Investigation of DNA repair genes in patients with obsessive-compulsive disorder

Results. Significant differences were found for XPD and genotype frequencies. Likewise, the frequency of the XPD Lys+ genotype was significantly increased in the patients as compared to the controls, and carriers of the Lys+ genotype had an increased risk for OCD (p = 0.027). The XPD Lys/Lys genotype frequency was also increased in the patients in comparison to the controls (p < 0.001). XPD Gln+ frequencies were higher in the controls than in the patients, and carriers of the Gln+ genotype showed decreased levels of OCD risk (p < 0.001). XPD Lys/Lys genotype frequency and XPD Gln+ frequency are also significantly associated even after Bonferroni correction (p < 0.008).

][3] There is considerable evidence suggesting that OCD may have a biological basis.[10][11] These studies have led to a better understanding of the genetic and environmental factors leading to the development of OCD.However, the impact of DNA repair genes on OCD development has not been investigated.DNA damage resulting from alkylation, deamination and oxidative stress is mainly reversed by the base excision repair (BER) pathway to ensure genome integrity.The human oxoguanine glycosylase 1 (HOGG1), AP endonuclease 1 (APE1) and X-ray repair cross-complementing 1 (XRCC1) genes are involved in the BER pathway.7][18] The XRCC genes are involved in different DNA repair processes contributing to genetic stability. 19,20XRCC1 acts as a scaffold for other BER enzymes, while XRCC3 encodes RAD-51-like proteins, and is necessary for homologous recombination DNA repair. 21he authors hypothesized that genetic alterations in the components of the DNA repair system could be used as a molecular marker for OCD prognosis.This study was aimed at evaluating the distribution of polymorphisms of the DNA repair genes XRCC1, XRCC3, XPD, XPG, APE1 and HOGG1 in OCD patients.

Material and methods
The study population OCD subjects were diagnosed according to the criteria in the 1994 American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders (DSM IV) and were recruited at the Psychiatric Department of Istanbul Erenkoy Psychiatric and Neurological Disorders Hospital (Turkey), which has an inpatient ward for acute psychiatric patients.All the patients' medical histories were taken upon their first admission.Assessments for the diagnosis of OCD were performed by 2 psychiatrists using cross-sectional interviews and case records.All the patients had active symptoms at the time of the study.Patients with a history of neurological or medical disorders that would affect neuropsychological function (i.e., seizures, head trauma, stroke, brain tumor, meningitis) or with a recent history of alcohol or psychoactive drug abuse were excluded.A total of 100 OCD patients were included in the study.
Normal control participants were recruited from a large medical outpatient clinic.Their demographic data, medical and psychiatric history were investigated, and subjects with a diagnosis of any DSM-IV axis I or axis II disorders were excluded.None of the control subjects had a history of medical illness, head injury, neurological disorder, psychiatric disorder or alcohol or substance abuse, and none had a family history of any psychiatric disorder.A total of 122 unrelated healthy controls were included the study.

Measurements, protocol and procedure
All the subjects were examined using a standardized interview.Assessments were done on a semi-structured socio-demographic form that required patient information regarding the demographic and personal details of the patients and informants, the patients' complaints, the history of the present illness, details of medical or surgical interventions, past history, family history, premorbid personality, physical examination details, a mental status examination, and a diagnostic formulation.The potential cases were interviewed by a psychiatrist, and their medical records were reviewed whenever relevant.Secondly, a consultant psychiatrist audited the 1 st phase results, and confirmed or rejected the diagnosis.
To minimize the effect of ethnic differences in gene frequencies, the study participants were chosen from the Turkish population living in the western region of Turkey.The study was approved by the Medical Ethics Committee of Istanbul Medical Faculty, and all the participants gave written informed consent.

Polymorphism analysis
Blood samples from all the study participants were collected in EDTA-containing tubes.Genomic DNA was extracted from peripheral whole blood using a commercially available kit according to the manufacturer's instructions (PureLink Genomic DNA Mini Kit, Thermo Fisher Scientific Inc., Waltham, USA).Polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis was performed to detect variations in DNA repair genes APE1, HOGG1, XRCC1, XRCC3, XPD and XPG.Appropriate primers were used to amplify the subjects' corresponding gene by PCR, and the reaction products were digested using the appropriate enzyme at 37°C.The digested products were analyzed on 2% agarose gel stained with ethidium bromide, and examined under transillumination.Two observers, blind to the subjects' diagnoses, read each gel.In case of any conflict, the experiments were repeated.

Statistical analysis
The statistical analyses were performed using SPSS software (v.11.5, SPSS Inc., Chicago, USA).Differences in the distribution of DNA repair genotypes or alleles between the patient group and the control group were tested using the χ 2 test; the data was expressed as means ± standard deviation (SD).Demographic data was compared using Student's ttest and the χ 2 test, as required.Relative risk with 95% confidence intervals (CI) was calculated as the odds ratio (OR).An OR > 1 is associated with higher odds of the outcome; an OR < 1 is associated with lower odds of the outcome; and an OR = 1 does not affect the odds of the outcome. 22n addition to this, minor allele frequency and Hardy-Weinberg equilibrium were calculated by Haploview 4.2 software (The Broad Institute, Cambridge, USA).Linkage disequilibrium among DNA repair gene polymophisms was assessed using D ' and r 2 values obtained through the Haploview program.Multiple comparisons were controlled using the Bonferroni correction.P-values < 0.05 were considered statistically significant.

Results
Table 1 shows the characteristics of the patient and control groups.The controls and patients were adjusted for age, sex, smoking habit, and alcohol use (p < 0.05).
Minor allele frequency (MAF) values are also given in Table 3.With the exception of the XRCC3 Thr241Met genotype, the distribution of all allele and genotype frequencies among the patients and the controls was in accordance with Hardy-Weinberg equilibrium.
When the results were analyzed in terms of haplotype frequencies, the only significant difference between the controls and the patients was found in the haplotype with XRCC1 Arg: XPG Asp (Table 4).There was a weak linkage disequilibrium between the XRCC1 and XPG polymorphisms (D': 0.198, logarithm of odds (LOD): 0.33, r 2 : 0.006).

Discussion
OCD is a widespread psychiatric disorder that is characterized by disabling obsessions and/or compulsions. 246][27] Although the exact causes of OCD are unknown, data from family studies suggests the presence of a heritable factor, and implies that genetic factors might account for around 50% of OCD symptoms.Moreover, polymorphisms of certain genes have been associated with OCD. 15,23,28The present study attempted to unravel potential association between DNA repair genes and OCD.To the authors' knowledge, this is the first study focused on the potential contribution of XRCC1, XRCC3, XPD, XPG, APE1 and HOGG1 genotypes to OCD pathogenesis.
The study found that certain XPD gene polymorphisms might have a facilitating or protective role in OCD development.These results not only suggest that DNA repair mechanisms somehow influence the neuronal pathways controlling behaviors, but also lend further support to a biological basis of OCD.
Exposure to certain chemicals such as hydrocarbons, aryl amines and nitrosamines might result in the formation of reactive oxygen species as by-products, which might ultimately affect DNA functions.DNA repair mechanisms are thus of paramount importance in reversing alterations to DNA and providing unmutated DNA during the replication process.0][31] Biological models of OCD propose anomalies in the dopamine and serotonin pathways of the brain. 32t least in some patients, OCD might plausibly be the end result of mesocortical pathway dysfunction caused by a variety of factors such as trauma, infections and autoimmunity.Increased generation of reactive oxygen species as a result of impaired DNA repair mechanisms might be rendering the dopaminergic mesocortical pathway more susceptible to these environmental factors.Recent findings on the association between DNA repair gene polymorphisms and Parkinson's disease, which is also related to dopaminergic pathways, support this hypothesis. 33It is also possible that XPD genes have some yet unknown function in dopamine and/or serotonin metabolism.][36][37][38] It is possible to say that there might be an important relationship between the DNA repair system and neurodegeneration.This area should be investigated further in large-scale studies to focus specifically on identifying which repair system is related to the disease mechanism.

Conclusions
The findings of the current study suggest that certain XPD genotypes are associated with the development of OCD.Further studies with larger sample groups are necessary to clarify the significance of these gene variants.Moreover, the mechanisms by which these genes contribute to OCD pathogenesis need to be clarified.

Table 1 .
Characteristics of the OCD patients and controls

Table 2 .
The distribution of the APE1, XRCC1, XRCC3, XPD, XPG and HOGG1 genotype frequencies in the OCD patient group and the control group

Table 3 .
Minor allele frequencies and the distribution of APE1, XRCC1, XRCC3, XPD, XPG and HOGG1 allele frequencies in the OCD patients and the controls

Table 4 .
The frequencies of haplotypes of DNA repair genes in the patients and the controls