Identification of the sphingolipid desaturase DEGS1 as a novel gene for a leukodystrophy with therapeutic hope Devesh Chandra Pant TESI DOCTORAL UPF / 2018 DIRECTOR DE LA TESI Dr. Aurora Pujol Onofre and Dr. Stéphane Fourcade DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD UNIVERSIDAD POMPEU FABRA [Pàgina en blanc] ii Dedicated to my parents… iii [Pàgina en blanc] Acknowledgement I wish to express my sincere gratitude to my supervisors, Professor Aurora Pujol and Dr. Stéphane Fourcade for their support, critical thinking and guidance throughout my PhD. I am deeply indebted to the patients and their families for their participation in the study. I would also like to thank the current and past members of Neurometabolic disease group, IDIBELL, the members of Developmental Biology group, UPF and ZeClinics, PRBB for their help, advice, valuable suggestions during my PhD. I also thank all our collaborators for their essential contribution to my research. I would also like to acknowledge the grants that supported this work. A special thanks to my precious family members, for encouraging my curiosity and instilling determination and perseverance in me. Thank you for giving me confidence and believing in me. I am forever grateful to all of you, and I could not have completed this PhD without you. I also thank all my friends who provided moral support and motivation during my stay in Barcelona. I thank you all… v “All that we are is the result of what we have thought. The mind is everything. What we think we become.” (Gautama Buddha, 563-483 B.C.) vii [Pàgina en blanc] viii Abstract In spite of recent advances in understanding the genetic bases of leukodystrophies, a large number of clinical cases remain unexplained, suggesting that many leukodystrophy- associated genes have yet to be identified. Here we report 18 patients from 12 families with biallelic deleterious variants in the DEGS1 gene identified via WES. DEGS1 encodes an enzyme which catalyzes the conversion of dihydroceramide to ceramide. Common features among the patients include severe cerebellum atrophy, thinning of the corpus callosum and hypomyelination suggesting a critical role of DEGS1 in the central nervous system. Using patient’s fibroblasts, we evidenced abnormal biochemical profiles. Knockdown of degs1 in zebrafish recapitulated the biochemical imbalance, showed impaired locomotor abilities and weak myelination. Moreover, a widely used drug for neurological disorders, fingolimod, able to normalized the toxic effects associated due to impaired DEGS1. These results pave the way to clinical translation, illustrating the transformative impact of genomics in patient care. ix ] Resum El hecho de que un gran número de casos clínicos de leucodistrofias sigan sin explicar sugiere que muchos de los genes asociados a esta enfermedad estarían aún por identificar. Secuenciando el exoma completo de 18 pacientes de 12 familias hemos encontrado variantes deletéreas bialélicas en el gen DEGS1, un gen que codifica para una enzima que cataliza la conversión de dihidroceramida a ceramida. Las características comunes entre estos pacientes incluyen atrofia grave del cerebelo, delgadez del cuerpo calloso e hipomielinización, sugiriendo un papel crítico de la proteína DEGS1 en el SNC. Sus fibroblastos envidencian un perfil bioquímico anormal. Hemos comprobado que un modelo de knockdown del gen degs1 en pez cebra muestra capacidades locomotoras alteradas, desequilibrio bioquímico y una mielinización débil. Hemos conseguido normalizar sus efectos tóxicos con fingolimod, un fármaco ampliamente usado para trastornos neurológicos. Son resultados que ilustran el impacto transformador de la genómica en la atención al paciente, abriendo un nuevo camino en la clínica traslacional. Preface Leukodystrophies are heritable myelin disorders affecting white matter of the central nervous system (CNS). All leukodystrophies result in destruction (demyelination) or failed development (dysmyelination) of myelin leads to a large clinical spectrum from rapidly fatal early infantile to slow late adult forms. There are about 80 genes responsible for leukodystrophies so far suggesting that novel forms still need to be characterized. Genetic research is evolving rapidly, with technologies like Next Generation Sequencing (NGS) generating vast amounts of genetic data. Part I of the thesis focusses on the genetic diagnosis of patients with leukodystrophy. Genome-wide association studies (GWAS) have been instrumental to link genes and pathways to the etiology of leukodystrophy. The identification of the novel disease gene, DEGS1, associated with early-onset hypomyelinating leukodystrophy was more than routine and required a collaborative effort of neurologists and medical geneticists from Spain, France, Canada and the United States we collected 18 patients from 12 families with a DEGS1 variant. Standardized phenotypic data were collected by review of the clinical histories and follow-up investigations. Affected individuals were examined by experienced neurologists at their primary care centers, and all available clinical and magnetic resonance image (MRI) data were collected and jointly reviewed. DEGS1 was previously not known to be associated with neurological disorders. However, understanding the effects of the variants identified remains a major problem in diseases. Bringing this research “from bench to bedside” requires intensive effort in translational research studies. In Part II, by taking advantage of human patient fibroblasts, we performed functional validation of a candidate gene DEGS1. To confirm the genetic analyses, we found abnormal biochemical measurements in patients fibroblasts which were explained by the altered lipidomics profile characterized by high dihydroceramide (DhCer) and low ceramide (Cer) in four affected individuals. Additionally, altered mRNA levels in patient fibroblasts further confirmed this. These results indicate loss of DEGS1 activity in the patients. We also found an increase in intracellular ROS levels in patients’ fibroblasts, which was rescued upon treatment with fingolimod, ceramide synthase xi inhibitor. However, we found exogenous long-chain DhCer increases the ROS levels in control fibroblasts suggesting the toxic effects of DhCer. Moreover, we found low mitochondria membrane potential in one patient fibroblasts and impaired autophagy in two patients fibroblasts. Determination of the pathogenicity of variants identified through screening of mutation has allowed clinicians to define DEGS1 related pathogenicity. In part III, a degs1 knockdown (KD) zebrafish model for the neurological disorder associated with DEGS1 was generated. A systemic knockout of Degs1 in mice ultimately failed to thrive, dying within 8 to 10 weeks of birth and fruitfly is believed to be embryonic lethal. In order to generate the degs1 KD zebrafish, antisense morpholino was used. Morpholino-mediated knockdown in a zebrafish model demonstrates that loss of the evolutionarily highly conserved DEGS1 alters lipidomics profiles, causes movement abnormalities and reduced number of mature oligodendrocytes. They developed signs of neuropathology at 4.5 days of age making this model suitable for therapeutic studies. Preliminary data suggests this gene is highly expressed in CNS. Intensive characterization of the degs1 zebrafish model found a resemblance to the human phenotype in several facets. The defects observed in the zebrafish model were rescued upon treatment with FTY720. Thus, the overlapping phenotype of degs1 knockdown zebrafish increases the potential impact of therapeutic studies. Collectively, the findings of this doctoral thesis show the utility of implementing genomic diagnosis in the clinic, with respect to providing simple and effective treatments in a timely manner to improve outcomes for patients with rare inborn errors of metabolism. xii [Pàgina en blanc] Table of Contents Abbreviations .............................................................................................................. xvii 1 INTRODUCTION ....................................................................................................... 3 1.2 Leukodystrophies.................................................................................................. 3 1.1.1 Overview ......................................................................................................... 3 1.1.2 MRI Pattern Recognition and Leukodystrophies ............................................ 5 1.1.3 Genetics and Leukodystrophies ....................................................................... 6 1.2 Technologies for Gene Identification .................................................................. 7 1.2.1 Next Generation Sequencing ........................................................................... 7 1.2.2 Whole Exome Sequencing............................................................................... 9 1.2.3 Whole Genome sequencing ........................................................................... 12 1.2.4 Human Gene Variant Databases and Interpretation ...................................... 13 1.3 Sphingolipids ....................................................................................................... 18 1.3.1 Introduction ................................................................................................... 18 1.3.2 Metabolism .................................................................................................... 21 1.4 Bioactive Sphingolipids .....................................................................................