September 2010
Volume 51, Issue 9
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Retina  |   September 2010
Polymorphisms of the DNA Repair Genes XPD and XRCC1 and the Risk of Age-Related Macular Degeneration
Author Affiliations & Notes
  • Ebru Görgün
    From the Department of Ophthalmology, Yeditepe University Medical Faculty, Istanbul, Turkey;
  • Mehmet Güven
    the Department of Medical Biology, Cerrahpasa Faculty of Medicine, University of Istanbul, Istanbul, Turkey;
  • Mustafa Ünal
    the Department of Ophthalmology, Akdeniz University Medical Faculty, Antalya, Turkey; and
  • Bahadır Batar
    the Department of Medical Biology, Cerrahpasa Faculty of Medicine, University of Istanbul, Istanbul, Turkey;
  • Gülgün S. Güven
    the Department of Medical Genetics, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey.
  • Melda Yenerel
    From the Department of Ophthalmology, Yeditepe University Medical Faculty, Istanbul, Turkey;
  • Sinan Tatlıpınar
    From the Department of Ophthalmology, Yeditepe University Medical Faculty, Istanbul, Turkey;
  • Mehmet Seven
    the Department of Medical Genetics, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey.
  • Adnan Yüksel
    the Department of Medical Genetics, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey.
  • Corresponding author: Mustafa Ünal, Pınarbaşı mah. 758 sokak Nazlıbahçe evleri, C Blok No: 18, Konyaaltı, Antalya, Istanbul, Turkey; mustafaunalmd@gmail.com
Investigative Ophthalmology & Visual Science September 2010, Vol.51, 4732-4737. doi:10.1167/iovs.09-4842
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      Ebru Görgün, Mehmet Güven, Mustafa Ünal, Bahadır Batar, Gülgün S. Güven, Melda Yenerel, Sinan Tatlıpınar, Mehmet Seven, Adnan Yüksel; Polymorphisms of the DNA Repair Genes XPD and XRCC1 and the Risk of Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2010;51(9):4732-4737. doi: 10.1167/iovs.09-4842.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Oxidative stress seems to be an important factor in the development of age-related macular degeneration (AMD). The role of DNA repair mechanisms has also received attention recently in AMD pathogenesis. This case–control study was conducted to determine the frequency of polymorphisms in two DNA repair enzyme genes, xeroderma pigmentosum complementation group D (XPD), codons 312 and 751, and x-ray cross-complementing group 1 (XRCC1), codons 194 and 399, in patients with AMD and in disease-free control subjects.

Methods.: Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to analyze XPD Asp312Asn and Lys751Gln and XRCC1 Arg194Trp and Arg399Gln in 120 patients with AMD (65 with dry type and 55 with wet type) and in age-matched 205 disease-free control subjects.

Results.: Genotypic and allelic distributions of the polymorphisms were detected. For the XPD polymorphism, although the allele frequencies were not different between the patients and healthy control subjects, there was a significant difference between frequencies for the XPD751 Gln/Gln genotype in AMD patients (9%) and healthy control subjects (19%; P = 0.02). The XPD751 Gln/Gln genotype seemed to have a protective effect against development of AMD (odds ratio, 0.41; 95% confidence interval, 0.19–0.88). Stratification by subtype of AMD revealed that the XPD751 Gln/Gln genotype was significantly lower only in the patients with dry type (P = 0.02). These interactions remained nearly significant after Bonferroni correction (P < 0.0125). Haplotype analysis for the two XPD polymorphisms revealed that the haplotype GC (312Asp-751Gln) was a protective haplotype against AMD. No statistically significant difference was found for the genotypic and allelic distributions of the polymorphisms in the XRCC1 gene between the patients and the control subjects.

Conclusions.: Polymorphism in XPD codon 751 may be associated with the development of AMD.

Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the elderly in industrialized nations. 1 It is attributable to degenerative tissue alterations occurring at the interface between the neural retina and underlying choroid. AMD can be divided into nonexudative and exudative forms. The nonexudative, atrophic, or dry form accounts for approximately 80% of AMD cases and is characterized by atrophic changes in the macula. Clinically, it has a slower deterioration and better preservation of visual acuity than does exudative AMD. 2 The hallmark of exudative, wet, or neovascular AMD is choroidal neovascularization (CNV), which is responsible for approximately 90% of cases of severe vision loss due to AMD. 2,3  
Questions remain as to the underlying pathogenesis of AMD. The most consistent major risk factor for AMD is age. Also, many others, including smoking, exposure to sunlight, hypertension, atherosclerosis, obesity, raised plasma fibrinogen levels, elevated C-reactive protein levels, increased body mass index (BMI), and dietary behavior, have been proposed. 4  
In recent years, single nucleotide polymorphisms (SNPs) associated with inflammation, oxidative stress, angiogenesis, and other pathologic processes have been linked to AMD. 5 SNPs in the gene encoding complement factor H (CFH) have been reported to confer major susceptibility to, or protection from, AMD. 6,7 CFH is a negative regulator of the complement system, and a major AMD-associated SNP is the Y402H (tyrosine to histidine substitution at amino acid 402) variant of CFH. CFH dysfunction may lead to excessive inflammation and tissue damage involved in the pathogenesis of AMD. 8 Also, the role of SNPs of many other genes including fibulin-5 (FBLN5), superoxide dismutase (SOD), apolipoprotein E (ApoE), and chemokine receptor 3 (CXCR3), have been investigated in AMD pathogenesis. 5,9,10  
Endogenous oxidative damage to proteins, lipids, and DNA has been hypothesized to be an important etiologic factor in aging and the development of systemic diseases, such as cancer, atherosclerosis, and ocular disorders including cataract, glaucoma, uveitis, and AMD. 11,12 Association between oxidative stress and DNA damage has been well known. 13 The retina is an obvious target for oxidative damage with the highest oxygen consumption of bodily tissues, 14 and several studies have indicated that oxidative stress leads to DNA damage and plays an important role in AMD. 5,9,1522  
DNA base modifications resulting in mutations and genetic instability are a major consequence of oxidative stress. Efficient DNA repair mechanisms are necessary to counterbalance the damaging effects of oxidizing species and are also vital in maintaining cellular integrity after oxidative damage has occurred. The disruption of DNA repair genes has been associated with the other degenerative and early-onset age-related diseases, such as the Werner and Bloom syndromes, as well as with the pathogenesis of aging. 21  
DNA repair enzymes continuously monitor chromosomes, to correct damaged nucleotide residues generated by exposure to cytotoxic compounds. 23 Repair of oxidative DNA damage is mediated by both base excision repair (BER) and nucleotide excision repair (NER) mechanisms. 24 Recently, it has been hypothesized in many studies that polymorphisms in DNA repair genes reduce their capacity to repair DNA damage and thereby lead to a greater susceptibility to cancer or age-related diseases. 25,26 Among them, polymorphisms of xeroderma pigmentosum complementation group D (XPD), x-ray cross-complementing group 1 (XRCC1), and x-ray cross-complementing group 3 (XRCC3) have been studied extensively. 25 X-ray cross-complementing group 1 (XRCC1), a DNA repair protein involved in single-strand breaks (SSBs) and the BER pathway, has been reported to be responsible for the efficient repair of DNA damage caused by active oxygen, ionization, and alkylating agents. 27 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 b, to repair SSBs. 27 Many polymorphisms have been detected in the XRCC1 gene, and three of them received most attention. These coding polymorphisms, resulting in amino acid substitutions, were detected at codons 399 (Arg-Gln), 194 (Arg-Trp), and 280 (Arg-His), with allelic frequencies of 27%, 13%, and 7%, respectively. 26  
XPD encodes a helicase, which participates in both NER and basal transcription as part of the transcription factor IIH. 25 Mutations destroying enzymatic function of the XPD protein are manifested clinically in combinations of three severe syndromes—Cockayne syndrome, xeroderma pigmentosum and trichothiodystrophy—depending on the location of the mutation. 25 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 SNPs in XPD gene exons have been identified; of them, Asp312Asn and Lys751Gln polymorphisms are the most common. 28 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. 28,29 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. 29,30  
We previously investigated the association between polymorphisms of DNA repair genes XPD and XRCC1 and risk of cataract and glaucoma development. 31,32 No studies have examined the possible relationship between the DNA repair enzyme XPD and XRCC1 polymorphisms and risk of AMD development. As the XPD Asp312Asn (rs1799793) and XPD Lys751Gln (rs13181) and XRCC1 Arg194Trp (rs1799782) and XRCC1 Arg399Gln (rs25487) polymorphisms are very common in the population and have immediate functional significance, we initiated this case-control study to determine the possible association with these polymorphisms and the development AMD. 
Materials and Methods
Patients and Control Subjects
This case–control study included a total of 120 patients with AMD and 205 disease-free control subjects. All subjects were of Turkish nationality belonging to the Turkish ethnic group. All subjects selected were nonsmokers and had not had diabetes mellitus or any other systemic diseases. Ethics approval was obtained from the Ethics Committee of Yeditepe University Medical Faculty. The research adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all subjects after explanation of the nature of the study. 
All participants were examined independently by two retinal specialists. Each subject underwent a complete ophthalmic examination including slit lamp biomicroscopy, funduscopy, contact lens biomicroscopic examination of the retina, fluorescein and indocyanine green fundus angiography, and optical coherence tomography. The inclusion criteria for AMD patients were as follows: (1) 50 years of age or older; (2) diagnosis of atrophic or exudative AMD in 1 or both eyes; and (3) no sign of other retinochoroidal diseases, such as pathologic myopia, geographic atrophy, central serous chorioretinopathy, angioid streaks, presumed ocular histoplasmosis syndrome, and choroidal rupture. Patients with any hereditary retinal diseases other than AMD and those who had undergone previous laser treatment for retinal conditions other than AMD were excluded. Any patient having choroidal neovascularization or a vascularized pigment epithelial detachment, which was confirmed with fluorescein angiography and indocyanine green angiography was included in the wet-type group. Fifty-five of the patients had wet-type AMD and 65 had dry type. The mean age of patients in the AMD group was 75 ± 8 years (range, 5–92 years), and 53 (44%) of them were men. 
Age-matched healthy volunteers presenting to our outpatient department with nonspecific ocular complaints, such as conjunctivitis, blepharitis, burning, itching, or presbyopia were selected randomly as a control group. They had no known family history of AMD and no clinical evidence of AMD after undergoing the same comprehensive ophthalmic examination that was used to confirm AMD in the patient group. The mean age of subjects in the control group was 73 ± 10 years (range, 50–92), and 91 (45%) of them were men. 
Blood Samples and DNA Isolation
We collected 3 mL of venous blood from all patients and control subjects. Immediately after collection, whole blood was stored in aliquots at −20οC until used. Genomic DNA was extracted from leukocytes with a DNA purification kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer's instructions. 
Genotyping of XPD Codons 312 and 751
XPD genotypes were detected by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. An Asp→Asn in exon 10 (codon 312) and a Lys→Gln in exon 23 (codon 751) were amplified to form undigested fragments of 751 and 436 bp, respectively, with primers described by Yu et al. 33 and Spitz et al., 34 respectively. 
Genotyping of XRCC1 Codons 194 and 399
XRCC1 genotypes were detected using a multiplex PCR-RFLP method. An Arg→Trp in exon 6 (codon 194) and Arg→Gln in exon 10 (codon 399) were amplified to form an undigested fragments of 491 and 615 bp, respectively, using primers described by Lunn et al. 35  
Statistical Analysis
Ages of the patient and the control groups were compared with Student's t-test. A χ2 analysis (χ2 tests) and Fisher's exact test were used to compare the distribution between the sexes and test the association between the genotypes and alleles in relation to the cases and controls and to test for deviation of genotype distribution from Hardy-Weinberg equilibrium (HWE). P < 0.05 was used as the criterion of significance. The odds ratio (OR) and 95% confidence interval (CI) were calculated to estimate the strength of the association between polymorphism genotype alleles and patients and control subjects. Bonferroni adjustments were made for P-value for the results of any SNP by multiplying the number of SNPs tested for the gene. 
Results
The demographic data of the patients and control subjects are shown in Table 1. The groups were not significantly different with respect to the age (P = 0.09) and sex (P = 0.94) of the subjects. 
Table 1.
 
Demographic Data of the Patients and Control Subjects
Table 1.
 
Demographic Data of the Patients and Control Subjects
AMD Group Control Group
Patients, n 120 205
Sex, n (%)
    Male 53 (44) 91 (45)
    Female 67 (56) 114 (55)
Age, y
    Mean ± SD 75 ± 8 73 ± 10
    Range (51–92) (50–92)
Table 2 shows the genotypic and allelic distributions of the polymorphisms in XPD and XRCC1 genes for both cases and control subjects. The distributions of the XPD-Asp312Asn, XPD-Lys751Gln, XRCC1-Arg194Gln, and XRCC1-Arg399Gln genotypes were in accordance with the Hardy-Weinberg equilibrium among the control subjects and the cases. 
Table 2.
 
Distribution of Genotype and Allele Frequencies of XPD and XRCC1 Polymorphisms in AMD Patients and Control Subjects
Table 2.
 
Distribution of Genotype and Allele Frequencies of XPD and XRCC1 Polymorphisms in AMD Patients and Control Subjects
Genotype/Allele Control, n (%) AMD, n (%) P OR (95% CI)
XPD 312
    Asp/Asp 72 (35) 46 (38) Ref.
    Asp/Asn 96 (47) 55 (46) 0.67 0.90 (0.55–1.47)
    Asn/Asn 37 (18) 19 (16) 0.52 0.80 (0.41–1.56)
    G (Asp) allele frequency 0.59 0.61
    A (Asn) allele frequency 0.41 0.39 0.77 0.92 (0.52–1.62)
XPD751
    Lys/Lys 72 (35) 51 (43) Ref.
    Lys/Gln 95 (46) 58 (48) 0.55 0.86 (0.53–1.40)
    Gln/Gln 38 (19) 11 (9) 0.02* 0.41 (0.19–0.88)
    A (Lys) allele frequency 0.58 0.67
    C (Gln) allele frequency 0.42 0.33 0.19 0.68 (0.38–1.21)
XRCC1 194
    Arg/Arg 180 (88) 98 (82) Ref.
    Arg/Trp 25 (12) 21 (17) 0.18 1.54 (0.82–2.90)
    Trp/Trp 0 (0) 1 (1) 0.36
    C (Arg) allele frequency 0.94 0.90
    T (Trp) allele frequency 0.06 0.10 0.30 1.74 (0.61–4.99)
XRCC1 399
    Arg/Arg 99 (48) 60 (50) Ref.
    Arg/Gln 85 (42) 46 (38) 0.65 0.89 (0.55–1.45)
    Gln/Gln 21 (10) 14 (12) 0.80 1.10 (0.52–2.33)
    G (Arg) allele frequency 0.69 0.69
    A (Gln) allele frequency 0.31 0.31 1.00 1.00 (0.55–1.82)
Although the allele frequencies were not different between the patients and healthy control subjects, there was a significant difference between frequencies for XPD751 Gln/Gln genotype in the AMD patients (9%) and healthy control subjects (19%; P = 0.02). The statistical analysis revealed a possible protective effect of XPD751 Gln/Gln genotype in development of AMD (OR, 0.41; 95% CI, 0.19–0.88). This difference remained nearly significant after Bonferroni correction (P < 0.0125). No statistically significant difference was found for the genotypic and allelic distributions of the polymorphisms in the XRCC1 gene (Arg194Trp and Arg399Gln) between the patients and the control subjects. 
The results for XPD were confirmed by haplotype analysis. We constructed four haplotypes (GA, AA, AC, and GC) according to the polymorphisms of XPD codons 312 and 751 (Table 3). The haplotypes were >5%. We observed that compared with the 312Asp–751Lys haplotype, the risk was most strongly reduced in carriers of the 312Asp–751Gln haplotype (P < 0.001). 
Table 3.
 
XPD Haplotype Frequencies in Control Subjects and Patients
Table 3.
 
XPD Haplotype Frequencies in Control Subjects and Patients
Haplotype Control, n (%) AMD, n (%) P OR (95% CI)
312Asp-751Lys (GA) 172 (41.9) 129 (53.7) Ref.
312Asp-751Gln (GC) 77 (18.8) 11 (4.6) <0.001 0.19 (0.09–0.38)
312Asn-751Lys (AA) 64 (15.6) 31 (12.9) 0.09 0.65 (0.38–1.08)
312Asn-751Gln (AC) 97 (23.7) 69 (28.8) 0.86 0.86 (0.52–1.41)
Table 4 shows the distribution of the allele and genotype frequencies of the XPD and XRCC1 polymorphisms in the healthy control subjects and AMD patients after they were stratified by AMD subtype. Stratification by subtype of AMD revealed that the XPD751 Gln/Gln genotype was significantly lower in the patients with dry-type AMD (6%) than in the control subjects (19%; P = 0.02) and may have a protective effect against the development of dry-type AMD (OR, 0.28; 95% CI, 0.09–0.86). This interaction was almost significant after Bonferroni correction (P < 0.0125). The frequencies of the XPD751 Gln/Gln genotype were not different between the patients with wet-type AMD (12%) and the control subjects (19%; P = 0.21). 
Table 4.
 
Distribution of Genotype and Allele Frequencies of XPD and XRCC1 Polymorphisms in Control Subjects and Patients with Different AMD Subtypes
Table 4.
 
Distribution of Genotype and Allele Frequencies of XPD and XRCC1 Polymorphisms in Control Subjects and Patients with Different AMD Subtypes
Genotype/Allele Control, n (%) AMD Subtypes P * OR (95% CI)* P OR (95% CI)†
Dry, n (%) Wet, n (%)
XPD312
    Asp/Asp 72 (35) 25 (39) 21 (38) Ref. Ref.
    Asp/Asn 96 (47) 30 (46) 25 (46) 0.74 0.90 (0.49–1.66) 0.74 0.90 (0.46–1.72)
    Asn/Asn 37 (18) 10 (15) 9 (16) 0.56 0.78 (0.34–1.79) 0.68 0.83 (0.35–2.00)
    G (Asp) allele frequency 0.59 0.62 0.61
    A (Asn) allele frequency 0.41 0.38 0.39 0.66 0.88 (0.50–1.56) 0.77 0.92 (0.52–1.62)
XPD751
    Lys/Lys 72 (35) 27 (42) 24 (44) Ref. Ref.
    Lys/Gln 95 (46) 34 (52) 24 (44) 0.88 0.95 (0.53–1.72) 0.40 0.76 (0.40–1.44)
    Gln/Gln 38 (19) 4 (6) 7 (12) 0.02‡ 0.28 (0.09–0.86) 0.21 0.55 (0.22–1.40)
    A (Lys) allele frequency 0.58 0.68 0.65
    C (Gln) allele frequency 0.42 0.32 0.35 0.14 0.65 (0.37–1.16) 0.31 0.74 (0.42–1.32)
XRCC1194
    Arg/Arg 180 (88) 50 (77) 48 (87) Ref. Ref.
    Arg/Trp 25 (12) 14 (21) 7 (13) 0.06 2.02 (0.98–4.16) 0.92 1.05 (0.43–2.57)
    Trp/Trp 0 (0) 1 (2) 0 (0) 0.22
    C (Arg) allele frequency 0.94 0.88 0.94
    T (Trp) allele frequency 0.06 0.12 0.06 0.14 2.14 (0.77–5.94) 1.00 1.00 (0.31–3.21)
XRCC1399
    Arg/Arg 99 (48) 32 (49) 28 (51) Ref. Ref.
    Arg/Gln 85 (42) 28 (43) 18 (33) 0.95 1.02 (0.57–1.83) 0.39 0.75 (0.39–1.45)
    Gln/Gln 21 (10) 5 (8) 9 (16) 0.57 0.74 (0.26–2.11) 0.36 1.52 (0.62–3.68)
    G (Arg) allele frequency 0.69 0.71 0.67
    A (Gln) allele frequency 0.31 0.29 0.33 0.76 0.91 (0.50–1.67) 0.76 1.10 (0.61–1.99)
No statistically significant difference was found for the genotypic and allelic distributions of the polymorphisms in the XRCC1 gene between the control subjects and patients, even after they were stratified by AMD subtype. 
Discussion
The oxidized bases of DNA arising from interactions with reactive oxygen species contribute significantly to aging and age-related disorders. 36 Age-related oxidative damage has been demonstrated in collagen, elastin, mucopolysaccharides, and nuclear and mitochondrial DNA, and lipid peroxidation has been shown to contribute to lipofuscinogenesis. 37  
The cellular response to DNA damage includes a variety of actions, including DNA repair, inhibition of replication and transcription, changes in cell cycle, apoptosis, senescence, necrosis, and other processes. DNA repair pathways are one of the most important defense systems against oxidative stress in the cell. Because damage to the nuclear and mitochondrial genomes is ongoing throughout life, efficient DNA repair mechanisms are necessary to counterbalance the damaging effects of oxidizing species and also to maintain cellular integrity after oxidative damage has occurred. A defect of DNA repair could allow rapid accumulation of damage that ultimately results in manifestation of an accelerated aging process. Such a mechanism has been associated with Cockayne syndrome, in which both the nucleotide excision repair and transcription-coupled repair are deficient. 38  
The eye is not an isolated organ, but rather one of many systems subject to the processes of aging. The age-related decrease in DNA repair capacity combines with SNPs, causing functional changes in DNA repair genes that may lead to increased levels of oxidatively damaged DNA and age-related ocular diseases such as glaucoma and cataract. 31,32,39,40  
Involvement of oxidative damage to DNA in AMD pathogenesis may also indicate the role of DNA repair genes. 5,9,21,22 Szaflik et al. 9 evaluated endogenous DNA damage, sensitivity to exogenous mutagens and DNA repair efficacy in AMD patients and age-matched subjects without visual disturbances. They used hydrogen peroxide and UV radiation as mutagens. They found that the cells taken from AMD patients displayed a higher extent of basal endogenous DNA damage than those from control subjects. Also, the extent of oxidative modification to DNA bases was greater in AMD patients than in control subjects. Lymphocytes from AMD patients displayed a higher sensitivity to the mutagens and repaired lesions induced by these factors less effectively than did the cells from the control individuals. They postulated that the impaired efficacy of DNA repair may combine with enhanced sensitivity of retina pigment epithelium (RPE) cells to mutagens, contributing to the pathogenesis of AMD. 9  
Tuo et al. 22 investigated the association between SNPs of ERCC6 gene and risk of AMD development. ERCC6, a DNA repair gene that is located on chromosome 10, at region q11 is involved in excision repair of DNA. Mice mutant for ERCC6 display some signs of premature ageing including stunted growth, neurologic dysfunction, retinal degeneration, cachexia, and kyphosis. 41 They concluded that ERCC6 C-6530>G was associated with AMD susceptibility, both independently and through interaction with a SNP (rs380390) in the CFH intron reported to be highly associated with AMD. A disease OR of 23 was conferred by homozygosity for risk alleles at both ERCC6 and CFH compared with homozygosity for nonrisk alleles. 22  
The human Ogg1 (hOgg1) gene is one of the DNA repair genes and encodes a DNA glycosylase that is involved in the base excision repair of 8-hydroxy-2′ deoxyguanin (8-OH-dG) from oxidatively damaged DNA. 42 It is closely associated with ERCC6. ERCC6 collaborates with hOgg1 to perform preferential DNA repair in eukaryotes and plays a role in the maintenance of efficient hOgg1 expression. Bojanowski et al. 21 investigated the hOgg1 S326C SNP in association with AMD. They did not find a statistically significant association between hOgg1 and AMD in their study. 
The present study is the first to examine the possible relationship between the DNA repair enzyme XPD and XRCC1 polymorphisms and the risk of AMD. As the XPD Asp312Asn and XPD Lys751Gln and XRCC1 Arg194Trp and XRCC1 Arg399Gln polymorphisms are very common in the population and have been shown to be associated with many diseases, we determined the frequencies of these polymorphisms in AMD patients. We found a possible protective effect of XPD751 Gln/Gln genotype in development of AMD, especially against dry-type AMD. No statistically significant difference was found for the genotypic and allelic distributions of the polymorphisms in XRCC1 gene between patients and control subjects. The protective effect of XPD751 Gln/Gln genotype remained nearly significant after Bonferroni correction. Reexamination of the original gels for this polymorphism revealed no genotyping errors, and so it seems likely that this result was due to chance. 
We confirmed the results for XPD by haplotype analysis. Haplotype analysis provides an advantage compared with single-locus analysis in terms of disease susceptibility by the assessment of the interaction between two or more genes that are located close to each other. To analyze the combined effect of two polymorphisms in XPD Asp312Asn (G>A) and Lys751Gln (A>C), we compared the haplotype frequencies between the patients and the control subjects. The haplotype GC (312Asp-751Gln) conferred a protective association against AMD susceptibility. The results showed that the haplotype effects reflect that of the 751Gln allele. 
We previously investigated the frequencies of the XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms in patients with primary open angle glaucoma (POAG) and senile cataract. 31,32 Senile cataract and POAG are two age-related ocular disorders that have been shown to be associated with oxidative DNA damage. Although we did not find any relationship between these polymorphisms and the risk of POAG, there was a statistically significant association between the XPD Lys751Gln polymorphism and the risk of cataract development. The frequency of the XPD751 Gln/Gln genotype was significantly lower in cataract patients than in healthy control subjects. This difference was also evident in cortical type cataract, even after stratification by the cataract subtypes. In accordance with the present study, we concluded that the XPD751 Gln/Gln genotype may have a protective effect in development of senile cataract, especially against cortical type. 32  
Association of XPD and XRCC1 polymorphisms with atherosclerosis, aging, and even longevity have been reported. 26,4345 Although there is an apparent divergence among the results, earlier studies have focused mainly on the relationship of XRCC1 and XPD polymorphisms with cancers. 31 Molecular epidemiologic studies have found that the XPD 751Gln allele is associated with increased risk of many types of cancers. However, inconsistent findings were also reported, including absence of any association with many others. 31 In the current study, a possible protective effect of the XPD751 Gln/Gln genotype in development of AMD was detected. Lunn et al. 29 reported that individuals homozygous for the wild-type Lys allele had higher levels of chromatid aberrations than did those with one or two Gln alleles. They also found that the Lys/Lys751 genotype was associated with suboptimal repair of DNA damage. Another study claimed a specific association with melanoma, suggesting that the 751Lys/Lys allele was associated with greater risk for cancer than 751Gln/Gln. 46 Dybdahl et al. 47 also reported that individuals with the common allele (Lys751) had an elevated risk of basal cell carcinoma. The XPD gene product is a DNA helicase, components of the basal transcription factor TFIIH complex. They are involved in transcription, NER, and apoptosis. Thus, one may expect that genetic variants in XPD could reduce NER capacity and be an important risk factor of impaired DNA damage. Vodicka et al. 48 investigated the potential links between genetic polymorphisms in genes coding DNA repair enzymes and the levels of chromosomal aberrations (CAs) and SSBs in DNA. They found that total CAs and SSBs were decreased in individuals with homozygous genotypes of the XPD exon 23 variant Gln allele in comparison with those with the wild-type Lys and heterozygous Gln/Lys genotypes. It is also possible that the 751 Gln allele has different effects in different DNA repair pathways. In addition, the effect of a given allele on repair may depend on the exposure and interaction with other genes participating in DNA damage recognition, repair, and cell cycle regulation. 
Identifying patients at risk of developing AMD through genotyping may allow a more personalized approach to moderate the risk of AMD development. One of the limitations of the study current is our small sample size. Another limitation is the requirement for large studies to overcome the statistical limitations of multiple testing. Even so, this preliminary study raises the possibility of an association of AMD development with Lys751Gln polymorphism in the XPD gene, which makes this gene a candidate for studies in susceptibility to AMD. However, additional studies with larger sample sizes and multiple SNPs are necessary to detect the small effects observed. Future case–control studies will aid in understanding the role of DNA repair gene polymorphisms in the etiology of AMD. 
Footnotes
 Supported by the Research Fund of The University of Istanbul, Project 1153. MU was supported by Akdeniz University Scientific Research Projects Unit.
Footnotes
 Disclosure: E. Görgün, None; M. Güven, None; M. Ünal, None; B. Batar, None; G.S. Guven, None; M. Yenerel, None; S. Tatlipinar, None; M. Seven, None; A. Yüksel, None
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Table 1.
 
Demographic Data of the Patients and Control Subjects
Table 1.
 
Demographic Data of the Patients and Control Subjects
AMD Group Control Group
Patients, n 120 205
Sex, n (%)
    Male 53 (44) 91 (45)
    Female 67 (56) 114 (55)
Age, y
    Mean ± SD 75 ± 8 73 ± 10
    Range (51–92) (50–92)
Table 2.
 
Distribution of Genotype and Allele Frequencies of XPD and XRCC1 Polymorphisms in AMD Patients and Control Subjects
Table 2.
 
Distribution of Genotype and Allele Frequencies of XPD and XRCC1 Polymorphisms in AMD Patients and Control Subjects
Genotype/Allele Control, n (%) AMD, n (%) P OR (95% CI)
XPD 312
    Asp/Asp 72 (35) 46 (38) Ref.
    Asp/Asn 96 (47) 55 (46) 0.67 0.90 (0.55–1.47)
    Asn/Asn 37 (18) 19 (16) 0.52 0.80 (0.41–1.56)
    G (Asp) allele frequency 0.59 0.61
    A (Asn) allele frequency 0.41 0.39 0.77 0.92 (0.52–1.62)
XPD751
    Lys/Lys 72 (35) 51 (43) Ref.
    Lys/Gln 95 (46) 58 (48) 0.55 0.86 (0.53–1.40)
    Gln/Gln 38 (19) 11 (9) 0.02* 0.41 (0.19–0.88)
    A (Lys) allele frequency 0.58 0.67
    C (Gln) allele frequency 0.42 0.33 0.19 0.68 (0.38–1.21)
XRCC1 194
    Arg/Arg 180 (88) 98 (82) Ref.
    Arg/Trp 25 (12) 21 (17) 0.18 1.54 (0.82–2.90)
    Trp/Trp 0 (0) 1 (1) 0.36
    C (Arg) allele frequency 0.94 0.90
    T (Trp) allele frequency 0.06 0.10 0.30 1.74 (0.61–4.99)
XRCC1 399
    Arg/Arg 99 (48) 60 (50) Ref.
    Arg/Gln 85 (42) 46 (38) 0.65 0.89 (0.55–1.45)
    Gln/Gln 21 (10) 14 (12) 0.80 1.10 (0.52–2.33)
    G (Arg) allele frequency 0.69 0.69
    A (Gln) allele frequency 0.31 0.31 1.00 1.00 (0.55–1.82)
Table 3.
 
XPD Haplotype Frequencies in Control Subjects and Patients
Table 3.
 
XPD Haplotype Frequencies in Control Subjects and Patients
Haplotype Control, n (%) AMD, n (%) P OR (95% CI)
312Asp-751Lys (GA) 172 (41.9) 129 (53.7) Ref.
312Asp-751Gln (GC) 77 (18.8) 11 (4.6) <0.001 0.19 (0.09–0.38)
312Asn-751Lys (AA) 64 (15.6) 31 (12.9) 0.09 0.65 (0.38–1.08)
312Asn-751Gln (AC) 97 (23.7) 69 (28.8) 0.86 0.86 (0.52–1.41)
Table 4.
 
Distribution of Genotype and Allele Frequencies of XPD and XRCC1 Polymorphisms in Control Subjects and Patients with Different AMD Subtypes
Table 4.
 
Distribution of Genotype and Allele Frequencies of XPD and XRCC1 Polymorphisms in Control Subjects and Patients with Different AMD Subtypes
Genotype/Allele Control, n (%) AMD Subtypes P * OR (95% CI)* P OR (95% CI)†
Dry, n (%) Wet, n (%)
XPD312
    Asp/Asp 72 (35) 25 (39) 21 (38) Ref. Ref.
    Asp/Asn 96 (47) 30 (46) 25 (46) 0.74 0.90 (0.49–1.66) 0.74 0.90 (0.46–1.72)
    Asn/Asn 37 (18) 10 (15) 9 (16) 0.56 0.78 (0.34–1.79) 0.68 0.83 (0.35–2.00)
    G (Asp) allele frequency 0.59 0.62 0.61
    A (Asn) allele frequency 0.41 0.38 0.39 0.66 0.88 (0.50–1.56) 0.77 0.92 (0.52–1.62)
XPD751
    Lys/Lys 72 (35) 27 (42) 24 (44) Ref. Ref.
    Lys/Gln 95 (46) 34 (52) 24 (44) 0.88 0.95 (0.53–1.72) 0.40 0.76 (0.40–1.44)
    Gln/Gln 38 (19) 4 (6) 7 (12) 0.02‡ 0.28 (0.09–0.86) 0.21 0.55 (0.22–1.40)
    A (Lys) allele frequency 0.58 0.68 0.65
    C (Gln) allele frequency 0.42 0.32 0.35 0.14 0.65 (0.37–1.16) 0.31 0.74 (0.42–1.32)
XRCC1194
    Arg/Arg 180 (88) 50 (77) 48 (87) Ref. Ref.
    Arg/Trp 25 (12) 14 (21) 7 (13) 0.06 2.02 (0.98–4.16) 0.92 1.05 (0.43–2.57)
    Trp/Trp 0 (0) 1 (2) 0 (0) 0.22
    C (Arg) allele frequency 0.94 0.88 0.94
    T (Trp) allele frequency 0.06 0.12 0.06 0.14 2.14 (0.77–5.94) 1.00 1.00 (0.31–3.21)
XRCC1399
    Arg/Arg 99 (48) 32 (49) 28 (51) Ref. Ref.
    Arg/Gln 85 (42) 28 (43) 18 (33) 0.95 1.02 (0.57–1.83) 0.39 0.75 (0.39–1.45)
    Gln/Gln 21 (10) 5 (8) 9 (16) 0.57 0.74 (0.26–2.11) 0.36 1.52 (0.62–3.68)
    G (Arg) allele frequency 0.69 0.71 0.67
    A (Gln) allele frequency 0.31 0.29 0.33 0.76 0.91 (0.50–1.67) 0.76 1.10 (0.61–1.99)
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