GENETIC POLYMORPHISM OF XRCC1 AND XRCC3 GENES AND RISK OF CERVICAL CANCER IN SENEGALESE POPULATION

cervical cancer. Also, no association was found between XRCC3 Thr241Met genotype and expressed risk of susceptibility to both cervical cancer in Senegalese population. This study indicates that variant types of DNA repair genes play an important role in modifying individual susceptibility to cervical cancer.

X-ray repair cross complementing 1 and 3 (XRCC1 and XRCC3) gene plays a key role in DNA repair, genetic instability and tumorigenesis. We hypothesized that single nucleotide polymorphisms (SNPs) in XRCC1 and XRCC3 gene might affect its expression and/or function which have an influence on the development of cervical cancer. The aim of our study was to assess the association of four polymorphisms was carried out in the following DNA repair genes: XRCC1 (Arg399Gln, Arg194Trp) (rs25487, rs1799782), XRCC3 (C241T and A316G) (rs861539, rs1799794). The study group included 505 Senegalese individuals (313 cervical cancer patients, and 192 healthy controls). From the cancer patients and controls, genomic DNA was extracted from blood and tissues samples. Genotype was carried out for four SNPs using Taqman genotyping assay method.
A significant association was found between XRCC1 194C>T and cervical cancer (OR= 2.696; 95% CI= 1.181-6.154; p= 0.018, using an additive model), (OR=2.989; 95% CI= 1.078-8.283; p= 0.035, using dominant model), while there is no significant association between Arg399Gln polymorphisms and cervical cancer. Also, no association was found between XRCC3 Thr241Met genotype and expressed risk of susceptibility to both cervical cancer in Senegalese population. This study indicates that variant types of DNA repair genes play an important role in modifying individual susceptibility to cervical cancer. 2

Background
Cervical cancer (CC) is one of the most common genital tract carcinomas which has become an exigent health concern and the first leading cause of cancer related death among women world over with more than 570 000 new cases and 311 365 deaths (Bray et al., 2018). Among these, 85% of the cervical cancer cases occur in developing countries (Jemal et al., 2011). Infection with high-risk types of human papillomavirus (HPV) is the main causative factor for developing cervical intraepithelial neoplasia (CIN) which is a precursor lesion for cervical cancer (Ahn et al., 2003). While, not all women who are infected with HPV will certainly progress into cervical cancer, suggesting that there are still other factors playing a role in the pathogenesis of cervical cancer such as genetic and environmental factors (Shi et al., 2016). In human body, DNA repair genes are considerable factors in the prevention of genomic injury and sequential carcinogenesis. Previous studies have shown that genomic instability is a crucial event in HPV associated cancers, with a significant increase in single and double strand breaks (Udumudi et al., 1998;Cortés-Gutiérrez et al., 2012).
DNA damage may contribute to both genetic instability and carcinogenesis, which could occur through different pathways (exogenous carcinogens, endogenously produced reactive oxygen metabolites). DNA damage repair systems play vital roles in maintaining normal physiological functions, particularly for sustaining genome integrity in humans (Pan et al., 2014). Exposure to different endogenous and exogenous mutagens and carcinogens can result in various types of DNA damages. These alterations, if not repaired, can cause genetic instability, mutagenesis and cancer (Goode et al., 2002). Importantly, to counteract the deleterious consequences of the DNA-damaging agents, evolution has moulded a number of DNA repair systems that as a whole take care of most of the insults inflicted on a cell's vital genetic information. The repairing of different types of DNA damages is important for safeguarding genomic integrity (Smith et al., 2003). There are six DNA repair pathways: direct repair (Eker et al., 2009) Among the main DNA maintenance mechanisms operating in humans, the base excision repair (BER) pathway is the primary defence against lesions and responsible for repairing the DNA single-strand breaks caused by oxidative stress (such as reactive oxygen species, ROS), thereby maintaining genomic integrity. Impaired BER pathways are considered the major cause of cervical cancer (Jiricny and Marra, 2003). The double-strand break DNA repair pathway, including XRCC3 genes, is implicated in maintaining genomic stability and therefore could affect the cancer risk (Tambini et al., 2010;Yuan et al., 2014).Genetic polymorphisms in homologous recombination repair (HRR) genes, which can lead to protein haploinsufficiency, have been associated with increased cancer risk (Areeshi, 2013).
We have selected two DNA repair genes, representing two different repair pathways, for this study: the X-ray repair cross-complementing group 1 (XRCC1) and the X-ray repair cross complementing group 3 (XRCC3) genes. XRCC1 gene, located on chromosome 19 (19q13.2-13.3) is a key mediator of single-strand break DNA repair, including nucleotide excision repair (NER) and base excision repair (BER) (Fan and Wilson, 2005;Al Zoubi, 2015). XRCC1 serves as the scaffold protein in the BER pathway, which recognizes DNA breaks and interacts with polynucleotide kinase enzyme, DNA polymerase β, PARP1 and DNA ligase IIIα (Caldecott et al., 1995;Pramanik et al., 2011). There are three most common polymorphisms in XRCC1, contributing to amino acid substitutions in XRCC1 at codon 194 (exon 6, base C to T, amino acid Arg to Trp), codon 280 (exon 9, base G to A, amino acid Arg to His), and codon 399 (exon 10, base G to A, amino acid Arg to Gln). Moreover, these variants eventually alter XRCC1 function. The associations of SNPs in DNA repair genes and various types of cancer and tumors have been extensively described. However, the evidence is frequently confusing, with some SNPs increasing the risk of certain types of cancer, but decreasing the risk of others.  Thus, our hereby was to investigate the relationship between XRCC1 and XRCC3 polymorphisms in women with cervical cancer and healthy controls. Then, we genotyped 4 variants of the two DNA repair genes XRCC1 (C194T and G399A) and XRCC3 (C241T and A316G), and assessed their contributions to cervical cancer susceptibility, and their association with other epidemiological risk factors in a Senegalese woman.

Materials and Methods Study population
The Ethical and Scientific committee of Cheikh Anta Diop of Dakar University approved the study under the number 0197/2016/CER/UCAD. Blood samples and tissues were obtained from patients attending at the center of cancerology Aristide le Dantec hospital (Joliot Curie center) and Gaspard Camara Health Center, from 313 cervical cancer patients (include 127 tissue and 186 blood samples) and 192 healthy controls (blood samples). All included cases were newly and histopathologically diagnosed with cervical cancer. None of the patients had received chemotherapy or radiotherapy prior to enrolment. Blood controls patients enrolled in this study were obtained from the Yeumbeul Health Center. All controls were age matched and recruited following physical examinations after diagnostic exclusion of cancer and who were histologically confirmed to have a normal cervix. Demographic data and clinical data of the patients were collected from their files, after obtaining informed and written consent from each patient ( Table 1).

DNA isolation
DNA was extracted from both whole blood and cervical tissue using Qiagen kit and based on the manufacturer's instructions (Qiagen, Germantown, Maryland, USA). Quantification of DNA was carried out by measuring absorbance using NanoDrop Lite Spectrophotometer (Thermo Scientific, USA) and were stored at −20°C until used for genotyping.

Single-nucleotide polymorphisms (SNP) genotyping
The SNPs included in this study were selected based on previous studies that demonstrated their association with cervical cancer (rs1799782, rs25487 of XRCC1 gene and rs861539, rs1799794 of XRCC3 gene). The polymorphisms were genotyped using a TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA, USA) and a 96-well ABI 7500HT real-time PCR system (Applied Biosystems, Foster City, CA, USA). The final volume for each reaction was 10 μL, containing 5 μL Quanta Mix, 0.5 μL TaqMan probe mix, 2.5 μL DNase/RNase-Free distilled water (Bio Basic Inc. CA) and 2 μL genomic DNA. The real-time PCR steps included an initial activation step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 1 min. To validate results from real-time PCR, around five percent of assays were repeated. Eventually, the frequencies of genotype or allele of XRCC1 and XRCC3 polymorphisms were determined based on the allelic discrimination plots. To ensure the accuracy of the results, 10% of samples were randomly chosen for repeated assays, and no inconsistent results 4 were found.

Statistical analysis
The comparison of two groups of categorical variables was performed using Student test or Fisher's exact test. Hardy-Weinberg equilibrium analysis was performed for each SNP. The statistical analysis was performed using multivariate logistic regression in the additive, dominant, recessive and genotypic model, adjusted for age, marital status, gestity, parity and oral contraceptive. Linkage disequilibrium (LD) was computed for each pair of polymorphisms. P < 0.05 was considered statistically significant. All statistical analyses were carried out using the PLINK software http://zzz.bwh.harvard.edu/plink/.

Characteristics of the study population
A total of 313 women diagnosed with cervical cancer (include 127 tissue and 186 blood samples) and 192 controls were included in this study. The demographic and clinical characteristics of cervical cancer patients and normal controls are summarized in Table 1 There was significant difference in age between controls and patients with CCB and CCT. Similarly, significant differences between the cases and controls were found in parity, gestity, oral contraception and the marital status. However, the smoking status, alcohol consumption was not significantly associated with cervical cancer risk.

XRCC1 and XRCC3 gene variations and structure in Senegalese population
In the present study, the polymorphism of the XRCC1 and XRCC3 gene were analysed in a population of 505 Senegalese, including 127 CCT, 186 CCB and 192 CTR individuals. The SNPs rs25487 and rs1799782 are in linkage disequilibrium (LD) with D' = 1 and r 2 = 0.006 in according to the 1000 Genomes African populations, while rs861539 and rs1799794 are not in linkage disequilibrium (LD) with D' = 0.54 and r 2 = 0.008. Table 2 summarizes the frequency of each polymorphism in the CCB, CCT and CTR groups. Allelic frequencies obtained in our study are similar to the data provided by 1000Genomes database with regard to African populations except rs861539. 5

Association of the XRCC1 and XRCC3 gene polymorphisms with CCB susceptibility
We found no significant association between the XRCC1 and XRCC3 polymorphism with the risk of cervical cancer in all the subjects in the multivariate logistic regression analyses ( Table 3). Similarly, by comparing 17 identical patients with blood and tumour we found that there is a mutation from the T allele in the blood to the C allele (3%) in the tumour and for the other patients they are homozygous (CC) in the blood and tumour.

Association of the
7 Table 3: Genotype frequency distribution of XRCC1

Discussion
Cervical cancer is still the leading cause of cancerrelated deaths among women. Carcinogenic compounds play an important role in causing direct or indirect DNA alteration. DNA repair is responsible for maintaining genomic stability in response to the assault of environmental carcinogens that causes DNA damage (Friedberg, 2003). If left unrepaired, such DNA damage could cause mutation fixation and initiation of carcinogenesis. Polymorphisms, which have an effect on the regulation of gene expression, can contribute to the differences between individuals in the susceptibility to a disease and its severity (Choi and Kim, 2007).
There are many opinions about influence of XRCC1 (Xray repair complementing defective repair in Chinese hamster cells 1) and XRCC3 (X-ray repair complementing defective repair in Chinese hamster cells 3) genes on different cancer types. These genes participate in excision repair of bases and repair of single and double strand breaks. In our study, we  (Jensen et al., 2013), seems to increase the risk of SCC of the cervix among HPV positive women. In our study, high parity was also associated significantly with CCB and CCT cases. Our study assessed the difference in age in cervical cancer cases and controls. There was a significant difference between cervical cancer cases and controls, whereas the difference in age was associated statistically significant with CCB and CCT cases.
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 non-invasive method to evaluate the risk of cervical cancer. 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.

Conclusion
In conclusion, the research of the relationship of XRCC1 and XRCC3 polymorphisms and cervical cancer is very popular but conflicting at present. This is the first molecular epidemiological study on XRCC1 (rs1799782, rs25487), XRCC3 (rs861539, rs1799194) polymorphism and cervical cancer risk in Senegalese.
Our study suggests that XRCC1 Arg194Trp polymorphism may be associated with cervical cancer 9 risk, while XRCC1 Arg399Gln and XRCC3 polymorphism didn't show an association with cervical cancer risk.