Implication of Polymorphisms in DNA Repair Genes in Prognosis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common primary cancer in men (523 000 cases, 7.9% of the total) and the seventh in women (226 000 cases, 6.5% of the total), and most of the burden is in developing countries (IARC, 2008). It is well known that chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) are the main risk factors for the pathogenesis of HCC. It is estimated 30% to 50% of the HBV related deaths are attributable to HCC, however, only less than 10% of HBV and HCV infected individuals developed HCC in their later life (Lavanchy, 2004; Bowen et al., 2005). It could be hypothesis that other factors might play a role in the development of HCC, such as environment and genetic factors. It is reported that individuals with HCC presented with DNA damaged by hepatitis virus, and this is a major underlying risk factors of HCC (Bowen et al., 2005). It is reported that majority of HCC cases result from DNA damage caused by hepatitis viruses, which is the main potential risk factors to the development of HCC (McKillop et al., 2006). Damage due to endogenuous or exogenous exposure may be repaired by enzymes encoded by the DNA repair pathways, such as base excision repair, nucleotide excision repair, mismatch repair, homologous recombination repair and non-homologous end joining. The nucleotide excision repair (NER) is the main DNA repair system, and constitutes the main


Introduction
Hepatocellular carcinoma (HCC) is the fifth most common primary cancer in men (523 000 cases, 7.9% of the total) and the seventh in women (226 000 cases, 6.5% of the total), and most of the burden is in developing countries (IARC, 2008). It is well known that chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) are the main risk factors for the pathogenesis of HCC. It is estimated 30% to 50% of the HBV related deaths are attributable to HCC, however, only less than 10% of HBV and HCV infected individuals developed HCC in their later life (Lavanchy, 2004;Bowen et al., 2005). It could be hypothesis that other factors might play a role in the development of HCC, such as environment and genetic factors. It is reported that individuals with HCC presented with DNA damaged by hepatitis virus, and this is a major underlying risk factors of HCC (Bowen et al., 2005).
It is reported that majority of HCC cases result from DNA damage caused by hepatitis viruses, which is the

Implication of Polymorphisms in DNA Repair Genes in Prognosis of Hepatocellular Carcinoma
Ai-Min Yue*, Zhen-Bin Xie, Shu-Ping Guo, Qi-Dong Wei, Xiao-Wei Yang defense against lesions generated by ionizing radiation and strong alkylating agents as well as lesions formed by endogenous DNA-damaging agents like viruses (Smith et al., 2003). The NER is reported to association with the development of several cancers (Han et al., 2012;Mandal et al., 2012). Defective DNA repair can lead to the accumulation of mutations and microsatellite instability in the genome, increasing the chance of malignant transformation (Hussain et al., 2007). Genetic variation may alter the function of DNA repair proteins, influence the development and clinical outcome of HCC. The role of XRCC1 and XPD polymorphisms has been reported to be associated with function of chemotherapy of several cancers, and the chemotherapy could induce the DNA damage of cancer cells, while the low activity of XRCC1 and XPD polymorphisms might influence the therapy outcome. There is still no study on the role of these two gene polymorphisms on the HCC prognosis in Chinese population. Therefore, we conducted a study to explore the role of selective SNPs of the two gene polymorphisms in the prognosis of HCC.

Subjects
A total of 242 cases with HCC were histological confirmed in Xinxiang Central Hospital between January 2008 and Dec. 2011. Finally, 231 patients finished the follow-up in our study, with the participation rate of 95.5%. Case with secondary or recurrent tumors was excluded. All the patients were followed up until the end of August 2012.
The diagnosis of HCC was made by liver biopsy or the combination of increased alpha-fetoprotein (AFP of ≥200 ng/ml) and the typical vascular pattern on angiography or dynamic imaging. The clinical information, such as tumor differentiation, tumor size, child-pugh class and surgery types were collected from medical records with patients' consent. All the patients were followed up until the end of April. 2012. Tumor progression was defined as postoperative tumor recurrence and distant metastasis. The overall survival time was defined as the data of tumor section or the first local treatment to the date of death. The ethics committee of the Xinxiang Central Hospital reviewed and approved the study.

DNA collection and genotyping
A 2 ml sample of blood was collected from each subject. DNA was extracted from frozen white blood cells using the buffy-coat fractions with TIANamp blood DNA kit (Tiangen Biotech, Beijing, China). Primers for polymerase chain reaction (PCR) amplification and single base extension (SBE) assays were designed by Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA, USA) according to the manufacturer's instructions (Table1).
PCR was carried out in a reaction volume of 20 μl containing 50ng of genomic DNA, 200μM dNTP, 2.5 units of Taq DNA polymerase (Promega Corporation, WI, USA), and 200μM primers. The parameters of PCR reaction used was as follows: 94°C for 2 minutes, 35 cycles of 94°C for 30 seconds, annealing temperature reduced to 64°C for 30 seconds, and 72°C for 1 minute seconds. PCR product was electrophoresis on 1.0% agrose gel to testify PCR products. For quality control, genotyping was performed without knowledge of the case/ control status of the subjects, and a random sample of 5% of cases and controls was genotyped again by different researchers. The reproducibility was 100%. For quality control, genotyping was performed without knowledge of the case/control status of the subjects, and a random sample of 5% of cases and controls was genotyped again by different researchers. The reproducibility was 100%.

Statistical analysis
The SPSS software package version 16.0 (SPSS Inc. Chicago, USA) was used for statistical analyses. Mean±standard deviation (SD) was used for continuous variables, and frequencies and percentages were used for categorical variables. The homozygous genotypes for these SNPs were used as reference. The Kaplan-Meier method was adopted to estimate survival curves, and the log-rank test was used to compare patients' survival time between genotype groups. Cox's proportional hazard model was used to assess the associations between these two gene polymorphisms and survival. Primary death from HCC was defined as the failure event, and the time of survival was the time between diagnosis and death. The cause of death was determined by specialists based on clinical documents from hospital or reports by patients' family members. If a patient died of causes other than HCC, he was censored at the date of death. P value was considered statistically significant which was less than or equal to 0.05.

Results
All the patients were followed up until the end of Dec. 2012. Among 242 patients, 11 patients were lost to follow-up due to migration, while the remaining 231 patients completed the study. The median follow-up time was 25.3 months (range: 2 to 53 months). A total of 195 patients (42.8%) died of HCC during the follow-up period. The demographic characteristics of subjects included and clinical features of HCC patients were shown in Table 2. The average age was 50.9±9.6 years in HCC cases, and was 51.2±9.1 years in controls. Most of the HCC patients had well differentiation. Almost 60% of the HCC patients were grade A of Child-Pugh class, and most of the HCC  (rs25487) 1st-primer 5 '-TTG TGC TTT CTC TGT GTC CA-3'  2nd-primer  5'-TCC TCC AGC CTT TTC TGA TA-3'  XPD Lys751Gln (rs13181) 1st-primer 5'-TTG TGC TTT CTC TGT GTC CA-3' 2nd-primer 5'-CTA TCA TCT CCT GGC CCC C-3' XPD Asp312Asn (rs1799793) 1st-primer 5'-CCCACCTGGCCAACCCCGTGCTGCCC-3' 2nd-primer 5'-ACGAAGTGCTGCAGGGTGAGCCCCG-3'    figure  2). The Cox's regression analysis showed individuals carrying XRCC1 399Trp/Trp genotype had 0.55 fold risk of death from HCC than Arg/Arg genotype. Meanwhile, XPD 751Gln/Gln had a strong decreased risk of death from HCC in comparison to XPD Lys/Lys carriers (HR=0.34, 95% CI=0.12-0.85). However, XRCC1 Arg194Trp and XPD Asp312Asn did not show significant association with survival of HCC.

Discussion
The present study evaluated the effect of polymorphisms in XRCC1 Arg399Gln, and XPD Lys751Gln on the prognosis of HCC risk in Chinese population. To the best of our knowledge, this is the first study to describe the association of XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms with the clinical outcomes of HCC. We demonstrated that XRCC1 399Gln/Gln and XPD 751Gln/ Gln genotypes were associated with a reduction of death from HCC (adjusted HR: 0.55 for XRCC1 399Gln/Gln; 0.34 for XPD 751Gln/Gln). These results suggest that polymorphisms in XRCC1 and XPD may have functional significance in the overall survival of HCC patients.
Various DNA damage may be caused during the endogenous metabolic processes or by environmental carcinogens. If not repaired for these DNA damages, genetic instability, mutagenesis, cell death and genome integrity as well as induce carcinogenesis might be occurred. NER is the main DNA damage repair pathway for the small base lesions from oxidation and alkylation's damage. XRCC1 protein is an important protein in the multistep NER pathway, and consists of three functional domains that interact with different enzymes to initiate DNA repair of different stages and types (16). HBV is known to be the primary risk factor for the development of HCC by inducing either chromosomal instability or insertional mutations (Pang et al., 2006). Any alteration in the activity of XRCC1 may be associated with polymorphisms may lead to modification of the clinical behavior of HBsAg positive patients.
A previous study conducted in South Korea indicated that A allele of XRCC1 Gln399Arg might be associated with an unfavorable prognosis in patients with HCC by multivariate Cox regression analysis, which suggested the SNP of XRCC1 rs25487 was significantly associated with reduced overall survival of HCC patients (Jung et al., 2012). Another study conducted in Taiwan indicated the A allele of XRCC1 Gln399Arg was associated with a dose-dependent increased risk of early-onset HCC patients less than 50 years old (Yu et al., 2003), and the SNP of XRCC1 Gln399Arg was significantly associated with a lower risk of distant metastasis and microsatellite nodule. In our study, Gln allele of XRCC1 Arg399Gln was more likely to reduce the overall survival of HCC, which was in line of previous studies (Jung et al., 2012;Yu et al., 2003). XPD protein, encoded by XPD gene, plays a role in NER pathway. During the NER, XPD participates in the opening of the DNA helix to allow the excision of the DNA fragment containing the damaged base (Benhamous and Sarasin, 2002;Manuguerra et al., 2006). 312 (Asp to Asn) and 751 (Lys to Gln) were the main two polymorphisms that induce amino acid changes in the proteins (Shen et al., 1998). The associations of the Lys751Gln polymorphism in XPD gene with the prognosis of various types of cancer have been widely reported in cancer epidemiologic studies, such as colorectal cancer, advanced lung cancer, oral cancer and pancreatic cancer (Giovannetti et al., 2011;Biason et al., 2012;Chen et al., 2012;Mahimkar et al., 2012;Slyskova et al., 2012). In our study, we have found polymorphism in XPD Lys751Gln is related to the prognosis of HCC, which has provided evidence that the XPD protein influences the overall survival of HCC through NER pathway.
Up to now, there are limited reports on the role of polymorphisms in XRCC1 and XPD on the survival of HCC. We have found modern decreased survival in XRCC1 399Gln/Gln or XPD 751Gln/Gln carriers, which is obvious because XRCC1 399Gln/Gln or XPD 751Gln/ Gln have reduced the enzyme activity and thus may have decrease DNA repair captivities. The chemotherapy for HCC is to induce the DNA damage of cancer cells, the low activity of XRCC1 and XPD polymorphisms would strengthen the efficacy of therapy. The role of XRCC1 and XPD polymorphisms has been reported to be associated with increased function of chemotherapy of several cancers, including non-small cell lung cancer, breast cancer, head and neck squamous cell cancer as well as pancreatic cancer (Giovannetti et al., 2011;Azad et al., 2012). Our study was in line with previous studies, which provided solid evidence for the above hypothesis. XRCC1 399Gln/Gln or XPD 751Gln/ Gln carriers had significant prognostic role on the survival of HCC patients in Chinese population. Moreover, the genetic information on the polymorphisms and gene expression could play an important role in creating successful pharmacogenetic-guided chemotherapy. The use of rapid and sensitive PCR assays for diagnostic screening, coupled with ready accessibility to peripherad blood from patients with HCC, will help facilitate application of our study.
In conclusion, our present data provide evidences to suggest that polymorphisms in XRCC1 Arg399Gln, and XPD Lys751Gln were related to HCC risk in Chinese population. Moreover, the genotype of XRCC1 399Gln/ Gln and XPD 751Gln/Gln are associated with a reduction of death from HCC. It is suggested that the XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms should be routine detected to HCC patients who are more likely benefit from radiotherapy and chemotherapy. Our finding are based on relative small numbers and limited by small number subjects. Larger sample studies from Chinese population are still needed.