Research team

Expertise

I use zebrafish as a model to study the genetics of human hereditary disorders. Therefore, I introduce mutations, identified in patients, into the zebrafish genome using the CRISPR/Cas9 technique. In order to select mutant zebrafish, I use a Zebrafish Embryo Genotyper, which allows me to genotype the fish without sacrificing them. Next, I perform Next Generation Sequencing in order to identify mutant alleles. Mutant fish are raised till adulthood and their offspring is used for phenotyping. Three day old zebrafish larvae are phenotyped in vivo using a light sheet microscope. Since my main interest lies in cardiac arrhythmia, I introduced a genetically encoded voltage sensor in the zebrafish heart. This voltage sensor allows us to visualize the electrical activity in the heart.

Elucidation of the TGF-b paradox in aortic aneurysmal disease using zebrafish. 01/04/2024 - 31/03/2025

Abstract

Impaired TGF-β signaling has been implied in thoracic aortic aneurysm and dissection (TAAD) related disorders such as Loeys-Dietz syndrome. Although pathogenic variants in genes coding for components of the TGF-β signaling pathway have been identified as causal for these diseases, the precise mechanisms by which these specific variants lead to pathology remain elusive. Since medial degeneration is the main pathological substrate for TAAD, vascular smooth muscle cell (VSMC) dysfunction is often considered as the main culprit, but the role of endothelial cells (ECs) is neglected. With this project, I will elucidate the TGF-β paradox by in vivo fluorescent light sheet imaging in zebrafish. I will use an innovative EC and VSMC specific fluorescent TGF-β reporter to study TGF-β signaling in real time in a zebrafish Tgfb2 knockout line. Elucidation of the true pathomechanisms will bring us closer to a curative therapy for life-threatening TAAD and pave the way for the identification of prognostic biomarkers of aortic disease severity.

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Research team(s)

Project type(s)

  • Research Project

Interrogation of the contribution of endothelial cells to aortic aneurysmal disease: unraveling the TGF-ß paradox and the role of nitric oxide. 01/10/2022 - 30/09/2026

Abstract

Impaired TGF-ß signaling has been implied in thoracic aortic aneurysm and dissection (TAAD) related disorders such as Loeys-Dietz syndrome. Although pathogenic variants in genes coding for components of the TGF-ß signaling pathway have been identified as causal for these diseases, the precise mechanisms by which these specific variants lead to pathology remain elusive. Since medial degeneration is the main pathological substrate for TAAD, vascular smooth muscle cell (VSMC) dysfunction is often considered as the main culprit, but the role of endothelial cells (ECs) is neglected. Dysregulated endothelial nitric oxide (NO) signaling contributes to aneurysm development, but its link to TGF-ß signaling remains vague. With this project, I will elucidate the TGF-ß paradox and investigate the effect of impaired TGF-ß signaling on NO regulation by in vivo fluorescent light sheet imaging in zebrafish. I will use an innovative EC and VSMC specific fluorescent TGF-ß reporter to study TGF-ß signaling in real time in a zebrafish Tgfb2 knockout line. Next, by using a novel genetically encoded eNO probe (geNOps), I will investigate how impaired TGF-ß signaling affects NO regulation by in vivo imaging. Finally, I will identify therapeutic targets using an RNA sequencing approach on the novel zebrafish models I developed. This will bring us closer to a curative therapy for life-threatening TAAD and pave the way for the identification of prognostic biomarkers of aortic disease severity.

Researcher(s)

Research team(s)

Project type(s)

  • Research Project

The role of the COL4A2 NC1-domain in cerebrovascular and aneurysmal disorders: a functional approach. 01/10/2021 - 30/09/2025

Abstract

COL4A1- and COL4A2-related disorders cause a broad spectrum of problems comprising abnormal brain development, brain hemorrhage at any age, aneurysms (local dilatations) of the brain arteries, but also eye or renal problems. In addition, COL4A1 was recently identified as a genetic modifier in Marfan syndrome. We studied the presence of COL4A1 and COL4A2 variants in two patient cohorts; a cerebral palsy (CP) and a TAA (thoracic aortic aneurysm) cohort. This led to a specific interest in the COL4A2 NC1 domain. A burden analysis demonstrated a statistically significant overrepresentation of COL4A2 NC1 variants in the CP cohort. Furthermore, we identified the NC1 variant p.Arg1662His in 3 TAA patients and 3 CP patients of Moroccan descent. In 5 cases in combination with the helical variant p.Met1355Thr. The latter is suggestive of a shared "risk haplotype". The p.Arg1662His variant was significantly overrepresented in Moroccan patients in our cohorts compared to a Moroccan control cohort. In addition, We will study the cellular effects of NC1 variants using patient fibroblasts in order to assess (1) the levels of endoplasmatic reticulum stress and activation of the unfolded protein response and (2) alterations in Akt-FAK-mTOR signaling and procaspase 8 and 9 expression. Fibroblasts were collected from patients harboring (1) the COL4A2 variant p.Arg1662His, (2) the COL4A2 variant p.Arg1662His in combination with the COL4A2 variant p.Met1355Thr and (3) the pathogenic COL4A2 p.Gly1353Ala as a positive control. Three wild-type fibroblasts are used as negative controls. Secondly, we will develop a zebrafish model to study the effect of COL4A2 NC1 variants. We will start with the introduction of the pathogenic COL4A2 p.Gly1353Ala variant and study the effect on zebrafish development using a fish that has fluorescent blood vessels in order to easily pick up abnormal vessels. We will study the occurrence of brain haemorrhage, changes in movement patterns and the basement membrane, a structure that stabilizes the wall of blood vessels and measure the aortic diameter. When a reliable read-out is identified, we will introduce NC1 variants in the zebrafish model to assess their effect. This project is the first study to investigate the contibution of specific COL4A2 NC1-domain variants in pathology. When our findings are corroborated by functional studies, it would also be the first identification of a population-specific COL4A2-related risk haplotype associated with cerebral and aortic vascular pathology, which is an important finding in the age of personalized medicine. Another novelty is the development and use of a zebrafish model to study functional effects of COL4A2 variants using (CRISPR)/Cas9 technology. The model would enable not only functional analysis of additional variants of unknown significance in cerebrovascular pathology and TAA, but additionally allows studies regarding the pathogenic mechanisms underlying different types of COL4A2-mutations. This will help in identifying potential therapeutic strategies. Eventually, the model is suited for testing of potential treatment strategies in vivo, enabling monitoring of the therapeutic effect, as well as unwanted side-effects.

Researcher(s)

Research team(s)

Project type(s)

  • Research Project

Elucidating the pathogenicity of genetic variants of uncertain significance in Brugada syndrome patients by functional modelling in hiPSC-derived cardiomyocytes and zebrafish. 01/11/2020 - 31/10/2024

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)

Research team(s)

Project type(s)

  • Research Project

The elucidation of pathogenesis and modes of inheritance of catecholaminergic polymorphic ventricular tachycardia using cardiac optical imaging in zebrafish. 01/04/2021 - 31/03/2022

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. One of these cardiac disorders is catecholaminergic polymorphic ventricular tachycardia (CPVT), characterized by abnormal calcium signaling in the heart. Both in the literature and in our own cardiogenetics clinic, several CPVT families with an uncertain inheritance pattern have been discovered. In order to investigate the effect of a presumed splice site mutation identified in the CASQ2 gene of a CPVT patient in our cardiogenetics clinic, I will perform a Minigene-assay. I have developed a zebrafish line in which cardiac electrical and chemical calcium signals are converted into fluorescent light signals, allowing in vivo imaging of cardiac action potentials and calcium transients. Using CRISPR/Cas9, I developed a casq2 knock-out zebrafish model for CPVT. By overexpressing mutant CASQ2 mRNA in these zebrafish, either alone or in combination with wildtype mRNA, I will be able to model human homozygous and heterozygous states. This will not only improve further diagnostic testing in CPVT but also ameliorate risk stratification and refine personalized management.

Researcher(s)

Research team(s)

    Project type(s)

    • Research Project

    Optical mapping of in vivo cardiac mechanics in zebrafish: exploring the pathogenesis and mode of inheritance in catecholaminergic polymorphic ventricular tachycardia. 01/10/2020 - 30/09/2022

    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)

    Research team(s)

      Project type(s)

      • Research Project

      A zebrafish model system to assess pathogenicity of genetic variants in patients with cardiac arrhythmias. 01/01/2020 - 31/12/2023

      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 fainting and sudden cardiac death. At present, over 50 ICA genes have been identified. With the advent of next generation sequencing technology it is possible to test all of these genes simultaneously in multiple ICA patients with a single test. This method proficiently identifies clear disease causing genetic alterations. However, as the number of genes involved increases through better mechanistic insight into disease modifier genes and polymo hisms, we are confronted with a high number of genetic alterations for which causality is unsure. These pose a major challenge for the management of ICA patients. Therefore, the aim of this project is to develop a functional tool that will allow to test the functional impact of variants of unknown significance. We will develop a zebrafish assay in which the electrical dynamics of the heart are reported by fluorescent light signals. As zebrafish are translucent in early development, this model lends itself perfectly to visualize these signals 'in vivo' and at an exceptional resolution. After validating this tool with known pathogenic alterations, we will apply this method to evaluate variants of unknown significance. This innovative approach will allow the clinicians to deliver true personalized medicine.

      Researcher(s)

      Research team(s)

      Project type(s)

      • Research Project

      Exploring the pathogenesis and mode of inheritance in catecholaminergic polymo hic ventricular tachycardia using optical mapping of in vivo cardiac mechanics in zebrafish. 01/01/2020 - 31/12/2022

      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 this project, we intend to create a new model to study the effects of these mutations on the heart in vivo. We 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. We will use the new zebrafish line to improve our understanding of one specific cardiac disorder, catecholaminergic polymo hic ventricular tachycardia (CPVT). This condition is characterized by abnormal calcium signaling in the heart, and as such our 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 we 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)

      Research team(s)

      Project type(s)

      • Research Project

      Elucidating the pathogenicity of genetic variants of uncertain significance in Brugada syndrome patients by functional modelling in hiPSC-derived cardiomyocytes and zebrafish. 01/11/2019 - 31/10/2020

      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)

      Research team(s)

        Project type(s)

        • Research Project

        Development of a functional assay to determine the pathogenicity of genetic variants with unknown significance identified in patients with cardiac arrhythmia. 01/10/2018 - 30/09/2022

        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 fainting and sudden cardiac death. At present, over 50 ICA genes have been identified. With the advent of next generation sequencing technology it is possible to test all of these genes simultaneously in multiple ICA patients with a single test. This method proficiently identifies clear disease causing genetic alterations. However, as the number of genes involved increases through better mechanistic insight into disease modifier genes and polymorphisms, we are confronted with a high number of genetic alterations for which causality is unsure. These pose a major challenge for the management of ICA patients. Therefore, the aim of this project is to develop a functional tool that will allow to test the functional impact of variants of unknown significance. We have developed a zebrafish assay in which the electrical dynamics of the heart are reported by fluorescent light signals. As zebrafish are translucent in early development, this model lends itself perfectly to visualize these signals 'in vivo' and at an exceptional resolution. After validating this tool with known pathogenic alterations, we will apply this method to evaluate variants of unknown significance and test the possible arrhythmogenic side effects of some drugs. This innovative approach will allow the clinicians to deliver true personalized medicine.

        Researcher(s)

        Research team(s)

        Project type(s)

        • Research Project

        Optical mapping of in vivo cardiac mechanics in zebrafish: exploring the pathogenesis and mode of inheritance in catecholaminergic polymorphic ventricular tachycardia. 01/10/2018 - 30/09/2020

        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)

        Research team(s)

          Project type(s)

          • Research Project

          Development of a functional assay to determine the pathogenicity of genetic variants with unknown significance identified in patients with cardiac arrhythmia. 01/01/2018 - 31/12/2020

          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 fainting and sudden cardiac death. At present, over 50 ICA genes have been identified. With the advent of next generation sequencing technology it is possible to test all of these genes simultaneously in multiple ICA patients with a single test. This method proficiently identifies clear disease causing genetic alterations. However, as the number of genes involved increases through better mechanistic insight into disease modifier genes and polymorphisms, we are confronted with a high number of genetic alterations for which causality is unsure. These pose a major challenge for the management of ICA patients. Therefore, the aim of this project is to develop a functional tool that will allow to test the functional impact of variants of unknown significance. We will develop a zebrafish assay in which the electrical dynamics of the heart are reported by fluorescent light signals. As zebrafish are translucent in early development, this model lends itself perfectly to visualize these signals 'in vivo' and at an exceptional resolution. After validating this tool with known pathogenic alterations, we will apply this method to evaluate variants of unknown significance. This innovative approach will allow the clinicians to deliver true personalized medicine.

          Researcher(s)

          Research team(s)

            Project type(s)

            • Research Project

            Development of a novel transgenic zebrafish model to determine the pathogenicity of genetic variants for cardiac arrhythmia. 01/04/2017 - 31/03/2018

            Abstract

            Inherited Cardiac Arrhythmia (ICA), such as long QT syndrome (LQTS) and Brugada syndrome (BrS), refers to a group of hereditary disorders in which patients present with irregular heart rhythm, caused by altered cardiac electrical dynamics. These episodes can remain asymptomatic, but also lead to sudden syncope and sudden death of the individual. Up to date, over 50 different genes have been identified that can cause ICA. Thanks to the advent of next generation sequencing it is possible to test all these genes simultaneously in multiple ICA patients in a single experiment, allowing the identification of pathogenic genetic alterations. However, we are also confronted with a high number of genetic alterations for which it is unsure whether they are causally involved in the disease or not, so-called variants of unknown significance. Therefore, there is a high need for a physiologically relevant functional tool to test the pathogenicity of these variants. By combining two state-of-the-art techniques, genetically encoded voltage indicators (GEVI) and selective plane illumination microscopy (SPIM), I will develop such a novel tool to study the cardiac conduction system and characterize its anatomical connectivity in zebrafish at an unprecedented resolution. By converting electrical dynamics of the zebrafish heart into fluorescent signals, this tool will enable me to optically map action potentials in the complete heart at single cell level. This will allow me to determine cardiac conduction speed and observe conduction delays, making it a novel and ideal tool to investigate the electro- and pathophysiological mechanisms underlying two arrhythmia syndromes, LQTS and especially BrS. Finally, using this functional assay, I will be able to evaluate the pathogenicity of genetic variants with an unknown clinical significance.

            Researcher(s)

            Research team(s)

              Project type(s)

              • Research Project

              Pathogenetic study of the intersection of two frequent monogenic diseases: the Marfan syndrome and autosomal dominant polycystic kidney disease. 01/01/2012 - 31/12/2015

              Abstract

              This project represents a research agreement between the UA and on the onther hand IWT. UA provides IWT research results mentioned in the title of the project under the conditions as stipulated in this contract.

              Researcher(s)

              Research team(s)

                Project type(s)

                • Research Project