Introduction

The X-ray repair cross-complementing group 1 (XRCC1) protein is essential for mammalian viability. XRCC1 deficiency in mice results in embryonic lethality, and it is required for the efficient repair of single-strand breaks and damaged bases in DNA. The XRCC1 has no known enzymatic activity and it is thought to act as a scaffold protein for both single-strand break repair and base excision repair activities. The XRCC1 protein complexes with three DNA repair enzymes: DNA ligase III, DNA polymerase β and poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) involved in excision and recombinational repair pathways [1].

The human XRCC1 gene has been mapped to the long arm of chromosome 19 and consists of 17 exons and encodes a protein of 633 amino acids. More than 60 validated single nucleotide polymorphisms in XRCC1 are listed in the Ensembl database, among which approximately 30 variants are located in exons or promoter regions. The most extensively studied single nucleotide polymorphisms are Arg194Trp on exon 6 (db SNP no. rs.1799782) and Arg399Gln on exon 10 (db SNP no. rs.25487) [2–4].

Because the Arg399Gln polymorphism is located in the region of the BRCT-I interaction domain of XRCC1 within a poly(ADP-ribose) polymerase binding region, this polymorphism has been extensively investigated both in its function and its association with cancer risk. The XRCC1 Arg194Trp variant occurs in the newly identified proliferating cell nuclear antigen binding region. The presences of the variant (399Gln and 194Trp) alleles have been shown to be associated with measurable reduced DNA repair capacity and increased risk of several types of cancers [2–4].

The distribution of serum proteins, blood groups, and red cell enzymes in Iranian populations has been studied by different investigators [5, 6]. Very recently we had reported the frequencies of some genetic polymorphisms in Shiraz population using DNA analysis [7–13]. In order to get more insight into the genetic structure of Iranian populations the present study was done.

Materials and methods

Subjects

A total of 707 healthy blood donors (304 female, 403 males) were included in the study. The study was done in Shiraz, Fars province (southern Iran). Because XRCC1 polymorphisms showed significant association with several multifactorial diseases [1–4, 14], we excluded the participants with history for diagnosed cancers, psychiatric disorders, and cardiovascular diseases. The mean age ±SD of participants was 29.1 ± 10.5 years, with age ranging between 15 and 70 years. The study group was unrelated Iranian Muslims. Informed consent was obtained from each subject before the study.

Extraction of DNA and genotyping analysis

Blood samples were obtained from the participants. Immediately after collection, whole blood was stored at −20°C until use. Genomic DNA for PCR was isolated from whole blood using the thawed blood samples. The XRCC1 genotypes were examined using PCR-RFLP assays as described previously [15]. Genotypic analysis for the XRCC1 gene was determined in DNA by PCR assay, using the PCR primers described by Lunn et al. for the codons 194 and 399 [16]. These primers generate 491 and 615 bp products containing the polymorphic sites at codons 194 and 399, respectively. The XRCC1 Arg399Gln polymorphism was genotyped by restriction enzyme digestion of the PCR product with the MspI enzyme. The MspI restricted products, Arg/Arg, Arg/Gln and Gln/Gln genotypes had band sizes of 374/221 bp, 615/374/221 bp, and 615 bp, respectively. The XRCC1 Arg/Trp polymorphism at codon 194 was genotyped by digestion of the PCR product with the PvuII restriction enzyme. The PvuII restricted products, Arg/Arg, Arg/Trp, and Trp/Trp genotypes had band sizes of 490, 490/294/196 and 294/196 bp [17].

To test for contamination, negative controls (tubes containing the PCR mixture, without the DNA template) were incubated in every run. Any sample with ambiguous result due to low yield was retested and a random selection of 15% of all samples was repeated. No discrepancies were discovered upon replicate testing.

Statistical analysis

A χ2 test was performed for each polymorphism to determine if the control sample demonstrated Hardy-Weinberg equilibrium. The software SNPAlyze(TM) ver. 6 Standard (Dynacom Co, Ltd. Kanagawa, Japan) was used to evaluate the status of pair wise linkage disequilibrium for the studied polymorphisms. A probability of P < 0.05 was considered statistically significant. All P-values were two-tailed.

Results and discussion

Table 1 shows the XRCC1 genotypes (at codons 194 and 399) of 707 subjects among two sex groups. There was no statistically significant differences between males and females for either polymorphism at codon 194 (χ2 = 1.73, df = 2, P = 0.421) or at codon 399 (χ2 = 1.86, df = 2, P = 0.395). Therefore, the sex groups were pooled.

Table 1 Frequencies (percentages) of Arg399Gln and Arg194Trp XRCC1 in Shiraz population (south of Iran)
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The allelic frequency of 194Arg and 194Trp was 90.95% and 9.05%, respectively. The study population was at Hardy-Weinberg equilibrium (χ2 = 0.018, df = 1, P = 0.893). Also the frequency of 399Arg and 399Gln was 66.05% and 33.95%, respectively. It should be noted that there was no obvious deviation from the Hardy-Weinberg equilibrium (χ2 = 1.003, df = 1, P = 0.316).

Table 2 shows the combination genotypes of the two polymorphisms. The observed frequencies of the combined genotypes were remarkably differing from the expected ones. The close physical proximity of these polymorphisms should place them in linkage disequilibrium. Based on the complete dataset, XRCC1 Arg194Trp and XRCC1 Arg399Gln show a linkage disequilibrium with D′ = 0.8986, r 2 = 0.0413 (P < 0.00001). The frequency for 194Arg-399Arg, 194Arg-399Gln, and 194Trp-399Gln haplotypes was equal to 0.573, 0.339, and 0.088, respectively. The haplotype 194Trp-399Gln was not observed.

Table 2 Combination genotypes of Arg194Trp and Arg399Gln of XRCC1
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Table 3 shows the allelic frequencies of 194Trp and 399Gln in several countries. The frequency of the polymorphic alleles varies among populations, suggesting an ethnic distribution. There were significant differences in terms of the 399Gln allele frequency between the three major ethnicities. Overall, the frequency of 399Gln was about 35% among European, 26% among Asian, and 13% among African populations [2]. The allelic frequency of 399Gln in our samples seems to be more similar to the Caucasians than the Asian populations.

Table 3 Distribution of Arg194Trp and Arg399Gl n polymorphisms of XRCC1 in African, Asian, and European countries
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The 194Trp allele showed high frequency in Asian populations and low frequency among Caucasians and Africans [2]. The 194Trp allele showed frequency about 9% in our samples, which is very lower than that reported from Asian. The allelic frequency of 194Trp in our samples is more similar to the Caucasians.

Overall, it should be mentioned that for alleles of other genetic polymorphisms, such as GSTM1, GSTT1, CC16, mutations and VNTR-polymorphism of PHA [7–13], Iranian populations showed intermediate frequency in comparison with European and Asian countries.