XRCC1 Arg399Gln Genetic Polymorphism in a Turkish Population

Abstract

Humans are routinely exposed to mutagenic and carcinogenic chemicals. These chemicals can form DNA adducts in vivo and thus lead to DNA damage. The integrity of most of the so-damaged DNAs is typically restored as a consequence of the action of certain DNA-repairing enzymes. In several DNA repair genes, polymorphisms may result in reduced repair capacity, which has been implicated as a risk factor for various types of cancer. XRCC1 is a base-excision repair protein that plays a central role in the repair of DNA base damage and strand breaks. Amongst the known genetic polymorphisms of the DNA-repair genes, X-ray repair cross-complementing groups 1 and 3 (XRCC1 and XRCC3) have been studied most commonly. Inconsistent results have been reported regarding the associations between the Arg399Gln (exon 10) polymorphism of XRCC1 and either functional significance or the risk of tobacco-associated cancers. The Gln allele of this polymorphism was associated with higher levels of DNA adducts. Therefore we genotyped one of the polymorphism of XRCC1, Gln allele. The frequency of the polymorphic alleles varies among populations, suggesting an ethnic distribution of genotypes. There has been no information on interindividual variability of Arg399Gln genotype in the Turkish population. Due to the association between the Arg399Gln polymorphism of XRCC1 and the risk of tobacco-associated cancers, we preferred to evaluate the allelic frequencies of Arg399Gln genotype than the other polymorphisms in XRCC1 gene in healthy Turkish population by polymerase chain reaction–restriction fragment polymorphism (PCR-RFLP) analysis to enable to show interindividual differences and compare to other populations.

Humans are routinely exposed to mutagenic and carcinogenic chemicals. These chemicals can form DNA adducts in vivo and thus lead to DNA damage. DNA repair enzymes continuously monitor chromosomes to correct damaged nucleotide residues generated by exposure to carcinogen and cytotoxic compounds (Wood et al. 2001). A variety of mutagenic and cytotoxic DNA lesions are formed by oxidative DNA damage, especially reactive oxygen species (ROS) (Krokan 1997; Lindahl 1997). The accumulation of ROS leads to oxidative stress, which is a risk factor for cancer development (Shirai et al. 1995). ROS can initiate lipid peroxidation, oxidize proteins, and cause damage to DNA directly or indirectly (Joenje 1989). Base damage and single-strand breaks (SSBs) are repaired through the base excision repair (BER) pathway. This pathway is a multistep process that requires the activity of several proteins (Dianov et al. 2001). Substantial evidence indicates an important role for X-ray repair cross complementing 1 (XRCC1) in single-strand break repair (SSBR) and BER. This protein is thought to act as a scaffolding protein for other repair factors. A large number of molecular epidemiological studies have analyzed the impact of polymorphisms in the human XRCC1 gene on cancer risk (Brem and Hall 2005). Amongst the known genetic polymorphisms of the DNA-repair genes, the xeroderma pigmentosum group D (XPD, also known as ERCC2) and X-ray repair cross-complementing groups 1 and 3 (XRCC1 and XRCC3) have been studied most commonly (Yeh et al. 2005). The gene XRCC1 encodes a protein involved in DNA base-excision repair. A common polymorphism (Arg → Gln) at codon 399 of the XRCC1 gene has been previously linked to functional changes of the gene product and risk of cancers.

A large number of studies have been conducted on the associations between cancer risk and XRCC1 Arg399Gln genetic polymorphisms (Kelsey et al. 2004). There has been no information on interindividual variability of Arg399Gln genotype in the Turkish population. Therefore, we aimed to determine the allelic frequencies of Arg399Gln genotype in healthy Turkish population by polymerase chain reaction–restriction fragment polymorphism (PCR-RFLP) analysis to enable to show interindividual differences and compare to other populations.

MATERIALS AND METHODS

Subjects

Whole blood samples (EDTA) were collected from 166 healthy volunteers after obtaining written informed consent, and detailed questionnaires were applied (age, smoking status, medication, etc). All subjects were apparently healthy and did not have any chronic disease. The age was ranged from 26 to 78 years. 60 subjects were smokers.

DNA Extraction

Whole blood samples (5 to 10 ml) were removed via venipuncture into tubes containing EDTA and samples were stored at −20°C until DNA extraction. Genomic DNA was extracted from whole blood using a sodium perchlorate/chloroform extraction as described by Karahalil and Kocabaş (2005). Lysis buffer (10 mM Tris-HCl, 320 mM sucrose, 5 mM MgCl₂, 1% Triton), Reagent B buffer (400 mM Tris-HCl, 60 mM EDTA, 150 mM NaCl, 1% sodium dodecyl sulfate [SDS]), and 5 mM Sodium perchlorate were used in extraction procedure. All reagents were obtained from Sigma and Merck.

XRCC1 Arg399Gln Genotype

The XRCC1 alleles were detected using PCR-RFLP technique. This technique is a modification of the method described by Sturgis et al. (1999). The sequences of the PCR primers were 5′-CAGTGGTGCTAACCTAATC-3′ for the forward and 5′-AGTAGTCTGCTGGCTCTGG-3′, which generate an 871-bp product. A 30-μl PCR reaction was performed using approximately 0.2 to 0.5 μg genomic DNA, 0.25 μM each primer, 200 μ M of each dNTP, 1.5 U Taq polymerase (Promega, Madison, WI) in the 1 × PCR buffer supplied by the manufacturer (50 mM KCl, 10 mM Tris-HCl, pH 9.0, 1% TritonX-100). After an initial melting step at 95°C for 5 min, amplification was carried out for 35 cycles by denaturing at 95°C for 30 s, annealing at 58°C for 45 s, extending at 72°C for 45 s, and a final extention at 72°C for 10 min for 1 cycle. The PCR products were digested with Nci I restriction enzyme (New England Biolabs) to distinguish the 28152 polymorphism of exon 10. The digestion of 6 μl of PCR products was carried out using 8U Nci I and the 1 × NEB4 buffer at 37°C overnight. The wild allele (i.e., 28152 G) has two Nci I restriction enzyme sites of 461 and 182 bp. The mutant allele (i.e., 28152 A) without the Nci I restriction enzyme sites has 593 bp. To analyze the restriction fragments, 3% 1:1 ratio of agarose: gamma micropore gel (Prone) was used. The digested products photographed using Genecam system (Spectronics GL-2000) (Sturgis et al. 1999).

RESULTS

We investigated the allelic frequencies of Arg399Gln genotype in 166 healthy Turkish population to enable to show interindividual differences and compare to other populations. We found that the XRCC1 gene allele frequencies were 0.60 for the 399Arg polymorphism and 0.40 for the 399Gln polymorphisms. Sixty subjects were smokers and rest of them (n = 106) was nonsmokers. We also compared smoker subjects to non-smoker subjects. We could not find any statistically significant difference (Table 1).

Table 2 shows the frequency distributions of the XRCC1 genotype in healthy individuals of our current study and compares these frequencies with those previously published for different ethnic groups. In our study, the allele frequency of the 399Arg polymorphism in Turkey (0.60) was similar to those previously published by Rossit et al. (2002) for 96 healthy Brazilians from the same region. The observed allele frequency in Turkish population was within the range described for Caucasians (American Caucasian, Italian, and Belgian populations). Wu et al. (2004) found that the frequency of Arg/Arg, Arg/Gln, and Gln/Gln in XRCC1 codon 399 in controls was 58% (114/196), 37% (73/196), and 5%, respectively. Ito et al. (2004) also observed similar results regarding the distribution of XRCC1 genotype in a Korean population (Table 2).

DISCUSSION

Different DNA repair systems maintain the integrity of the human genome, so deficiency in the repair capacity due to mutations or polymorphisms in genes involved in DNA repair can lead to genomic instability that, in turn, is related to chromosomal instability syndromes and increased risk of developing various types of cancer (Duarte et al. 2005). The frequency of the polymorphic alleles varies among populations, suggesting an ethnic distribution of genotypes.

XRCC1, a protein directly involved in the repair of DNA base damage, contains at least three common polymorphisms. One of these, the codon 399 Arg→ Gln variant, has been associated with several cancer-related biomarkers, suggesting it may have functional significance in exposure-induced cancers (Nelson et al. 2002; Patel et al. 2005). The genotype distribution of the XRCC1 gene does not vary between sexes, but differences between ethnic groups have been suggested (Park et al. 2002; Olshan et al. 2002).

The XRCC1 gene contains 17 exons and is located on chromosome 19q13.2. Although many polymorphisms have been documented, two nonsynonymous polymorphisms in XRCC1 (Arg194Trp [C → T allelic change] and Arg399Gln [G → A allelic change]) have been shown to alter DNA repair capacity in some phenotypic studies and have received considerable attention (Patel et al. 2005).

Three gene polymorphisms resulting in nonconservative amino acid substitutions (Arg194Trp, Arg280His, and Arg399Gln) have been identified in the XRCC1 gene. Two of these (Arg280His and Arg399Gln) were recently predicted to be likely to affect the function of the protein based on the conservation of the amino acids among protein family members. The XRCC1 Arg280His polymorphism lies in between the DNA polymerase β and poly (ADP-ribose) polymerase (PARP) binding areas (Metsola et al. 2005).

The proportions of individuals homozygous for 399Arg allele, heterozygous and homozygous for the 399Gln allele were 85.8%, 13.7%, and 0.5% among the control group (Jeon et al. 2005). XRCC1 Arg399Gln polymorphism is a genetic polymorphism at codon 399 was frequently found in the Korean population.

We have been preparing to publish polymorphisms of the DNA repair genes XRCC1 and hOGG1, relation to environmental exposures, and bladder cancer risk in a case-control study in Turkey. In conclusion, it seems that still there is a growing need for expanding genotype studies with respect to XRCC1 gene because of its importance in various cancer types.