Gülgün S. Güven

DNA repair gene XRCC1 and XPD polymorphisms and their association with coronary artery disease risks and micronucleus frequency

Coronary artery disease (CAD) is a multifactorial process that appears to be caused by the interaction of environmental risk factors with multiple predisposing genes. In this study, we investigated the effects of the XPD Lys751Gln and XRCC1 Arg399Gln polymorphisms on the presence and the severity of CAD. We also investigated the presence of DNA damage in the peripheral lymphocytes of patients with CAD by using the micronucleus (MN) test and the effect of XPD Lys751Gln and XRCC1 Arg399Gln polymorphisms on this damage. The study population consisted of 147 patients with angiographically documented CAD and 48 healthy controls. No association between XPD Lys751Gln or XRCC1 Arg399Gln polymorphisms and the presence or the severity of CAD was observed. On the other hand, a significantly higher frequency of MN was observed in CAD patients compared with controls (5.7 ± 1.9 vs 5.0 ± 2.1, respectively, P = 0.018). We found an elevated frequency of MN in CAD patients with the XPD 751Gln allele (Gln/Gln genotype) or the XRCC1 399Gln (Arg/Gln or Gln/Gln genotypes) allele compared with the XPD 751Lys (Lys/Lys genotype) allele or XRCC1 399 Arg (Arg /Arg genotype) allele, respectively. These preliminary results suggest that XPD Lys751Gln and XRCC1 Arg399Gln polymorphisms may not be a significant risk factor for developing CAD. In addition, our results indicate that the MN frequency is associated with presence, but not severity, of CAD and is related to the XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms, suggesting an elevated frequency of MN in CAD patients with the XPD 751Gln or XRCC1 399Gln alleles.

Polymorphisms of the DNA repair genes XPD and XRCC1 and the risk of age-related macular degeneration.

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-(751)Gln) 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.

Glutathione S-Transferase M1, GSTT1 and GSTP1 Genetic Polymorphisms and the Risk of Age-Related Macular Degeneration

Purpose: To determine the possible effects of glutathione S-transferase (GST) M1, GSTT1 and GSTP1 genetic polymorphisms on the risk of developing age-related macular degeneration (AMD). Patients and Methods: This case-control study included a total of 120 patients with AMD (65 with dry-type AMD and 55 with wet-type AMD) and 198 disease-free controls. GSTM1 and GSTT1 polymorphisms were analyzed by using a multiplex polymerase chain reaction (PCR), and GSTP1 polymorphism was detected by real-time PCR assay. Results:GSTM1-null genotype was significantly associated with the development of AMD (p = 0.01, OR = 1.82, 95% CI = 1.14–2.91). Stratification by AMD subtypes revealed a significant relationship between GSTM1-null genotype and dry-type AMD (p = 0.02, OR = 1.98, 95% CI = 1.10–3.53). In a stepwise regression model, only GSTM1-null genotype was significantly associated with the development of AMD (p = 0.01, OR = 1.77, 95% CI = 1.11–2.81). Conclusions: Our findings suggest that genetic polymorphisms of GST may have a role in the development of AMD.

Characterization of Stem-Like Cells Directly Isolated from Freshly Resected Laryngeal Squamous Cell Carcinoma Specimens

Larynx cancer (LCa) is an aggressive malignancy, which is the second most common malignant neoplasm of head and neck squamous cell carcinoma. Its incidences have been reported to increase and therapeutic options mostly fail to give positive clinical response especially for the advanced LCa cases. In this study we aimed to isolate stem-like cells from freshly resected LCa tumor specimens and characterize them by quantitative real time PCR (qRT-PCR) for expression of cancer stem cell markers including SOX2, OCT4, KLF4, ABCG2, CXCR4 and CD44. Our results showed that CD133(high) cells directly isolated from freshly resected tumor specimens exhibit elevated levels of SOX2, OCT4 and KLF4, and have increased expression levels of ABCG2 and CXCR4, which were associated with resistance of tumors to regular chemotherapeutic reagents. In conclusion, this study offers a useful approach utilizing CD133 to isolate stem cells directly from fresh tissues, which gives the opportunity to develop novel therapeutic tools specifically targeting these cells through their further characterization.

Enhanced Sensitivity to Oxidant-Induced Micronucleus Frequency in Elderly Individuals Is Not Associated with Glutathione S- Transferase M1 (GSTM1) Null Genotype in Lymphocytes

Background: A large number of studies have demonstrated that various kinds of DNA damage accumulate during aging and that oxidative stress possibly contributes to this process. Glutathione S-transferase M1 (GSTM1) can prevent their possible effects on DNA via detoxification of reactive substances that induced oxidative stress. Objective: To investigate the relationship between GSTM1 polymorphism and DNA sensitivity to oxidative stress with age, we used micronucleus (MN) frequency as a marker of DNA damage in lymphocytes from young and elderly subjects. Methods: This study was performed in 30 young (age range 20–36 years) and 30 elderly (age range 66–87 years) healthy individuals who were chosen on the basis of their GSTM1 genotype (15 GSTM1 null and 15 GSTM1 positive for each group). Lymphocytes were cultured after Ficoll isolation and treated for 48 h with a 30-µM dose of cumene hydroperoxide (CumOOH), a dose that does not decrease cell viability. Results: There was no significant difference in the MN frequency observed in control cultures from young and elderly individuals. However, the MN frequency in CumOOH-treated cultures was significantly higher in the elderly group than the young group (p < 0.001). No association was found between the GSTM1 phenotype and CumOOH-induced MN frequency. Conclusions: The results suggest that lymphocytes of elderly individuals are more susceptible to in vitro MN induction by CumOOH. However, this difference in susceptibility is not explained by the lack of GSTM1.

Decreased DNA repair gene XRCC1 expression is associated with radiotherapy-induced acute side effects in breast cancer patients

DNA repair plays a critical role in response to ionizing radiation (IR) and developing of radiotherapy induced normal tissue reactions. In our study, we investigated the association of radiotherapy related acute side effects, with X-ray repair cross complementing group 1 (XRCC1) and Poly (ADP-ribose) polymerase 1 (PARP1) DNA repair gene expression levels, their changes in protein expression and DNA damage levels in breast cancer patients. The study included 40 women with newly diagnosed breast cancer; an experimental case group (n=20) with acute side effects and the control group (n=20) without side effects. For gene and protein expression analysis, lymphocytes were cultured for 72 h and followed by in vitro 2 Gray (Gy) gamma-irradiation. For detection of DNA damage levels, lymphocytes were irradiated with in vitro 2 Gy gamma-rays and followed by incubation for 72 h. XRCC1 mRNA and protein expression levels were significantly higher in controls than in experimental cases (P=0.020). In terms of DNA damage levels, an increased frequency of micronucleus (MN) was observed in experimental cases versus controls, but this association was not significant (P=0.206). We also observed a significant negative correlation between MN frequency and XRCC1 protein levels in experimental (r=-0.469, P=0.037) vs control (r=-0.734, P<0.001). Our results suggested that decreased XRCC1 expression levels might be associated with the increased risk of therapeutic IR-related acute side effects in patients with breast cancer.

CYTOGENETIC CLONAL EVOLUTION IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA

ABSTRACT Chronic myeloid leukaemia (CML) is a clonal haematological disease characterised by t(9;22)(q34;q11) which is called Philadelphia (Ph) chromosome. Highly improved haematological and cytogenetic results were obtained in chronic phase CML with the introduction of imatinib. Occurrence of additional cytogenetic abnormalities in Ph(+) cells is defined as clonal evolution (CE) and considered to be a preceding sign for acceleration. The most common additional chromosomal changes are +8, +Ph, i(17q), +19, -Y, +21, +17 and-7. The aims of this study were to delineate the occurrence pattern of cytogenetic clonal evolution in our cohort of CML patients and to investigate the impact on the course and prognosis of CML. Additional clonal chromosomal changes in Ph(+) cells were observed in 20 cases (19%). The abnormalities seen were monosomy 21 (%35), -17 (%30), -19 and +8 in (%25), -7, -8, -13, -15, -22, +Ph, and different marker chromosomes (%20), -Y, -12, -14, -16, -20 (%15), +Y, -10, -18 (%10), and -X, -3, -9, +20, der(7;17)(q10;q10), der3?, der12? (%5). The findings of-Y, -7, -8, +8, -14, -15, -17, -18, -21, +Ph, der(7;17)(q10;q10) and del(7)(q11) have been recorded in more than one samples in at least one case. The clinical data of the 20 cases with CE were compared to 7 cases with no or minor cytogenetic response without CE and no statistically significant differences found. Considering their high frequency and persistence in successive samples, we recommend to trace -21 and -17 with FISH in addition to classical cytogenetics in CML cases.

Expression of aphidicolin, FUdR and caffeine-induced fragile sites in lymphocytes of healthy Turkish individuals.

The expression of common fragile sites (c‐fra) and frequency of chromosomal aberrations were studied in peripheral lymphocytes of 50 healthy Turkish individuals (26 males and 24 females from 1 to 87 years of age) after induction with aphidicolin (APC), 5′‐fluorodeoxyuridine (FUdR), and caffeine. A correlation was seen between age and the frequency of chromosomal aberrations in APC and caffeine treated cultures, but there were no significant differences in the frequencies of chromosomal aberrations between males and females in any of the treatments. The mean frequency of aberrations induced by FUdR was significantly higher than that induced by APC and caffeine. A chromosome aberration is defined as a fragile site when present in 1% of the cells analyzed from each culture and in at least 50% of the individuals studied. Using these criteria, 12 c‐fra were observed in the three treatments: 1p21, 1q21, 2p11‐q11, 3p14, 4q31, 6q26, 7q22, 7q32, 8q24, 11q23, 16q23, and Xp22. Sites 3p14, 16q23, and Xp22 were the most frequently observed c‐fra, with only the frequency of Xp22 being significantly increased in females in APC treated cultures. The results of these studies are important as a base against which the effects of other clastogenic and environmental agents, as well as genetic background, can be compared.

A novel frameshift mutation and infrequent clinical findings in two cases with Dyggve-Melchior-Clausen syndrome.

Dyggve–Melchior–Clausen syndrome (DMC) (MIM #223800) is a rare autosomal-recessive type of skeletal dysplasia accompanied by variable degrees of intellectual disability (ID). It is characterized by progressive spondyloepimetaphyseal dysplasia leading to disproportionate short stature, microcephaly, and coarse facies. The radiographic appearance of generalized platyspondyly with double-humped end plates and the lace-like appearance of iliac crests are pathognomonic in this syndrome. The disorder results from mutations in the dymeclin (DYM) mapped to the 18q12–12.1 chromosomal region. Here, we report two cases with DMC: one with disproportionate short stature, developmental delay, and severe ID with a novel frameshift mutation (c.1028_1056del29) leading to a premature stop codon, and the second patient with classical clinical and radiological features of DMC with mild ID and rectal prolapse, which is very rare. The clinical diagnosis was confirmed with molecular analysis of DYM with a known mutation at c.580C>T (p.R194X). The parents and sibling of the second patient were heterozygous carriers with mild skeletal changes and short stature.

Relationship between genomic damage and clinical features in dialysis patients.

Patients with end-stage renal disease display enhanced genomic damage. We investigated the presence of genomic damage in the peripheral lymphocytes by using the micronucleus (MN) test and the factors associated with the MN frequency in hemodialysis (HD) and peritoneal dialysis (PD) patients. We studied 121 dialysis patients (60 HD and 61 PD) and 129 age- and gender-matched healthy controls. The MN analysis, used as a biomarker of chromosomal/DNA damage, was performed in peripheral lymphocytes by the cytokinesis-block method. Univariate analysis showed a significantly higher MN frequency in all patients in comparison with the controls (7.6% ± 0.3% vs. 4.9% ± 0.2%, respectively, p<0.001). Significantly higher frequency of MN was observed in both HD and PD patients compared to controls (7.7% ± 0.5% vs. 4.9% ± 0.2%, p<0.001 and 7.5% ± 0.5% vs. 4.9% ± 0.2%, p<0.001, respectively). Multivariate analysis was performed, and it showed that the low-density lipoprotein level was the only independent determinant of increasing MN frequency in our patients (β=0.16, t=2.172, p<0.05). There is no significant difference in terms of genomic damage between two dialysis modalities, which suggests that PD may not be a more reliable choice in terms of genomic damage.