Anna Bielecka-Kowalska

Genetic polymorphisms in DNA base excision repair gene XRCC1 and the risk of squamous cell carcinoma of the head and neck

BackgroundThe genes of base excision repair (BER) pathway have been extensively studied in the association with various human cancers. We performed a case-control study to test the association between two common single nucleotide polymorphisms (SNPs) of XRCC1 gene with human head and neck squamous cell carcinoma (HNSCC).MethodsThe genotype analysis of Arg194Trp and Arg399Gln gene polymorphisms for 92 HNSCC patients and 124 controls of cancer free subjects, in Polish population were performed using the PCR-based restriction fragment length polymorphism (PCR-RFLP) with endonuclease Msp I.ResultsNo altered risk has been found individually for these SNPs, however haplotypes analysis showed high association with head and neck cancer. The highest frequency, according to wild-type of Arg194Arg and Arg399Arg genotypes, was identified for Arg194Trp-Arg399Arg haplotype (OR, 2.96; 95% CI, 1.01–8.80).ConclusionFinally, we identified the combined Arg194Trp-Arg399Arg genotype of base excision repair gene XRCC1 that was associated with HNSCC and may have an impact on identification of a high-risk cancer population.

Elevated Level of DNA Damage and Impaired Repair of Oxidative DNA Damage in Patients with Recurrent Depressive Disorder

Background Depressive disorder (DD), including recurrent DD (rDD), is a severe psychological disease, which affects a large percentage of the world population. Although pathogenesis of the disease is not known, a growing body of evidence shows that inflammation together with oxidative stress may contribute to development of DD. Since reactive oxygen species produced during stress may damage DNA, we wanted to evaluate the extent of DNA damage and efficiency of DNA repair in patients with depression. Material/Methods We measured and compared the extent of endogenous DNA damage – single- and double-strand breaks, alkali-labile sites, and oxidative damage of the pyrimidines and purines – in peripheral blood mononuclear cells isolated from rDD patients (n=40) and healthy controls (n=46) using comet assay. We also measured DNA damage evoked by hydrogen peroxide and monitored changes in DNA damage during repair incubation. Results We found an increased number DNA breaks, alkali-labile sites, and oxidative modification of DNA bases in the patients compared to the controls. Exposure to hydrogen peroxide evoked the same increased damage in both groups. Examination of the repair kinetics of both groups revealed that the lesions were more efficiently repaired in the controls than in the patients. Conclusions For the first time we showed that patients with depression, compared with non-depresses individuals, had more DNA breaks, alkali-labile sites, and oxidative DNA damage, and that those lesions may be accumulated by impairments of the DNA repair systems. More studies must be conducted to elucidate the role of DNA damage and repair in depression.

Serum MicroRNAs as Potential Biomarkers of AMD

Background Age-related macular degeneration (AMD) is a major cause of blindness worldwide. Circulating microRNAs (miRNAs) in serum have emerged as novel candidate biomarkers for many diseases. The aim of the present study was to identify a serum microRNA (miRNA) expression profile specific for dry and wet forms of AMD. Material/Methods Serum miRNA expression was first screened using TaqMan® Human MicroRNA Array A (Applied Biosystems). An extensive, self-validated, individual, quantitative RT-PCR (qRT-PCR) study was then performed on a cohort of 300 AMD patients (150 wet form and 150 dry form) and 200 controls. The Mann-Whitney U test and nonparametric Spearman’s rank correlation coefficient were used for statistical analysis. Results miRNA expression analysis revealed increased expression of miR661 and miR3121 in serum of patients with dry AMD and miR4258, miR889, and Let7 in patients with wet form. Expression of analyzed miRNA was not observed or remained at low level in controls. Conclusions Differences in miRNA serum profile exist between patients with wet and dry form of AMD, which indicates miRNAs as potential biomarkers of AMD. Further studies should be performed to confirm its significance in clinical practice.

Association between single nucleotide polymorphisms of MUTYH, hOGG1 and NEIL1 genes, and depression

BACKGROUND An elevated levels oxidative modified DNA bases and a decreased efficiency of oxidative DNA damage repair were found in patients with depression disorders, including recurrent type (rDD). The glycosylases are involved in base excision repair (BER), which eliminates oxidative DNA damage. Therefore, we genotyped the single nucleotide polymorphisms (SNPs) of genes encoding three glycosylases: hOGG1, MUTYH and NEIL1. METHODS We selected three polymorphisms: c.977C > G - hOGG1 (rs1052133), c.972G > C - MUTYH (rs3219489) and c.*589G > C - NEIL1 (rs4462560). A total of 555 DNA samples (257 cases and 298 controls) were genotyped using TaqMan probes. RESULTS The C/C genotype and allele C of the c.*589G > C decreased the risk of rDD occurrence, while the G/G genotype and allele G of the same SNP increased the risk. This polymorphism had a stronger association with early-onset depression (patients with first episode <35 years of age) than with late-onset depression (first episode ≥ 35 years of age). We did not find any significant differences in distribution of alleles and genotypes of other SNPs; however, the G/G genotype of the c.972G > C increased the risk of late-onset rDD. We also found that combined genotype C/C-C/C of c.977C > G and c.*589G > C significantly reduced the risk of rDD. LIMITATIONS Limited sample size and ethnic homogeneity of the studied population. CONCLUSION This is the first study to show that SNPs of genes involved in DNA repair, particularly in BER pathway, may modulate the risk of rDD. These results further support the hypothesis on the involvement of DNA repair mechanisms in pathogenesis of depression.

Variants of Base Excision Repair Genes MUTYH , PARP1 and XRCC1 in Alzheimer's Disease Risk

Background: Many clinical studies have shown that oxidative stress pathways and the efficiency of the oxidative DNA damage base excision repair (BER) system are associated with the pathogenesis of Alzheimers disease (AD). Reduced BER efficiency may result from polymorphisms of BER-related genes. In the present study, we examine whether single nucleotide polymorphisms (SNPs) of BER genes are associated with increased risk of AD. Methods: SNP genotyping was carried out on DNA isolated from peripheral blood mononuclear cells obtained from 120 patients with AD and 110 healthy volunteers. Samples were genotyped for the presence of BER-related SNPs, i.e. XRCC1-rs1799782, rs25487; MUTYH-rs3219489, and PARP1-rs1136410. Results: We found a positive association between AD risk and the presence of G/A genotype variant of the XRCC1 rs25487 polymorphism [odds ratio (OR) = 3.762, 95% CI: 1.793-7.891]. The presence of the A/A genotype of this polymorphism reduced the risk of AD (OR = 0.485, 95% CI: 0.271-0.870). In cases of the PARP1 gene rs1136410 polymorphism, we observed that the T/C variant increases (OR = 4.159, 95% CI: 1.978-8.745) while the T/T variant reduces risk (OR = 0.240, 95% CI: 0.114-0.556) of AD. Conclusions: We conclude that BER gene polymorphisms may play an important role in the etiology of AD. Diagnosing the presence or absence of particular genetic variants may be an important marker of AD. Further research on a larger population is needed. There is also a need to examine polymorphisms of other BER in the context of AD risk.

Alterations in expression profile of iron-related genes in colorectal cancer

Abstract Iron can play a role in colorectal cancer (CRC) development. The expression of genes involved in iron metabolism and its regulation in CRC has not been investigated well. Also the correlation between the level of iron-related genes expression and cancer progression is not known. In this study we collected paired samples of primary adenocarcinoma and adjacent normal mucosa from 73 patients. We assessed the mRNA or miRNA levels of 21 genes and verify their association with clinicopathological characteristics of CRC patients. Our experiments revealed, that the level of divalent metal transporter 1 transcript is well correlated with mRNA levels of iron regulatory proteins (IRPs) in tumor specimens. We have shown, that IRP2 can also be engaged in the mRNA stabilization of other iron transporter–transferrin receptor 1 (TfR1) in early stage of disease, however, in more advanced stages of CRC, mRNA level of TfR1 is related to miR-31 level. For the first time we have shown, that ferroportin concentration is significantly associated with miR-194 level, causing the reduction of this transporter amount in tumor tissues of patients with more advanced stages of CRC. We have also shown the alterations in expressing profile of miR-31, miR-133a, miR-141, miR-145, miR-149, miR-182 and miR-194, which were observed even in the early stage of disease, and identified a set of genes, which take place in correct assigning of patients in dependence of CRC stage. These iron-related genes could become potential diagnostic or prognostic indicators for patients with CRC.

Single-Nucleotide Polymorphisms of Genes Involved in Repair of Oxidative DNA Damage and the Risk of Recurrent Depressive Disorder

Background Depressive disorder, including recurrent type (rDD), is accompanied by increased oxidative stress and activation of inflammatory pathways, which may induce DNA damage. This thesis is supported by the presence of increased levels of DNA damage in depressed patients. Such DNA damage is repaired by the base excision repair (BER) pathway. BER efficiency may be influenced by polymorphisms in BER-related genes. Therefore, we genotyped nine single-nucleotide polymorphisms (SNPs) in six genes encoding BER proteins. Material/Methods Using TaqMan, we selected and genotyped the following SNPs: c.-441G>A (rs174538) of FEN1, c.2285T>C (rs1136410) of PARP1, c.580C>T (rs1799782) and c.1196A>G (rs25487) of XRCC1, c.*83A>C (rs4796030) and c.*50C>T (rs1052536) of LIG3, c.-7C>T (rs20579) of LIG1, and c.-468T>G (rs1760944) and c.444T>G (rs1130409) of APEX1 in 599 samples (288 rDD patients and 311 controls). Results We found a strong correlation between rDD and both SNPs of LIG3, their haplotypes, as well as a weaker association with the c.-468T>G of APEXI which diminished after Nyholt correction. Polymorphisms of LIG3 were also associated with early onset versus late onset depression, whereas the c.-468T>G polymorphism showed the opposite association. Conclusions The SNPs of genes involved in the repair of oxidative DNA damage may modulate rDD risk. Since this is an exploratory study, the results should to be treated with caution and further work needs to be done to elucidate the exact involvement of DNA damage and repair mechanisms in the development of this disease.

Role of base-excision repair in the treatment of childhood acute lymphoblastic leukaemia with 6-mercaptopurine and high doses of methotrexate

Methotrexate (MTX) and 6-mercaptopurine (6MP) are the most commonly used drugs in the therapy of childhood acute lymphoblastic leukaemia (ALL). The main genotoxic effect of MTX resulting from inhibition of thymidylate synthase is mis-incorporation of uracil into DNA, which is considered essential for the effectiveness of the Protocol M in ALL IC BFM 2002/EURO LB 2002 regimens. In this study, we investigated the level of basal and induced DNA damage as well as the effectiveness of DNA repair in lymphocytes of children with ALL at four time-points during therapy with MTX and 6MP. To assess DNA damage and the efficacy of DNA repair we used the modified alkaline comet assay with uracil DNA glycosylase (Udg) and endonuclease III (EndoIII). In addition, we examined the induction of apoptosis in the lymphocytes of the patients during treatment. Finally, we compared the activity of base-excision repair (BER), involved in removal of both uracil and oxidized bases from DNA in lymphocytes of children with ALL and lymphocytes of healthy children. BER efficiency was estimated in an in vitro assay with cellular extracts and plasmid substrates of heteroduplex DNA with an AP-site. Our results indicate that there is a significant decrease in the efficacy of DNA repair associated with an increased level of uracil in DNA and induction of apoptosis during therapy. Moreover, it was found that the BER capacity was decreased in the lymphocytes of ALL patients in contrast to that in lymphocytes of healthy children. Thus, we suggest that an impairment of the BER pathway may play a role in the pathogenesis and therapy of childhood ALL.

Association between Single-Nucleotide Polymorphisms of the hOGG1,NEIL1,APEX1, FEN1,LIG1, and LIG3 Genes and Alzheimer's Disease Risk.

Background: One of the factors that contribute to Alzheimers disease (AD) is the DNA damage caused by oxidative stress and inflammation that occurs in nerve cells. It has been suggested that the risk of AD may be associated with an age-dependent reduction of the DNA repair efficiency. Base excision repair (BER) is, among other things, a main repair system of oxidative DNA damage. One of the reasons for the reduced efficiency of this system may be single-nucleotide polymorphisms (SNP) of the genes encoding its proteins. Methods: DNA for genotyping was obtained from the peripheral blood of 281 patients and 150 controls. In the present study, we evaluated the impact of 8 polymorphisms of 6 BER genes on the AD risk. We analyzed the following SNP: c.-468T>G and c.444T>G of APEX1, c.*50C>T and c.*83A>C of LIG3, c.977C>G of OGG1, c.*283C>G of NEIL1, c.-441G>A of FEN1, and c.-7C>T of LIG1. Results: We showed that the LIG1 c.-7C>T A/A and LIG3 c.*83A>C A/C variants increased, while the APEX1 c.444T>G G/T, LIG1 c.-7C>T G/, LIG3 c.*83A>C C/C variants reduced, the AD risk. We also evaluated the relation between gene-gene interactions and the AD risk. We showed that combinations of certain BER gene variants such as c.977C>G×c.*50C>T CC/CT, c.444T>G×c.*50C>T GG/CT, c.-468T>G×c.*50C>T GG/CT, c.-441G>Ac.*50C>T×c.*50C>T GG/CT, c.*83A>C× c.*50C>T CT/AC, and c.-7C>T×c.*50C>T CT/GG can substantially positively modulate the risk of AD. Conclusions: In conclusion, we revealed that polymorphisms of BER genes may have a significant effect on the AD risk, and the presence of polymorphic variants may be an important marker for AD.

Ferric Reducing Ability of Tears in Healthy Subjects—Diurnal Variation and Dependence on Defined Demographic Data

Purpose: The assay of the ferric reducing ability of tears (FRAT) can be useful for monitoring ocular antioxidant capacity in clinical settings. FRAT diurnal variation was evaluated in healthy subjects and its relation with age, sex, body mass index, and ferric reducing ability of plasma (FRAP) were studied. Material and Methods: FRAT of 10-μ l tear samples collected with capillary tubes from 68 healthy subjects (age 10 to 92 yrs, 46 women, 22 men) were measured after 0, 3, 5, and 10 min of incubation with Fe3 +. FRAT diurnal variation was estimated in 11 subjects, with tear samples collected every 4 hr during the day. Comparison of FRAT versus FRAP was determined in 20 subjects. Results: Mean FRAT after 10-min incubation reached 207.7 ± 136.8 μ mol/l. No differences were found between FRAT of men and women. Only donor age correlated with FRAT (ρ = 0.40, p < 0.01). Subjects ≤ 59 years had lower FRAT levels than those age > 59 yrs (179.4 ± 95.4 versus 237.2 ± 151.7 μ mol/l, p < 0.01). For all investigated times of incubation, FRAT was approximately two times lower than FRAP (p < 0.01). FRAT revealed diurnal variation, with the highest value at 08:00 hours, upon awakening. Conclusion: The significance of age-matched controls and the specific time of the day for tears collection should be considered in the trial design and investigations with FRAT as a marker of the ocular antioxidant defense system.