Evaluation of the promoter methylation status of hypoxia factor 3A and interleukin-6 genes and expression levels of mir-130b and mir-146b in childhood obesity.

OBJECTIVE: Obesity, which causes many serious diseases, is increasing exponentially in childhood across the world. Epigenetic changes, as well as genetics, play an important role in the process of adipogenesis. Therefore, we aimed to examine the expression levels of obesity-related MicroRNA-130b and MicroRNA-146b and the methylation status of hypoxia factor 3A and interleukin-6 genes associated with obesity in children. METHODS: This study was performed with 98 individuals (49 obese children and 49 controls) whose DNA was isolated from peripheral blood. Gene promoter methylations were analyzed by methylation-specific Polymerase chain reaction. In addition, expression levels of MicroRNAs were determined by quantitative real-time Polymerase chain reaction in 30 children (15 obese children and 15 controls). RESULTS: Methylation status of interleukin-6 gene was 93.9% in obese children (n=46/49) and 100% (n=49/49) in control group (p>0.05). There was no methylation for hypoxia factor 3A gene (p>0.05). As a result of the study, there was no statistically significant difference in terms of methylation status for hypoxia factor 3A and interleukin-6 genes in the obese group compared to the control group. However, we found that expression levels of MicroRNA-130b (p<0.01) and MicroRNA-146b (p<0.001) were higher in the obese group. CONCLUSIONS: Results support that MicroRNA-130b and MicroRNA-146b are potential biomarkers for the prevention and early diagnosis of obesity. This is the first study on childhood obesity in the Middle Black Sea region of Turkey. We believe that the results obtained by expanding the studies in our country and neighboring countries will be more decisive.

Evaluation of the promoter methylation status of hypoxia factor 3A and interleukin-6 genes and expression levels of mir-130b and mir-146b in childhood obesity

SUMMARY OBJECTIVE: Obesity, which causes many serious diseases, is increasing exponentially in childhood across the world. Epigenetic changes, as well as genetics, play an important role in the process of adipogenesis. Therefore, we aimed to examine the expression levels of obesity-related MicroRNA-130b and MicroRNA-146b and the methylation status of hypoxia factor 3A and interleukin-6 genes associated with obesity in children. METHODS: This study was performed with 98 individuals (49 obese children and 49 controls) whose DNA was isolated from peripheral blood. Gene promoter methylations were analyzed by methylation-specific Polymerase chain reaction. In addition, expression levels of MicroRNAs were determined by quantitative real-time Polymerase chain reaction in 30 children (15 obese children and 15 controls). RESULTS: Methylation status of interleukin-6 gene was 93.9% in obese children (n=46/49) and 100% (n=49/49) in control group (p>0.05). There was no methylation for hypoxia factor 3A gene (p>0.05). As a result of the study, there was no statistically significant difference in terms of methylation status for hypoxia factor 3A and interleukin-6 genes in the obese group compared to the control group. However, we found that expression levels of MicroRNA-130b (p<0.01) and MicroRNA-146b (p<0.001) were higher in the obese group. CONCLUSIONS: Results support that MicroRNA-130b and MicroRNA-146b are potential biomarkers for the prevention and early diagnosis of obesity. This is the first study on childhood obesity in the Middle Black Sea region of Turkey. We believe that the results obtained by expanding the studies in our country and neighboring countries will be more decisive.

SAHA attenuates rotenone-induced toxicity in primary microglia and HT-22 cells.

Rotenone is an industrial and environmental toxicant that has been strongly associated with neurodegeneration. It is clear that rotenone induces inflammatory and oxidative stress; however, information on the role of histone acetylation in neurotoxicity is limited. Epigenetic alterations, neuroinflammation, and oxidative stress play a role in the progression of neurodegeneration and can be caused by exposure to environmental chemicals, such as rotenone. Histone modifications, such as methylation and acetylation, play an important role in mediating epigenetic changes. Therefore, we here investigated the effects of histone acetylation on rotenone-induced inflammation and oxidative stress in both primary mouse microglia and hippocampal HT-22 cells using the pan-histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA). Our results showed that SAHA suppressed the inflammatory response by decreasing nuclear factor kappa B and inducible nitric oxide synthase expression. Additionally, SAHA inhibited the rotenone-induced elevation of interleukin 6 and tumor necrosis factor α levels in both cell lines. Furthermore, SAHA improved the rotenone-induced antioxidant status by mitigating the decrease in cellular glutathione levels. Additionally, SAHA prevented the rotenone-induced increase in the HDAC activity in microglial and hippocampal HT-22 cells. Together, our results showed that SAHA reduced rotenone-induced inflammatory and oxidative stress, suggesting a role for histone deacetylation in environmental-related neurotoxicity.

Novel RET Proto-oncogene variants identified in Turkish patients with thyroid carcinoma.

Thyroid cancer is one of the few malignancies whose incidence is increasing in the last decades. Advances in understanding the molecular mechanisms lead to provide opportunity for prevention, effective early identification and targeted therapies for management. A total of 63 patients with participated in this study Genomic DNA samples were obtained from the samples formalin- embedded tissue and peripheral blood. Following polymerase chain reaction amplification of the 6 RET key exons (10, 11, 13, 14, 15, and 16) were applied and PCR products were subjected to next generation DNA sequencing (ABI 3730). Results revealed that; genotype frequencies were for rs1800961 (G > T) , GG 6 (%9.5), GT 17 (%27) TT40 (%63.5) for rs2472732 (G > A), GG31 (%49.2) GA29 (%46) AA3 (%4.8,) for rs1799939, (G > A) GG42 (%66.7) GA19 (%30.2) AA2 (%3.2), for rs1800962, (C > T) CC54 (%85.7) CT9 (%14.3), for rs1800863 (C > G), CC39 (%61.9) CG22 (%34.9) GG2 (%3.2), for rs3026272 (C > G) CC 13 (%20.6) CG 50 (%79.4). Additionally 15 potential novel genetic variants were identified in these key exons. Detailed information was given both known and new detected variants in supplementary table. Genetic variants distribution frequencies and new variants represented in Turkish thyroid cancer patients for RET proto-oncogene and that results would provide contribution to the literature.

Association of the MTHFR Gene C677T Polymorphism with Breast Cancer in a Turkish Population.

BACKGROUND: Breast cancer is the most common cancer among women. 1 in every 8 women in the United States have a lifetime risk of getting breast cancer. MTHFR is a key enzyme that regulates the folate metabolism which has an important role in DNA synthesis, repair, and methylation. The aim of the current study was to analyze the association between the MTHFR gene C677T (Ala222Val, rs1801133) polymorphism and breast cancer. PATIENTS AND METHODS: 199 breast cancer patients and 195 healthy controls were included in this study. The MTHFR gene C677T polymorphism was analyzed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) methods. A meta-analysis including 707 breast cancer patients and 880 controls from Turkish populations was also carried out. Statistical analyses were performed using the χ2 test. RESULTS: No statistically significant differences in allele and genotype frequencies were observed between patients and controls (p > 0.05). Although not statistically significant, TT homozygous variants were encountered more frequently in patients than in controls. A statistically significant association was observed between the MTHFR gene C677T polymorphism and the tumor histology of breast cancer patients (p = 0.038). The results of the meta-analysis suggested that there was a high association between breast cancer and the MTHFR gene C677T polymorphism in Turkish populations (p < 0.0001). CONCLUSION: In our study, we did not find any association between the MTHFR gene C677T polymorphism and breast cancer. However, a meta-analysis of the 6 association studies carried out in Turkish populations with 707 patients and 880 controls showed a significant association between breast cancer and the MTHFR gene C677T polymorphism.

Evaluation of CYP17A1 and LEP Gene Polymorphisms in Breast Cancer.

BACKGROUND: Polymorphisms of estrogen synthesis- and adiposity-related genes can contribute to the development of breast cancer. The purpose of the current study was to analyze the association between CYP17A1 T27C (rs743572) and LEP -2548G>A (rs7799039) gene polymorphisms and breast cancer. MATERIAL AND METHODS: 199 breast cancer patients and 197 healthy controls were included in the study. The CYP17A1 and LEP gene polymorphisms were determined using polymerase chain reaction-based restriction fragment length polymorphism analysis. RESULTS: No statistically significant association was found between these polymorphisms and breast cancer risk among a Turkish population. However, stratified analysis of these polymorphisms in relation to different clinicopathological characteristics of breast cancer revealed an association between breast cancer diagnosis and the CYP17A1 T27C polymorphism (p = 0.024). CONCLUSION: Our study suggests no strong association between the CYP17A1 T27C and LEP -2548G>A polymorphisms and the incidence of breast cancer in Turkish women. The potential association between CYP17A1 T27C and the type of breast cancer deserves further consideration.

Genotoxic effects of prenatal exposure to levetiracetam during pregnancy on rat offsprings.

Levetiracetam is a new-generation antiepileptic drug initially approved as an adjunctive treatment for patients with refractory partial seizures and is now also used as a monotherapy. The aim of this study was to evaluate the genotoxic effects of levetiracetam exposure during pregnancy on rat offsprings. In this study, we used the newborn pups of rats exposed to levetiracetam during pregnancy. Thirty Sprague-Dawley rats were divided into three groups. The mother rats of groups 1 and 2 were treated with different doses of levetiracetam (25 mg/kg/d and 50 mg/kg/d) from gestational days 1 to 18 during pregnancy. Group 3 (control group) was not treated with any drug. In vivo sister chromatid exchange (SCE) induction and in vivo micronucleus formation were assessed. Bone marrow from rat pups were used for investigation. As a result of this study, levetiracetam exposure did not alter SCE frequencies or the mean of number of micronuclei in the prenatal period (p>0.05). Levetiracetam did not cause miscarriage during pregnancy in mother rats. The present study highlighted fetal safety after prenatal exposure to levetiracetam.

Effects of leptin and leptin receptor gene polymorphisms on lung cancer.

Leptin (LEP), an adipocyte-derived cytokine, has been reported to participate in carcinogenesis. Elevated levels of systemic and pulmonary LEP are associated with diseases related to lung injury and lung cancer. The purpose of the present study was to investigate if the LEP and leptin receptor (LEPR) gene polymorphisms are associated with lung cancer in a cohort of Turkish population. One hundred and sixty-two lung cancer patients and 130 healthy controls were included in the study. The genotypes of LEP gene -2548G > A and LEPR gene Q223R polymorphisms were determined using polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) analysis. The genotype frequencies of LEP -2548G > A polymorphism showed statistically significant differences between lung cancer patients and controls (p = 0.007). GA + AA genotypes and A allele of LEP -2548G > A polymorphism was found to be susceptibility factors for lung cancer (p = 0.003, odds ratio (OR) 2.32, 95 % confidence interval (CI) 1.32-4.10; p = 0.003, OR 1.65, 95 % CI 1.18-2.29, respectively). The genotype and allele frequencies of LEPR Q223R polymorphism did not show any statistically significant differences between lung cancer patients and controls (p = 0.782 and p = 0.762, respectively). Although AA-QQ and AA-QR combined genotypes of LEP -2548G > A-LEPR Q223R loci were significantly higher in lung cancer patients (p = 0.020 and p = 0.047, respectively), GG-QQ, GG-QR, and AA-RR combined genotypes were significantly higher in control group. As a result, susceptibility effects of LEP -2548G > A polymorphism alone or in combination with LEPR Q223R polymorphism on lung cancer were observed. Further studies are necessary to prove the association of LEP and LEPR gene polymorphisms with lung cancer.

Keratinocyte growth factor-2 and autologous serum potentiate the regenerative effect of mesenchymal stem cells in cornea damage in rats.

AIM: To investigate the healing process after severe corneal epithelial damage in rats treated with mesenchymal stem cells (MSCs) cultured with or without keratinocyte growth factor (KGF-2) and autologous serum (AS) on amniotic membrane (AM). Many patients are blind and devastated by severe ocular surface diseases due to limbal stem cell deficiency. Bone marrow-derived MSCs are potential sources for cell-based tissue engineering to repair or replace the corneal tissue, having the potential to differentiate to epithelial cells. METHODS: The study included 5 groups each including 10 female "Sprague Dawley" rats in addition to 20 male rats used as bone marrow donors. Group I rats received AM+MSCs, Group II rats AM+MSCs cultured with KGF-2, Group III rats AM+MSCs cultured with KGF-2+AS, Group IV rats only AM and Group V rats, none. AS was derived from blood drawn from male rats and bone marrow was obtained from the femur and tibia bones of the same animals. Therapeutic effect was evaluated with clinical, histopathological and immunohistochemical assessment. MSC engraftment was demonstrated via detection of donor genotype (Y+) in the recipient tissue (X) with polymerase chain reaction. RESULTS: Corneal healing was significantly better in Groups I-III rats treated with MSC transplantation compared to Group IV and Group V rats with supportive treatment only. The best results were obtained in Group III rats with 90% transparency, 70% lack of neovascularization, and 100% epithelium damage limited to less than 1/4 of cornea. CONCLUSION: We suggest that culture of MSCs with KGF-2 and AS on AM is effective in corneal repair in case of irreversible damage to limbal stem cells.

Association between osteoporosis and polymorphisms of the bone Gla protein, estrogen receptor 1, collagen 1-A1 and calcitonin receptor genes in Turkish postmenopausal women.

In this study, we have investigated the association between osteoporosis and osteocalcin (BGLAP) -298 C>T, estrogen receptor 1 (ER1) 397 T>C, collagen type1 alpha 1 (Col1A1) 2046 G>T and calcitonin receptor (CALCR) 1340 T>C polymorphisms. Genomic DNA was obtained from 266 persons (158 osteoporotic and 108 healthy controls). Genomic DNA was extracted from EDTA-preserved peripheral venous blood of patients and controls by a salting-out method and analyzed by PCR-RFLP. As a result, there was no statistically significant difference in the genotype and allele frequencies of patients and controls for BGLAP -298 C>T, Col1A1 2046 G>T, ER1 397 T>C and CALCR 1340 T>C polymorphisms. However, ER1 CC genotype compared with TT+TC genotypes was found to increase the two fold the risk of osteoporosis [p=0.039, OR=2.156, 95% CI (1.083-4.293)] and CALCR CC genotype compared with TT+TC genotypes was found to have protective effect against osteoporosis [p=0.045, OR=0.471, 95% CI (0.237-0.9372)]. In the combined genotype analysis, ER1/CALCR TCCC combined genotype was estimated to have protective effect against osteoporosis [p=0.0125, OR=0.323, 95% CI (0.1383-0.755)] whereas BGLAP/Col1A1 CCTT and ER1/CALCR CCTT combined genotypes were estimated as risk factors for osteoporosis in Turkish population (p=0.027, p=0.009 respectively).

Tumor necrosis factor alpha and beta and interferon gamma gene polymorphisms in Turkish breast cancer patients.

Cytokine genes are important for researching cancer predisposition to cancers that elicit anti-tumor immune response. In this study, we investigated the association between breast cancer and tumor necrosis factor alpha (TNF-α) -308 (G>A), TNF-ß +252 (A>G), and interferon gamma (IFN-γ) +874 (T>A) gene polymorphisms in a Turkish population. This study involved 204 female breast cancer patients and 204 healthy female controls. Genomic DNA was extracted from EDTA-preserved peripheral venous blood of patients and controls by a salting-out method and analyzed by polymerase chain reaction, allele-specific oligonucleotide polymerase chain reaction, and restriction fragment length polymorphism. TNF-α -308 genotype was found to have no effect on breast cancer susceptibility. However, there were statistically significant differences between the genotype frequencies of patients and controls for TNF-ß polymorphism (p = 0.016) and the allele and genotype frequencies for the IFN-γ polymorphism (p = 0.0312 and p = 0.001, respectively). In the composite genotype analysis, the TNF-α/ß GAAG composite genotype (p = 0.0424), the TNF-α/IFN-γ GGTT and GATT composite genotypes (p = 0.0296 and p = 0.0129, respectively), the TNF-ß/IFN-γ AGTT composite genotype (p = 0.0003), and the TNF-α/ß/IFN-γ GGAGTT and GAAGTT composite genotypes (p = 0.0437 and p = 0.0038, respectively) were estimated to have a protective effect against breast cancer. However, the TNF-α/IFN-γ GGTA composite genotype is a risk factor for breast cancer (p = 0.0156). In conclusion, TNF-ß +252GG genotype was found more frequent in Turkish breast cancer patients than controls and IFN-γ TA+AA genotypes were estimated to increase breast cancer risk significantly in Turkish population.

P53 codon 72 and HER2 codon 655 polymorphisms in Turkish breast cancer patients.

The polymorphisms in codon 72 of the tumor suppressor protein p53 (P53) gene and codon 655 of the human epidermal growth factor receptor 2 (HER2) gene have been suggested to play roles in most cancers. The purpose of this study was to investigate the association between common variants of HER-2 and P53 genes with breast cancer risk. Blood samples collected from 204 women with primary breast carcinoma and 192 healthy female controls were analyzed through polymerase chain reaction-restriction fragment length polymorphism methods. The frequencies of Arg/Arg, Arg/Pro, and Pro/Pro genotypes for P53 codon 72 were 51.7%, 41.4%, and 6.9% in patients and 42.6%, 47.3%, and 10.1% in controls, respectively. The frequencies of Ile/Ile, Ile/Val, and Val/Val genotypes for HER2 codon 655 were 75.0%, 22.5%, and 2.5% in patients and 73.4%, 25.0%, and 1.6% in controls, respectively. The genotype and allele frequencies between patient and control groups for P53 gene polymorphism were not significantly different (p = 0.177 and p = 0.07, respectively). Similarly, the genotype and allele frequencies between patient and control groups for HER2 gene polymorphism were not significantly different (p = 0.716 and p = 0.891, respectively). With the exception of association between the P53 codon 72 polymorphism and tumor stages (p = 0.026), there was no significant association between the studied polymorphisms and clinicopathological characteristics. The P53 gene codon 72 Arg/Pro and Her2 gene Ile655Val polymorphisms were not associated with the risk of breast cancer in Turkish women. However, significant associations between the P53 codon 72 and the homozygote and heterozygote Pro genotypes with tumor stages were found.

Lack of association between prohibitin 3' untranslated region C-->T polymorphism and breast cancer in a Turkish population.

The 3' untranslated region (3'UTR) of the prohibitin gene has a positive effect on arresting cell proliferation between G1 and S phases and inhibits DNA synthesis. A C-to-T transition within this region creates a variant that alters mRNA function and has been shown to be associated with an increased breast cancer risk among young North Americans who are under 50 years and have at least one first-degree relative with breast cancer. We carried out a population-based case-control study to assess whether this association exists in Turkish women. We examined 106 breast cancer patients and 154 healthy controls by PCR and restriction fragment length polymorphism analysis. In the prohibitin 3'UTR, we did not detect a difference in CT/TT genotype frequency (p = 0.694; odds ratio [OR], 1.106; 95% confidence intervals [CI], 0.659-1.86) or in C/T allele frequency (p = 0.850; OR, 1.043; 95% CI, 0.667-1.62) between the all breast cancer patients and the controls. The results did not change in subgroups defined by age or family history. Hence our results do not lend support to the hypothesis that this polymorphism contributes to risk of breast cancer. The prohibitin T variant is not associated with the risk of breast cancer in Turkish women.

Polymorphisms of the serotonin-2A receptor and catechol-O-methyltransferase genes: a study on fibromyalgia susceptibility.

Genetic and environmental factors are thought to play roles in the etiopathology of fibromyalgia syndrome (FMS). The objective of this study was to determine the potential effects of single nucleotide polymorphisms (SNPs) in catechol-O-methyltransferase (COMT) (rs4680) and 5-hydroxytryptamine (serotonin) 2A (5-HT2A) receptor (rs6313 and rs6311) genes on susceptibility to FMS. One hundred seventy-one women (80 FMS, 91 control) were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used for the genotyping analyses. Genotype and allele frequencies were calculated by the chi-square test. Beck depression inventory, state and trait anxiety inventory and symptom checklist-90 revised (SCL-90-R) tests were applied to both patients and controls. There were no observed differences in the frequencies of alleles and genotypes between patients and controls for the COMT, and the two 5-HT2A receptor gene polymorphisms (P>0.05). Our results suggest that the investigated polymorphisms seem not to be the susceptibility factors in etiology of FMS.

Prostate-specific antigen and 17-hydroxylase polymorphic genotypes in patients with prostate cancer and benign prostatic hyperplasia.

We investigated the association of prostate cancer (PCa) and benign prostatic hyperplasia (BPH) with genetic polymorphisms in prostate-specific antigen (PSA) (-158 G/A) and 17-hydroxylase (CYP17) (-34 T/C) genes in a Turkish population. In this study, we investigated the distribution of these polymorphisms in 148 PCa patients, 136 BPH patients, and 102 healthy individuals as controls. The polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism assay. Genotype and allele frequencies were calculated, and their associations with PCa or BPH risk are assayed. The frequency of PSA gene GA and GG genotypes was significantly higher in PCa patients than in controls (p = 0.017 and p = 0.019, respectively). GG genotype was also associated with BPH (p = 0.033). In a case analysis, according to Gleason score, the association of PSA gene GG genotype with Gleason score >7 was near to statistical significance (odds ratio, 2.94; 95% confidence interval, 0.95-9.28). There was also an association between CYP17 polymorphism and BPH (p = 0.004). No association was observed between PCa and CYP17 gene polymorphism. These data demonstrate that PSA gene promoter variation may play a significant role in the development of PCa and BPH, and that CYP17 gene polymorphism may be associated with BPH in the Turkish population studied.

Association of serotonin 2A receptor and lack of association of CYP1A2 gene polymorphism with tardive dyskinesia in a Turkish population.

The aim of this study was to investigate the possible association of serotonin 2A receptor gene (HTR2A) -1438 G/A polymorphism and CYP1A2 gene 163C/A polymorphism with tardive dyskinesia (TD) in a Turkish population. A total of 47 patients with persistent TD, 80 patients who were consistently without TD, and 100 healthy controls were included in this study. The polymorphic regions of -1438 G/A polymorphism of HTR2A receptor gene (rs6311) and 163C/A of CYP1A2 (rs762551) gene were amplified using polymerase chain reaction (PCR), followed by digestion with restriction enzymes MspI and Bsp1201. Genotype and allele frequencies were calculated by the chi(2)-test. Crude and adjusted odds ratios (ORs) were estimated, and 95% confidence intervals (CIs) were computed by multivariate logistic regression analysis. The genotype and allele frequencies of HTR2A and CYP1A2 gene were similar in schizophrenia with TD, schizophrenia without TD, and healthy controls. The logistic regression analysis showed that cumulative exposure to antipsychotic drugs for every year (p = 0.003; OR = 1.15; CI = 1.07-1.23), and AA genotype of HTR2A gene (p = 0.0258; OR = 4.34; CI = 1.19-15.81) are risk factors for TD. The same logistic regression model showed no association between CYP1A2 polymorphism and TD. The results of the present study seem to indicate that HTR2A gene polymorphism influences the tendency to express TD following prolonged antipsychotic drug exposure in Turkish schizophrenia patients.