Research Maaike Alaerts

Abstract

The attrition rate of novel drug candidates due to cardiotoxic adverse events remains a big challenge in the preclinical and clinical drug development. As such, it is pivotal for the pharmaceutical industry to identify these liabilities at early stages by applying sensitive and translatable assaysto predict potential harmful effects to the human cardiovascular system. The current project aims to develop and optimize an assay in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) that combines impedance and multi-electrode array (MEA) measurements with cardiotoxicity biomarkers, in particular dysregulated microRNAs (miRNAs). To achieve this goal, commercially available hiPSC-CM were treated with a large set of drugs at clinically relevant concentrations, and drug responses were monitored for 72h on XCELLigence Real Time Cell Analysis (RTCA) Cardio ExtraCellular Recording (ECR) instrument measuring impedance and electrical changes. RT-qPCR on RNA extracted from the cell pellets was employed to study the upregulation of previously identified miRNAs candidates (Gryshkova et al. Arch of Toxicology, 2022). Several miRNAs were found to be upregulated in hiPSC-CM, especially in response to anthracycline drugs. hsa-miR-187-3p, hsa-miR-182-5p, hsa-miR-365a-5p, and hsa-miR-133b were upregulated with the highest fold changes in response to several treatments. In the past decade, several studies have confirmed the expression of dysregulated miRNAs in the blood/serum of patients with various diseases, spacing from oncological disorders to cardiovascular liabilities. Therefore, the investigation of miRNAs released in the supernatant of hiPSC-CM culture could confirm their utility as potential novel biomarkers of cardiotoxicity in the clinic. To assess the translatability of the already generated data and the effect of existing genetic cardiac liabilities on cardiotoxic drug exposure, the assays will be applied to in-house created hiPSC-CMs from patients carrying TTN and SCN5A mutations, causing cardiomyopathy and the cardiac arrhythmia Brugada syndrome respectively, and healthy control individuals (isogenic and unrelated controls). Impedance and electrophysiology will be measured on these cell lines by RTCA CardioECR and alteration in expression level of the selected miRNAs will be analyzed both in the cell pellet and the supernatant. In addition, expression level of the selected miRNAs will be analyzed in blood samples collected from the same individuals as well as a selection of cardio-oncology patients.

Researcher(s)

• Promoter: Alaerts Maaike
• Co-promoter: Guns Pieter-Jan

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Inherited Cardiac Arrhythmia (ICA) refers to a group of genetic disorders in which patients present with abnormal and potentially harmful heart rhythm. These episodes often go unnoticed, but can lead to sudden cardiac death. At present, over 60 ICA genes have been identified. Using novel next-generation sequencing technology it is possible to screen all ICA genes in a single molecular diagnostic test. This analysis allows the identification of clear disease-causing variants in patients, but also results in detection of a high number of genetic variants for which causality is unsure. These pose a major burden for the management of ICA patients. Therefore, the aim of this project is to develop a functional tool that allows to test the functional impact of these so-called 'variants of uncertain significance' (VUS). We will create an advanced model of 'human induced pluripotent stem cells (hiPSC)' with built-in special fluorescent proteins that report on calcium and voltage signals. Starting from these hiPSCs we will generate cardiomyocytes, cardiac fibroblasts and endothelial cells that we grow in a controlled mixture into cardiac microtissues (cMT). The electrical activity and calcium handling of these cMTs can then be monitored with a specialized confocal fluorescence microscope. To validate our tool, we will first introduce known disease-causing alterations into the genome of these transgenic hiPSCs and study the effect on the electrical activity of the derived cMTs. Next, we will apply this method to evaluate the functional effect of VUS identified in patients. This innovative approach will improve the molecular diagnostics of inherited cardiac arrhythmias and allow clinicians to deliver true personalized medicine.

Researcher(s)

• Promoter: Alaerts Maaike
• Fellow: Akdeniz Dogan

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Anthracyclines are the mainstay of chemotherapeutic treatment in a wide range of malignancies, including breast cancer. Cardiotoxicity is a well-known and feared adverse effect of anthracyclin therapy and due to the growing population of cancer survivors, cardiovascular disease in these patients is expected to escalate. Unless we can identify high-risk patients for anthracycline therapy, today's breast cancer patients may become tomorrow 's heart failure patients. However, there is an important inte individual susceptibility for the development of cardiotoxicity and at present, it is not possible to predict which patients will develop cardiotoxicity. It was recently shown that genetic variants in titin, an import anchoring protein in the cardiomyocytes, can cause a predisposition to dilated cardiomyopathies and are also more prevalent in chemotherapy-induced cardiotoxicity. In this research project we investigate if mutations in titin increase the susceptibility for cardiotoxicity to anthracyclines, in order to identify high -risk patients. If this can be confirmed, the impact on both the individual patient (morbidity, mortality) and on society will be huge. The development of an hiPSC-CM model harbouring different TTNtv will allow us to test different possible therapeutic and preventive measures for this high risk population.

Researcher(s)

• Promoter: Van Craenenbroeck Emeline
• Co-promoter: Alaerts Maaike

Research team(s)

• Cardiovascular diseases (CARDIOVASC)

Project type(s)

• Research Project

Abstract

In this application, we request financing for three benchtop CERO 3D Cell Culture Bioreactor units for the culture of 3D cell cultures, including spheroids and organoids, that are increasingly being used in biomedical research. Currently, 3D organoids and spheroids are cultured in traditional cell culture plates under static or shaking (using orbital shaker) conditions in a standard CO2 cell culture incubator, which is suboptimal for long-term and large-scale culture of spheroids and organoids. A bioreactor system would take organoid and spheroid culture at the campus to a next level in terms of quality (improved viability, maturation and homogeneity) as well as quantity. Each CERO 3D Cell culture bioreactor unit can maintain four 50 mL organoid cultures, including monitoring and control of temperature, pH and carbon dioxide levels. In total, the envisaged bioreactor infrastructure will be able to accommodate twelve simultaneous organoid cultures under highly controlled conditions. The envisaged CERO 3D Bioreactor units will be applied for multiple research domains at the University of Antwerp, and more specifically for upscaled culture of stem cell-derived spheroids and organoids, tumoroids derived from primary tumour material of patients, stem cell-derived cardiomyocytes, stem cell-derived cartilage tissue and intestinal organoids. Furthermore, based on our own experience in upscaled organoid culture, the instalment of bioreactor units has become an urgent need to progress towards future valorisation activities.

Researcher(s)

• Promoter: Ponsaerts Peter
• Co-promoter: Alaerts Maaike
• Co-promoter: Lardon Filip
• Co-promoter: Verstraeten Aline

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Loeys-Dietz syndrome (LDS) is a genetic disorder presenting with thoracic aortic aneurysm (TAA), causing abnormal widening of the aorta, which leads to aortic rupture or dissection, a life-threatening complication that occurs unexpectedly. LDS is caused by genetic defects in six different genes of the TGF? pathway (TGFBR1/2, SMAD2/3, TGFB2/3), which is vital in the proper development of the body's connective tissue. Despite the progress in unraveling its genetic basis, there is a lack of understanding of the wide range of severity of cardiovascular involvement. In my project, I will focus on patients within families, carrying pathogenic SMAD3 variants, which show either no or early-onset aortic aneurysmal disease. I hypothesize that genetic modifiers of the primary SMAD3 mutation are the main contributors to the striking aortopathy variability in LDS-SMAD3 families. In this project, an innovative strategy will be used to identify genetic modifiers. I will perform genome-wide single nucleotide polymorphism-based linkage analysis on two large SMAD3 families and whole-genome sequencing on selected individuals, combined with SMAD3 iPSC-VSMC (induced pluripotent stem cell-derived vascular smooth muscle cells) model creation and characterization and subsequent CRISPR/Cas9-based validation of the identified modifier(s). The predicted outcomes will advance the LDS and TAA knowledge, contributing to the discovery and development of novel therapeutic targets and personalized medicine.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Alaerts Maaike
• Co-promoter: Verstraeten Aline
• Fellow: Velchev Joe Davis

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Brugada syndrome (BrS) is an inherited arrhythmic disorder and is estimated to account for up to 12% of all sudden cardiac death cases, especially in the young (< 40 years old). Only in circa 30% of BrS patients the underlying genetic cause can be identified with current diagnostic arrhythmia gene panels. Moreover, the use of these panels result in detection of numerous genetic "Variants of Uncertain Significance" (so called VUS), but currently functional models to prove their causality are lacking. Therefore, in my project I will create two proof-of-concept models for a known pathogenic CACNA1C mutation associated with BrS: a cardiomyocyte cell model, created from human stem cells, and a novel transgenic zebrafish model with built-in fluorescent calcium and voltage indicators. By functionally characterising these models with innovative imaging and electrophysiological techniques, I will assess the mutation's effect on a cellular level and in the whole heart, proving its contribution to disease causation. After validating these models, I will apply this strategy to functionally assess the pathogenicity of two VUS identified in two BrS patients. Ultimately, by establishing the use of these state-of-the-art study models to predict the pathogenicity of BrS-related VUS, a more accurate risk stratification and proficient use of specialized prevention strategies can be implemented in the future, potentially also for other electrical disorders of the heart.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Alaerts Maaike
• Co-promoter: Knapen Dries
• Co-promoter: Labro Alain
• Co-promoter: Schepers Dorien
• Co-promoter: Snyders Dirk
• Fellow: Vandendriessche Bert

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Sudden death in the young is primarily caused by inherited diseases of the heart. These conditions are frequently caused by mutations in genes responsible for maintaining a regular heartbeat. Many genes that can cause sudden death have already been identified. However, for an important portion of patients, the genetic test reveals a genetic variant with unknown significance. With my project, I intend to create a new model to study the effects of these mutations on the heart in vivo. For this purpose, I will generate a new zebrafish line, in which cardiac electrical and chemical calcium signals will be converted into fluorescent light signals. As zebrafish are translucent during the first days of development, this animal model lends itself perfectly to visualize these signals in vivo. I will use the new zebrafish line to improve our understanding of one specific cardiac disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT). This condition is characterized by abnormal calcium signaling in the heart, and as such my method will be highly suitable to study CPVT. Both in the literature and in our own cardiogenetics clinic, several CPVT families with an uncertain inheritance pattern have been discovered. With my assay I intend to expose the mechanisms of CPVT in these families and hereby clarify the results of the genetic tests and contribute to future diagnostic testing in CPVT.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Alaerts Maaike
• Co-promoter: Schepers Dorien
• Fellow: Sieliwonczyk Ewa

Research team(s)

Project type(s)

• Research Project

Abstract

Brugada syndrome (BrS) is an inherited arrhythmic disorder and is estimated to account for up to 12% of all sudden cardiac death cases, especially in the young (< 40 years old). Only in circa 30% of BrS patients the underlying genetic cause can be identified with current diagnostic arrhythmia gene panels. Moreover, the use of these panels result in detection of numerous genetic "Variants of Uncertain Significance" (so called VUS), but currently functional models to proof their causality are lacking. Therefore, in my project I will create two proof-of-concept models for a known pathogenic CACNA1C mutation associated with BrS: a cardiac muscle cell-model, created from human stem cells, and a novel transgenic zebrafish model with built-in fluorescent calcium and voltage indicators. By functionally characterising these models with innovative imaging techniques, I will assess the mutation's effect on a cellular level and in the whole heart, proving it's contribution to disease causation. After validating these models, I will apply this strategy to functionally assess the pathogenicity of two VUS identified in two BrS patients. Ultimately, by establishing the use of these state-of-the-art study models to predict the pathogenicity of BrS-related VUS, a more accurate risk stratification and proficient use of specialized prevention strategies can be implemented in the future, potentially also for other electrical disorders of the heart.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Alaerts Maaike
• Co-promoter: Schepers Dorien
• Fellow: Vandendriessche Bert

Research team(s)

Project type(s)

• Research Project

Abstract

Brugada syndrome (BrS) is an autosomal dominantly inherited cardiac electrical disorder, characterized by ventricular arrhythmias and a significant risk for sudden cardiac death (SCD). It accounts for up to 20% of SCD cases in young individuals (<45 years) with structurally normal hearts. At present over 25 genes, including SCN5A, have been associated with BrS, but mutations in these genes explain only 30% of the cases. One other major unresolved aspect of BrS concerns the significant variability in disease expression, from completely asymptomatic over mild arrhythmia to SCD, observed even within families with an established disease-causative mutation. Genetic modifiers must play an important role in this phenomenon, and the identification of such modifiers is the aim of this project. Hereto, I will use a unique collection of BrS families recruited through our cardiogenetics clinic, sharing a Belgian SCN5A founder mutation and displaying remarkable variable expressivity. I will perform whole genome sequencing and RNA-sequencing on the advanced model of induced pluripotent stem cell (iPSC)-derived cardiomyocytes from eight carefully selected mutation carriers, four at each end of the disease severity spectrum. An in-depth combined analysis of the resulting genome and transcriptome data will certainly reveal the modifier gene(s) underlying intra-familial phenotypic variability in BrS. This will lead to a significantly improved insight into the mechanisms causing BrS, drive the development of novel therapies and result in more accurate risk prediction and personalized management of BrS patients.

Researcher(s)

• Promoter: Alaerts Maaike

Research team(s)

Project type(s)

• Research Project

Abstract

Sudden death in the young is primarily caused by inherited diseases of the heart. These conditions are frequently caused by mutations in genes responsible for maintaining a regular heartbeat. Many genes that can cause sudden death have already been identified. However, for an important portion of patients, the genetic test reveals a genetic variant with unknown significance. With my project, I intend to create a new model to study the effects of these mutations on the heart in vivo. For this purpose, I will generate a new zebrafish line, in which cardiac electrical and chemical calcium signals will be converted into fluorescent light signals. As zebrafish are translucent during the first days of development, this animal model lends itself perfectly to visualize these signals in vivo. I will use the new zebrafish line to improve our understanding of one specific cardiac disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT). This condition is characterized by abnormal calcium signaling in the heart, and as such my method will be highly suitable to study CPVT. Both in the literature and in our own cardiogenetics clinic, several CPVT families with an uncertain inheritance pattern have been discovered. With my assay I intend to expose the mechanisms of CPVT in these families and hereby clarify the results of the genetic tests and contribute to future diagnostic testing in CPVT.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Alaerts Maaike
• Co-promoter: Schepers Dorien
• Fellow: Sieliwonczyk Ewa

Research team(s)

Project type(s)

• Research Project

Abstract

Brugada syndrome (BrS) is an inherited electrical disorder of the heart, presenting in patients with an irregular heart rhythm. This can go unnoticed throughout life, but also lead to sudden cardiac death, typically in patients between age 25-55. At present, more than 25 genes have been identified that can explain about 30% of BrS cases. The question remains why in the same family individuals with the identical genetic alteration can live without symptoms, have few or many arrhythmia episodes or even experience sudden death. I will look for an answer to this question in a group of families with a known error in the SCN5A gene causing BrS. I will study the exact effect of this genetic error and I will compare the difference in genetic signals between a selection of patients without symptoms and patients with severe symptoms, using whole genome and RNA sequencing techniques. Since it is important to work with heart cells in this study, I will use an advanced method that allows me to create heart cells from the patient's own skin or blood cells. Analysis of the genetic signals will lead us to the 'modifier genes' responsible for the differences in BrS disease severity. Identification of these modifiers will lead to a better insight into the mechanisms causing BrS, drive the development of novel therapies and result in more accurate risk prediction and personalized management of BrS patients.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Alaerts Maaike
• Fellow: Simons Eline

Research team(s)

Project type(s)

• Research Project

Abstract

Brugada syndrome (BrS) is an inherited cardiac electrical disorder, presented in patients by irregular heart rhythm. It is often asymptomatic, therefore it can be unnoticed. However it can also cause sudden cardiac death, typically in patients between age 25-55. First degree relatives have a 50% chance to develop BrS, putting a high burden on a family. Although some genes have been causally involved, for roughly 75% of the patients the genetic background is unknown. This project aims to fill this knowledge gap, by searching for novel genetic alterations implicated in BrS. We have gathered DNA samples from individuals from 10 genetically unresolved BrS families. We will sequence the whole genome of 3 selected patients per family. For the 3 largest families, we will combine sequencing with a dedicated linkage approach to identify genomic regions shared by patients. After genetic identification of new disease-causing mutations, their effects will be investigated in an advanced cell model, consisting of heart cells created from the patient's own skin cells. This allows us to mimic the environment of the heart in vitro and study what is happening at the molecular level. This will lead to better insight into mechanisms causing BrS, driving the development of novel therapies and ultimately resulting in more accurate risk prediction and personalized management of the BrS patients.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Alaerts Maaike
• Fellow: Nijak Aleksandra

Research team(s)

Project type(s)

• Research Project

Abstract

Brugada syndrome (BrS) is an autosomal dominant heart rhythm disorder associated with a high risk for sudden cardiac death. It has a prevalence of 1:2000 in the general population. Since clinical diagnosis is imperfect, genetic testing has an important added value, but for roughly 70% of the patients the genetic causality is still unknown. Therefore, the objective of this project is to identify novel genetic variants implicated in BrS. Eleven BrS patients and 15 unaffected relatives from two well-characterized family that are negative for all currently known arrhythmia genes will be subjected to SNP-array genotyping followed by linkage analysis. We will apply whole-genome sequencing to DNA of five of the patients to allow a comprehensive interrogation of coding, non-coding and structural DNA variation, focusing on shared variants in the linked candidate regions. This powerful approach in combination with the best suitable bioinformatics pipelines will enable us to identify novel causal variants related to BrS within the timeframe of this project. The novel BrS genes or variants will immediately be incorporated into our existing diagnostic arrhythmia gene panel, allowing direct translation into clinical care and improved risk prediction, genetic counselling and prevention of sudden cardiac death. Investigation of the function of the gene at pathway and cellular level will provide new insights into the pathogenesis of BrS and likely other arrhythmogenic disorders and ultimately drive the development of novel therapies.

Researcher(s)

• Promoter: Alaerts Maaike

Research team(s)

Project type(s)

• Research Project

Abstract

This PhD project aims to identify susceptibility genes that play a role in the etiology of the psychiatric disorders schizophrenia (SZ) and bipolar disorder (BP). Both are severe psychiatric disorders with a worldwide prevalence of about 1%. Bipolar disorder is a mood disorder characterized by the cyclic alteration of manic and depressive periods, schizophrenia is characterized by the occurrence of psychoses (periods in which contact with reality is disturbed), affective and cognitive symptoms. These are complex disorders, meaning that they are caused by an interaction of different genetic and environmental factors. Despite the high prevalence, morbidity and socio-economical costs, the pathophysiology and etiology of BP and SZ are still unknown. In this project we will use a positional cloning strategy. By performing a complete genome scan we will be able to determine chromosomal regions that show linkage with the disorders and positional candidate genes will be evaluated for their contribution to the etiology of BP and SZ through association studies. Also already known functional candidate genes will be analyzed using population based association studies. For the genome scan we have access to a unique family-based patient population from the Skellefteå region in the province of Västerbotten (northern Sweden). This Skellefteå population is a geographically isolated population, founded in 1320 and characterized by low immigration and emigration and a high expansion rate. For the association studies we have patient-control populations existing of 276 SZ patients and 500 non-related healthy controls and 276 BP patients and 500 non-related healthy controls.

Researcher(s)

• Promoter: Del-Favero Jurgen
• Fellow: Alaerts Maaike

Research team(s)

Project type(s)

• Research Project

Abstract

This PhD project aims to identify susceptibility genes that play a role in the etiology of the psychiatric disorders schizophrenia (SZ) and bipolar disorder (BP). Both are severe psychiatric disorders with a worldwide prevalence of about 1%. Bipolar disorder is a mood disorder characterized by the cyclic alteration of manic and depressive periods, schizophrenia is characterized by the occurrence of psychoses (periods in which contact with reality is disturbed), affective and cognitive symptoms. These are complex disorders, meaning that they are caused by an interaction of different genetic and environmental factors. Despite the high prevalence, morbidity and socio-economical costs, the pathophysiology and etiology of BP and SZ are still unknown. In this project we will use a positional cloning strategy. By performing a complete genome scan we will be able to determine chromosomal regions that show linkage with the disorders and positional candidate genes will be evaluated for their contribution to the etiology of BP and SZ through association studies. Also already known functional candidate genes will be analyzed using population based association studies. For the genome scan we have access to a unique family-based patient population from the Skellefteå region in the province of Västerbotten (northern Sweden). This Skellefteå population is a geographically isolated population, founded in 1320 and characterized by low immigration and emigration and a high expansion rate. For the association studies we have patient-control populations existing of 276 SZ patients and 500 non-related healthy controls and 276 BP patients and 500 non-related healthy controls.

Researcher(s)

• Promoter: Del-Favero Jurgen
• Co-promoter: Van Broeckhoven Christine
• Fellow: Alaerts Maaike

Research team(s)

Project type(s)

• Research Project