Effects of DMSO, glycerol, betaine and their combinations in detecting single nucleotide polymorphisms of epidermal growth factor receptor (EGFR) gene promoter sequence in non-small-cell lung cancer (NSCLC) patients

The aim of the study was to examine the effects of frequently used polymerase chain reaction (PCR) additives DMSO, glycerol and betaine on amplification of GC-rich epidermal growth factor receptor (EGFR) gene promoter region, in order to detect the presence of -216G gt T and -191C gt A gene variations in non-small-cell lung cancer (NSCLC) patients. PCR products and restriction fragments were detected by electrophoresis on 8% polyacrylamide gel and 3% agarose gel. Our analysis shows that single used additives including DMSO in concentration of 7% and 10%, glycerol in concentration of 10%, 15% and 20%, as well as betaine in concentration of 1 M, 1.5 M and 2 M significantly enhanced the yield and specificity of PCR reaction. In addition, the combination of 10% DMSO with 15% glycerol has shown positive effects, whereas other analyzed combinations of additives failed to amplify the EGFR promoter region.This is the peer reviewed version of the paper: Jurišić, V., Obradović, J., Tošić, N., Pavlović, S., Kulić, M., & Djordjević, N. (2016). Effects of DMSO, glycerol, betaine and their combinations in detecting single nucleotide polymorphisms of epidermal growth factor receptor (EGFR) gene promoter sequence in non-small-cell lung cancer (NSCLC) patients. Journal of Pharmaceutical and Biomedical Analysis, 128, 275–279. [https://doi.org/10.1016/j.jpba.2016.05.010][https://www.sciencedirect.com/science/article/abs/pii/S0731708516302473?via%3Dihub

long noncoding rna gaS as a new biomarker in oncology

Growth arrest specific 5 (GAS5) je duga nekodirajuća RNK koja zaustavlja ćelijski ciklus i promoviše apoptozu. Ponašajući se kao signalni protein, kao mamac za druge molekule ili kao transportni molekul, ova regulatorna RNK utiče na niz puteva i molekula koji su bitni za rast ćelije i apoptozu, među kojima se ističu p53 mreža, mTOR signalni put, AKT signalni put, kao i molekuli mikro RNK, PTEN i slični. Brojne studije na različitim tipovima karcinoma su pokazale da nivo ekspresije GAS5 utiče na razvoj i tok bolesti kod hematoloških maligniteta, ginekoloških karcinoma, glioma, karcinoma dojke, karcinoma gastrointestinalnog trakta, bubrega, bešike, prostate i pluća. Shodno tome, GAS5 je novi biomarker u onkologiji, koji ima dijagnostički i prognostički značaj.Growth arrest specific 5 (GAS5) is a long noncoding RNA which halts the cell cycle and promotes apoptosis. Acting as a signal protein, as a decoy for other molecules or as a transport molecule, this regulatory RNA influences a number of pathways and molecules relevant for the growth of the cell and apoptosis, among them the most important being the p53 network, the mTOR signal pathway, the AKT signal pathway, as well as molecules of microRNA, PTEN and others. Numerous studies on diverse cancer types have confirmed that the expression of GAS5 influences the development and the course of hematological malignancies, gynecologic carcinoma, gliomas, breast cancer, gastrointestinal cancer, kidney cancer, bladder cancer, prostate cancer and lung cancer. Therefore, GAS5 is a promising new diagnostic and prognostic biomarker in oncology

Reversal of FLT3 Mutational Status and Sustained Expression of NPM1 Mutation in Paired Presentation, and Relapse Samples in a Patient with Acute Myeloid Leukemia

We report a case of de novo acute myeloid leukemia (AML) with unstable FLT3 gene mutations and stable NPM1 mutation. FLT3/D835 and NPM1 (Type A) mutations were detected upon diagnosis. During the relapse, the FLT3/D835 mutation changed to an FLT3/ITD mutation while the NPM1 (Type A) mutation was retained. Cytogenetic analyses showed the normal karyotype at diagnosis and relapse. Our findings raise interesting questions about the significance of these mutations in the leukemogenic process, about their stability during the evolution of the disease, and regarding the selection of appropriate molecular markers for the monitoring of minimal residual disease

Optimization of PCR Conditions for Amplification of GC-Rich EGFR Promoter Sequence

BackgroundPolymerase chain reaction (PCR) is an extremely sensitive method that often demands optimization, especially when difficult templates need to be amplified. The aim of the present study was to optimize the PCR conditions for amplification of the epidermal growth factor receptor (EGFR) promoter sequence featuring an extremely high guanine-cytosine (GC) content in order to detect single nucleotide polymorphisms -216G gt T and -191C gt A. MethodsGenomic DNA used for amplification was extracted from formalin-fixed paraffin-embedded lung tumor tissue and PCR products were detected by agarose gel electrophoresis. ResultsResults showed that addition of 5% dimethyl sulfoxide (DMSO), as well as DNA concentration in PCR reaction of at least 2 g/ml, were necessary for successful amplification. Due to high GC content, optimal annealing temperature was 7 degrees C higher than calculated, while adequate MgCl2 concentration ranged from 1.5 to 2.0 mM. ConclusionIn conclusion, EGFR promoter region is a difficult PCR target, but it could be amplified after optimization of MgCl2 concentration and annealing temperature in the presence of DMSO and the DNA template of acceptable concentration

Effect of steroids on transcription and secretion of Gal-1 by the human trophoblast cell line in vitro

Galectin-1 (Gal-1) is a lectin with recently documented pro-invasive function in trophoblasts in vitro, whose regulation is currently insufficiently known. The potential involvement of steroid hormones, synthetic glucocorticoid dexamethasone (DEX), the sex steroid progesterone (PRG) and mifepristone (RU486) in the regulation of Gal-1 in the trophoblast-derived cell line HTR-8/SVneo was investigated. Gal-1 mRNA levels were assessed by real-time PCR. The effect on secretion of Gal-1 into the culture media was followed using the SELDI-TOF protein chip array. We present evidence that DEX and RU486 significantly reduced Gal-1 in the HTR-8/SVneo cell line at the mRNA level. In addition, trophoblast-derived HTR-8/SVneo cells were shown to secrete detectable Gal-1 protein, which was only slightly increased by PRG. The potential clinical relevance of these findings remains to be determined. [Projekat Ministarstva nauke Republike Srbije, br. 173004

Application of Next-Generation Sequencing Technology and Establishment of Biobanks

Institute of Molecular Genetics and Genetic Engineering has become widely recognized as an expert centre for rare diseases (RD). It is the first institution is Serbia that applied NGS methodology in research and diagnostics of RD. We are also the institution with RD biobank collections containing DNA, RNA, mononuclear cells and tissue samples from over 2000 of patients affected with 50 different diseases. An accurate diagnosis was provided to over than 100 RD patients who were undiagnosed for years. We used Clinical-Exome Sequencing TruSightOne Gene Panel (4813 clinicaly-relevant genes), Illumina MiSeq instrument and Illumina VariantStudio. For monogenic diseases, filtration and prioritization of variants were performed according to “in-house” pipeline, using virtual gene panels. Variants were analyzed by various in silico softwares and classified according to ACMG guidelines. Variants selected by these criteria were confirmed by conventional Sanger sequencing and parents’ samples were analyzed whenever available. Furthermore, novel variants in DNAI1, MUT, PAH, PCCB, SLC37A4, SPAG16 and SPAG17 genes were functionally characterized in adequate in vitro systems such as immortalized patients’ fibroblasts or CRISPR /Cas9 edited commercial cell lines. Clinical-exome sequencing enabled diagnosis of more than 50 different diagnosis (hematological, metabolic, endocrinological, pulmonary, immunological, orthopedic, dermatological, ophthalmological, cardiological, epileptic encephalopathies etc.). It was particularly important for genetically heterogeneous diseases, such as glycogen storage diseases, branched-chain organic acidurias, primary ciliary dyskinesia, MODY or mitochondriopathies. Moreover, different diseases with overlapping clinical manifestations were accurately diagnosed. Also, we used TruSeq-Amplicon Cancer Panel to analyse different childhood and adult rare hematological malignancies. Besides studying diagnostic and prognostic malignancy markers, we designed “in-house” virtual pharamocogenomic panel, and performed association studies of pharmacogenomic markers and the course and outcome of rare hematological malignancies, resulting in recommendations for therapeutic modalities in accordance with genomic profile of the patien

Association of Bax expression and Bcl2/Bax ratio with clinical and molecular prognostic markers in chronic lymphocytic leukemia

Uvod: Rezistencija na apoptozu koja karakteriše maligne B limfocite in vivo u hroničnoj limfocitnoj leukemiji (HLL) delimično je uzrokovana unutrašnjim poremecajima apoptotske mašinerije u ovim celijama. Ti poremecaji su rezultat genetičkih promena i aberantne ekspresije regulatora procesa apoptoze, među kojima ključnu ulogu imaju članovi Bcl2 familije. Cilj: Cilj ove studije je bio da se ispita udruženost nivoa ekspresije proapoptotskog Bax gena, kao i Bcl2/Bax odnosa, sa kliničkim karakteristikama bolesnika sa HLL kao i molekularnim prognostičkim markerima, i to mutacionim statusom rearanžiranih gena za teške lance imunoglobulina (IGHV) i ekspresijom gena za lipoproteinsku lipazu (LPL). Metode: Analizirana je ekspresija Bax iRNK i Bcl2/Bax iRNK odnos u mononuklearnim celijama periferne krvi 58 bolesnika sa HLL i 10 zdravih kontrola metodom reverzne transkripcije i lančane reakcije polimeraze u realnom vremenu (qRT-PCR). Rezultati: Detektovana je povišena ekspresija Bax gena u HLL uzorcima u odnosu na kontrolne uzorke (p=0,003), kao i povišen Bcl2/Bax odnos (p= lt 0,001). Kada je u pitanju udruženost sa prognostičkim markerima, Bcl2/Bax odnos je ispoljio negativnu korelaciju sa vremenom udvostručavanja broja limfocita (r=-0,307; p=0,0451), dok je visoka ekspresija Bax bila povezana sa LPL-pozitivnim statusom (p=0,035). I ekspresija Bax gena i Bcl2/Bax odnos su bili viši kod bolesnika sa nemutiranim u odnosu na bolesnike sa mutiranim IGHV genima, ali nije dostignuta statistička značajnost. Zaključak: Rezultati ove studije ukazuju na mogucu ulogu poremecene ekspresije Bcl2 i Bax gena, koja dovodi do visokog Bcl2/Bax odnosa u leukemijskim celijama, u patogenezi i kliničkom toku HLL.Background: In chronic lymphocytic leukemia (CLL), in vivo apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the Bcl2 family members play a central role. Aim: The aim of this study was to investigate the association of pro-apoptotic Bax gene expression and Bcl2/Bax ratio with the clinical features of CLL patients as well as with molecular prognostic markers, namely the mutational status of rearranged immunoglobulin heavy variable (IGHV) genes and lipoprotein lipase (LPL) gene expression. Methods: We analyzed the expression of Bax mRNA and Bcl2/Bax mRNA ratio in the peripheral blood mononuclear cells of 58 unselected CLL patients and 10 healthy controls by the quantitative reverse-transcriptase polymerase chain reaction. Results: We detected significant Bax gene overexpression in CLL samples compared to non-leukemic samples (p=0.003), as well as an elevated Bcl2/Bax ratio (p= lt 0.001). Regarding the association with prognostic markers, the Bcl2/Bax ratio showed a negative correlation to lymphocyte doubling time (r=-0.307; p=0.0451), while high-level Bax expression was associated with LPL-positive status (p=0.035). Both the expression of Bax and Bcl2/Bax ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance. Conclusions: Our results suggest that dysregulated expression of Bcl2 and Bax, which leads to a high Bcl2/Bax ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL

Impact of alterations in X-linked IRAK1gene and miR-146a on susceptibility and clinical manifestations in patients with systemic sclerosis

Systemic sclerosis (SSc) is a heterogeneous multisystem autoimmune disease with unknown etiology. Numerous studies have indicated that the disease heterogeneity implies various genetic abnormalities. Considering that SSc is characterized by a strong sex bias and that the position of IRAK1 gene is on the X chromosome, we assume that variations in IRAK1 gene could explain female predominance of SSc. It was previously described that miR-146a has a role in 'fine-tuning' regulation of the TLR/NF-kB signaling pathway through down-regulation of IRAK1 gene. The aim of the present study was to analyze both variants and expression level of IRAK1 and miR-146a genes in terms of susceptibility to SSc and clinical presentation of SSc patients. We analyzed variants IRAK1 rs3027898 C gt A and miR-146a rs2910164 C gt G in 102 SSc patients and 66 healthy subjects. Genotyping was performed by Sanger sequencing. Expression level of IRAK1 mRNA and miR-146a in PBMCs was performed in subset of 50 patients and 13 healthy controls by RT-qPCR. Our results showed that there was no association between IRAK1 rs3027898 and the risk of SSc in women. However, the analysis of genotype distribution of the mir-146a rs2910164 C gt G variant indicated that CC genotype shows strong association with lung fibrosis and active form of the disease. When expression level of IRAK1 gene was analyzed, we detected significant downregulation of IRAK1 mRNA in SSc patients compared to controls, as well as in male compared to female patients, in patients with ACAS autoantibodies and in patients with severe skin involvement. Regarding the expression level of miR-146a, we have found significantly reduced expression in SSc patients, in patients with skin involvement and in male SSc patients. The results from this study indicate that expression of IRAK1 gene could explain phenotypic heterogeneity of SSc and may be involved in the pathogenesis of SSc due to its differential expression in certain subgroups. Our results also suggested that miR-146a rs2910164 CC genotype may be predisposing factor for development lung fibrosis and more progressive form of SSc. Results from relative expression analysis of miR-146a demonstrated that changes in the level of this miRNA may have an impact on development and clinical course of SSc.This is the peer reviewed version of the paper: Vreca, M., Andjelkovic, M., Tosic, N., Zekovic, A., Damjanov, N., Pavlovic, S., & Spasovski, V. (2018). Impact of alterations in X-linked IRAK1gene and miR-146a on susceptibility and clinical manifestations in patients with systemic sclerosis. Immunology Letters, 204, 1–8. [https://doi.org/10.1016/j.imlet.2018.10.002