Mehri Igci

Differential Expression of a Set of Genes in Follicular and Classic Variants of Papillary Thyroid Carcinoma

Fine-needle aspiration biopsy (FNA) is currently the best initial diagnostic test for evaluation of a thyroid nodule. FNA cytology cannot discriminate between benign and malignant thyroid nodules in up to 30% of thyroid nodules. Therefore, an adjunct to FNA is needed to clarify these lesions as benign or malignant. Using differential display-polymerase chain reaction method, the gene expression differences between follicular and classic variants of papillary thyroid carcinoma (PTC) and benign thyroid nodules were evaluated in a group of 42 patients. Computational gene function analyses via Cytoscape, FuncBASE, and GeneMANIA led us to a functional network of 17 genes in which a core sub-network of five genes coexists. Although the exact mechanisms underlying in thyroid cancer biogenesis are not currently known, our data suggest that the pattern of transformation from healthy cells to cancer cells of PTC is different in follicular variant than in classic variant.

The association of the expression of miR-122-5p and its target ADAM10 with human breast cancer

MicroRNAs can regulate many biological functions. miR-122-5p has a tumor suppressor function through different molecular pathways. Also, our second hit, ADAM10, targeted by miR-122-5p, is a major determinant of HER2 shedding causing that trastuzumab cannot bind to HER2 receptors. Therefore, our analysis upon ADAM10 expression and miR-122-5p was a good point to understand molecular mechanism of breast cancer. In our study, we investigated the expression profiles of miR-122-5p and its target ADAM10 in 71 breast cancer patients. Immunohistochemical analysis of ER, PR and HER2 gene products was used to categorize tumors in patients. Expression data and immunohistochemical findings were evaluated to comment on the relationship between miR-122-5p and ADAM10. ADAM10 expression was higher in tumor than that of normal tissue but miR-122-5p expression was lower in tumor than that of normal tissue. The expression pattern in HER2+ patients was reverse of the overall result. It can be explained like that miR-122-5p expression increases especially in HER2+ cancer cell to suppress ADAM10 shedding activity on HER2 receptor. However, increase in expression of tumor suppressor miR-122-5p is not enough to inhibit ADAM10. All in all, we can think miR-122-5p as potential regulator of ADAM10 and trastuzumab resistance. Since if we increase miR-122-5p activity together with trastuzumab administration, then HER2+ breast cancer cells may overcome trastuzumab resistance by inhibiting ADAM10 shedding activity on HER2 receptors and increase the efficiency of trastuzumab.

Gene expression profiles of autophagy-related genes in multiple sclerosis

Multiple sclerosis (MS) is an imflammatory disease of central nervous system caused by genetic and environmental factors that remain largely unknown. Autophagy is the process of degradation and recycling of damaged cytoplasmic organelles, macromolecular aggregates, and long-lived proteins. Malfunction of autophagy contributes to the pathogenesis of neurological diseases, and autophagy genes may modulate the T cell survival. We aimed to examine the expression levels of autophagy-related genes. The blood samples of 95 unrelated patients (aged 17-65years, 37 male, 58 female) diagnosed as MS and 95 healthy controls were used to extract the RNA samples. After conversion to single stranded cDNA using polyT priming: the targeted genes were pre-amplified, and 96×78 (samples×primers) qRT-PCR reactions were performed for each primer pair on each sample on a 96.96 array of Fluidigm BioMark™. Compared to age- and sex-matched controls, gene expression levels of ATG16L2, ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, HTT were significantly altered (false discovery rate<0.05). Thus, altered expression levels of several autophagy related genes may affect protein levels, which in turn would influence the activity of autophagy, or most probably, those genes might be acting independent of autophagy and contributing to MS pathogenesis as risk factors. The indeterminate genetic causes leading to alterations in gene expressions require further analysis.

Association Between the T-593A and C6982T Polymorphisms of the Osteopontin Gene and Risk of Developing Nephrolithiasis

BACKGROUND AND AIMS Increased synthesis of several urinary proteins including osteopontin (OPN) has been shown to be associated with stone formation within the urinary tract. The objective of this study was to analyze the genotype distributions and allele frequencies for OPN gene promoter T-593A and C6982T (in exon 7) polymorphisms among patients with kidney stones. METHODS In this case-control study, the study group consisted of 121 patients with radiologically confirmed nephrolithiasis. Genomic DNA from patients and control cases (n = 100) was analyzed by single-strand conformation polymorphism method and nucleotide sequence analysis. RESULTS Homozygous carriers of the T-593T genotype were more frequent, but carriers of the A-593A genotype were less frequent in patients than in controls. There was also an increase in -593T allele (88% in patients vs. 79% in controls) and decrease in -593A allele frequencies (21% in control vs. 12% in patients) in the nephrolithiasis groups (p = 0.013). The carriers of C6982C genotype were less frequent, but marked increases in T6982T genotype (25.6% in patients vs. 7% in controls, p = 0.001) and 6982T allele frequency (53.3% in patients vs. 37.5% in controls, p = 0.001) were noted in patients of Turkish ancestry. CONCLUSIONS These results are the first to demonstrate the existence of T-593A promoter polymorphism of the OPN gene and significant association with risk of developing nephrolithiasis. Our results showed marked associations between polymorphisms (C6982T and T-593A) of the OPN gene and the stone-forming phenotypes in the Turkish population.

MCM3 Protein Expression in Follicular and Classical Variants of Papillary Thyroid Carcinoma

Minichromosome maintenance (MCM) proteins are needed as licensors in the DNA replication of eukaryotic cells and transcriptional control of MCM genes has critical role in the regulation of MCM functions. Different MCM protein family members are proposed as diagnostic or prognostic markers in various cancers due to their increased proliferative potential. Among MCM family members, minichromosome maintenance protein 3 (MCM3) expressions in both mRNA and protein levels were shown to be associated with papillary thyroid carcinoma (PTC). But, the usability of MCM3 in some histological variants of PTC might be controversial due to tissue specific molecular heterogeneities. In follicular variant of papillary thyroid carcinoma (FVPTC), a number of genes including MCM3 were shown to be differentially expressed which were specific to this kind of variant. Using immunohistochemistry method, MCM3 protein expression levels were compared in FVPTC, classic variant of papillary thyroid carcinoma (CVPTC), and multi-nodular goiter (MNG) tissues in a group of 32 cases. There was meaningful differences between MNG vs. FVPTC (p = 0.016) and MNG vs. CVPTC (p = 0.019) while there was no significant difference in the comparison FVPTC vs. CVPTC (p = 0.15). Four of the 5 CVPTC cases having surrounding tissue invasion had high expression values. For FVPTC and CVPTC, MCM3 protein expression results were parallel to our previous mRNA expression study while there was downregulation in protein expression despite the increased expression of MCM3 mRNA in MNG suggesting tissue-specific post-transcriptional events in benign thyroid neoplasms of which should be focused on. Moreover, the relatively lower MCM3 protein expression in FVPTC comparing to CVPTC could be due to a different tumorigenic pathway favored in this type of tissue.

DNA Methylation of BDNF Gene in Schizophrenia

Background Although genetic factors are risk factors for schizophrenia, some environmental factors are thought to be required for the manifestation of disease. Epigenetic mechanisms regulate gene functions without causing a change in the nucleotide sequence of DNA. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic transmission and plasticity. It has been suggested that BDNF may play a role in the pathophysiology of schizophrenia. It is established that methylation status of the BDNF gene is associated with fear learning, memory, and stressful social interactions. In this study, we aimed to investigate the DNA methylation status of BDNF gene in patients with schizophrenia. Material/Methods The study included 49 patients (33 male and 16 female) with schizophrenia and 65 unrelated healthy controls (46 male and 19 female). Determination of methylation pattern of CpG islands was based on the principle that bisulfite treatment of DNA results in conversion of unmethylated cytosine residues into uracil, whereas methylated cytosine residues remain unmodified. Methylation-specific PCR was performed with primers specific for either methylated or unmethylated DNA. Results There was no significant difference in methylated or un-methylated status for BDNF promoters between schizophrenia patients and controls. The mean duration of illness was significantly lower in the hemi-methylated group compared to the non-methylated group for BDNF gene CpG island-1 in schizophrenia patients. Conclusions Although there were no differences in BDNF gene methylation status between schizophrenia patients and healthy controls, there was an association between duration of illness and DNA methylation.

High-throughput screening of Sirtuin family of genes in breast cancer.

Mammalian Sirtuins have been shown to perform distinct cellular functions and deregulated expression of these genes was reported to be involved in the development of various malignancies including breast cancer. An increasing number of evidence indicates that Sirtuins have both tumor promoter and tumor suppressor functions. However, the roles of Sirtuins have not been well-reported in breast cancer. In the present study, quantitative expression levels of Sirtuins (SIRT1-7) in breast cancer patients and breast cancer cell lines (MCF-7 and SKBR3) and control cell line (CRL-4010) were assessed by using a high-throughput real-time PCR method. As a result, Sirtuins were found to be differentially expressed in breast cancer tissues and cancer cell lines. Particularly, expressions of SIRT1 and SIRT4 were found to be significantly down-regulated in breast cancer tissues and SKBR3 breast cancer cells. In contrast, SIRT2, SIRT3, and SIRT5 genes were shown to be up-regulated in our study. Although SIRT6 and SIRT7 were also up-regulated in breast cancer tissues, these expression changes were statistically insignificant. Additionally, SIRT2, SIRT3, SIRT5, SIRT6 and SIRT7 were found to be differentially expressed in breast cancer cell lines. Yet, these changes were not well-correlated with tissue expression levels. In conclusion, Sirtuin family of genes shows differential expressions in breast cancer tissues and cells and SIRT1 and SIRT4 seem to play key tumor suppressor roles in breast cancer development. Herein, we report expression levels of Sirtuin family of genes in both breast cancer tissues and cancer cell lines simultaneously.

Mutational screening of the SOCS3 gene promoter in metastatic colorectal cancer patients.

Cytokine-induced expression of suppressors of cytokine signalling (SOCS) molecules is important for the negative feedback control of STAT-dependent cytokine signalling. The aim of this study was to investigate possible association between the promoter region polymorphisms of the SOCS3 gene and metastatic colorectal carcinoma in a Turkish population. The DNA samples obtained from 103 patients and 109 healthy individuals were analyzed by polymerase chain reaction/single-strand conformation polymorphism (SSCP), and nucleotide sequence analysis. Five sets of primers designed for the SOCS3 gene were used, and we did not detect significant differences in genotype frequencies for any of these polymorphisms between the study groups. Only the S3P1 region showed polymorphism and displayed three (1,2,4, 2,3,4 and 2,4) genotypes. Interestingly, 2,3,4 genotype was observed in 3 patients, but not in controls. Moreover, the sequence analysis revealed that the nucleotides positioned at -914 and -1031 nt had the polymorphisms. Nucleotide sequence analysis of SSCP band 1 and band 3 revealed C-914A (rs12953258) and T-1031C (rs111033850) polymorphisms, respectively. The T-1031C polymorphism lies in the border of the STAT-binding site. The T-1031C polymorphism (rs111033850) is a newly identified single nucleotide polymorphism with this study, and we submitted this to the NCBI database. However, these results suggested that there is no marked association between SOCS3 gene promoter region polymorphisms and the risk of developing metastatic colorectal cancer.

Bikunin and α1-microglobulin/bikunin precursor (AMBP) gene mutational screening in patients with kidney stones: a case-control study.

Abstract Objective. Bikunin is an inhibitor of kidney stone formation synthesized in the liver together with α1-microglobulin from the α1-microglobulin/bikunin precursor (AMBP) gene. The aim of this study was to investigate the possible association between bikunin/AMBP gene polymorphisms and urinary stone formation. Material and methods. To analyse the DNA, blood samples were taken from 75 kidney stone formers who had a familial stone history, 35 sporadic stone formers and 101 healthy individuals. Four exons of bikunin gene and five parts of the promoter region of the AMBP gene were screened using single-strand conformation polymorphism and nucleotide sequence analysis. Results. The Init-2 region of the promoter of AMBP gene had polymorphisms at positions -218 and -189 nt giving three different genotypes having 1,3, 2,4 and 1,2,3,4 alleles with frequencies of 17.06%, 60.19% and 22.75%, respectively, in all groups. Therefore, the Init-2 region appears to be polymorphic. As a result, the 1,3 allele has -218G and -189T complying with the reference database sequence, the 2,4 allele has -218G and T-189C substitution and the allele 1,2,3,4 genotype has substitutions at positions G-218C and T-189C. Conclusions. There were no significant differences in allele distribution between patients and controls. These common alleles exist in the Turkish population independent of stone formation. These results are the first to demonstrate the existence of bikunin and AMBP promoter polymorphism. Although the Init-2 region of the AMBP gene is the binding site for various transcription factors, the results showed no association between these observed genotypes and stone-forming phenotypes.

Gene Expression and Promoter Region Polymorphisms of Interleukin-10 in Meningitis Patients

Meningitis is an inflammatory disease caused by bacteria, fungi, and viruses with various clinical symptoms. Interleukin-10 (IL-10) levels have been shown to be increased in blood or cerebrospinal fluid of patients with meningitis, but the association of IL-10 gene promoter polymorphisms or gene expression with meningitis has not been evaluated. IL-10 gene promoter polymorphisms A-592C, T-819C, and A-1082G in 61 patients with meningitis and 64 healthy controls were determined by real-time polymerase chain reaction analysis. mRNA from blood and cerebrospinal fluid samples was extracted, and real-time polymerase chain reaction was performed for IL-10 gene expression. No statistically significant differences were found in the allele and genotypic frequencies between patients and control subjects. Expression of IL-10 in meningitis at mRNA levels was detected in the infiltrating leukocytes. IL-10 gene expression in blood from patients was significantly higher than the control group. Our results suggest that there was no association between promoter polymorphisms of IL-10 and meningitis, but a significant increase of IL-10 gene expression was present in patients with meningitis.