Title Promoter Affiliations Abstract "Deep familial phenotyping and genotyping to resolve phenotypic variability of inherited pathogenic genetic variants" "Jeroen Breckpot" "Department of Human Genetics" "The high burden of de novo pathogenic genetic variants in sporadic patients with rare developmental disorders (DD) has led to a de novo paradigm in genetic research and diagnostics. Genetic variants inherited from an unaffected parent are typically disregarded in variant interpretation pipelines. However, we are confronted with a growing list of rare inherited variants, that are enriched in patient versus control cohorts. Mechanisms underlying reduced penetrance of these variants are poorly understood. Whole genome sequencing (WGS) is particularly suited to explore genetic modifiers underlying this phenotypic variability, given its ability to detect both coding and non-coding variants. We aim to bridge the knowledge gap of phenotypic variability by combining deep phenotyping, genotyping and hi-C sequencing in affected and unaffected relatives of patients with inherited pathogenic copy number variants for DD. This will be crucial for improved diagnosis and counseling of genetic risk variants in DD." "Development of an innovative hiPSC-derived cardiac-microtissue-based functional assay to determine the pathogenicity of genetic variants with uncertain significance identified in patients with inherited cardiac arrhythmia;" "Maaike Alaerts" "Medical Genetics (MEDGEN), Medical Genetics (MEDGEN)" "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." "Chromatin structure analysis of rare likely pathogenic inherited Copy Number Variants" "Catia Attanasio" "Laboratory of Gene Regulation and Disease, Department of Human Genetics" "Chromatin structure has been shown to play important roles in the orchestration of gene expression programs during development. Spatio-temporal specific cis-regulatory sequences often lie at a long distance from the gene(s) they regulate, requiring spatial chromatin folding to move them in close proximity of their target promoters and fine-tune the time, place and level of gene expression. Interestingly, several whole-genome chromatin interaction analyses have also recently revealed the existence of topologically associated domains (TADs) which seem to act as fundamental regulatory units of the genome by delimiting the scope of action of regulatory elements in the 3D nuclear space. In other words, TADs organize the genome into regulatory islands by defining regions that interact more frequently with themselves than with the rest of the genome. Few studies have reported that the alteration of TADs by structural genomic rearrangements could disrupt the normal activity of TADs and play significant roles in human diseases. Copy-Number Variants (CNVs) are DNA sequence deletions or insertions of ≥50bp which have been shown to play important roles in human biology. The overall contribution of CNVs to human phenotypic variability and diseases is, however, still largely unclear. Few studies have now reported that deletion or duplication of DNA segments could alter gene expression regulation by disrupting, duplicating or shifting TADs boundaries causing gene(s) misexpression and diseases. Here, we propose to investigate the contribution of rare likely pathogenic CNVs into human disorders by analyzing theirs effect on chromatin architecture, and by extension on gene regulatory programs, in patients’ and controls’ derived cells. This project is part of a larger research effort that will combine our analyses with deep phenotyping, whole genome sequencing and evolutionary analyses in selected families to assess the contribution of rare likely pathogenic CNVs to phenotypic variability of developmental disorders." "Inherited cardiac arrhythmias: identification of novel genes and development of a new diagnostic tool for translating genetic diagnosis into precision medicine." "Bart Loeys" "Molecular, Cellular and Network Excitability (MCNE), Cardiovascular diseases (CARDIOVASC), Translational Pathophysiological Research (TPR), Molecular biophysics, physiology and pharmacology, Medical Genetics (MEDGEN)" "Inherited cardiac arrhythmias (ICA) are a group of predominantly autosomal dominant disorders characterized by a disturbed cardiac action potential that can lead to sudden cardiac death at a young age. Although currently more than 50 genes have been associated with ICA, in roughly 70% of the patients the precise genetic cause is still unknown. Moreover, this group of diseases is genetically and phenotypically heterogeneous and in a molecular diagnostic setting many variants of unknown pathogenic significance are detected, hampering proper risk stratification and efficient patient management. In a unique interfaculty collaboration between the Centre of Medical Genetics, the Cardiology department, the Laboratory of Experimental Hematology and Laboratory for Molecular Biophysics, Physiology and Pharmacology, we envision to address these needs in a project with two major aims: the identification of novel genes implicated in ICA and the development of a new diagnostic tool that allows functional phenotypic evaluation of the effect of genetic variants detected in ICA patients and family members. The first aim will be achieved using linkage analysis and state-of-the-art whole-genome sequencing in phenotypically well-characterized but genetically unresolved families, followed by functional characterization of the identified candidate variants. The second aim will be accomplished by the construction and electrophysiological characterization of patient-specific induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs). Focusing on the Brugada syndrome (BrS) as a proof-of-principle, iPSC-CMs will be created from fibroblasts of family members carrying an identical BrS-causing mutation but with different phenotypic expression of disease severity, and of BrS patients with a variant of unknown significance in the SCN5A gen. These powerful approaches in combination with the existing expertise in the different collaborating teams, will definitely allow accomplishing the envisioned ultimate goals of the project. As a result, a genetic diagnosis in a larger proportion of ICA families will be reached and can be translated into a personalized functional interpretation of the genetic result in patients and relatives. This will introduce the concept of precision medicine, tailoring proper risk stratification and efficient use of preventive and therapeutic measures for the individual patient." "IMAGica: an Integrative personalised Medical Approach for Genetic diseases, Inherited Cardiac Arrhythmias as a model" "Ann Nowe" "Basic (bio-) Medical Sciences, Public Health Sciences, Clinical sciences, Informatics and Applied Informatics, Electronics and Informatics" "More and more scientists are convinced that to conquer a prediction and an efficient and comprehensive diagnosis of a disease, a more integrative approach is needed in which all contextual factors are taken into account. The participating teams are already in a great deal working within this new paradigm. The aim of the proposed interdisciplinary network is to combine their efforts to study persons with inherited genetic diseases in a multi- perspective way, including clinical-physiological, genomic, psycho-social and environmental factors. The ultimate goal of the aforementioned research proposal is to enable researchers for analyzing and predicting complex inherited (and later on acquired) genetic diseases in a multi-dimensional data integrative platform. COMO-AI lab along with CMG, VUB-UZ Brussel have already developed a platform (in the Innoviris supported BridgeIRIS project) for storage and analysis of patients' genomic and clinical/phenotypic data for a cardiac arrhythmia, namely the Brugada syndrome [Sengupta et al., 2015]. The monogenic nature of this syndrome with incomplete genetic penetrance and various expression has been questioned, and more support has recently been gained for the hypothesis of a complex, oligo- over polygenic inheritance to even a multifactorial disease [Bezzina et al. 2013; Béziau et al. 2014]. Therefore, the Brugada syndrome will be an ideal model to accomplish the general aims of this project. The focus shall be laid on this inherited complex, multifactorial syndrome, since not only genetic susceptibility, but also clinical-physiological, psycho-social and environmental factors might influence the development of disease as well as the appropriate treatment. This aim will be envisioned by further enrichment of the platform towards these physiological, psycho-social and environmental dimensions. We also aim to enrich the platform with information discovery feature based on ensemble approaches (black box + white box) to analyse the multi-dimensional data and build a predictive model. The flexible architecture of the CliniPhenome database will allow easy expansion of the platform in the future towards other inherited and acquired genetic disease information and implementation of such integrative research aiming at a personalized medical approach, for i.e. cancer patients." "Understanding inherited and acquired genetic variation in Parkinson's disease through single-cell multi-omics analysis: a unique data resource." "Thierry Voet" "Translational Research in GastroIntestinal Disorders, Department of Human Genetics" "Understanding gene expression pertubation by inheritde genetic variants of GWAS PD-risk loci. Identifying nature and roles of somatic CNV's in PD-etiopathology. Functional characterisation of eQTL and somatically mutated genes" "Molecular genetic analysis of genes responsible for inherited axonal peripheral neuropathies." "Vincent Timmerman" "Peripheral Neuropathies Group" "In this project, we aim to elucidate the pathomechanisms involved in hereditary sensory neuropathies. Hereditary sensory neuropathy (HSN) is a rare variant of hereditary peripheral neuropathies, characterized by progressive sensory loss in the distal parts of the limbs. Therefore, a genotype-phenotype correlation analysis was performed in a vast HSN-cohort of the known HSN-genes (SPTLC1, RAB7, HSN2, NTRK1, NGFB and CCT5) to provide better counseling, to gain more insight in the underlying disease mechanisms and to select mutations for further functional research. The second aim of this project is to identify novel genes for HSN. This is performed by screening functional and positional candidate genes." "Prenatal Chromosomal Microarray Analysis and Identification of Genetic Variants in Congenital Diaphragmatic Hernia." "Joris Vermeesch" "Laboratory for Cytogenetics and Genome Research, Department of Human Genetics, Woman and Child" "Chromosomal microarray analysis has gradually replaced conventional karyotyping over recent years in the postnatal setting which has revolutionized whole genomescreening for genomic imbalances in patients.  We sought to evaluate the benefits and the challenges of applying chromosomal microarrays to prenatal diagnosis for referrals with abnormal ultrasound findings.  Our findings, presented in Chapter 3, demonstrate a diagnostic yieldof ~10%.  Importantly, ~3% are caused by submicroscopic CNVs whichwould go undetected by conventional karyotyping alone.  Furthermore, the higher resolution offered by chromosomal microarray analysis led to important additional information in ~4% of patients.  Of particular interest we discover a novel and unexpected advantage of arrays; a 500kb paternal insertional translocation is the likely driver of a de novo unbalanced translocation, thus improving recurrence risk calculation in this family.  Our study has, in part, paved the way for the recent Summary Guidelines for Prenatal Chromosomal Microarray Analysis and Genetic Counselling from the Belgian Society for Human Genetics [http://www.beshg.be/download/annex_2_summary_of_array_and_prenatal_guidelines_20130502.pdf].  The implementation of prenatal chromosomal microarray analysis as the first tier test in place of conventional karyotyping brings the standard of prenatal diagnosis in line with that which is provided for postnatal genetic diagnosis.Congenital diaphragmatic hernia(CDH) is a life-threatening prenatal disorder detectable by ultrasound during pregnancy.  We sought to further unravel the genetic factorsunderlying isolated CDH by the design of a custom microarray covering genomic loci recurrently associated with CDH and candidate CDH genes.  Our retrospective screen of 79 isolated CDH patients using this custom microarray is presented in Chapter 4.  This study identified a novel duplication of the EFNB1 gene in a male patient which was consideredlikely to be pathogenic.  Since our publication, a second case of a male CDH patient with duplication of EFNB1 was reported, thus reinforcing EFNB1 dosage sensitivity as a cause of isolated CDH.  In order to further identify (novel) CNVs and genes associated with isolated CDH,we undertook a prospective prenatal study using chromosomal microarrayswith genome-wide coverage in 75 foetuses with isolated CDH, which is presented in Chapter 4.  This study revealed submicroscopic de novo pathogenic CNVs in 9.3% and rare inherited variants which may be involvedin CDH in a further 4% of foetuses.  This diagnostic yield is significantly higher than the ~3% rate of pathogenic submicroscopic CNVs which we observed in our prenatal study using the same microarray platform.  Isolated CDH thus represents a valid cohort for CNV screening in the prenatal phase, and suggests that the clinical utility of conventional karyotyping is questionable for this group of patients.  This study allowed us to further refine the critical region at 15q26 to only 2 genes, pinpointing NR2F2 as the causal gene.  We add further evidence for the 15q25.2 and 16p11.2 recurrent microdeletions as CDH loci, andwe identify novel CNVs not previously observed in association with CDH,including a duplication of 4p15.2-p14.We next evaluated the use of exome sequencing for the investigation of isolated CDH and non-isolated CDH where a genetic cause was suspected.  Our results show that exome sequencing represents an effective technique with which to investigate familial CDH, described in Chapter 5.  In the first family studied, we identified a nonsense mutation in ZFPM2 in 2 individuals with isolated CDH, as well as a sibling with a congenital heart defect.  Surprisingly, the mutation was shown to be transmitted from the unaffected mother, and is also carried by the maternal grandfather and the maternalsister, both of whom are also asymptomatic.  This intriguing finding highlights the complexity of CDH, reinforcing the involvement of additional as yet unidentified (epi)genetic factors in CDH penetrance. In a second family with 2 male foetuses with MCA, we identify a mutation in the X-linked PORCN gene inherited from an unaffected mother who wasshown to have extreme skewing of X-inactivation.  This further implicates Wnt signaling as playing a role in CDH, as well as multiple aspects of foetal development.  In a third consanguineous family we identify a mutation in PIGN in a foetus with MCA, inherited from carrier parents.  PIGN is involved in GPI anchor synthesis and our finding adds to a growing body of evidence that defective GPI anchor synthesis causes multiple phenotypes in humans.Given the variation in severity ofherniation and thus pulmonary hypoplasia, as well as differences in responses to foetal and / or neonatal therapy for CDH patients, we sought to explore whether gene expression analysis of amniotic fluid cells from CDH foetuses could identify dysregulated genes and biological pathways which may act as predictive biomarkers.  In this exploratory study we applied RNA-sequencing to investigate gene expression in cultured cells sourced from amniotic fluid of isolated CDH patients, described in Chapter 6.  This analysis identifies 2 potential molecular subtypes ofisolated CDH, one of which is characterized by downregulation of TGFB1 and CTGF, and upregulation of TNF, IL6 and IL8.  This highlights downregulation of TGFB signalling as a likely cause of much of of the downstream dysregulation in gene expression observed, including that of CTGFwhich has been previously implicated in the nitrofen rodent model of CDH.  Furthermore, this group of patients shows an apparent inflammatory response indicated by the upregulation of TNF, IL6 and IL8 which mayin turn exacerbate postnatal pulmonary hypertension.  These findings direct future targeted studies in a larger cohort of isolated CDH patients to determine the clinical significance and therapeutic potential." "New perspectives on familial clustering of Alzheimer's disease: the role of rare genetic risk variants." "Kristel Sleegers" "VIB CMN - Neurodegenerative Brain Diseases Group" "Alzheimer's disease (AD) is an important threat to both personal and publich health. Familial clustering of AD has long been known, and in the past few decades, important progress has been made in unraveling the genetics of AD. For patients and their relatives today, the most tangible benefit of this progress is the ability to genetically diagnose or predict the disease. Nevertheless, this only applies to a minority of families, in which a mutation in APP, PSEN1 or PSEN2 has been identified that causes a rare autosomal dominant form of AD. For the majority of patients, the disease has a complex genetic background, which cannot be readily translated into accurate risk prediction. Remarkable in this light is our recent observation that rare predicted loss-of-function mutations in the AD risk gene ABCA7 are associated with autosomal dominant pattern of inheritance of AD. Multiplex AD families segregating such rare variants represent a distinct subgroup in which some of the benefits of genetic testing may hold true.The key aim of this PhD project is to get a better understanding of the role of these rare risk variants in familial clustering of AD, with the ultimate goal to assess if and how the knowledge of carrier status of such a variant can be used in genetic risk prediction. A variety of state-of-the-art approaches will be used to address this aim. Molecular characterization of rare variants through RNA sequencing analysis of lymphoblast cell lines and brain will contribute to a better understanding of the impact of the mutations, as well as a delineation of neutral and pathogenic variants. Genetic-epidemiological characterization through DNA sequencing on additional cohorts will shed further light on the frequency and penetrance of these mutations. The mode of action and causes of phenotypic heterogeneity will be investigated in derivatives of induced pluripotent stem cells of mutation carriers. Finally, this patient-derived model will be used to investigate the potential of compounds to modify the effect of the mutation, eventually enabling targeted treatment.This PhD project covers an uncharted but promising territory in the battle against AD, particularly considering the increasing focus in this battle on patient stratification, early detection and precision medicine." "Development of a novel transgenic zebrafish model to determine the pathogenicity of genetic variants for cardiac arrhythmia." "Dorien Schepers" "Molecular, Cellular and Network Excitability (MCNE), Medical Genetics (MEDGEN)" "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."