GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 16, Number 6, 2012 ª Mary Ann Liebert, Inc. Pp. 504–507 DOI: 10.1089/gtmb.2011.0252
DNA Repair Genes in Parkinson’s Disease Mehmet Gencer,1 Selcuk Dasdemir,2 Bedia Cakmakoglu,2 Yilmaz Cetinkaya,1 Figen Varlibas,1 Hulya Tireli,1 Cem Ismail Kucukali,3 Elif Ozkok,3 and Makbule Aydin3
Aims: There is a growing interest in the understanding of a possible role of DNA repair systems in ageing and neurodegenerative diseases after DNA damage is observed in the brain of individuals affected by neurodegenerative diseases. In the light of these findings, we investigated whether DNA repair gene polymorphisms (XRCC1 Arg399Gln, XRCC3 Thr241Met XPD Lys751Gln, XPG Asp1104His, APE1 Asp148Glu, and HOGG1 Ser326Cys) account for an increased risk of Parkinson’s disease (PD). Methods: The present analyses are based on 60 case subjects with PD and 108 unrelated healthy controls. Genotyping of DNA repair gene polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism. Results: We, for the first time, demonstrated the positive association of APE1, XRCC1, and XRCC3 DNA repair gene variants with PD risk. In our study, the frequencies of Glu/Glu genotype in APE1, Gln + genotype of XRCC1, and Thr + genotype of XRCC3 are higher in patients than in controls ( p = 0.028, p = 0.002 and p = 0.046, respectively). Conclusions: In conclusion, our findings have suggested that APE1, XRCC1, and XRCC3 genetic variants may be a risk factor by increasing oxidative stress that might cause the loss of dopaminergic cells in the substantiata nigra and locus caeruleus, leading to abnormal signal transmittion, and ultimately, the development of PD. In addition, generation of reactive oxygen species from dopamine might affect the other DNA repair pathway proteins that we did not examine in the current study. Further studies with larger sample groups are necessary to clarify the role of DNA repair genes and the development of PD.
Introduction
P
arkinson’s disease (PD) is a common neurodegenerative disease associated with progressive loss of dopaminergic cells in the substantiate nigra and locus caeruleus. Multiple genetic and environmental factors are involved in the pathological process underlying the neurodegeneration associated with PD (Pankratz and Foroud, 2004). The etiology of the underlying neurodegenerative process is widely unknown; however, oxidative stress is a unifying factor in the current theories of PD pathogenesis (Olanow and Tatton, 1999). Postmortem studies of PD brains demonstrate increased indices of oxidative stress, including increased levels of iron, increased lipid peroxidation, decreased mitochondrial complex I activity, and decreased levels of glutathione in the substantiate nigra (Hirsch et al.,1991). This may be the result of the combined presence of a high dopamine metabolism generating reactive oxygen species (ROS), low levels of antioxidant glutathione, and increased levels of iron catalyzing ROS formation (Chinta and Andersen, 2008). Several studies indicate that the formation of ROS is a key step in selective neuronal vulnerability in PD (Kolesnick and Golde, 1997).
ROS can directly damage neurons by alteration of biological molecules, that is, proteins, nucleic acids, and may also take part in signal transduction pathways leading to apoptosis (Zhou et al., 2008). Excess ROS may induce oxidative DNA damage, DNA strand breaks, base modifications, and chromosomal aberrations (Marnett, 2000). Defects in the response to DNA damage, whether it is repair or signal transduction defects, underpin many human diseases, including cancer, immune dysfunction, radiosensitivity disorders, and neurodegenerative diseases. For repair of oxidative DNA damage, human cells have five DNA repair systems: Direct reversal, mismatch repair, double-strand break repair, base excision repair (BER), and nucleotide excision repair (Wood et al., 2001). Nearly all oxidatively induced DNA lesions, as well as single-strand breaks, are repaired via the BER pathway in organisms ranging from E. coli to mammals (Krokan et al., 2000). Although it has been suggested that some DNA repair gene variants may affect the repair capacity and influence the development of PD, there is no report describing an association between DNA repair gene polymorphisms (XRCC1 Arg399Gln, XRCC3 Thr241Met XPD Lys751Gln, XPG
1 Department of Neurology, Haydarpasa Numune Training and Research Hospital, Istanbul, Turkey. Departments of 2Molecular Medicine and 3Neuroscience, Institute of Experimental Medicine Research, Istanbul University, Istanbul, Turkey.
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DNA REPAIR GENES IN PARKINSON’S DISEASE Asp1104His, APE1 Asp148Glu, and HOGG1 Ser326Cys) and PD risk.
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Materials and Methods
(4) Surgery or neuroleptic drugs in the past few months. The healthy control subjects were unrelated and randomly selected among volunteer blood donors. None of the controls had a family history of PD.
Subject selection
Polymorphism analysis
In this study, polymorphisms of six DNA repair genes were analyzed in 60 PD patients referred to Istanbul Haydarpasa Numune Training and Research Hospital, and in 108 controls. The mean age was 64.70 – 9.36 for PD patients, and 61.50 – 9.16 years for the controls. There were no significant differences among the study and control groups in terms of mean age and sex distribution. PD was diagnosed according to the criteria of the UK Parkinson’s Disease Society Brain Bank (Lees, 1988). All patients had at least two of four cardinal features of PD (bradykinesia, rigidity, tremor, and postural instability). Parkinsonian symptoms were assessed by Unified Parkinson’s Disease Rating Scale Part III (UPDRS) (Fahn et al., 1987) and HoehnYahr scale (Hoehn and Yahr, 1967). The UPDRS is composed of 42 items that include four sections (I. Mentation, behavior, and mood; II. Activities of daily living; III. Motor examination; and IV. Complications of therapy) and 42 items. Each item in the first three sections are rated from 0 (normal) to 4 (severe). The Part III of the UPDRS has 14 items for evaluating bradykinesia, tremor, rigidity, speech, facial expression, postural stability, and gait. Patients were evaluated for cognitive state by using Turkish version of Ko¨kmen’s Short Test of Mental Status (STMS) (Ko¨kmen et al., 1987). The cut-off score for the STMS is 29, and the patients below this score were considered demented and were excluded from the study. The study was approved by the Medical Ethics Committee of Istanbul Medical Faculty, and all participants (i.e., controls, patients, or unaffected family members (on behalf of some patients) gave written informed consent.
DNA was extracted from white blood cells according to the method of Miller et al. (1988). Polymerase chain reaction (PCR)/restriction fragment length polymorphism analysis was performed for the detection of the variations in these regions (Sturgis et al., 1999; Hu et al., 2001; Le Marchand et al., 2002; Mort et al., 2003; Yeh et al., 2005). Initially, PCR was performed to determine the polymorphic regions using suitable primers. PCR products of XRCC3 Thr241Met, APE1 Asp148Glu, HOGG1 Ser326Cys, XRCC1 Arg399Gln, XPG Asp1104His, and XPD Lys751Gln were further subjected to digestion with Hsp92II, FspBI, Fnu4HI, PvuII, Hsp92II, and PstI restriction enzymes, respectively. The PCR products were visualized by electrophoresis through a 3% agarose gel. The relative size of the PCR products was determined by comparison of the migration of a 50–1000 bp DNA molecular weight ladder (Invitrogen). A permanent visual image was obtained using a UV illuminator. All genotypes were read by 2 independent researchers. In case of any conflicts, the genotypes were repeated.
Measurements, protocol, and procedure All subjects were examined according to a standardized interview and examination. Assessment was done in a form that required patient information regarding demographic and personal details of the patients and informants, complaints of the patients, history of present illness, details of medical or surgical interventions, past history, family history, personal history, premorbid personality, details of physical examination, mental status examination, and diagnostic formulation. Inclusion and exclusion criteria Inclusion criteria. Parkinsonism of no known cause, defined as two or more signs (resting tremor, bradykinesia, rigidity, and postural reflex impairment) (Lees, 1988), was included. Exclusion criteria. (1) History of stroke, transient ischemic attack, hypertension, syphilis, encephalitis, epilepsy, cerebral tumor, alcoholism, diabetes mellitus, or head injury resulting in loss of consciousness. (2) Presence or history of any neurological sign not compatible with a diagnosis of PD (such as cerebellar signs, supranuclear gaze palsy, and oculogyric crises). (3) Any illness associated with chronic confusional states or of any chronic neurodegenerative disease other than PD.
Statistical analysis Statistical analyses were performed using the SPSS software package (revision 11.5; SPSS, Inc., Chicago, IL). Data are expressed as means – SD. Differences in the distribution of genotypes or alleles of DNA repair gene between cases and controls were tested using the v2 statistic. Linkage disequilibrium among DNA repair gene polymorphisms was assessed using D0 and r2 values obtained through the Haploview program (www.broad.mit.edu/mpg/haploview/ documentation.php). Values p < 0.05 were considered statistically significant. Results Table 1 summarizes the distributions of genotypes and alleles of APE1, XRCC1, XRCC3, XPD, XPG, and HOGG1 genes in patients with PD and controls. The distributions of the genotypes and alleles of the study groups were in Hardy– Weinberg equilibrium. We did not find any significant differences for XPD, XPG, and HOGG1 genotype and allele frequencies between patients with PD and controls. There were statistically significant differences in APE1 Asp148Glu, XRCC1 Arg399Gln, and XRCC3 Thr241 genotypes between the controls and patients. APE1 Glu/Glu genotype was significantly increased in patients compared with controls ( p = 0.028, v2:4.80, OR: 2.75, 95% CI = 1.085–6.97). Frequencies of Asp + genotype in controls were higher than the patients (91.7.0% and 80.0%, respectively). It seems that there is a protective role of APE1 ASP + genotype against PD ( p = 0.028, v2: 4.80, OR: 0.36, 95% CI = 0.143–0.922). Frequencies of XRCC1 Gln + genotype in patients were higher than in the controls (31.7% and 12.0% respectively p = 0.002, v2: 9.63, OR: 3.38, 95% CI = 1.52–7.49) The individuals who had XRCC1 Gln/Gln, Arg/Gln genotypes, or Gln allele had an increased risk for PD ( p = 0.044 Fisher’s exact
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Table 1. Distribution of APE1, XRCC1, XRCC3, XPD, XPG, and HOGG1 Genotype Frequencies in Patients with Parkinson’s Disease and Control Groups Controls Polymorphism
n
Parkinson’s disease
%
APE1 Asp148Glu rs 3136820 Asp/Asp 57 52.8 Asp/Glu 42 38.9 Glu/Glu 9 8.3 Asp 156 72.2 Glu 60 27.7 XRCC1 Arg399Gln rs25487 Arg/Arg 95 88.0 Arg/Gln 13 12.0 Gln/Gln 0 0 Arg 203 93.98 Gln 12.5 6.01 XRCC3 Thr241Met rs 861539 Thr/Thr 24 22.2 Thr/Met 53 49.1 Met/Met 31 28.7 Thr 101 46.75 Met 115 53.24 XPD Lys751Gln rs 1052559 Lys/Lys 37 34.3 Lys/Gln 48 44.4 Gln/Gln 23 21.3 Lys 67 55.83 Gln 53 44.16 XPG Asp1104His rs 17655 Asp/Asp 58 53.7 Asp/His 43 39.8 His/His 7 6.5 Asp 159 73.61 His 57 26.37 HOGG1 Ser326Cys rs 1052133 Ser/Ser 81 75.0 Ser/Cys 25 23.1 Cys/Cys 2 1.9 Ser 187 86.57 Cys 29 13.42
n
%
p-Value
35 13 12 83 37
58.3 21.7 20.0 68.16 30.83
0.019
41 16 3 98 22
68.3 26.7 5 81.66 18.3
0.002
19 32 9 70 50
31.7 53.3 15.0 58.33 41.66
0.104
17 33 10 122 94
28.3 55.0 16.7 56.48 43.51
0.420
36 17 7 89 31
60.0 28.3 11.7 74.16 25.83
0.220
46 14 0 106 14
76.6 23.3 0 88.33 11.66
0.570
0.55
0.0004
0.042
0.908
0.91
0.643
test; p = 0.016, v2: 5.78; p = 0.0004, v2: 12.53, respectively), and individuals with Arg/Arg genotype seemed to be protected from PD ( p = 0.002, v2: 9.63, OR: 0.295, 95% CI = 0.133–0.654). Individuals carrying XRCC3 Met/Met genotype had a decreased risk for PD ( p = 0.046, v2: 3.99, OR: 0.43, 95% CI = 0.19– 0.99). Frequencies of Thr + genotype in patients were higher than in the controls (85.0% and 71.3%, respectively), and carriers of Thr + genotype had a 2.28-fold increased risk for PD ( p = 0.046, v2: 3.99, OR: 2.28, 95% CI = 1.00–5.19).
In addition to SNP analyses, haplotypes were evaluated for association with PD (Table 2). Haplotype analysis confirmed the association of APE1 and XRCC1 gene variants with PD and revealed that the frequencies of APE1 Asp:XRCC1 Gln and APE1 Glu:XRCC1 Gln haplotype frequencies were significantly higher in patients as compared with those of the controls ( p < 0.05). There was a weak linkage disequilibrium between APE1 and XRCC1 polymorphisms (D¢: 0.128, r2: 0.005, LOD: 0.21). Discussion There is a growing interest in the understanding of a possible role of DNA repair systems in ageing and neurodegenerative diseases after DNA damage is observed in the brain of individuals affected by neurodegenerative diseases (Coppede` and Migliore, 2010). We, for the first time, demonstrated the positive association of APE1, XRCC1, and XRCC3 DNA repairing gene variants with PD risk. In our study, the frequencies of Glu/ Glu genotype in APE1, Gln + genotype of XRCC1, and Thr + genotype of XRCC3 are more prevalent in patients than in controls. Molecular epidemiological studies have also showed the association of XRCC3 and APE1 T1349G genetic variants with different kinds of cancer, such as lung cancer ( Jacobsen et al., 2004) and colorectal cancer (Mort et al., 2003). Although it is not known how the genetic variations cause diseases, there are some explanations related to genetic variants responsible for modulation of DNA repair capacity, leading to diseases. A recent study reported that the XRCC1-Arg280His variant protein is defective in its efficient localization to a damaged site in the chromosome, thereby reducing the cellular BER efficiency (Takanami et al., 2005). Furthermore, functional studies about DNA repair gene polymorphism have suggested that genetic variant or allele of genes may alter endonuclease, DNA-binding activity, and reduced ability to communicate with BER proteins. On the other hand, it has been reported that stresses, such as ultraviolet radiation, oxidative agents (e.g., H2O2 and ROS), can generate injuries, promoting a transient APE1 induction which correlates with an increase of endonuclease activity (Tell et al., 2005). Individuals carrying the Ape1 Glu allele are thought to have a higher sensitivity to ionizing radiation than those who carry the Ape1 Asp allele (Hu et al., 2001). Similarly, our patients carrying Glu/Glu genotype of APE1, Gln + genotype of XRCC1, and/or Thr + genotype of XRCC3 might be more sensitive to oxidative stress than those with the other genotypes of these DNA repairing genes. At present, it is hard to explain by which mechanisms genotypes of DNA repair enzyme result in oxidative stress. Nevertheless, we can make some plausible interpretations depending on the previous study findings. Genetic variants may directly affect the function of BER or APE1, which might affect each other, or the other BER regulatory proteins, leading
Table 2. The Frequencies of Haplotypes of DNA Repair Gene in Patients and Controls Frequency Number of haplotype
Haplotype associations
Overall
All patients
Control
w2
p-Value
1 2 3 4
APEAsp:XRCC1Arg APEGlu:XRCC1Arg APEAsp:XRCC1Gln APEGlu:XRCC1Gln
0.671 0.225 0.067 0.037
0.623 0.194 0.119 0.064
0.698 0.242 0.038 0.022
1.968 1.025 8.013 3.906
0.1607 0.3114 0.0046 0.0481
DNA REPAIR GENES IN PARKINSON’S DISEASE to oxidative stress. APE1 is thought to be one of the ratelimiting steps in the BER pathway under a variety of conditions. These genotypes of the BER enzyme, the major pathway responsible for removing oxidative DNA damage and restoring the integrity of the genome (Rao, 2007), might indirectly cause dopamine oxidation because of insufficient repairing activity. In the BER pathway, XRCC1 is recruited to the site of repair till the last stage of ligation, regulating and coordinating the whole process. The XRCC3 gene codes a protein involved in homologous recombinational repair of double-strand DNA and is required for genomic stability (Griffin et al., 2000). Decrease in BER repairing activity might cause accumulation of oxidative damage, the situation becoming a vicious cycle in a cell or organ. These genetic variants might also affect proteins or other kinds of molecules involved in the whole DNA repairing system. It has been suggested that the XRCC3 gene has a sequence variation in exon 7 (C18067T), which results in an amino acid substitution at codon 241 (Thr241Met) that may affect the enzyme’s function and/or its interaction with other proteins involved in DNA damage and repair (Matullo et al., 2001). XRCC1 Arg399Gln polymorphism is thought to be ‘‘possibly damaging’’ to XRCC1 function based on the conservation of the sequences in mammalian orthologs (Metsola et al., 2005). Although the current study has some novel findings, there are some limitations of our study. Oxidative stress markers, such as 8-OH-guanosine, and the activity of DNA repair enzymes were not measured. In conclusion, our findings have suggested that APE1, XRCC1, and XRCC3 genetic variants may be a risk factor by increasing the oxidative stress that might cause the loss of dopaminergic cells in the substantiate nigra and locus caeruleus, leading to abnormal signal transmittion, and ultimately development of PD. In addition, the generation of ROS from dopamine, highly redox reactive molecules, and production of ROS during normal neurotransmission might affect the other DNA repair pathway proteins that we did not examine in the current study. Further studies with larger sample groups are necessary to clarify the role of DNA repair genes and the development of PD. Disclosure Statement No competing financial interests exist. References Chinta SJ, Andersen JK (2008) Redox imbalance in Parkinson’s disease. Biochim Biophys Acta 1780:1362–1367. Coppede` F, Migliore L (2010) DNA repair in premature aging disorders and neurodegeneration. Curr Aging Sci 3:3–19. Fahn S, Marsden CD, Goldstein M, Calne DB (1987) Recent Developments in Parkinson’s Disease. Mc Millan, New York. Griffin CS, Simpson PJ, Wilson CR, Thacker J (2000) Mammalian recombination- repair genes XRCC2 and XRCC3 promote correct chromosome segregation. Nat Cell Biol 2:757–761. Hirsch EC, Brandel JP, Galle P, et al. (1991) Iron and aluminum increase in the substantia nigra of patients with Parkinson’s disease: an X-ray microanalysis. J Neurochem 56:446–451. Hoehn MM, Yahr MD (1967) Parkinsonism: onset, progression and mortality. Neurology 17:427–442. Hu JJ, Smith TR, Miller MS, et al. (2001) Amino acid substitution variants of APE1 and XRCC1 genes associated with ionizing radiation sensitivity. Carcinogenesis 22:917–922.
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Address correspondence to: Elif Ozkok, Ph.D. Department of Neuroscience Institute of Experimental Medicine Research Istanbul University Capa 34390 Istanbul Turkey E-mail:
[email protected]
This article has been cited by: 1. Laurie H. Sanders, J. Timothy Greenamyre. 2013. Oxidative damage to macromolecules in human Parkinson disease and the rotenone model. Free Radical Biology and Medicine 62, 111-120. [CrossRef] 2. Mengxia Li, David M. Wilson , III. Human Apurinic/Apyrimidinic Endonuclease 1. Antioxidants & Redox Signaling, ahead of print. [Abstract] [Full Text HTML] [Full Text PDF] [Full Text PDF with Links] 3. Chao Wei, Zhe Jian, Lin Wang, Huini Qiang, Qiong Shi, Sen Guo, Kai Li, Ye Huang, Ling Liu, Qiang Li, Qi Luan, Xiuli Yi, Xia Li, Gang Wang, Tianwen Gao, Chunying Li. 2013. Genetic variants of the APE1 gene and the risk of vitiligo in a Chinese population: A genotype–phenotype correlation study. Free Radical Biology and Medicine 58, 64-72. [CrossRef]
