Browsing by Author "Radojković, Dragica (6602844151)"

The primary role of alpha-1-antitrypsin (AAT), encoded by the highly polymorphic SERPINA1 gene, is to protect the lung parenchyma from proteolysis by neutrophil elastase. AAT deficiency (AATD) is an autosomal recessive disease, considered as the most important genetic cause of liver disease in children and emphysema in adults. According to frequency, deficient alleles can be classified as common (Z and S) and rare (Mmalton, Mheerlen, Mprocida etc). Type, intensity and onset of clinical disease associated with AATD occur as a result of interaction between AATD and additional genetic and acquired factors (tobacco smoking, air pollution exposure etc). The most frequent clinical manifestations include premature emphysema, chronic hepatitis, cirrhosis and hepatocellular carcinoma. Epidemiological studies highlight the need for improvement in diagnostic efficiency for AATD. It is recommended for a diagnostic approach to integrate precise, internationally recognized clinical criteria and a standardized laboratory protocol, based on a combination of biochemical and molecular methods. The predilection site of clinical manifestations guides the therapeutic approach. Augmentation therapy is possible in lung disease, while currently the only specific measure in patients with severe liver failure due to AATD is transplantation. In all patients, preventive measures, ammeliorating the deleterious effects of habits and environmental factors are recommended. Introduction of gene therapy is expected to additionally improve health outcomes in affected persons. Current results with an integrative AATD diagnostic strategy in the Serbian population are highly encouraging, prompting towards its further implementation in common medical practice with the ultimate goal to establish a national register of affected individuals.

The primary role of alpha-1-antitrypsin (AAT), encoded by the highly polymorphic SERPINA1 gene, is to protect the lung parenchyma from proteolysis by neutrophil elastase. AAT deficiency (AATD) is an autosomal recessive disease, considered as the most important genetic cause of liver disease in children and emphysema in adults. According to frequency, deficient alleles can be classified as common (Z and S) and rare (Mmalton, Mheerlen, Mprocida etc). Type, intensity and onset of clinical disease associated with AATD occur as a result of interaction between AATD and additional genetic and acquired factors (tobacco smoking, air pollution exposure etc). The most frequent clinical manifestations include premature emphysema, chronic hepatitis, cirrhosis and hepatocellular carcinoma. Epidemiological studies highlight the need for improvement in diagnostic efficiency for AATD. It is recommended for a diagnostic approach to integrate precise, internationally recognized clinical criteria and a standardized laboratory protocol, based on a combination of biochemical and molecular methods. The predilection site of clinical manifestations guides the therapeutic approach. Augmentation therapy is possible in lung disease, while currently the only specific measure in patients with severe liver failure due to AATD is transplantation. In all patients, preventive measures, ammeliorating the deleterious effects of habits and environmental factors are recommended. Introduction of gene therapy is expected to additionally improve health outcomes in affected persons. Current results with an integrative AATD diagnostic strategy in the Serbian population are highly encouraging, prompting towards its further implementation in common medical practice with the ultimate goal to establish a national register of affected individuals.

Background. Promoter hypermethylation of the SMAD4 gene has been registered in some cancer types, but in general doesn’t appear to be a frequent event in carcinogenesis. However, only a few published studies deal with this topic and not many cancer types have been analyzed. The aim of this study was to establish SMAD4 gene promoter methylation status in pancreatic and endometrial cancers. Methods. Patients included in the study (62 subjects) were diagnosed and surgically treated at the University of Belgrade, Clinical Center of Serbia. Patients with pancreatic carcinoma (17 subjects) underwent surgical removal of the pancreatic adenocarcinoma at the First Surgical Clinic, while the patients with endometrial carcinoma (45 subjects) underwent hysterectomy with adnexectomy at the Institute for Gynecology and Obstetrics. Extraction of DNA from fresh tissue samples was performed and the methylation status of the SMAD4 gene promoter was studied by a previously designed PCR-based HpaII and MspI restriction enzyme assay. The resulting PCR products were analyzed by electrophoresis in 2% agarose gels. Results. Neither of the analyzed samples was found to be hypermethylated. Conclusion. This is the first report on SMAD4 methylation status in pancreatic and endometrial tumor specimens, and supports the viewpoint that SMAD4 hypermethylation is not a common event in malignant tumors. Nevertheless, promoter hypermethylation remains a candidate mechanism for SMAD4 inactivation in malignant tissue as a potential cause of decreased or lost SMAD4 expression in certain tumor types, and should be further investigated in different tumor types and larger cohorts of patients. © 2018, Institute of Oncology Sremska Kamenica. All rights reserved.

Background: Chronic obstructive pulmonary disease (COPD) is a complex disorder characterized by increased oxidative stress. Functional genetic variants of phase I and II genes are implicated in oxidants-antioxidants imbalance and may be involved in COPD development. In this study, we aimed to investigate the role of cytochrome P450 (CYP), glutathione S-transferase (GST) and microsomal epoxide hydrolase (mEH) functional variants in the pathogenesis of COPD in a Serbian population. Methods: The genotypes of 122 COPD patients and 100 controls with normal lung function were determined for CYP1A1 ∗1A/∗2A, CYP2E1 ∗1A/∗5B, GSTM1 null, GSTT1 null GSTP1 Ile105Val, mEH Tyr113His and mEH His139Arg gene variants. Results: Results obtained showed that GSTM1 null variant was significantly more represented in COPD patients than in controls (61.5% vs. 47.0%; OR=1.80; p=0.042). Also, a significant difference was observed for combinations of GSTM1 null and GSTP1 105Val/(Val) (38.5% vs. 24.0%; OR=1.98; p=0.029), as well as for CYP1A1 ∗1A/∗2A, GSTM1 null and mEH 113His/(His) genotypes (7.4% vs. 1.0%; OR=7.88; p=0.025). Conclusions: These are the first data concerning the analysis of the variants of phase I and II genes in the pathogenesis of COPD in a Serbian population. Results obtained in this study open up the possibility for thorough analyses of the role of genetic factors in COPD on larger cohorts. Also, they implicate the importance of previously described genetic associations with COPD in our population, as well as reveal a new one, not reported so far. © by Marija Stanković 2015.

Background: Chronic obstructive pulmonary disease (COPD) is a complex disorder characterized by increased oxidative stress. Functional genetic variants of phase I and II genes are implicated in oxidants-antioxidants imbalance and may be involved in COPD development. In this study, we aimed to investigate the role of cytochrome P450 (CYP), glutathione S-transferase (GST) and microsomal epoxide hydrolase (mEH) functional variants in the pathogenesis of COPD in a Serbian population. Methods: The genotypes of 122 COPD patients and 100 controls with normal lung function were determined for CYP1A1 ∗1A/∗2A, CYP2E1 ∗1A/∗5B, GSTM1 null, GSTT1 null GSTP1 Ile105Val, mEH Tyr113His and mEH His139Arg gene variants. Results: Results obtained showed that GSTM1 null variant was significantly more represented in COPD patients than in controls (61.5% vs. 47.0%; OR=1.80; p=0.042). Also, a significant difference was observed for combinations of GSTM1 null and GSTP1 105Val/(Val) (38.5% vs. 24.0%; OR=1.98; p=0.029), as well as for CYP1A1 ∗1A/∗2A, GSTM1 null and mEH 113His/(His) genotypes (7.4% vs. 1.0%; OR=7.88; p=0.025). Conclusions: These are the first data concerning the analysis of the variants of phase I and II genes in the pathogenesis of COPD in a Serbian population. Results obtained in this study open up the possibility for thorough analyses of the role of genetic factors in COPD on larger cohorts. Also, they implicate the importance of previously described genetic associations with COPD in our population, as well as reveal a new one, not reported so far. © by Marija Stanković 2015.

Liquid biopsy and cell-free DNA (cfDNA) show great promise in cancer diagnostics. In this study, we designed a custom droplet digital PCR (ddPCR) assay for the quantification and quality control of cfDNA isolated from serum. The assay was validated on a group of locally advanced colorectal cancer (CRC) patients and two control groups—patients with hemorrhoids and healthy individuals. The assay shows a high correlation with Qubit measurement (r = 0.976) but offers a higher dynamic range. Mean concentrations of cfDNA were 12.36 ng/µL, 5.17 ng/µL, and 0.29 ng/µL for CRC, hemorrhoid patients, and healthy controls, respectively. The quality of cfDNA was assessed with the measurement of B-cell DNA contamination. On a subset of CRC patients, we compared the mutation status on KRAS (G12A, G12D, G12V, G13D) and BRAF (V600E) genes in the primary tumor and cfDNA isolated from the serum. A total of 70.6% of primary tumor samples were mutated, and the mean fractional abundance of mutations was 9.50%. The matching serum samples were mutated in 38% cases with an average fractional abundance of 0.23%. We conclude that any decisions based solely on the amount of cfDNA present in patient serum must be inter-preted carefully and in the context of co-morbidities. This study explores the potential of ddPCR somatic mutations detection from liquid biopsy as a supplement to tissue biopsy in targeted per-sonalized CRC patient management. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Liquid biopsy and cell-free DNA (cfDNA) show great promise in cancer diagnostics. In this study, we designed a custom droplet digital PCR (ddPCR) assay for the quantification and quality control of cfDNA isolated from serum. The assay was validated on a group of locally advanced colorectal cancer (CRC) patients and two control groups—patients with hemorrhoids and healthy individuals. The assay shows a high correlation with Qubit measurement (r = 0.976) but offers a higher dynamic range. Mean concentrations of cfDNA were 12.36 ng/µL, 5.17 ng/µL, and 0.29 ng/µL for CRC, hemorrhoid patients, and healthy controls, respectively. The quality of cfDNA was assessed with the measurement of B-cell DNA contamination. On a subset of CRC patients, we compared the mutation status on KRAS (G12A, G12D, G12V, G13D) and BRAF (V600E) genes in the primary tumor and cfDNA isolated from the serum. A total of 70.6% of primary tumor samples were mutated, and the mean fractional abundance of mutations was 9.50%. The matching serum samples were mutated in 38% cases with an average fractional abundance of 0.23%. We conclude that any decisions based solely on the amount of cfDNA present in patient serum must be inter-preted carefully and in the context of co-morbidities. This study explores the potential of ddPCR somatic mutations detection from liquid biopsy as a supplement to tissue biopsy in targeted per-sonalized CRC patient management. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Introduction/Objective Coumarin therapy represents one of the best models for applying pharmacogenetics. The contribution of factors influencing coumarin therapy can vary significantly between ethnic groups, which justifies conducting population-specific studies. The aim of this study was to analyze the influence of the most important genetic factors (VKORC1 and CYP2C9 genes) that affect coumarin therapy in patients from Serbia. Methods A retrospective study involving 207 patients on acenocoumarol therapy was conducted. Genetic analyses were performed by direct sequencing. Influence on acenocoumarol dose of variants (VKORC1, CYP2C9*2, CYP2C9*3) causing hypersensitivity and VKORC1 variants causing resistance to acenocoumarol were analyzed. Multiple regression analysis was used to design a mathematical model for predicting individual drug dosage based on clinical-demographic and genetic data. Results The study confirmed significant influence of the analyzed genetic factors on acenocoumarol maintenance dose. We designed mathematical model for predicting individual acenocoumarol dose and its unadjusted R2 was 61.8. In the testing cohort, our model gave R2 value of 42.6 and showed better prediction in comparison with model given by other authors. In the analyzed patients, nine different variants in the VKORC1 coding region were found. Among carriers of these variants 78% were completely resistant, and it was not possible to achieve therapeutic effect even with high doses of acenocoumarol. Conclusions Population-specific model for prediction individual dose of acenocoumarol, may show advantages over protocols that are used in a generalized manner. Also, VKORC1 variants which cause coumarin resistance should be considered when planning therapy. © 2022, Serbia Medical Society. All rights reserved.