Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We showed that alterations of the expression of RNA that was isolated from peripheral blood leukocytes allow the identification of genes whose expression is significantly altered in the acute phase of MI.
• We discovered that the identified alterations of gene expression at the early phase of AMI allow the differentiation of patients who develop heart failure from those who do not, and such alterations may serve as a convenient tool to contribute to the prognosis of HF.
• We demonstrated the prognostic value of circulating miR-30a-5p and its association with LV dysfunction and symptoms of HF after AMI.

Opis badań

• Current projects

Project 1

Heart failure (HF) is the most common cause of morbidity and mortality in developed countries. Cardiac gene expression plays an important role in the pathogenesis and clinical manifestations of HF. Specifically, the downregulation of mRNA in myocardial tissue in HF patients suggests the importance of molecular mechanisms that suppress steady-state levels of mRNA. The main aims during the report period were (1) to establish alterations of gene expression patterns in leukocytes that are associated with acute myocardial infarction (AMI) and (2) to identify distinct biomarkers that correlate with the development of HF. We collected peripheral blood samples from patients with ST-segment elevation myocardial infarction (STEMI) from study and validation groups. Control groups comprised patients with stable coronary artery disease without a history of MI. Based on plasma NT-proBNP levels and left ventricular ejection fraction parameters, the STEMI patients were divided into HF and non-HF groups. Microarrays were used to analyze mRNA levels in peripheral blood mononuclear cells that were isolated from the study group at four time points and the control group. The microarray results were validated by real-time quantitative polymerase chain reaction (RT-qPCR) using whole-blood RNA from the validation group. We identified biologically relevant transcripts that were significantly altered in the early phase of MI and were associated with the development of post-MI HF.

We also sought to identify prognostic miRNAs that are associated with left ventricular (LV) dysfunction following AMI. Patients were divided into subgroups of patients who developed or did not develop LV dysfunction within 6 months of the infarction. miRNA profiles were determined in plasma and serum samples from the patients on the first day of AMI. Levels of 14 plasma miRNAs and 16 serum miRNAs were significantly different in samples from AMI patients who later developed LV dysfunction compared with MI patients who did not. Two miRNAs were upregulated in both types of material. Validation in an independent group of patients using droplet digital PCR (ddPCR) confirmed that miR-30a-5p was significantly elevated on admission in patients who developed LV dysfunction and HF symptoms 6 months after AMI. A bioinformatics analysis indicated that miR-30a-5p may regulate genes that are involved in the pathogenesis of cardiovascular disease.

We recently focused on studying the prognostic value of circulating miRNAs that correlate with the risk of atrial fibrillation (AF) recurrence after cardiac ablation. Atrial fibrillation is the most common and persistent cardiac arrhythmia that increases the risk of death, stroke, and other thromboembolic incidents. Atrial fibrillation patients more frequently develop heart failure and have a higher rate of hospitalization. Approximately one-third of patients may not experience symptoms of arrhythmia. Such an asymptomatic course impedes and delays diagnosis and the implementation of proper treatment. Early diagnosis provides an opportunity to prescribe treatments that can protect against such devastating consequences. Thus, our next goal will be the detection of specific microRNAs as predictors of successful AF ablation.

Project 2

The main objective of this project is to determine the molecular background of congenital hemolytic anemias (CHAs) in selected patients with anemia of unclear etiology. The term CHAs encompasses a heterogeneous group of hereditary disorders that are associated with mutations of more than 70 genes and characterized by the destruction of abnormal red blood cells (RBCs), with presentations that range from asymptomatic to severe and life-threatening. Congenital hemolytic anemias are associated with ineffective erythropoiesis and the dysregulation of iron metabolism. The pathogenesis of CHAs is still only partially understood. An accurate diagnosis of CHAs is often challenging because the clinical presentation of genetically distinct diseases can be quite similar, and standard medical procedures are incapable of distinguishing individual nosological entities. This study investigates genetic determinants of CHAs that are caused by impairments in enzyme activity in RBCs, disturbances in membrane proteins, and abnormalities in the structure and synthesis of hemoglobin. We plan to perform whole-exome sequencing for selected patients with hemolytic anemia who lack an accurate diagnosis. Moreover, the analysis of previously uncharacterized variants, both novel and already reported, using bioinformatic tools and molecular modelling will be performed.

• Past projects

Project 3

The yeast Saccharomyces cerevisiae is a useful model for studying cellular mechanisms that are related to sterol synthesis in humans because of the high similarity of the mevalonate pathway between these organisms. In this project, we evaluated the effects of four statins that are widely used in clinical practice on mevalonate and associated pathways.

Project 4

Thyroid peroxidase is a major autoantigen in thyroid autoimmune disease, in which pathogenic autoantibodies recognize immunodominant conformational epitopes that are localized on the protein surface. To elucidate the basis of this autoantigenicity, we performed complex structural, functional, and antigenic characteristics of thyroid peroxidase. The project was undertaken in collaboration with Monash University (Australia) and the Medical Centre of Postgraduate Education (Poland).

Metodologia

We conduct research using microarray analyses (with cooperation with the Laboratory of Microarrays Analyses). We perform mutation analysis using Sanger sequencing and whole exome sequencing. For gene expression analysis, we utilize qPCR and ddPCR. In our laboratory, we have a ddPCR system (Bio-Rad) that enables the precise quantification of nucleic acids. The digital PCR concept has many potential advantages over classic RT-PCR, including the ability to obtain absolute quantification without external references and robustness to variations. In view of the fact that ddPCR has demonstrated a high degree of linearity and quantitative correlation in measuring miRNAs, we used this method to confirm the results from small RNA sequencing experiments.

Wybrane publikacje

• Maciejak A, Kiliszek M, Michalak M, Tulacz D, Opolski G, Matlak K, Dobrzycki S, Segiet A, Gora M, Burzynska B. Gene expression profiling reveals potential prognostic biomarkers associated with the progression of heart failure. Genome Med. 2015 Mar 14;7(1):26. doi: 10.1186/s13073-015-0149-z.
• Warchol I, Gora M, Wysocka-Kapcinska M, Komaszylo J, Swiezewska E, Sojka M, Danikiewicz W, Plochocka D, Maciejak A, Tulacz D, Leszczynska A, Kapur S, Burzynska B. Genetic engineering and molecular characterization of yeast strain expressing hybrid human-yeast squalene synthase as a tool for anti-cholesterol drug assessment. J Appl Microbiol. 2016 Apr;120(4):877-88. doi: 10.1111/jam.13053.
• Maciejak A, Kostarska-Srokosz E, Gierlak W, Dluzniewski M, Kuch M, Marchel M, Opolski G, Kiliszek M, Matlak K, Dobrzycki S, Lukasik A, Segiet A, Sygitowicz G, Sitkiewicz D, Gora M, Burzynska B. Circulating miR-30a-5p as a prognostic biomarker of left ventricular dysfunction after acute myocardial infarction. Sci Rep. 2018 Jun 29;8(1):9883. doi: 10.1038/s41598-018-28118-1.
• Maciak K, Adamowicz-Salach A, Siwicka A, Poznanski J, Urasinski T, Plochocka D, Gora M, Burzynska B. Hereditary xerocytosis – spectrum and clinical manifestations of variants in the PIEZO1 gene, including co-occurrence with a novel β-globin mutation. Blood Cells Mol Dis. 2020 Feb;80:102378. doi: 10.1016/j.bcmd.2019.102378.

Współpraca

• Dr hab. n. med. Marek Kiliszek Department of Cardiology and Internal Diseases, Military Institute of Medicine, Warsaw
• Dr hab. n. med. Anna Adamowicz-Salach Department of Paediatrics, Haematology and Oncology, Medical University of Warsaw
• Dr hab. Beata Hukowska-Szematowicz Molecular Biology and Biotechnology Center, University of Szczecin
• Dr hab. Grażyna Sygitowicz, Dr Agata Maciejak Department of Clinical Chemistry and Laboratory Diagnostics, Medical University of Warsaw, Warsaw, Poland.
• Dr George Patrinos Erasmus University Medical Center, Faculty of Medicine, Department of Bioinformatics, Rotterdam, the Netherlands and University of Patras School of Health Sciences, Department of Pharmacy, Patras, Greece
• Dr Ashley Buckle Biomedicine Discovery Institute, Monash University, Clayton, Australia

Nagrody i wyróżnienia

• Beata Burzynska, Agata Leszczynska 2017 Medical University of Warsaw, 1st degree award for didactic achievement.