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Volume 16 - 2014


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1-9

Contribution of MLPA to routine testing to detect the prognostic chromosomal abnormalities in chronic lymphocytic leukemia

Author(s): Dr. Emre Tepeli,

Abstract: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease and chromosomal abnormalities with prognostic impact are frequently detected in CLL patients. There are a variety of characterized chromosomal abnormalities detected by conventional cytogenetics in CLL. These abnormalities are valuable prognostic indicators and important key strategies for making treatment decisions. Deletions of 13q14, 11q22, and 17p13, and trisomy 12 are the most frequent chromosomal abnormalities in CLL. In this study, multiplex ligation probe amplification (MLPA) results using SALSA® MLPA kit P037-A2/P038-A2 were compared with results from conventional cytogenetics and fluorescence in-situ hybridization (FISH) and we assessed the suitability of MLPA technology as a method for detecting a variety of known chromosomal abnormalities in 41 CLL patients. DSP30+IL-2 combination was used as the mitotic stimulating agents because of the low mitotic index of CLL cells in conventional cytogenetics. Locus-specific probes for 11q22.3 (ATM), 13q14.3, and 17p13 (p53), and centromeric probe for chromosome 12 were used for FISH analysis.Informative results were obtained from 80.04% of peripheral blood and bone marrow cultures. Among the 13 positive patients for trisomy 12 by conventional cytogenetics and FISH, 5 patients were normal by MLPA. The 13q14 deletions were detected in 20 patients by FISH, however, of these, 6 patients were normal by MLPA. In contrast, the 17p13 and 13q14 deletions were detected by MLPA but not by conventional cytogenetics and FISH. In this study, we found that MLPA was not as sensitive as conventional cytogenetics and FISH at detecting mosaicisms below 25-30%. Although MLPA is a simple and cost-effective technique, it may give false negative results in patients with low level mosaicism for any abnormalities. We suggest that MLPA should be used with conventional cytogenetics and FISH in detection of chromosomal abnormalities with potential clinical significance in CLL.

Keywords: MLPA, CCL, DSP30



10-29

Survival assessment and Optimization of BCR/ABL-KD amplification protocol for detection of Imatinib resistant mutations in Ph+ Chronic Myeloid Leukemia patients from Pakistan

Author(s): Dr. Afia Muhammad,

Abstract: Translocation between parts of BCR and ABL genes is the baseline abnormality for chronic myeloid leukemia (CML) genesis. To overcome this malignancy, Imatinib mesylate, a tyrosine kinase inhibitor (TKI) is being used as the first line treatment option. Certain point mutations arising in the kinase domain of ABL part constitute resistance against drug therapy. By knowing the underlying mutation, resistance can be addressed either by dose adjustment or choosing second generation TKI’s. The present studies aimed to investigate survival probability of patients in relation to their clinical features and to optimize an efficient as well as reliable protocol for RT-PCR based amplification of BCR-ABL kinase domain, and its direct sequencing analysis for mutation detection. Since this procedure has been established for the first time in Pakistan, reproducible and amplifiable products of 1306bp (b2a2) and 1380bp (b3a2), carrying BCR-ABL KD were successfully achieved after trial and error. Their sequencing analysis revealed a total of fourteen point mutations, six in Imatinib responders and eight in resistant CML patients. Thus the mutation detection supported the usefulness of this protocol in both, Imatinib sensitive and resistant patients of chronic myeloid leukemia.

Keywords: CML, Imatinib resistance, nested RT-PCR, ABL kinase domain mutations, CML survival



30-34

A comparison between two different qPCR-based strategies for expression analysis of microRNAs

Author(s): Dr. Seyed Javad Mowla,

Abstract: MicroRNAs (miRNAs) are naturally conserved families of non-coding transcripts which are processed from larger hairpin precursors. There are two major qPCR-based strategies (stem- loop and Poly-A based) for miRNA quantification in different samples. In this study, we reviewed and compared the efficiency of these specific miRNA named miR-886-5p.

Keywords: miRNA, qPCR, Real time PCR



35-43

The Protective Role Of N-Acetylcysteine Against Acrylamide-Induced Genotoxicity And Oxidative Stress In Rats

Author(s): Dr. Eyup Altinoz, Karabuk,

Abstract: Acrylamide is neurotoxic, genotoxic and highly carcinogenic in humans and animals. The aim of this study was to research the protective role of N-acetylcysteine against acrylamide-induced genotoxicity and oxidative stress in rats. In the present study, male wistar albino rats were divided into four groups, each containing 10 as follows: Control (C) group, Acrylamide (AA) group, N-acetylcysteine (NAC) group, Acrylamide + N-acetylcysteine (AA+NAC) group. At the end of 21 days, Malondialdehyde (MDA) and Glutathione (GSH) levels were measured in plasma and DNA damage was observed in lymphocytes. Plasma GSH level decreased significantly in AA group compared to C group (P < 0.05). However, GSH level increased significantly in AA+NAC group compared to AA group (p< 0.05). MDA levels increased significantly in AA group compared to C group (p< 0.05). But AA+NAC administration decreased MDA levels as compared to AA group (p< 0.05). Comet analysis results showed that lymphocyte DNAs were normal appearance in C and NAC groups. DNA damage was observed at a highest level in AA group. Furthermore some lymphocytes exhibited apoptotic appearance. However this damage was prevented by NAC administration on lymphocyte DNAs significantly. Our results demonstrated that high level of AA caused oxidative stress and DNA damage, but NAC could have a protective role on acrylamide-induced toxicity.

Keywords: Oxidative stress, lymphocyte, genotoxicity, acrylamide, N- acetylcysteine



44-54

GADD45? Methylation Is More Common In Benign Prostatic Hyperplasia Than In Prostate Cancer

Author(s): Dr. Vildan Caner,

Abstract: Prostate cancer (PCa) is one of the most common cancer types worldwide in men. Although Growth Arrest DNA Damage-Inducible 45 (GADD45) family members, GADD45?, GADD45? and GADD45?, are responsible for the activation of several molecules in certain cellular pathways affecting cell fate, including tumorigenesis, as stress sensors, the role of GADD45? in PCa is still not clear. In this study, our aim was to detect the methylation and protein expression profiles of GADD45? in benign prostate hyperplasia (BPH) (60 patients) and PCa (56 patients). The methylation of GADD45? was determined by methylation-sensitive high resolution melting analysis. Immunohistochemistry was used for evaluating GADD45? protein expression. The methylation frequency for GADD45? was as low as 1.8% in PCa compared with BPH (P=0.000). GADD45? protein was overexpressed in PCa cases (53.6%) compared with the BPH cases (33.3%), and the difference was statistically different (P=0.028). There was no correlation between GADD45? methylation and protein expression in both groups. This study shows that in contrast to hematological malignencies, GADD45? methylation is not one of the common epigenetic changes in PCa. In addition, we suggest that the loss of important regulatory mechanisms involved in GADD45? might play a role in the pathogenesis of BPH.

Keywords:



55-60

Association of Vitamin D Receptor Gene Polymorphisms in Children With Atopic Diseases

Author(s): Naci Topalo?lu, MD,

Abstract: Interaction of environmental factors and the gene polymorphisms has been shown to increase the risk of resulting allergic diseases. In addition to vitamin D forming the cellular immune response, polymorphisms of vitamin D receptors (VDR) has been shown to contribute to asthma and atopy. Our aim is to show the VDR gene polymorphisms in children in our region with atopic constitutions. A total of 46 children between 2-16 years with diagnosis of atopic dermatitis were included in the study. Blood samples were used to investigate polymorphisms of VDR genotypes FokI C>T (rs2228570), ApaI A>C (rs7975232), BsmI G>A (rs1544410) and TaqI C>T (rs731236). Comparing heterozygous mutation of FokI and ApaI in patients with those with no mutation (wild), the atopy risk was increased 2.13 and 3.33 times, respectively (p<0.05). In addition comparing patients with ApaI heterozygous and homozygote mutation with wild patients, the atopy risk was increased 2.88 times (p<0.05). The role of VDR gene polymorphisms in development of atopy is not fully understood. We believe that different approaches to the timing and amount of vitamin D added to the diet, especially of children and individuals at risk of atopy, should be developed and this may help reduce the incidence of asthma and atopy in the population.

Keywords: Polymorphism, Genetic; Dermatitis, Atopic; Vitamin D



61-76

Design and assessment of safety-oriented retroviral vectors for the gene transfer of an active glucocerebrosidase gene in human hematopoietic CD34+ progenitor cells

Author(s): Stavros Polyviou MD, PhD,

Abstract: Quantitative or qualitative deficiency of the lysosomal enzyme glucocerebrosidase results in the accumulation of its substrate, glucocerebroside, in macrophages, leading to the pathology of Gaucher disease. For more than two decades of research, gammaretroviral vectors have been used for gene transfer of the glucocerebrosidase gene for the correction of the enzyme’s deficit. It has been shown, even on clinical level, that the design of efficient as well as safe gammaretroviral vectors, aiming mainly at eliminating “insertional mutagenesis”, is an imperative need. The present study aims at the design and assessment of the efficiency of six new and safety-oriented gammaretroviral vectors for the gene transfer of the glucocerebrosidase gene into human hematopoietic progenitor cells. All vectors are Self-Inactivating and bear sequences for the improvement of transcriptional termination; either the WPRE (“Woodchuck hepatitis virus Post-transcriptional Regulatory Element”) or the 2xSV40 USE (two copies of “Simian Virus 40 Upstream Sequence Element”). It is shown that these new gammaretroviral vectors are efficient in transferring an active wild type glucocerebrosidase gene into a CD34+ human primary hematopoietic progenitor cell population. All six vectors showed increased enzyme activity, as compared to the untransduced cell population, albeit with differences amongst them, partly due to differences in the transduction efficiency.

Keywords: Gaucher disease, glucocerebrosidase, lysosomal storage disease, gammaretroviral vector, WPRE, EF1 alpha promoter



77-87

Variable R.Msp1 fragmentation in genomic DNA due to DNA hypomethylation in CRF patients with MTHFR C677Tgene polymorphism: from genetics to epigenetics

Author(s): Prof. Ozturk OZDEMIR, PhD.,

Abstract: The role of inflammation, hyperhomocysteinemia, germ-line genetic markers and epimutations haven’t been understood completely in chronic renal failure (CRF). DNA methylation is a post- replicative modification mechanism that is strongly involved in the physiological control of epimutations and gene expression. In the current study it was aimed to find out the possible role of epigenetic alterations in renal failure due to functional MTHFR deficiency in CRF patients requiring long-term haemodialysis. Current cohort includes 228 CRF patients and 212 healthy individuals from same ethnicity. The MTHFR C677T SNP analysis was genotyped by real-time PCR analysis. Genomic DNA fragmentation sizes were correlated for wild, heterozygous and homozygous mutated CRF patients after methyl marker cognate enzyme of R.Msp1 digestion. The digested DNA fragmentation profiles were also compared by Scion Image histogram plot analysis. Increased T allele frequency was detected in CRF patients, the MTHFR 677TT genotype was found 6.1% and the T allele frequency 2.53-fold increased in CRF when compared with healthy individuals. Distinct global DNA methyl patterns that showed variable R.Msp1 fragmentations were also detected in current MTHFR gene mutated CRF patients. The current results indicate that individuals with germ-line MTHFR C677T mutations have a risk for CRF pathogenesis due to the reduced enzyme activity and global DNA hypomethylation that alters the allelic expression of distinct systemic genes. Results needs to be confirmed by a larger scale of sample size.

Keywords: SNP; chronic renal failure; MTHFR C677T; DNA hypomethylation; R.Msp1 fragmentation; epigenetics



88-94

Recombinant OmpC Subunit Vaccine Facilitate Satisfactory Protection Against Challenge Dose Of Salmonella Typhimurium In Mice Model

Author(s): Dr. Porteen Kannan,

Abstract:

Keywords: ompC, Salmonella Typhimurium, subunit vaccine, mice model



95-104

The effect of hysteroscopic polypectomy on the gene expression of endometrial receptivity markers: HOXA10, HOXA11 and LIF

Author(s): Engin Y?ld?r?m, PhD.,

Abstract: The aim of this study is to determine the effect of hysteroscopic polypectomy on the mRNA expression levels of the endometrial receptivity markers, namely, homeobox A10 (HOXA10), homeobox A11 (HOXA11) and leukemia inhibitory factor (LIF). Twenty-five reproductive-aged women with endometrial polyps underwent hysteroscopy. Samples were taken at the mid-secretory phase using hysteroscopic polypectomy and 4 months after polypectomy, and the change in mRNA expression levels of normalized HOXA10, HOXA11 and LIF genes were determined using Reverse Transcription Quantitative Real Time–Polymerase Chain Reaction (RT-qPCR). The results show that mRNA levels of HOXA10 and HOXA11 taken prior to surgery and 4 months after the complete hysteroscopic removal of polyps were not significantly different (P=0.79 and P=0.14, respectively). Moreover, a marked difference could not be obtained between preoperative and postoperative endometrial LIF mRNA expression levels (P=0.86). As a conclusion, these results indicate that mRNA levels of HOXA10, HOXA11 and LIF genes, three of molecular markers of endometrial receptivity, are not affected by hysteroscopic polypectomy.

Keywords: homeobox A10, homeobox A11, Endometrialpolyp, RT-qPCR, HOXA10, HOXA11 and LIF



105-114

A molecular modeling method for protein functional site recognition with dipeptide blind docking

Author(s): Dr. Yong-Bo Song,

Abstract: Many computational methods are being developed to predict protein functional positions. Here, we present a novel two-step dipeptide blind docking method aimed to recognize protein functional sites. Twenty amino acids were first docked to the target protein, and the top 30% of amino acids with the lowest binding energies were then selected as bricks to generate dipeptide ligands, which were then used to identify the functional sites on target proteins via automated blind docking. The efficiency and accuracy of this method was validated by comparing with the reported experimental results and dinucleotide docking using the scorpion toxin protein BmkM1 and human fibroblast growth factor 2 (h-FGF2). The results indicated that the method is suitable to predict protein functional sites. Compared with regular full dipeptide blind docking, this method can also significantly reduce the total workload without sacrificing the calculation accuracy.

Keywords: Protein functional site recognition; Dipeptide; Blind docking; Molecular modeling



105-114

A molecular modeling method for protein functional site recognition with dipeptide blind docking

Author(s): Dr. Yong-Bo Song,

Abstract: Many computational methods are being developed to predict protein functional positions. Here, we present a novel two-step dipeptide blind docking method aimed to recognize protein functional sites. Twenty amino acids were first docked to the target protein, and the top 30% of amino acids with the lowest binding energies were then selected as bricks to generate dipeptide ligands, which were then used to identify the functional sites on target proteins via automated blind docking. The efficiency and accuracy of this method was validated by comparing with the reported experimental results and dinucleotide docking using the scorpion toxin protein BmkM1 and human fibroblast growth factor 2 (h-FGF2). The results indicated that the method is suitable to predict protein functional sites. Compared with regular full dipeptide blind docking, this method can also significantly reduce the total workload without sacrificing the calculation accuracy.

Keywords: Protein functional site recognition; Dipeptide; Blind docking; Molecular modeling



115-129

Prevalence of MTHFR, MTR and MTRR Gene Polymorphisms in Turkish Patients with Nonsyndromic Cleft Lip and Palate

Author(s): Dr, Deniz Aslar,

Abstract: Objective: To assess the association between the MTHFR gene (Al298C), MTR gene (A2756G) and MTRR gene (A66G) polymorphisms and nonsyndromic cleft lip with or without cleft palate(nsCL/P), we investigated patients in the Turkish population. Methodology: Analysis of gene polymorphisms was carried out using polymerase chain reactions and restriction enzyme digestions. Our study included 100 patients with nsCL/P and 125 age matched healty individuals. Results: We found that MTHFR Al298C polymorphism and MTR A2756G polymorphism are significant risk factors for nsCL/P. The frequency of MTRR A66G homozygotes was higher compared to control group, however the difference was not statistically significant. Conclusions: We revealed a statistical significant association between the MTHFR Al298C and MTR A2756G polymorphism and nsCL/P.

Keywords: Cleft lip; cleft palate; MTHFR; polymorphism



130-138

Production of pantropic retrovirus by ecotropic packaging cell line

Author(s): Dr. Yan-Yi Wang,

Abstract: The range of infectivity (tropism) of the packaged retrovirus is generally determined by env protein expressed in packaging cell line. Therefore, conversion of the env component of existing packaging cell line to another one can alter the tropism of packaged retrovirus. To achieve this, the VSV-G vector encoding the VSV-G env glycoprotein was cotransfected with the retroviral GFP expression vector into BOSC23 ecotropic packaging cell line which stably express the MMLV env protein. Unfortunately, the produced virus could only infect few human 293F cells, indicating the VSV-G can not totally replace the MMLV env as an env component of the packaged virus in BOSC23 cells. Therefore, RNAi technology was used to silence the MMLV env gene expression. The high-titer pantropic retrovirus were obtained by cotransfection of BOSC23 cells with MMLV env silencing vector, VSV-G vector, and retroviral GFP expression vector. When the pantropic retrovirus was applied to gene transfer of human umbilical cord blood CD34+ cells, the enriched CD34+ cells were successfully transduced. These results suggest that a method by which different tropic (pantropic) retrovirus can be packaged using an existing, tropism-fixed (ecotropic) packaging cell line has been successfully developed. This method is especially useful for researchers in developing countries who have one packaging cell line with tropism not compatible with the target cells and can not easily obtain the comercial packaging cell lines because there might not be enough suppliers to provide the commercial products of packaging cell lines in developing countries.

Keywords: retrovirus; packaging cell line; tropism; ecotropic; pantropic; envelope protein; RNAi



139-148

High Frequency of Chromosomal Anomalies and a Novel Chromosomal Insertion Associated with Infertility and Recurrent Miscarriages (Reproductive Failure) in West Turkey

Author(s): Prof. Dr. Fatma Silan,

Abstract: Numerical and/or structural chromosomal abnormalities may be a reason of high infertility rates and recurrent pregnancy losses (RPLs) in humans. Karyotype and karyogram profiles of patients with RPLs are presented in current results. A total of 722 patients; 161(44.5%) infertile and 200(55.5%) RPL couples were included in the study. Karyotype and structural chromosome analyses of both patient groups in Canakkale population were made between May 2011- December 2013, using peripheral lymphocyte cell culture and GTG banding technique. High frequency of chromosomal abnormalities(%7.45) were detected in 24 patients of the infertility group(n:322). 10 patients(42%) of this group(n:24) had numerical and 14 patients(58%) had balanced structural choromosomal abnormalities. A novel choromosomal insertion was found in an infertile male, one of the 22th choromosome was totally inserted in 9th choromosome [ins(9;22)(9pter-q12::22q11.1-q13.33::9q12-9qter)]. This is the first report of germline total inserion of a chromosome. Interestingly, this insertion was inherited from father. Balanced structural chromosomal abnormalities was also detected in 17 patients (4.25%) of RPL group without any numerical abnormalities. Current results constitute the first report on the high incidence of structural chromosomal aberrations in RPLs and infertile couples in Canakkale district.

Keywords: Chromosome, Cytogenetics, Insertion, Inversion, Translocation



149-159

An in vivo investigation on the effect of gene-delivered human serine hydrolase KIAA1363 on chemical warfare nerve agent intoxication

Author(s): Dr. Nageswararao Chilukuri,

Abstract: The ability of human serine hydrolase KIAA1363 as a chemical warfare nerve agent detoxifying enzyme was investigated in vivo in mice. Using an adenovirus containing the gene for human KIAA1363, the enzyme was expressed as a fusion protein with a hemaglutinin tag (Ad-KIAA1363-HA) in vitro in HEK-293A cells and in vivo in mice. Recombinant KIAA1363 (rKIAA1363-HA) expressed in vitro in HEK-293A cells is membrane-associated with a molecular weight of ~50 kDa. Following intravenous injection of Ad-KIAA1363-HA into mice, rKIAA1363-HA expression was observed in liver and diaphragm but not in circulation or in any other tissues. Expression of the enzyme was first noted on day 2 post-virus injection, reached peak levels on day 4 and declined thereafter. Compared to the livers of control virus- injected mice, the livers of mice injected with Ad-KIAA1363-HA contained ~25-50-folds higher levels of rKIAA1363-HA on day 4. Despite such high levels of rKIAA1363-HA in their livers, mice challenged with lethal doses of diisopropylfluorophosphate, VX or soman did not gain any protection relative to control animals. These results suggest that wild-type human KIAA1363 is not a suitable enzyme for development as a pretreatment candidate against chemical warfare nerve agents.

Keywords: Human serine hydrolase KIAA1363, chemical warfare nerve agents, bioscavenger, adenovirus



160-171

Protective effect of saffron (its active constituent, crocin) on oxidative stress and hepatic injury in streptozotocin induced diabetic rats

Author(s): Eyup Altinoz,

Abstract: The objective: the reactive oxygen species (ROS) take role in pathogenesis of many diseases like diabetes. Saffron extract, crocin and safranal are remarkable ROS scavenging as antioxidant agents. Methods and results: rats were divided into three groups each containing 10 as follows: control group, Diabetes Mellitus (DM) group, and Diabetes Mellitus+crocin (DM+crocin) group. Tissue samples were processed by routine histological and biochemical procedurs. Liver tissue of control group showed normal histological appearance. Sinusoidal dilatation, sinusoidal congestion, infiltration and vacuolization were observed in hepatocytes of DM group. These findings were reduced in DM+crocin group. The MDA and XO levels in DM group were higher than the other groups (P<0.01), and GSH levels in DM+crocin group were higher than DM group (P<0.01). Blood glucose concentration in DM group increased (p=0.002) compared to control group, but decreased in DM+crocin group (p=0.002) compared to DM group. Serum alanine aminotransferase (ALT) and aspartat aminotransferase (AST) levels increased remarkably (P?0.01) in DM group compared to control group. When DM+crocin group was compared with DM group, serum ALT levels decreased (p?0.05); however, decrease was lower in serum AST level (p>0.05). Conclusion: we observed in our study that crocin decreased blood glucose level of STZ induced diabetic rats and protected the liver tissue by decreasing the oxidative stress.

Keywords: Diabetes mellitus, oxidative stress, saffron, crocin, liver, blood glucose.



172-181

H19: a long non-coding RNA with different roles in cancer progression

Author(s): Mohammadreza Hajjari,

Abstract: Long non-coding RNAs (lncRNAs) are pervasively transcribed and critical RNA molecules with different roles in the cell. The association of some lncRNAs with different diseases especially cancers has been documented. One of these lncRNAs is H19 which its dysregulation is reported in different cancers. In this review, we describe the molecular function and regulation of H19 in human cells. We also point to the H19 dysregulation in different types of cancer.

Keywords: Long non-coding RNA; Cancer; Genomic Imprinting; Epigenetics




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