Polymorphisms of base-excision repair genes and the hepatocarcinogenesis

Accepted Manuscript Polymorphisms of base-excision hepatocarcinogenesis

repair

genes

and

the

Manar-Aleslam M. Mattar, Abdel-Rahman N. Zekri, Nehal Hussein, Heba Morsy, Gamal Esmat, Magdy A. Amin PII: DOI: Reference:

S0378-1119(18)30712-1 doi:10.1016/j.gene.2018.06.056 GENE 42989

To appear in:

Gene

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29 March 2018 6 June 2018 18 June 2018

Please cite this article as: Manar-Aleslam M. Mattar, Abdel-Rahman N. Zekri, Nehal Hussein, Heba Morsy, Gamal Esmat, Magdy A. Amin , Polymorphisms of base-excision repair genes and the hepatocarcinogenesis. Gene (2018), doi:10.1016/j.gene.2018.06.056

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ACCEPTED MANUSCRIPT Title: Polymorphisms of base-excision repair genes and the hepatocarcinogenesis Manar-Aleslam M. Mattar1, Abdel-Rahman N. Zekri2*, Nehal Hussein2, Heba Morsy2, Gamal Esmat4, Magdy A. Amin3 Clinical Pharmacy department, National Cancer Institute, Cairo University, Cairo, Egypt.

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Virology and Immunology Unit, Cancer Biology Department, National Cancer Institute,

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Cairo University, Cairo, Egypt.

Department of Endemic Medicine and Hepatology, Faculty of Medicine, Cairo University

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Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.

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Email:

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Manar Aleslam M. Mattar, B.Sc.: [email protected] Abdel-Rahman N Zekri, PhD: [email protected] Nehal Hussein Msc: [email protected]

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Heba Morsy Msc: [email protected] Gamal Esmat, PhD: [email protected] Magdy A. Amin, PhD: [email protected]

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*Correspondence to: Abdel-Rahman N Zekri, Ph.D., Molecular Virology and Immunology

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Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Kasr Al-Aini St., Fom El-Khaleeg, Cairo 11976, Egypt. [email protected] Email:

Abdel-Rahman N Zekri: [email protected] List of Declarations: Conflict of interest Declaration: The Authors declare that there is no conflict of interest.

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ACCEPTED MANUSCRIPT Funding: This work was supported by Science and Technology Development Fund #5193 to ARNZ and Egypt National Cancer Institute. Ethical approval: The study was ethically approved by the Institutional Review Boards (IRB) of the National

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Cancer Institute, Cairo University. Organization No.IORG0003381. (IRB NO.IRB00004025)

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ACCEPTED MANUSCRIPT Abstract Aim: To determine the possible association between polymorphisms of DNA repair genes, including XRCC1 Arg194Tryp, Arg280His, and Arg399Glu, APE1 Asp148Glu, and NEIL2 Arg257Leu, and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Methods: A total of 264 subjects were recruited in this retrospective case-control study and

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were categorized into four groups: 88 control subjects (CR), 53 chronic hepatitis C patients (CHC), 36 liver cirrhotic patients (LC), and 87 HCC patients. The XRCC1 Arg194Tryp,

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Arg280His, and Arg399Glu polymorphisms were detected using PCR-RFLP, while real-time

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PCR was used to genotype APE1 Asp148Glu and NEIL2 Arg257Leu.

Results: Our data revealed that, compared with the healthy controls, for those subjects with

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the XRCC1 Arg194Trp genotype, the risk of developing CHC, LC, and HCC was increased by 6.66- (odds ratio (OR)=6.667; 95% confidence interval (CI)=3.244-13.701; P>0.01), 3.85(OR=3.852; 95% CI=1.797-8.256; P>0.01), and 2.14-fold (OR=2.14; 95% CI=1.13-4.06;

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P>0.05), respectively. There was no association between the risk of HCC development and the XRCC1 Arg280His or XRCC1 Arg399Gln genotypes. Moreover, the analysis showed a

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lack of association between APE1 Asp148Glu and the risk of HCC development. The analysis of clinicopathological parameters showed that the HCC patients with the XRCC1

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Arg280His polymorphism were 2.9 fold more likely to have hepatic lesions in both hepatic lobes (OR: 2.9; 95% CI: 1.15–7.29). Notably, in the HCC patients, the prevalence of the APE1 polymorphism in the males was four times higher than that in the females (OR=4; 95%

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CI=1.129-14.175; P>0.05).

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Conclusion: Our results indicate that the XRCC1 Arg194Trp polymorphism could be a risk factor for HCV-related HCC development in Egypt.

Keywords: Polymorphism, HCC, XRCC1, APE1, NEIL2, Base Excision DNA repair.

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ACCEPTED MANUSCRIPT 1. Introduction Hepatocellular carcinoma (HCC) ranks fifth and seventh among all malignancies in men and women, respectively, and is the third most common cause of mortality among cancer patients worldwide [1]. Hepatitis C virus (HCV) remains a common risk factor for HCC, while other risk factors include aflatoxin, alcoholism and hepatitis B virus [2,3] Egypt is a country with a

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high prevalence of HCV [1]. Most HCV-infected patients (55–85%) develop chronic

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infection and exhibit an increased risk of developing liver cirrhosis or HCC several years

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after initial infection [4]. HCV induces HCC through various mechanisms, primarily involving chronic inflammation, which causes oxidative stress. In particular, the HCV core

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and NS5A proteins increase the formation of reactive oxygen species (ROS) and nitrogen species in vitro, leading to chromosomal damage [5,6]. Various DNA repair systems correct

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DNA damage and prevent cancer development [7]. However, if these damages exceed the repair capacity, it will produce somatic mutations. Also, when these mutations occur in genes

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responsible for genetic stability, they result in a cascade of mutations and cancer development

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[8]. The primary defence mechanism for lesions generated by alkylating agents, ionizing radiation, is base excision repair [9]. X-ray repair cross-complementing group 1 (XRCC1),

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AP endonuclease 1 (APE1) and Nei-like 2 (NEIL2) are the major enzymes involved in base excision repair. Although the genetic variations of the genes of these enzymes like non-

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synonymous single nucleotide polymorphisms (SNPs) that result in amino acid alteration at the

mutated site, may cause changes in the structure or the function of the mutated protein [10]. The presence of these SNPs in the repair genes could cause reduction of the DNA repair capacity that can result in higher mutation rate and increased the risk of cancer [11]. XRCC1 is a repair protein that participates in the repair of individual damaged bases and single-strand breaks. XRCC1 has no recognized enzymatic activity but forms a complex with PARP, ligase III and β-pol [12], and the encoding gene of XRCC1 is located on chromosome 19q13.2. The 4

ACCEPTED MANUSCRIPT most common three variants that extensively studied in XRCC1 are Arg194Trp on exon 6, Arg280His on exon 9 and Arg399Gln on exon 10. These polymorphisms may affect the XRCC1 activity, reduce repair kinetics, and influence susceptibility to disease like cancer [10]. The AP endonuclease 1 (APE1) gene has been mapped to chromosome 14q11.2–q12 and encodes a repair enzyme that plays a crucial role in the BER pathway by cleaving

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apurinic/apyrimidinic (AP) sites resulting from the incision of a damaged base. This process

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leaves a free 3' hydroxyl group and 5' deoxyribose phosphate group, which facilitates gap

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filling to complete the repair [12]. The most common sequence variant identified in APE1 is a G/T alteration in exon 5, which causes a change from aspartic acid to glutamic acid

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(Asp148Glu). This substitution does not lead to a defect in the endonuclease activity of APE1 but may reduce its communication with other enzymes, leading to decreased BER efficiency

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[13]. Human NEIL2 is a DNA glycosylase involved in repairing oxidized bases from transcribed genes. There are two common variants in NEIL2, among them Arg257Leu

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polymorphism results in a lower repair capacity compared with the wild type due to a lower

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affinity for other repair proteins, especially Polβ [14]. Genetic polymorphisms of those genes may decrease their DNA repair efficiency [15]. Therefore, current studies are focused on

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studying the effect of single-nucleotide polymorphisms (SNPs) in DNA repair genes, due to their crucial role in maintaining genome integrity [12].

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To the best of our knowledge, there have been no previous studies of the genotype distribution and association of XRCC1, APE1, and NEIL2 with HCV-associated HCC in Egypt.

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ACCEPTED MANUSCRIPT 2. Patients and methods 2.1.

Study design and grouping

This retrospective case-control study was approved by the Investigation and Ethics Committee of the National Cancer Institute (NCI). Two hundred sixty-four subjects were

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enrolled in this study and were categorized into four groups. Group 1 consisted of healthy individuals, serving as controls (CR, n=88), and were recruited from the relatives of patients

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attending the NCI of Egypt. These individuals were negative for HCV and HBV antibodies,

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as confirmed by enzyme-linked immunosorbent assay (ELISA) and PCR and their liver enzymes were normal. Group 2 consisted of chronically infected HCV patients without

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cirrhosis (CHC, n=53), as verified by ELISA and PCR. These patients were characterized by

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a persistent increase in alanine aminotransferase (ALT) to levels three-fold greater than normal levels for at least six months. Group 3 consisted of liver cirrhotic patients (LC, n=36)

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diagnosed by abdominal ultrasonography. Group 4 consisted of HCV related HCC patients

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(n=87) recruited from the NCI of Egypt. The HCC patients were diagnosed via abdominal ultrasonography, triphasic CT of the abdomen and serum AFP level and were confirmed via histopathology. All patients included in groups 2, 3, 4 were positive for HCV antibodies, as

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confirmed by ELISA and PCR. Informed consent was obtained from all subjects before their

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involvement in the study. The clinicopathological characteristics of the study groups are summarized in Table 1.

2.2.

Sample collection and DNA Isolation

Human whole blood was collected in EDTA-coated tubes for genomic DNA extraction using the QIAamp DNA Blood Mini Kit (Cat. No. 51104) according to the manufacturer’s instructions. DNA samples were stored at -80°C until use.

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ACCEPTED MANUSCRIPT 2.3. Genotyping SNPs in DNA repair genes were genotyped using either polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) or the TaqMan allelic discrimination assay. 2.3.1 PCR-RFLP

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The PCR primers and specific conditions (annealing temperature, and restriction enzymes)

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for XRCC1 polymorphisms Arg194Trp, Arg280His, and Arg399Gln are illustrated in table 2.

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The analysis of XRCC1 SNPs (Arg194Trp (rs1799782) codon 194; Arg280His (rs25489) codon 280; Arg399Gln (rs25487) codon 399) was performed in a final volume of 25 µl,

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containing 20 ng of genomic DNA, 1X buffer, 10 µm each dNTP, 0.2 µM primers, and 5 U/ml Taq DNA polymerase. The cycling conditions were as follows: 94ºC for 5 min for

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initial denaturation, followed by 35 cycles of 95ºC for 30 s, an appropriate annealing temperature for 30 s and then a final extension at 72ºC for 10 min. The amplified DNA

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product was treated with a particular restriction enzyme and separated in a 3% agarose gel,

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which was stained with ethidium bromide for UV visualization, the RFLP pattern of XRCC1 Arg280His is showed in Fig. (1).

TaqMan allelic discrimination assay

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2.3.2

SNPs of NEIL2 (Arg257 Leu (rs8191664)) and APE1 SNP (Asp148Glu (rs1130409)) were

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studied using the TaqMan allelic discrimination assay (Applied Biosystems, Assay-on Demand, SNP genotyping products: C_25987256_10 for NEIL2 and C_ 8921503_10 for APE1)[16]. TaqMan probes used to genotype both NEIL2 Arg257Leu and APE1 Asp148Glu are showed in table 2. Amplification was performed using the 7500 real-time PCR system with the following cycling conditions: 95ºC for 10 min, 92ºC for 15 s, and 60ºC for 1 min for 40 cycles. Fig 2 showed the results of TaqMan allelic discrimination assay for APE1 Asp148Glu on the basis of fluorescence signal intensity of FAM and VIC.

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2.4.

Statistical analysis

Statistical analysis was performed using IBM© SPSS© Statistics version 22 (IBM© Corp., Armonk, NY, USA). The Kolmogorov–Smirnov test was used as a test for normality.

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Continuous data were expressed as the mean and standard deviation, or median and range when appropriate. Categorical data were expressed as the frequency and percentage. Analysis

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of variance (ANOVA) followed by the post hoc Tukey HSD test was employed to analyse

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normally distributed continuous data. For continuous data that were not normally distributed, the Kruskal-Wallis test was used, followed by the post hoc Scheffe test for pair-wise

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comparisons based on the Kruskal-Wallis distribution. The chi-square test was employed to

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examine the relationship between categorical variables. The odds ratio (OR) with a 95% confidence interval (CI) was used for risk estimation. All tests were two-tailed. P value<0.05

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was considered significant.

3. Results

The clinicopathological characteristics of the patients and the control subjects

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3.1.

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are presented in Table 1. The mean age of the patients with HCC and liver cirrhosis (58 years) was significantly higher than that of the chronic hepatitis patients (50.2 years) and the control subjects (43.6 years) (P<0.001), in accord with the fact that cirrhosis and HCC develop in older age. The higher percentage of males (75.9%) than that of females (24.1%) in the HCC group demonstrates the male predominance of HCC in the Egyptian population. In addition, the level of alpha-fetoprotein was significantly higher in the HCC group than that in the HCV and control groups (P<0.001). 8

ACCEPTED MANUSCRIPT 3.2.

.Primary observations of the genotypic distribution of XRCC1, APE1, and

NEIL2 SNPs in Egypt The genotype distribution of various polymorphisms XRCC1 (Arg194Trp, Arg280His and Arg399Gln), APE1Asp148Glu and NEIL2 Arg257Leu are elucidated in Table 3. Among all

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the groups, the most frequent genotype for the XRCC1 Arg280His was Arg280Arg (CR, 47.7%; CHC, 54.7%; LC, 72.2%; HCC, 58.6%), while that for the APE1Asp148Glu was

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Asp148Glu (CR, 51.1%; CHC, 47.2%; LC, 63.9% %; HCC, 48.3%).

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The least frequent genotype among all the groups for XRCC1 Arg280His was His280His

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(CR, 10.2%; CHC, 7.5%; LC, 5.6%; HCC, 13.8%), while that for Arg399Gln was Arg399Arg (CR, 3.4%; CHC, 11.3%; LC, 13.9%; HCC, 4.6%), and least frequent genotype

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among all the groups for APE1 was Asp/Asp (CR, 19.3%; CHC, 20.8%; LC, 16.7%; HCC, 13.8%). Wild-type Arg257Leu (NEIL2) was observed in all the groups (100%).

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For the XRCC1 Arg194Trp, Arg194Arg was the most frequent genotype in the control

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(72.7%) and HCC (56.3%) groups. The Arg194Trp genotype was found to present at the highest frequency in the CHC (84.9%) and LC (72.2%) patients. Trp194Trp was only found

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in the HCC group (3.4%). For codon Arg399Gln, Arg399Gln was the most frequent genotype among the control (67.0%), CHC (56.6%) and HCC (65.5%) groups. In addition, Gln399Gln

3.3.

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was the most common genotype among the LC patients (47.2%). ORs of the various genotypes of XRCC1 codons (194, 280, and 399) and

APE1 Asp148Glu to induce CHC, LC and HCC using the controls as a reference The risk factors exerted by the various genotypes of XRCC1 codons (194, 280, and 399) and APE1 Asp148Glu for the development of HCV-related liver disease are illustrated in Table 3.

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ACCEPTED MANUSCRIPT According to our data for the CR group, the heterozygous genotype of XRCC1 Arg194Trp polymorphism increased the risk of CHC 6.66-fold (OR=6.667; 95% CI=3.244-13.701; P>0.01), the risk of LC 3.85-fold (OR=3.852; 95% CI=1.797-8.256; P>0.01), and the risk of HCC 2.14-fold (OR=2.14; 95% CI=1.13-4.06; P>0.05).

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The heterozygous genotypes of XRCC1 Arg280His and Arg399Gln polymorphisms were found to be protective for LC risk in healthy controls (OR=0.40; 95% CI=0.17-0.91; P>0.05)

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(OR=0.12; 95% CI=0.02-0.53; P>0.01). However, there was no association between these

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genotypes and liver cancer (OR=0.53; 95% CI=0.27-1.03; P˃0.05) (OR=0.72; 95% CI=0.153.38; P˃0.05).For the APE1 Asp148Glu polymorphism, both the homozygous and

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heterozygous genotypes exhibited a non-significant association with HCC (OR: 1.79; 95%

3.4.

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CI: 0.73–.42), OR: 1.32; 95% CI: 0.56–.09) in the CR subjects. Association between XRCC1 (194, 280, and 399) and APE1 Asp148Glu

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polymorphisms and clinico-pathological parameters of the HCC subjects

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The association between XRCC1 and APE1 genotypes and the different parameters of HCC patients are presented in tables (4, 5, 6 and 7).The parameters analysed for their association

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with HCC susceptibility were age, gender, lobe, number of lesions and alpha fetoprotein (AFP) levels. AFP serum level is a reliable biomarker for the diagnosis of HCC especially if

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it is combined with imaging approaches. Also, it is used in HCC early detection as high serum level of AFP usually indicates a high risk for the development of HCC [17].The HCC patients carrying the XRCC1 Arg280His polymorphism were 2.9-fold more susceptible to have hepatic lesions in both hepatic lobes (OR: 2.9; 95% CI: 1.15–7.29; P>0.05). In addition, the prevalence of APE1 polymorphisms was four fold higher in the males than that in the females among the HCC patients (OR: 4; 95% CI: 1.129-14.175). No P value was calculated

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ACCEPTED MANUSCRIPT for XRCC1399 because there were only four patients within the wild-type subgroup of HCC patients, which is considered a very small number for this calculation. 4. Discussion SNPs in critical repair genes may affect enzyme activity and alter cancer susceptibility. Previous studies have demonstrated the effect of genetic polymorphisms in BER genes and

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their correlation with the risk of various malignancies [12,18–21] A few case-control studies

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have focused on the association between SNPs in several DNA repair genes and the risk of

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HCC, with conflicting results [22]. Therefore, we examined the association between HCC risk and common polymorphisms in XRCC1 Arg194Tryp, Arg280His, and Arg399Glu,

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APE1 Asp148Glu and NEIL Arg257Leu in Egypt. The XRCC1 gene encodes a pivotal scaffold protein in the BER pathway. The most common non-synonymous SNPs of the

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XRCC1 gene, such as XRCC1 Arg194Trp, Arg280His, and Arg399Gln, alter the amino acid sequence and may affect protein activity [9]. Previous studies have shown that those

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polymorphisms were associated with an increased risk for the development of several types

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of cancers, including HCC [18,23–25]. The Arg399Gln polymorphism produces significant conformational changes at several sites in the BRCT1 domain, and these changes may be

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critical for protein-protein interactions [26]. Irradiation-specific DNA repair rates are significantly decreased in individuals with the XRCC1 Gln399Gln genotype compared with

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those in individuals harbouring the wild-type genotype [27]. Our results showed a higher frequency of the heterozygous variant XRCC1 Arg194Trp in HCC group (40.2%) when compared to the control subjects (27.3%), which comes in agreement with earlier report by Yang et al. in Chinese population [28]. However, lower frequency were observed in Indian population reported in Kiran et al. study [9]

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ACCEPTED MANUSCRIPT Besides, a significant association was showed between the heterozygous Arg194Trp variant and HCC risk in the healthy controls. This result was consistent with earlier reports indicating such an association with the risk of non-small-cell lung cancer NSCLC [29,30] cervical cancer [31], gastric cancer in an Asian population [32], and colorectal cancer (CRC) in a Kashmiri population [33]. In contrast to our results, Huang et al. showed no association

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between the Arg194Trp genotype and the risk of CRC with HCC development in a Chinese

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population [34,35]. The XRCC1 Arg280His genotype distribution was consistent with

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previous results illustrated in Yuan et al. study [2] and was higher than that studied in Americans [36]. The same genotype showed no influence on the development of HCC.

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Similar to our findings, earlier studies showed the same lack of an association with the risk of CRC [34], gastric cancer [37], and pancreatic cancer [38]. In addition, Tao et al. reported the

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absence of an association between HCC susceptibility and the XRCC1 Arg280His genotype [2].

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In addition, genotype distribution of XRCC1 Arg399Gln polymorphism in Egypt (65%), was

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higher compared to Italy [39], Brazil [40] Turkey [41] and in Northern Spain [42].However, there was no association detected between the Arg399Glu genotype and HCC risk. This result

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was supported by another study from India [43] showing that the XRCC1 Arg399Glu genotype was not associated with HCC. Many previous studies have indicated a similar lack

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of association between this genotype and other malignancies, including colorectal cancer [15], cervical cancer [31], gastric cancer [37], and pancreatic cancer [38]. Furthermore, earlier reports on APE1 Asp148Glu frequency distribution were compatible with our observation [44,45] while had a lower frequency distribution reported by Yang and Zhao [28,46]. Moreover, our data indicated that the APE1 polymorphism was not associated with the risk of HCC development. In accord with our results, earlier reports by Xiaoyun et al. [47], Paul et al. [48], and Misra et al. [49] found no association between the Asp148Glu

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ACCEPTED MANUSCRIPT polymorphism and cancer susceptibility. Other authors have reported an apparently decreased cancer risk associated with this polymorphism [45,50] Regarding the NEIL Arg257Leu polymorphism, frequency distribution analysis showed that all the groups exhibited the wild-type genotype NEIL2 Arg257Arg. Contrary to our results, Sanjib Dey et al. found that the Arg257Leu variant was frequently present in lung cancer

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patients. The Arg257Leu polymorphism results in a lower repair capacity compared with the

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wild type due to a lower affinity for other repair proteins, especially Polβ [14].

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The differences between our results and other studies may be due to ethnic variation, and environmental exposure also plays a vital role in the distribution of genetic polymorphisms.

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Moreover, the difference may be due to the comparison between different cancers or due to the small sample size, which suggests future studies involving large sample sizes and more

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ethnic groups may confirm our observations. In conclusion:

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Our results suggest that the Arg194Trp polymorphism in XRCC1 gene is significantly

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associated with the risk of HCV associated HCC development in the Egyptian population. However, further studies on a larger number of patients are needed to confirm our data before

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this polymorphism can be employed as a biomarker for HCC.

Competing interests:

We declare that we don’t have any competing interests. Acknowledgements:

The authors are deeply grateful to Dr Amira Salah El-Din Youssef and Nature Research Editing Service for reviewing my manuscript for English language. This work was supported

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ACCEPTED MANUSCRIPT by Science and Technology Development Fund #5193 to ARNZ and Egypt National Cancer Institute. Author contributions: AZ and MMM designed the experiments while MMM, HM and NH performed all

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experiments (PCR RFLP and RT PCR). AZ, GE, MA, and MMM carried out interpretation of the results. AZ and MMM conceived of the experiments. All authors participated in

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coordination and helped to draft the manuscript. All authors read and approved the final

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manuscript.

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ACCEPTED MANUSCRIPT Abbreviations list:

AFP

hepatitis C virus

HCV

chronic hepatitis C

CHC

liver cirrhotic

LC

control

CR

odds ratio

OR

confidence interval

CI

RI

PT

Alpha-fetoprotein

HCC

SC

hepatocellular carcinoma reactive oxygen species

ROS

AC

CE

PT E

D

MA

NU

single-nucleotide polymorphisms

21

SNPs

ACCEPTED MANUSCRIPT Table 1: Clinicopathological characteristics of the study subjects

Parameters

CR

CHC

LC

HCC

(n=88)

(n=53)

(n=36)

(n=87)

58.6±7.2

58.1±8.7 (27-78)

P-value

Age 43.6±9.9

50.2±10.8

Range

(27-72)

(21-69)

(40-69)

50 (56.8%)

32 (60.3%)

23 (63.8%)

66 (75.9%)

21 (39.6%)

13 (36.1%)

21 (24.1%)

Female

38 (34.2%)

SC

Male

4.3

5

Range

(1-10)

(1-87)

NU

AFP Median

RI

Sex

PT

Mean ± SD

10

42

(2-146)

(2.5-9538.0)

<0.001

<0.001

AC

CE

PT E

D

MA

Control: CR, Chronic Hepatitis C: CHC, Liver Cirrhotic: LC, Hepatocellular Carcinoma: HCC, Alphafetoprotein (AFP)

22

ACCEPTED MANUSCRIPT

Table 2: PCR primers and conditions used for XRCC1 codons (194, 280, and 399), and Taqman assay probes for both APE1 Asp148Glu and NEIL2 Arg257Leu

Forward primer

Reverse primer

(5ʼ -3ʼ)

XRCC1 Arg399Gln

XRCC1 Arg194Trp

XRCC1 Arg280His

APE1 Asp148Glu probe

NEIL2 Arg257Leu probe

T P

I R

Annealing

PCR product

Restriction

RFLP products

(5ʼ -3ʼ)

temperature (°C)

(bp)

enzyme

(bp)

GCATCGTGCGTAAGGAGTG

CCTTCCCTCATCTGGAGTC

55

236 Gln allele

HpaII

(177+59) Arg allele

[12]

GTTCCGTGTAAGGAGGAGA

CGAGTCTAGTAACCCTACTCACT

60

138 Arg allele

PvuII

(63+75) Trp allele

[13]

58

304 His allele

RsaI

(246+58) Arg allele

[2]

Codon

D E

M

PT

CCCCAGTGGTGCTAACCTAA

N A

CTACATGAGTGCGTGCGT

C S U

C A

E C

AATTCTGTTTCATTTCTATAGGCGA[G/T]GAGGAGCATGATCAGGAAGGCCGGG [VIC/FAM]

GGTTCAGTCCTGAGTGCCTCGCGTC[G/T]GGAGGTCCTGGTGGATCACGTGGTG [VIC/FAM]

1

Reference

ACCEPTED MANUSCRIPT Table 3: Genotype frequencies of XRCC1, APE1, and NEIL2 SNPs in the chronic hepatitis, liver cirrhosis, and HCC groups and their associations with the control group

Xrcc1

Arg /His

Arg280His His/His

Arg/Arg

Xrcc1

Arg/Gln

Arg399Gln Gln/Gln

Asp/Asp

APE1

Asp/Glu

(%)

(%)

(%)

(%)

64

8

10

49

72.7%

15.1%

27.8%

56.3%

24

45

26

35

6.66**

3.85**

2.14*

27.3%

84.9%

72.2%

40.2%

(3.24-13.7)

(1.79-8.25)

(1.13-4.06)

0

0

0

3

0.0%

0.0%

0.0%

3.4%

-

-

-

42

29

26

51

47.7%

54.7%

72.2%

58.6%

37

20

8

24

42.0%

37.7%

22.2%

9

4

2

10.2%

7.5%

5.6%

3

6

3.4%

Glu/Glu

NEIL2

Arg/Arg

Arg257Leu

27.6%

OR a

OR b

OR c

(95% CI)

(95% CI)

(95% CI)

PT

Reference

Reference

0.80

0.40*

0.53

(0.40-1.58)

(0.17-0.91)

(0.27-1.03)

12

0.68

0.30

1.09

13.8%

(0.21-2.24)

(0.06-1.49)

(0.42-2.85)

5

4

11.3%

13.9%

4.6%

59

30

14

57

0.24

0.12**

0.72

67.0%

56.6%

65.5%

(0.06-1.01)

(0.02-0.53)

(0.15-3.38)

38.9%

Reference

17

17

26

0.36

0.33

0.75

29.5%

32.1%

47.2%

29.9%

(0.08-1.57)

(0.07-1.43)

(0.15-3.68)

17

11

6

12

19.3%

20.8%

16.7%

13.8%

45

25

23

42

0.97

1.457

1.32

51.1%

47.2%

63.9%

48.3%

(0.40-2.32)

(0.53-3.96)

(0.56-3.09)

26

17

7

33

1.09

0.93

1.79

29.5%

32.1%

19.4%

37.9%

(0.42-2.79)

(0.29-2.93)

(0.73-4.42)

82

47

36

69

100%

100%

100%

100%

26

AC

Asp148Glu

(n=87)

RI

Arg /Arg

(n=36)

SC

Trp/Trp

(n=53)

NU

Arg194Trp

Arg/Trp

(n=88)

MA

Xrcc1

HCC,

D

Arg/Arg

LC,

PT E

Genotype

CHC,

CE

Codon

CR,

Reference

a

* P<0.05; ** P>0.01; OR for CHC with reference to the controls; b OR for LC with reference to the controls; c OR for HCC with reference to the controls.

1

ACCEPTED MANUSCRIPT Table 4 Risk estimates for the XRCC1 Arg194Tryp polymorphism in association with clinicopathological features XRCC1 194 polymorphism genotypes Parameters

Number AA n (%)

AT/TT n (%)

OR

P-value

(95% CI)

Age 33

21 (63.6%)

12 (36.4%)

0.80

>55

54

17 (31.5)

17 (68.5%)

(0.32- 2.00)

Female

21

14 (66.7)

7 (33.3%)

Male

66

44 (66.7%)

Single

58

37 (63.8%)

Both

29

21 (72.4%)

36

23 (63.9%)

PT

≤ 55

MA

Number of lesions

Single lesion

51

Multiple lesions

62

PT E

≥200

D

AFP >200

SC

1.00

21 (36.2%)

0.67

8 (27.4%)

(0.25-1.77)

13 (36.1%)

0.80 (0.32-1.99)

39 (62.9%)

23 (37.1%)

0.53

19 (76.0%)

6 (24%)

(0.18-1.53)

AC

0.42

0.64

16 (31.4%)

2

1.0

(0.35-2.83)

35 (68.6%)

CE

25

22 (33.3%)

NU

Lobe

RI

Gender

0.63

0.24

ACCEPTED MANUSCRIPT Table 5: Risk estimates for the XRCC1 Arg280His polymorphism in association with clinicopathological features XRCC1 280 polymorphism genotypes Parameters

Number

AA n (%)

AH/HH n (%)

OR

P-value

(95% CI)

Age 33

18 (54.5%)

15 (45.5%)

0.76

>55

54

33 (61.1%)

21 (38.9%)

(0.31-1.83)

0.54

Female

21

11 (52.4%)

10 (47.6%)

0.71

0.50

Male

66

40 (60.6%)

Single

58

39 (67.2%)

Both

29

12 (41.4%)

36

19 (52.8%)

17 (47.2%)

32 (62.7%)

19 (37.3%)

38 (61.3%)

24 (38.7%)

1.46

13 (52.0%)

12 (48.0%)

(0.57- 3.72)

PT

≤ 55

MA

Number of lesions

Single lesion

AFP >200

PT E

62

D

51

Multiple lesions

≥200

AC

CE

25

SC

26 (39.4%)

NU

Lobe

RI

Gender

3

19 (32.8%) 17 (58.6%)

(0.26 -1.92)

2.90

0.02

(1.15-7.29)

0.35 0.66 (0.27-1.57)

0.42

ACCEPTED MANUSCRIPT Table 6: Risk estimates for the XRCC1 Arg399Glu polymorphism in association with clinicopathological features XRCC1 399 polymorphism genotypes Parameters

Number

AA n (%)

AG/GG n (%)

OR (95% CI)

≤ 55

33

1 (3.0%)

32 (97.0)

0.53

>55

54

3 (5.6%)

51 (94.4%)

(0.05-5.33)

Age

21

3 (14.3%)

66

1 (1.5%)

Single

58

2 (3.4%)

Both

29

2 (6.9%)

Male

NU

1 (2.8%)

MA

36

Single lesion

51

Multiple lesions

≥200

25

10.83

65 (98.5%)

(1.06-110.52)

56 (96.6%)

0.45

27 (93.1%)

(0.04-4.58)

35 (97.2%) 0.457

3 (5.9%)

48 (94.1%)

4 (6.5%)

58 (93.5%)

0 (0.0%)

25 (100)

(0.046- 4.581)

-

AC

CE

PT E

62

D

AFP >200

SC

Lobe

Number of lesions

18 (85.7%)

RI

Female

PT

Gender

4

P-value

ACCEPTED MANUSCRIPT Table 7: Risk estimates for the APE1 Asp148Glu polymorphism in association with clinicopathological features APE1 148 polymorphism genotypes Parameter

Number

AA n (%)

AT/TT n (%)

≤ 55

33

6 (18.2%)

27 (81.8%)

>55

54

6 (11.1%)

48 (88.9%)

21

6 (28.6%)

15 (71.4%)

OR (95% CI)

P-value

Age

6 (9.1%)

Single

58

9 (15.5%)

Both

29

3 (10.3%)

36

7 (19.4%)

PT

26 (89.7%)

(0.396 -6.394)

MA

29 (80.6%)

51

5 (9.8%)

>200

62

12 (19.4%)

50 (80.6%)

≥200

25

0 (0%)

25 (100%)

Multiple lesions

AC

CE

PT E

AFP

(1.129 -14.175)

1.592

D

Single lesion

4

49 (84.5%)

NU

Lobe

Number of lesions

(0.522-6.057)

0.353

0.024

60 (90.9%)

SC

66

Male

RI

Gender Female

1.778

5

46 (90.2%)

2.221 (0.644-7.660)

0.510

0.199

0.08

ACCEPTED MANUSCRIPT Fig. 1. RFLP analysis of XRCC1 codon 280. Lane (1): 100 bp marker; lane (2): homozygous wildtype (246 bp); lane (3, 4): heterozygous genotype (246 + 304 bp). Fig. 2. Fluorescent signals for individuals with APE1 Asp148Glu genotypes: Panel A represents heterozygote genotype Asp148Glu while B and C panel show homozygous genotypes, B is

AC

CE

PT E

D

MA

NU

SC

RI

PT

Glu148Glu (variant) and C is Asp148Asp (wild-type).

6

ACCEPTED MANUSCRIPT Highlights

AC

CE

PT E

D

MA

NU

SC

RI

PT

1- XRCC1 Arg194Trp genotype may increase the risk of HCV related liver disease. 2- No association of XRCC1 codons 280,399 or APE1 codon148 with HCC risk. 3- NEIL2 Arg257Arg wild-type genotype is present in the Egyptian population.

7

Figure 1

Figure 2