Introduction

Patients with end-stage renal disease (ESRD) display enhanced genomic damage [1]. If genomic damage is left unrepaired or is repaired with errors, mutations of critical genes may occur and may result in an enhanced cancer risk. Genomic damage may also be involved in initiation as well as progression of cardiovascular diseases [2]. End-stage renal disease is associated with a high incidence of cancers and cardiovascular diseases [3, 4]. Among the several pathogenic mechanisms suggested to explain these phenomena there are uremia per se, micro inflammation and oxidative stress [5, 6], which involves the whole cell structure (proteins, membrane lipids, carbohydrates and DNA) [7]. Oxidative stress is enhanced in patients with ESRD [8]. It has been reported that oxidative stress can induce DNA damage, such as base modifications and strand breaks [9, 10].

DNA repair enzymes continuously monitor chromosomes to correct damaged nucleotide residues generated by exposure to cytotoxic compounds or carcinogens. Repair of oxidative DNA damage is mediated by both base excision repair (BER) and nucleotide excision repair (NER) mechanisms. It has been hypothesized in many studies that polymorphisms in DNA repair genes reduce their capacity to repair DNA damage and thereby lead to increased cancer or other disease susceptibility [11, 12]. Although hundreds of polymorphisms in DNA repair genes have been identified, their effects on repair function have not been well characterized.

Xeroderma pigmentosum complementation group D (XPD) encodes a helicase, which participates in both NER and basal transcription as part of the transcription factor IIH. Because XPD is important in multiple cellular tasks and rare XPD mutations result in genetic diseases, XPD polymorphisms may operate as genetic susceptibility factors. Several single nucleotide polymorphisms (SNPs) in XPD gene exons have been identified, of them Asp312Asn and Lys751Gln polymorphisms are the most common [13]. XPD Asp312Asn in exon 10 causes an amino acid substitution in a conserved region of XPD. XPD Lys751Gln in exon 23 also causes an amino acid substitution in the C-terminal part of the protein [13, 14]. These polymorphisms may produce the most relevant change in XPD function and affect different protein interactions, diminish the activity of TFIIH complexes, influence DNA repair capacity, and alter the genetic susceptibility for diseases [14, 15].

X-ray cross-complementing group 1 (XRCC1), a DNA repair protein involved in single-strand breaks and BER pathway, has been reported to be responsible for the efficient repair of DNA damage caused by active oxygen, ionization, and alkylating agents [16]. It is a multidomain protein that interacts with the nicked DNA and participates with at least three different enzymes, poly-ADP-ribose polymerase (PARP), DNA ligase III, and DNA polymerase β, to repair single-strand breaks [16]. Three coding polymorphisms were identified in the XRCC1 gene at the codons 194 (Arg to Trp), 280 (Arg to His), and 399 (Arg to Gln) [13]. Whereas the functional effects of these polymorphisms in XRCC1 have not been well known, amino acid changes at evolutionary conserved regions may alter its function. Two polymorphisms: Arg194Trp and Arg399Gln have been widely studied in the literature.

No studies have examined the possible relationship between DNA repair enzymes polymorphisms and the risk of development ESRD. As the XPD Asp312Asn and XPD Lys751Gln and XRCC1 Arg194Trp and XRCC1 Arg399Gln polymorphisms have immediate functional significance and are very common in the population, we initiated this case control study to determine the possible association with these polymorphisms and the development ESRD.

Materials and methods

Subjects

Between October 2006 and November 2007, 136 patients with ESRD (85 females and 51 males, mean age 47 ± 13) were recruited consecutively to this controlled study in our out-patient clinic. All subjects were of Turkish nationality belonging to the Turkish ethnic group. Of these 136 patients, 71 were undergoing chronic hemodialysis (HD) and 65 were undergoing peritoneal dialysis (PD). Patients who had been undergoing HD or PD more than 6 months were enrolled in this study. Time on dialysis of patients was 49.9 ± 45.0 months. Control group was formed by 147 healthy individuals who do not smoke regularly (92 females and 55 males, mean age 48 ± 15).

The causes of ESRD were chronic glomerulonephritis in 61 patients (45%), diabetes mellitus in 23 patients (17%), hypertensive nephrosclerosis in 17 patients (12%), chronic interstitial nephritis in eight patients (6%), polycystic kidney disease in eight patients (6%), amyloidosis in five patients (4%), multiple myeloma in two patients (1%), and unknown in 12 patients (9%). In eight of 136 patients, there were different types of malignancy: multiple myeloma in one patient, colon cancer in one patient, colon cancer with multiple myeloma in one patient, breast cancer in two patients, lymphoma in one patients, cervix cancer in one patient, and larynx cancer in one patient. Seven patients were receiving immunosuppressive drugs.

The study was approved by the Ethics Committee of Istanbul University Cerrahpasa Medical Faculty and conducted in accordance with the current version of the Declaration of Helsinki. All participants gave their written informed consent prior to participation in the study.

Extraction of DNA and genotyping analysis

We collected 3 ml of venous blood from patients and controls. Immediately after collection, whole blood was stored in aliquots at −20°C until use. Genomic DNA was extracted from leukocytes using Roche DNA purification kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions.

XPD genotypes were detected using a polymerase chain reaction-restriction fragment lenght polymorphism (PCR–RFLP) method. An Asp → Asn in exon 10 (codon 312; rs.1799793) and a Lys → Gln in exon 23 (codon 751; rs.13181) were amplified to form an undigested fragments of 751 and 436 bp, respectively, using primers described by Yu et al. [17] and Spitz et al. [18] respectively.

XRCC1 genotypes were detected using a multiplex PCR–RFLP method. An Arg → Trp in exon 6 (codon 194; rs.1799782) and Arg → Gln in exon 10 (codon 399; rs.25487) were amplified to form an undigested fragments of 491 and 615 bp, respectively, using primers described by Lunn et al. [14].

Statistical analysis

Mean and standard deviations (SD) are presented in case of continuous variables. Chi-square analysis (χ2 tests) and Fisher’s exact test were used to compare the gender distribution and test the association between the genotypes and alleles in relation to the cases and controls and test for deviation of genotype distribution from Hardy Weinberg equilibrium (HWE). The odds ratio (OR) and their 95% confidence intervals (CI) were calculated to estimate the strength of the association between polymorphism genotype alleles and patients and controls. Bonferroni adjustments were made for p value for the results of any SNP by multiplying the number of SNPs tested for the gene. Differences between the means of the two continuous variables were evaluated by the Student’s t-test. A value of P < 0.05 was considered statistically significant. Analysis was performed with SPSS 11.5 statistical software.

Results

The patient and control groups were not statistically different with respect to age (P = 0.47) and gender (P = 0.80). Clinical characteristics of patients with ESRD are outlined in Table 1.

Table 1 Clinical characteristics of patients with ESRD
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Table 2 shows the genotypic and allelic distributions of the polymorphisms in XPD and XRCC1 genes for both patients and controls. The distributions of the XPD-Asp312Asn, XPD-Lys751Gln, XRCC1-Arg194Gln and XRCC1-Arg399Gln genotypes were in accordance with the HWE among the cases and controls. There were a significant difference between frequencies for XRCC1-399 Arg/Gln genotype and XRCC1-399 Gln/Gln genotype in patients and controls (P = 0.007 and P = 0.005, respectively). We found that the patients with XRCC1-399 Arg/Gln (OR:1.98; 95% CI: 1.21–3.25) or XRCC1-399 Gln/Gln (OR: 3.95; 95% CI: 1.45–10.76) genotype had a significantly higher risk of ESRD than those with XRCC1-399 Arg/Arg genotype. After Bonferroni correction for multiple testing, these associations remained significant (P = 0.028 and P = 0.02, respectively). In addition, the Gln allele of this polymorphism was associated with increased risk of ESRD (OR:2.03; 95% CI: 1.08–3.81, P = 0.03). No statistically significant differences were observed in the alleles or in the genotype frequencies of the XRCC1-Arg194Trp, XPD-Asp312Asn and XPD-Lys751Gln gene polymorphisms between the control group and the patients group.

Table 2 Distribution of genotype and allele frequencies of XPD and XRCC1 polymorphisms in patients and controls
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Regarding the effect of combined polymorphisms of XPD Asp312Asn, XPD Lys751Gln, XRCC1 Arg194Trp and XRCC1 Arg399Gln on the risk of ESRD development, the wild type genotypes for each gene were taken as references (Table 3). The analysis showed that combination of Arg/Gln or Gln/Gln genotypes of XRCC1 Arg399Gln polymorphism with the two possible genotypes of XPD-Asp312Asn polymorphism (Asp/Asn and Asn/Asn or Asp/Asp) was significantly associated with the development of ESRD (p values <0.05 for both). Combination of Arg/Gln or Gln/Gln genotypes of XRCC1 Arg399Gln polymorphism with Lys/Gln or Gln/Gln genotypes of XPD Lys751Gln polymorphism was also found to increase the risk of ESRD development (P < 0.05). However, no significant association was found between other compound polymorphisms and the risk of development ESRD.

Table 3 Distribution of combined XPD and XRCC1 genotypes among ESRD patients and control
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Discussion

Various studies have shown the existence of a large interindividual variation in repair of DNA damage induced by endogenous and exogenous insults and the individuals with less dramatic reduction in the capacity to repair DNA are observed at polymorphic frequency [19]. Such individuals with repair capacity below the population mean can be at increased risk of developing several chronic diseases. In our study, all the examined subjects were genotyped for two repair genes, XRCC1 and XPD in order to analyze the possible influence of the genetically determined variations on susceptibility to ESRD. We found that the XRCC1 codon 399 polymorphism was associated with susceptibility to ESRD. However, the XRCC1 codon 194, XPD codon 312 and 751 polymorphisms were not associated with ESRD.

To our knowledge this is the first study to examine the effect of DNA repair gene polymorphisms on risk of ESRD. The major finding of this study is that the presence of a common genetic polymorphism of XRCC1 codon 399, the 399Gln variant, is associated with ESRD. Our finding is consistent with the published functional studies that reported some associations between XRCC1 Arg399Gln polymorphism and markers of DNA damage. In these studies it has shown that the XRCC1 399Gln polymorphic variant is associated with higher levels of DNA adducts, somatic mutations, micronuclei, sister chromatid exchanges and chromosomal damages.

In patients with ESRD, DNA damage has been shown by numerous biomarkers, such as the analysis of sister chromatid exchange and chromosomal aberrations [20, 21], micronucleus frequency [22–24], comet assay (single-cell gel electrophoresis) in peripheral lymphocytes [16, 25], 8-hydroxy 2-deoxyguanosine content in leukocytes [26, 27], and mitochondrial DNA deletions in skeletal muscle tissue and hair follicles [28].

Moreover, the XRCC1 399Gln gene variant has been associated with artherosclerotic coronary artery disease, schizophrenia, pterygium, cataracts and systemic lupus erythematosus [2, 29–32] as well as various cancer types such as breast, lung, prostate, renal, carcinomas of head and neck, stomach, colon and acute lymphoblastic leukemia [33–40].

Combinations of common genetic polymorphisms may increase or decrease the susceptibility to certain diseases [41]. To investigate the presence of such an effect we also made an association analysis between genotype combinations and ESRD. It was found that combination of Arg/Gln or Gln/Gln genotypes of XRCC1 Arg399Gln polymorphism (at least one Gln carriers) with all genotypes of XPD-Asp312Asn polymorphism and XPD Lys751Gln polymorphism (except Lys/Lys genotype) may increase the risk of ESRD development. The only exception was the combination with Lys/Lys genotype of XPD Lys751Gln polymorphism, which has been reported previously to have an protective effect against certain diseases [42, 43]. It may be hypothesized that increased risk associated with Gln allele at XRCC1 codon 399 was decreased with Lys/Lys genotype of XPD Lys751Gln polymorphism.

The XRCC1 protein plays an important role in the BER pathway. XRCC1 is thought to act as a scaffold protein, play a coordinating role for consecutive stages of the BER system [44], interact with several proteins of BER and single-strand break repair machinery including OGG1, NEIL1, NEIL2, ANPG, NTH1, as well as APE1, PNK, TDP1, PARP1, PARP2, POLβ, LIG3a, and exert function in recognizing and binding to single-strand breaks. The XRCC1 Arg399Gln polymorphism is located within the XRCC1 BRCA1 carboxyl-terminal domain (BRCT I) and is hypothesized to have functional significance because it is located within a well-conserved region and encodes a nonconservative amino acid change. Chinese hamster ovary cells with no functional XRCC1 protein are hypersensitive to a broad range of DNA damage, such as that induced by alkylating agents, ROS, or ionizing radiation, suggesting an essential role for XRCC1 in DNA repair [16].

In conclusion, our current study demonstrated that XRCC1 Arg399Gln polymorphism may contribute to individual susceptibility to ESRD. In accordance with our knowledge, this is the first report showing an association between DNA repair gene polymorphisms and ESRD susceptibility. Thus, BER genes are suggested to be used as a predictive factor for ESRD. However, further studies are needed to evaluate the influence of their polymorphisms on the risk of ESRD.