Association Between Genetic Polymorphism of XRCC 1 Gene and Risk of Glioma in а Chinese Population

BACKGROUND
Gliomas are the most common type of primary brain tumor in adults, and the X-ray repair complementing group 1 gene (XRCC1) is an important candidate gene influencing its risk. The objective of this study was to detect the influence of XRCC1 genetic polymorphisms on glioma risk.


MATERIALS AND METHODS
A total of 629 glioma patients and 641 cancer-free subjects were enrolled in this case-control study. The genotypes of the c.1471G>A genetic polymorphism were determined by created restriction site-polymerase chain reaction (CRS-PCR) and DNA sequencing methods. The influence of the XRCC1 genetic polymorphism on glioma risk was evaluated by association analysis.


RESULTS
Our data indicated that the alleles/genotype of this genetic variant was statistically associated with glioma risk. The AA genotype was statistically associated with the increased risk of glioma compared to the GG wild genotype (odds ratios (OR) = 1.89, 95% CI 1.25-2.87, P = 0.003). The allele-A may contribute to increased the susceptibility to glioma (OR = 1.23, 95% CI 1.04-1.46, P = 0.017).


CONCLUSIONS
These preliminary findings indicate that the c.1471G>A genetic polymorphism of XRCC1 has the potential to influence glioma susceptibility, and might be used as molecular marker for assessing glioma risk.


Study subjects
This case-control study was conducted in Chinese Han populations from the First Affiliated Hospital of Dalian Medical University.The 629 patients (males = 376, females = 253) were enrolled, which newly diagnosed and histologically confirmed with glioma by doctors.The 641 cancer-free subjects (males = 398, females = 243) were recruited as controls, which free from any cancer and matched with age and gender.All subjects were genetically unrelated Chinese of Han ethnicity.Table 1 shows the general characteristics on age, gender, tobacco smoking, alcohol drinking, histology types, ionizing radiation (IR) exposure history and family history of cancer.Approval to conduct this study was granted by the Ethics Committees of the First Affiliated Hospital of Dalian Medical University.The written informed consent form was obtained from all recruited subjects.

Genotyping
Genomic DNA was extracted by using the DNA Blood Mini kit (QIAGEN, Valencia, CA).The specific polymerase chain reaction (PCR) primers were designed through Primer Premier 5.0 software.Table 2 shows the detailed information of primers sequences, Annealing temperature, PCR amplification region and fragment sizes.The PCR amplifications was carried out in a reaction volume of 20 μL containing 50 ng of genomic DNA, 10 pM of each primer, 0.20 mM dNTP, 2.5 mM MgCl 2 and 0.5 U Taq DNA polymerase (TaKaRa, Dalian, China).The condition of PCR reactions began with 95°C for 5 minutes, 35 cycles of 94°C for 30 seconds, followed by annealing temperature to 63.2°C for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 8 minutes.The genotypes of c.1471G>A genetic polymorphism of XRCC1 gene was determined by the created restriction site-polymerase chain reaction (CRS-PCR) method, with one of the primers containing a nucleotide mismatch, which enables the use of restriction enzymes for discriminating sequence variations (Haliassos et al., 1989;Zhao et al., 2003;Yuan et al., 2012;Yuan et al., 2013;Yuan et al., 2013).The PCR amplified products (5 μL) were digested with 2U selected restriction enzymes (Table 2) at 37°C for 10 hours, following the manufacturer's instructions.The digested products were separated by electrophoresis in 2.0% agarose gel, and observed different genotypes on the UV light.In order to verify the genotype results from CRS-PCR method, 15% of random samples were re-analyzed by DNA sequencing method (ABI3730xl DNA Analyzer, Applied Biosystems, Foster City, CA).

Statistical analyses
All statistical analyses were performed by the SPSS software (Windows version release 16.0; SPSS Inc.; Chicago, IL, USA).The differences between cases and controls in the frequencies of alleles/genotypes, general characteristics, and risk factors were assessed by the chi-square (χ 2 ) test.The Hardy-Weinberg equilibrium (HWE) was determined for compatibility between cases and controls using the chi-square (χ 2 ) test.The association between the XRCC1 genetic polymorphism and the risk of glioma was analyzed by calculating the odds ratios (ORs) and their 95% confidence intervals (CIs) using unconditional logistic regression.P-values less than 0.05 were regarded as statistically significant.

General characteristics
In totally, 1270 Chinese Han subjects were enrolled in this case-control study, containing of 629 glioma subjects and 641 cancer-free controls.The general characteristics of cases and controls subjects are summarized in Table 1.The mean ages were 53.22 years old (standard deviation (SD) ± 13.66) and 54.75 years old (SD ± 14.11) for the cases and controls, respectively.The frequency-matching on age and gender between the cases and controls appeared to be adequate (P = 0.2842 and 0.3982, respectively).There were no statistically significant differences among the cases and controls in terms of tobacco smoking and alcohol drinking (P = 0.0617 and 0.0528, respectively).Those subjects who have more family history of cancer and higher IR exposure, were more likely to have higher risk of glioma (P = 0.0122 and 0.0380, respectively).

XRCC1 genetic polymorphism identification
In this study, the genotypes of c.1471G>A genetic polymorphism of XRCC1 gene were determined by CRS-PCR and DNA sequencing methods in a Chinese Han population.Based on the human XRCC1 gene DNA sequences (GenBank ID: NC_000019.9),mRNA sequences (GenBank ID: NM_006297.2),and protein sequences (GenBank ID: NP_006288.2),the results from our DNA sequence analyses suggested that the c.1471G>A genetic polymorphism is a non-synonymous mutation, which corresponding to the GgA mutations and Glutamic (Glu) to Lysine (Lys) amino acid replacement (p.Glu491Lys) in the exon13 of XRCC1 gene.The AlwNI restriction enzyme has been selected and utilized to digest the PCR products of c.1471G>A genetic polymorphism.

Discussion
Recently, previous studies indicate that the XRCC1 gene is one of the most important candidate genes for influecning the risk of glioma, and analysis of genetic polymorphisms in XRCC1 gene allows to effectively screen for the susceptibility to glioma (Wang et al., 2004;Kiuru et al., 2008;Liu et al., 2009;Yosunkaya et al., 2010;Zhou et al., 2011;Zhang et al., 2012;Jiang et al., 2013;Luo et al., 2013;Wei et al., 2013).In the present study, we firstly investigated the distribution of the c.1471G>A genetic polymorphism of XRCC1 gene using CRS-PCR and verified by DNA sequencing methods and evaluated the relationship of this genetic polymorphism with respect to glioma risk in a Chinese Han population by an association analysis on the basis of analysis of 629 glioma patients and 641 cancer-free controls.Our data indicated that the allele and genotype frequencies of this genetic variant in glioma patients were significantly different from those of cancer-free controls (All P-values < 0.05, Table 3).The genotype-AA was statistically associated with the increased risk of glioma compared to wild genotype-GG and GA/GG carriers (OR = 1.89, 95% CI 1.25-2.87,P = 0.003 and OR = 1.83, 95% CI 1.23-2.74,P = 0.003, Table 4).The A allele might be an increased risk factor for glioma susceptibility (A vs. G: OR = 1.23, 95% CI 1.04-1.46,P = 0.017, Table 4).Results from this study suggested that this genetic polymorphism has a statistically significant association with glioma risk and may affect the subjects susceptibility toward glioma in Chinese Han population.It could be used as molecular markers for evaluating glioma risk.Up to now, several similar studies concerned the influence of other genetic polymorphisms in the XRCC1 gene on glioma risk (Wang et al., 2004;Felini et al., 2007;Kiuru et al., 2008;Liu et al., 2009;Rajaraman et al., 2010;Yosunkaya et al., 2010;Hu et al., 2011;Melin, 2011;Zhou et al., 2011;Jacobs et al., 2012;Liu et al., 2012;Sun et al., 2012;Wang et al., 2012;Zhang et al., 2012;Jiang et al., 2013;Luo et al., 2013;Pan et al., 2013;Wei et al., 2013).Results from these observations are in accordance with our conclusion that the genetic polymorphisms of XRCC1 gene may contribute to influences on glioma risk (Kiuru et al., 2008;Liu et al., 2009;Rajaraman et al., 2010;Yosunkaya et al., 2010;Hu et al., 2011;Zhou et al., 2011;Liu et al., 2012;Sun et al., 2012;Wang et al., 2012;Jiang et al., 2013;Luo et al., 2013;Pan et al., 2013;Wei et al., 2013).Previous studies demonstrated that many of other non-synonymous genetic polymorphisms (for example, Arg194Trp, Arg280His and Arg399Gln) have been approved significantly associated with the risk of glioma and influenced the function of XRCC1 protein (Kiuru et al., 2008;Liu et al., 2009;Rajaraman et al., 2010;Yosunkaya et al., 2010;Hu et al., 2011;Zhou et al., 2011;Liu et al., 2012;Wang et al., 2012;Luo et al., 2013;Pan et al., 2013).In our study, DNA sequence analyses suggested that the c.1471G>A genetic polymorphism is also a non-synonymous mutation and causes Glu to Lys amino acid replacement (p.Glu491Lys).This c.1471G>A genetic polymorphism might be linked to those other nonsynonymous genetic polymorphisms and play the similar function on the development of glioma.Our findings could provide more evidence to explain the role of XRCC1 gene in the development of glioma.Further functional studies on larger different populations analyzing the c.1471G>A and other genetic polymorphisms spanning the whole XRCC1 gene region are still necessary to elucidate the underlying biological mechanisms underlying XRCC1mediated autism susceptibility.

Table 2 . The Primer Sequences, PCR and CRS-PCR Analysis for c.1471 G>A Genetic Polymorphism of XRCC1 Gene
, polymerase chain reaction; CRS-PCR, created restriction site-polymerase chain reaction; Underlined nucleotides mark nucleotide mismatches enabling the use of the selected restriction enzymes for discriminating sequence variations at CRS-PCR analysis PCR