in the patient population did not show any trend. This could be because of already high baseline HPV positivity in the patient samples. Our study also showed that both blood and tumor samples can act as sources to detect polymorphism in DNA repair genes as findings of both blood and tissue samples matched 100%. Therefore, this interesting finding of a complete correlation between tissue and blood samples suggests that gene polymorphism in blood may be used as a noninvasive method to evaluate the risk of cervical cancer. Additional work is required to determine whether polymorphisms do indeed lead to a reduced DNA repair capacity in vivo and to identify the effects of these phenotypes in different environmental conditions. The identification of polymorphisms in many genes and the determination of their functional importance in cervical cancer will enable the design of susceptibility–risk models for de-novo and therapy-related disease. In summary, the present study suggested that XRCC1, ERCC2, and ERCC4 SNPs act as predisposing factors in cervical precancer and increased risk of invasive cervical cancer. This perhaps indicates that reduced DNA repair capacity of these gene polymorphisms modulated the SCC and SILs. Given that the sample size of our study was comparatively small, modest effects cannot be ruled out. However, like most association studies, larger studies involving more SNPs in one gene or genes in the same biologic pathway are warranted to replicate the study in different populations.
Acknowledgements The authors are grateful to all the study participants, without whom this study would not have been possible. Deepti Bajpai is grateful to the Indian Council of Medical Research for providing the Senior Research Fellowship. Conflicts of interest
There are no conflicts of interest.
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