Antioxidant effect of lactic acid bacteria in human bronchial epithelial cells exposed to cigarette smoke.

AIMS: Chronic lung diseases are a major and increasing global health problem, commonly caused by cigarette smoke. We aimed to explore the antioxidant effects of lactic acid bacteria (LAB) against cigarette smoke in bronchial epithelial cells. METHODS AND RESULTS: The antioxidant effects of 21 heat-killed (HK) LAB strains were tested in cigarette smoke-stimulated BEAS-2B cells and 3-D bronchospheres organoids. We showed that HK Lactiplantibacillus plantarum BGPKM22 possesses antioxidant activity against cigarette smoke, resistance to hydrogen peroxide, and free radical neutralizing activity. We demonstrated that HK BGPKM22 inhibited cigarette smoke-induced expression of the Aryl hydrocarbon receptor (AhR) and Nuclear factor erythroid 2 related factor 2 (Nrf2) genes. The cell-free supernatant (SN) of BGPKM22 fully confirmed the effects of HK BGPKM22. CONCLUSIONS: For the first time, we revealed that HK and SN of Lactip. plantarum BGPKM22 possess antioxidant activity and modulate AhR and Nrf2 gene expression in bronchial epithelial cells exposed to cigarette smoke.

Interactions of the eNOS and ACE genes and cigarette smoking in chronic obstructive pulmonary disease.

Background: Chronic obstructive pulmonary disease (COPD) is a complex disorder with unexplained heritability. Interactions of genetic and environmental factors are thought to be crucial in COPD. So, we aim to examine interactions of the endothelial nitric oxide synthase (eNOS) and angiotensin converting enzyme (ACE) genes and cigarette smoking in COPD. Methods: The eNOS G 894T and ACE ID variants were analyzed in 122 COPD patients and 200 controls from Serbia. The effect of the variants on COPD was assessed by logistic regression. Interactions between eNOS, ACE and cigarette smoking in COPD were evaluated using a case-control model. Interaction between the genes was analyzed in silico. Results: No effect of the eNOS G 894T and ACE ID variants on COPD was found in our study. Gene-gene interaction between the eN OS T T and A CE D was identified (p=0.033) in COPD. The interaction is realized within the complex network of biochemical pathways. Gene-environment interactions between the eNOS T and cigarette smoking (p=0.013), and the ACE II and cigarette smoking (p=0.009) were detected in COPD in our study. Conclusions: This is the first research to reveal interactions of the eNOS and ACE genes and cigarette smoking in COPD progressing our understanding of COPD heritability and contributing to the development of appropriate treatments.

Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22 Exhibit Anti-Inflammatory Effect by Attenuation of NF-κB and MAPK Signaling in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are exposed to environmental influences, microbiota, and pathogens and also serve as a powerful effector that initiate and propagate inflammation by the release of pro-inflammatory mediators. Recent studies suggested that lung microbiota differ between inflammatory lung diseases and healthy lungs implicating their contribution in the modulation of lung immunity. Lactic acid bacteria (LAB) are natural inhabitants of healthy human lungs and also possess immunomodulatory effects, but so far, there are no studies investigating their anti-inflammatory potential in respiratory cells. In this study, we investigated immunomodulatory features of 21 natural LAB strains in lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Our results show that several LAB strains reduced the expression of pro-inflammatory cytokine and chemokine genes. We also demonstrated that two LAB strains, Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22, effectively attenuated LPS-induced nuclear factor-κB (NF-κB) nuclear translocation. Moreover, BGZLS10-17 and BGPKM22 reduced the activation of p38, extracellular signal-related kinase (ERK), and c-Jun amino-terminal kinase (JNK) signaling cascade resulting in a reduction of pro-inflammatory mediator expressions in BEAS-2B cells. Collectively, the LAB strains BGZLS10-17 and BGPKM22 exhibited anti-inflammatory effects in BEAS-2B cells and could be employed to balance immune response in lungs and replenish diminished lung microbiota in chronic lung diseases.

The stress responsive gene ankrd1a is dynamically regulated during skeletal muscle development and upregulated following cardiac injury in border zone cardiomyocytes in adult zebrafish.

Ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic protein found in the nuclei and sarcomeres of cardiac and skeletal muscles, with a proposed role in linking myofibrilar stress and transcriptional regulation. Rapid upregulation of its expression in response to both physiological and pathological stress supports the involvement of ANKRD1 in muscle tissue adaptation and remodeling. However, the exact role of ANKRD1 remains poorly understood. To begin to investigate its function at higher resolution, we have generated and characterized a TgBAC(ankrd1a:EGFP) zebrafish line. This reporter line displays transgene expression in slow skeletal muscle fibers during development and exercise responsiveness in adult cardiac muscle. To better understand the role of Ankrd1a in pathological conditions in adult zebrafish, we assessed ankrd1a expression after cardiac ventricle cryoinjury and observed localized upregulation in cardiomyocytes in the border zone. We show that this expression in injured hearts is recapitulated by the TgBAC(ankrd1a:EGFP) reporter. Our results identify novel expression domains of ankrd1a and suggest an important role for Ankrd1a in the early stress response and regeneration of cardiac tissue. This new reporter line will help decipher the role of Ankrd1a in striated muscle stress response, including after cardiac injury.

Detection of Somatic Mutations with ddPCR from Liquid Biopsy of Colorectal Cancer Patients.

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 interpreted 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 personalized CRC patient management.

Cloning and expression profiling of muscle regulator ANKRD2 in domestic chicken Gallus gallus.

Striated muscle signaling protein and transcriptional regulator ANKRD2 participates in myogenesis, myogenic differentiation, muscle adaptation and stress response. It is preferentially expressed in slow, oxidative fibers of mammalian skeletal muscle. In this study, we report on characterization of chicken ANKRD2. The chicken ANKRD2 coding region contains 1002 bp and encodes a 334-amino acid protein which shares approximately 58% identity with human and mouse orthologs, mostly in the conserved region of ankyrin repeats. Comprehensive analysis of the ANKRD2 gene and protein expression in adult chicken demonstrated its predominant expression in red muscles of thigh and drumstick, compared to white muscle. It was not detected in heart and white pectoral muscle. Uneven expression of ANKRD2 in chicken skeletal muscles, observed by immunohistochemistry, was attributed to its selective expression in slow, oxidative, type I and fast, oxidative-glycolytic, type IIA myofibers. Association of chicken ANKRD2 with phenotypic differences between red and white muscles points to its potential role in the process of myofiber-type specification. In addition to expression in slow oxidative myofibers, as demonstrated for mammalian protein, chicken ANKRD2 was also detected in fast fibers with mixed oxidative and glycolytic metabolism. This finding suggests that ANKRD2 is responsive to metabolic differences between types of avian myofibers and orientates future studies towards investigation of its role in molecular mechanisms of myofiber-type-specific gene expression.

Laboratory reporting on the clinical spectrum of CFTR p.Arg117His: Still room for improvement.

BACKGROUND: The clinical spectrum associated with cystic fibrosis transmembrane conductance regulator (CFTR) variant p.Arg117His is highly variable, ranging from full-blown cystic fibrosis (CF) in a small number of cases to CFTR-related disorders (CFTR-RDs) or no symptoms at all. Therefore, taking into account phenotype variability is essential for interpretation. External quality assessment (EQA) schemes can help laboratories to objectively assess the quality of genotyping and reporting by the laboratory. METHODS: We performed a retrospective longitudinal data analysis on laboratory performance regarding the interpretation of p.Arg117His during CF EQA scheme participation. Completeness and accuracy of reporting on two mock clinical cases were each compared over time (case 1: 2005, 2007 and 2012; case 2: 2015 and 2018). These cases concerned subjects compound heterozygous for p.Phe508del and p.Arg117His in cis with 7T, but with different clinical backgrounds (family planning (case 1) versus diagnostic testing for a child (case 2)). Furthermore, we analyzed the influence of previous participations, annual test volume, accreditation status and laboratory setting on overall performance. RESULTS: Overall performance improved over time, except during the 2007 CF EQA scheme. In addition, previous participations had a beneficial effect on laboratory performance. Accreditation status, annual test volume and laboratory setting did not significantly influence total interpretation scores. CONCLUSIONS: In general, laboratories performed well on both cases, although reporting on the variable clinical spectrum of p.Arg117His in cis with 7T and on the disease liability of individual CFTR variants can still improve. Moreover, this study underlined the educational role of CF EQA schemes.

Molecular Diagnostics of Cystic Fibrosis in Serbia: Our Approach to Meet the Diagnostic Challenges.

Background: High heterogeneity levels of cystic fibrosis transmembrane regulator (CFTR) are manifested in different populations. The aim of this study was to analyze comprehensively all mutations in the CFTR gene in Serbian patients with cystic fibrosis (CF) and to use the findings to propose a testing algorithm for the Serbian population. Materials and Methods: Cascade screening was employed to detect mutations in the CFTR gene of 90 patients suspected of having CF, using polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism or PCR-mediated site directed mutagenesis, Sanger sequencing, and/or next-generation sequencing. Results: This is the first report for the Serbian CF population where single nucleotide polymorphisms, small insertions and deletions, large genome rearrangements, and copy number variants were analyzed in detail. A high degree of heterogeneity within the CFTR was documented among our cohort of 90 patients. We identified 19 CF-causing mutations and 3 with varying consequences, including a previously unreported deletion of the entire exon 11. Conclusion: Considering the spectrum and frequency of mutations found, we recommend a multistep sequencing algorithm in combination with evaluation of large rearrangements for future analyses of the CFTR gene in the Serbian population.

The Silence Speaks, but We Do Not Listen: Synonymous c.1824C>T Gene Variant in the Last Exon of the Prothrombin Gene as a New Prothrombotic Risk Factor.

BACKGROUND: Thrombosis is a major global disease burden with almost 60% of cases related to underlying heredity and most cases still idiopathic. Synonymous single nucleotide polymorphisms (sSNPs) are considered silent and phenotypically neutral. Our previous study revealed a novel synonymous FII c.1824C>T variant as a potential risk factor for pregnancy loss, but it has not yet been associated with thrombotic diseases. METHODS: To determine the frequency of the FII c.1824C>T variant we have sequenced patients' DNA. Prothrombin RNA expression was measured by quantitative PCR. Functional analyses included routine hemostasis tests, western blotting and ELISA to determine prothrombin levels in plasma, and global hemostasis assays for thrombin and fibrin generation in carriers of the FII c.1824C>T variant. Scanning electron microscopy was used to examine the structure of fibrin clots. RESULTS: Frequency of the FII c.1824C>T variant was significantly increased in patients with venous thromboembolism and cerebrovascular insult. Examination in vitro demonstrated increased expression of prothrombin mRNA in FII c.1824T transfected cells. Our ex vivo study of FII c.1824C>T carriers showed that the presence of this variant was associated with hyperprothrombinemia, hypofibrinolysis, and formation of densely packed fibrin clots resistant to fibrinolysis. CONCLUSION: Our data indicate that FII c.1824C>T, although a synonymous variant, leads to the development of a prothrombotic phenotype and could represent a new prothrombotic risk factor. As a silent variant, FII c.1824C>T would probably be overlooked during genetic screening, and our results show that it could not be detected in routine laboratory tests.

Neutrophil extracellular traps-associated markers are elevated in patients with systemic lupus erythematosus.

Neutrophil extracellular traps (NETs) are the main source of autoantigens in systemic lupus erythematosus (SLE). The aim of this study was to evaluate the clinical importance of NETs-associated markers in SLE. We compared NETs-associated markers in SLE patients (n = 111) with healthy controls (n = 50). Moreover, in 35 patients with drug-naïve SLE (n = 35), we investigated correlation between NETs-associated markers [DNase I concentration, myeloperoxidase (MPO) activity, anti-MPO antibodies, cell-free DNA (cfDNA), NETolytic activity] with serological parameters [anti-dsDNA antibodies, C3, C4 and B-cell activating factor (BAFF) levels] and disease activity measured by modified SLE Disease Activity Index (M-SLEDAI-2K). In comparison with healthy controls, SLE patients had higher cfDNA, MPO activity, anti-MPO antibodies (p < 0.001), BAFF and DNase I concentration (p < 0.01). Contrary, NETolytic activity was lower in SLE patients (p < 0.05), despite higher concentration of DNase I. MPO activity and cfDNA levels showed correlation with DNase I concentration (p < 0.001, p < 0.01, respectively). BAFF levels correlated with cfDNA, DNase I concentration and MPO activity (p < 0.05). Anti-dsDNA antibodies showed correlation with MPO activity (p < 0.01), cfDNA and BAFF levels (p < 0.001). Anti-dsDNA and C3 levels were independent predictors of M-SLEDAI-2K in multivariate analysis (p < 0.01). We demonstrated that sera of SLE patients have decreased NETolytic activity, leading to increased levels of various NETs-associated markers, which correlate with anti-dsDNA antibodies in drug-naïve SLE. We showed that BAFF participates in a complex relationship between NETosis and anti-dsDNA antibodies production. These findings have important implications for a better understanding of SLE pathogenesis and development of therapy that inhibits NETs persistence and disease progression.

European recommendations integrating genetic testing into multidisciplinary management of sudden cardiac death.

Sudden cardiac death (SCD) accounts for 10-20% of total mortality, i.e., one in five individuals will eventually die suddenly. Given the substantial genetic component of SCD in younger cases, postmortem genetic testing may be particularly useful in elucidating etiological factors in the cause of death in this subset. The identification of genes responsible for inherited cardiac diseases have led to the organization of cardiogenetic consultations in many countries worldwide. Expert recommendations are available, emphasizing the importance of genetic testing and appropriate information provision of affected individuals, as well as their relatives. However, the context of postmortem genetic testing raises some particular ethical, legal, and practical (including economic or financial) challenges. The Public and Professional Policy Committee of the European Society of Human Genetics (ESHG), together with international experts, developed recommendations on management of SCD after a workshop sponsored by the Brocher Foundation and ESHG in November 2016. These recommendations have been endorsed by the ESHG Board, the European Council of Legal Medicine, the European Society of Cardiology working group on myocardial and pericardial diseases, the ERN GUARD-HEART, and the Association for European Cardiovascular Pathology. They emphasize the importance of increasing the proportion of both medical and medicolegal autopsies and educating the professionals. Multidisciplinary collaboration is of utmost importance. Public funding should be allocated to reach these goals and allow public health evaluation.

Comparative toxicity evaluation of targeted anticancer therapeutics in embryonic zebrafish and sea urchin models.

Cancer drug resistance and poor selectivity towards cancer cells demand the constant search for new therapeutics. PI3K-Akt-mTOR and RAS-MAPK-ERK signaling pathways are key mechanisms involved in cell survival, proliferation, differentiation, and metabolism and their deregulation in cancer can promote development of therapy resistance. We investigated the effects of targeted inhibitors (wortmannin, GSK690693, AZD2014 and tipifarnib) towards these two pathways on early zebrafish and sea urchin development to assess their toxicity in normal, fast proliferating cells. PI3K inhibitor wortmannin and RAS inhibitor tipifarnib displayed highest toxicity while GSK690693, a pan-Akt kinase inhibitor, exhibited a less significant impact on embryo survival and development. Moreover, inhibition of the upstream part of the PI3K-Akt-mTOR pathway (wortmannin/GSK690693 co-treatment) produced a synergistic effect and impacted zebrafish embryo survival and development at much lower concentrations. Dual mTORC1/mTORC2 inhibitor AZD2014 showed no considerable effects on embryonic cells of zebrafish in concentrations substantially toxic in cancer cells. AZD2014 also caused the least prominent effects on sea urchin embryo development compared to other inhibitors. Significant toxicity of AZD2014 in human cancer cells, its capacity to sensitize resistant cancers, lower antiproliferative activity against human normal cell lines and fast proliferating embryonic cells could make this agent a promising candidate for anticancer therapy.

Characterization of zebrafish (Danio rerio) muscle ankyrin repeat proteins reveals their conserved response to endurance exercise.

Muscle proteins with ankyrin repeats (MARPs) ANKRD1 and ANKRD2 are titin-associated proteins with a putative role as transcriptional co-regulators in striated muscle, involved in the cellular response to mechanical, oxidative and metabolic stress. Since many aspects of the biology of MARPs, particularly exact mechanisms of their action, in striated muscle are still elusive, research in this field will benefit from novel animal model system. Here we investigated the MARPs found in zebrafish for protein structure, evolutionary conservation, spatiotemporal expression profiles and response to increased muscle activity. Ankrd1 and Ankrd2 show overall moderate conservation at the protein level, more pronounced in the region of ankyrin repeats, motifs indispensable for their function. The two zebrafish genes, ankrd1a and ankrd1b, counterparts of mammalian ANKRD1/Ankrd1, have different expression profiles during first seven days of development. Mild increase of ankrd1a transcript levels was detected at 72 hpf (1.74±0.24 fold increase relative to 24 hpf time point), while ankrd1b expression was markedly upregulated from 24 hpf onward and peaked at 72 hpf (92.18±36.95 fold increase relative to 24 hpf time point). Spatially, they exhibited non-overlapping expression patterns during skeletal muscle development in trunk (ankrd1a) and tail (ankrd1b) somites. Expression of ankrd2 was barely detectable. Zebrafish MARPs, expressed at a relatively low level in adult striated muscle, were found to be responsive to endurance exercise training consisting of two bouts of 3 hours of forced swimming daily, for five consecutive days. Three hours after the last exercise bout, ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change), while ankrd1b and ankrd2 were upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively). This study provides the foundation to establish zebrafish as a novel in vivo model for further investigation of MARPs function in striated muscle.

Correction to: Analysis of the CYP2C19 genotype associated with bleeding in Serbian STEMI patients who have undergone primary PCI and treatment with clopidogrel.

The correct Author names are shown in this paper.

Human germline gene editing: Recommendations of ESHG and ESHRE.

Technological developments in gene editing raise high expectations for clinical applications, first of all for somatic gene editing but in theory also for germline gene editing (GLGE). GLGE is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if GLGE would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique can help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? The European Society of Human Reproduction and Embryology (ESHRE) and the European Society of Human Genetics (ESHG) together developed a Background document and Recommendations to inform and stimulate ongoing societal debates. After consulting its membership and experts, this final version of the Recommendations was endorsed by the Executive Committee and the Board of the respective Societies in May 2017. Taking account of ethical arguments, we argue that both basic and pre-clinical research regarding GLGE can be justified, with conditions. Furthermore, while clinical GLGE would be totally premature, it might become a responsible intervention in the future, but only after adequate pre-clinical research. Safety of the child and future generations is a major concern. Future discussions must also address priorities among reproductive and potential non-reproductive alternatives, such as PGD and somatic editing, if that would be safe and successful. The prohibition of human germline modification, however, needs renewed discussion among relevant stakeholders, including the general public and legislators.

Responsible innovation in human germline gene editing: Background document to the recommendations of ESHG and ESHRE.

Technological developments in gene editing raise high expectations for clinical applications, including editing of the germline. The European Society of Human Reproduction and Embryology (ESHRE) and the European Society of Human Genetics (ESHG) together developed a Background document and Recommendations to inform and stimulate ongoing societal debates. This document provides the background to the Recommendations. Germline gene editing is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if germline gene editing would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique could help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? This Background document summarizes the scientific developments and expectations regarding germline gene editing, legal regulations at the European level, and ethics for three different settings (basic research, preclinical research and clinical applications). In ethical terms, we argue that the deontological objections (e.g., gene editing goes against nature) do not seem convincing while consequentialist objections (e.g., safety for the children thus conceived and following generations) require research, not all of which is allowed in the current legal situation in European countries. Development of this Background document and Recommendations reflects the responsibility to help society understand and debate the full range of possible implications of the new technologies, and to contribute to regulations that are adapted to the dynamics of the field while taking account of ethical considerations and societal concerns.

Responsible innovation in human germline gene editing. Background document to the recommendations of ESHG and ESHRE.

Technological developments in gene editing raise high expectations for clinical applications, including editing of the germline. The European Society of Human Reproduction and Embryology (ESHRE) and the European Society of Human Genetics (ESHG) together developed a Background document and Recommendations to inform and stimulate ongoing societal debates. This document provides the background to the Recommendations. Germline gene editing is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if germline gene editing would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique could help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? This Background document summarizes the scientific developments and expectations regarding germline gene editing, legal regulations at the European level, and ethics for three different settings (basic research, pre-clinical research and clinical applications). In ethical terms, we argue that the deontological objections (e.g. gene editing goes against nature) do not seem convincing while consequentialist objections (e.g. safety for the children thus conceived and following generations) require research, not all of which is allowed in the current legal situation in European countries. Development of this Background document and Recommendations reflects the responsibility to help society understand and debate the full range of possible implications of the new technologies, and to contribute to regulations that are adapted to the dynamics of the field while taking account of ethical considerations and societal concerns.

Human germline gene editing. Recommendations of ESHG and ESHRE.

Technological developments in gene editing raise high expectations for clinical applications, first of all for somatic gene editing but in theory also for germline gene editing (GLGE). GLGE is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if GLGE would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique can help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? The European Society of Human Reproduction and Embryology (ESHRE) and the European Society of Human Genetics (ESHG) together developed a Background document and Recommendations to inform and stimulate ongoing societal debates. After consulting its membership and experts, this final version of the Recommendations was endorsed by the Executive Committee and the Board of the respective Societies in May 2017. Taking account of ethical arguments, we argue that both basic and pre-clinical research regarding human GLGE can be justified, with conditions. Furthermore, while clinical GLGE would be totally premature, it might become a responsible intervention in the future, but only after adequate pre-clinical research. Safety of the child and future generations is a major concern. Future discussions must also address priorities among reproductive and potential non-reproductive alternatives, such as PGD and somatic editing, if that would be safe and successful. The prohibition of human germline modification, however, needs renewed discussion among relevant stakeholders, including the general public and legislators.

Analysis of the CYP2C19 genotype associated with bleeding in Serbian STEMI patients who have undergone primary PCI and treatment with clopidogrel.

PURPOSE: Bleeding is one of the possible adverse events during clopidogrel therapy. The CYP2C19 gene is the most significant genetic factor which influences response to clopidogrel treatment. We aimed to examine the contribution of the CYP2C19 gene to bleeding occurrence during clopidogrel therapy in Serbian patients with ST segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI). METHODS: This case-control study included 53 patients who experienced bleeding and 55 patients without bleeding. Bleeding events were defined and classified using the Bleeding Academic Research Consortium (BARC) criteria. All patients were prescribed daily doses of clopidogrel during the 1-year follow-up after PCI. The CYP2C19*17 (c.-806C>T, rs12248560), rs11568732 (c.-889T>G, CYP2C19*20), CYP2C19*2 (c.681G>A; rs4244285) and CYP2C19*3 (c.636G>A; rs4986893) variants were analysed in all 108 patients. Additionally, sequencing of all nine exons, 5'UTR and 3'UTR in the rs11568732 carriers was performed. RESULTS: Association between bleeding (BARC type ≥ 2) and the CYP2C19*17 variant was not observed [odds ratio (OR), 0.53; 95% confidence interval (CI), 0.2-1.1; p = 0.107). The rs11568732 variant showed significant association with bleeding (OR, 3.7; 95% CI, 1.12-12.44; p = 0.025). Also, we found that the rs11568732 variant appears independently of haplotype CYP2C19*3B, which is contrary to the previous findings. CONCLUSIONS: Our results indicate the absence of CYP2C19*17 influence and turn the attention to the potential significance of the rs11568732 variant in terms of adverse effects of clopidogrel. However, it is necessary to conduct an independent conformation study in order to verify this finding. Also, an analysis of the functional implication of the rs11568732 variant is necessary in order to confirm the significance of this variant, both in relation to its influence on gene expression and in relation to its medical significance.