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Investigating Vitamin B6-Dependent Epileptic Encephalopathies in Human Patients and a Mouse Model

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Investigating Vitamin B6-Dependent Epileptic Encephalopathies in Human Patients and a Mouse Model INVESTIGATING VITAMIN B6-DEPENDENT EPILEPTIC ENCEPHALOPATHIES IN HUMAN PATIENTS AND A MOUSE MODEL by Hilal Hamed Al Shekaili BSc, Sultan Qaboos University, 2008 MSc, Sultan Qaboos University, 2011 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2020 © Hilal Hamed Al Shekaili, 2020 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Investigating Vitamin B6-Dependent Epileptic Encephalopathies in Human Patients and a Mouse Model submitted by Hilal Al Shekaili in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Medical Genetics Examining Committee: Dr. Jan M. Friedman, Department of Medical Genetics Supervisor Dr. Blair R. Leavitt, Department of Medical Genetics Co-supervisor Dr. Lorne Clarke, Department of Medical Genetics Supervisory Committee Member Dr. Sylvia Stockler, Department of Pediatrics University Examiner Dr. Daniel Goldowitz, Department of Medical Genetics University Examiner Additional Supervisory Committee Members: Dr. William T. Gibson, Department of Medical Genetics Supervisory Committee Member Dr. Clara van Karnebeek, Department of Pediatrics Supervisory Committee Member ii Abstract PLPHP deficiency is a recently discovered form of vitamin B6-dependent epilepsies (B6Es) that is caused by recessive mutations in PLPBP. PLPHP is involved in pyridoxal 5’-phosphate (PLP) homeostatic regulation. However, the mechanism by which PLPHP dysfunction disrupts PLP homeostasis and leads to the observed encephalopathy in patients was still elusive. We characterized the clinical, genomic and biochemical abnormalities in a new series of 12 PLPHP deficiency patients. Our results identified previously undescribed clinical features of PLPHP deficiency, including non-epileptic movement disorder, fatal mitochondrial encephalopathy and folinic acid-responsive seizures. We characterized the pathogenicity of patients’ PLPBP variants using in silico tools and 3D modelling of PLPHP and developed a system of clinical severity score. We generated and characterized PLPBP knockout models in HEK293 cells, yeast and zebrafish. Our plpbp-KO zebrafish model replicated the clinical phenotype of PLPHP-deficient patients by showing vitamin B6-dependent seizures and death in untreated KO larvae. Consistent with the biochemical picture in patients, Plphp-deficient fish displayed decreased systemic levels of PLP. In the future this model can be utilized as a tool for investigating the disease pathophysiology, drug screening and identifying diagnostic biomarkers. Pyridoxine-dependent epilepsy (PDE- ALDH7A1) is another form of B6Es that is caused by mutations in ALDH7A1, a gene which encodes an enzyme within the lysine catabolism pathway. We have successfully generated and characterized transgenic mouse strain with constitutive genetic ablation of Aldh7a1. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid (PIP), similar to the biochemical picture in ALDH7A1-defiecint patients. KO mice fed the regular diet (0.9% lysine) did not exhibit seizures based on EEG analysis. When KO mice are iii switched to a diet containing higher amount of lysine (4.7%), they developed severe recurrent seizures which led to their quick death. In analogy to the patients’ picture also, treating KO mice under high lysine diet with pyridoxine injections prevented seizures and prolonged their survival. This study provides a proof-of-concept for the utility of the model to study PDE-ALDH7A1 biochemistry and to test new therapeutics. iv Lay Summary Vitamin B6-dependent epilepsies (B6Es) are rare genetic diseases that cause frequent seizures that are resistant to conventional drugs but are well-controlled by administration of vitamin B6 (vitB6). We investigated two types of B6Es. The first one is known as PLPHP deficiency in which we studied 12 new patients and characterized their clinical, genetic and biochemical abnormalities. Our results showed vitB6 deficiency in patient cells. The zebrafish model that we developed for this disease replicated the patient symptoms and therefore can be used for future investigations and drug screening. The other form of B6Es that we studied is known as ALDH7A1 deficiency. We generated and characterized a mouse model for this disease. Results showed that the model reproduced the patients’ clinical and biochemical features including accumulation of chemical compounds in the brain and seizures. This model can be used as a tool to study the disease biology and test new treatments. v Preface All of the work performed by the candidate was conducted at the Center for Molecular Medicine and Therapeutics (CMMT) and the British Columbia Children’s Hospital Research Institute at the University of British Columbia, Children’s and Women’s Hospital campus. Studies in human patients were approved by the Clinical Research Ethics Board of the British Columbia Children's and Women's Hospital, University of British Columbia (certificate # H12-00067). Ethical approval for studies in mice was obtained from the University of British Columbia’s Animal Care Committee (Animal Protocols # A15-0200 and A15-0180). Chapter 2: A version of this chapter has been published as Johnstone, D. L.*, Al-Shekaili, H. H.*, Maja Tarailo-Graovac, Nicole I. Wolf, Autumn S. Ivy, Scott Demarest, Yann Roussel, Jolita Ciapaite, Carlo W.T. van Roermund, Kristin D. Kernohan, Ceres Kosuta, Kevin Ban, Yoko Ito, Skye McBride, Khalid Al-Thihli, Rana A. Abdelrahim, Roshan Koul, Amna Al Futaisi, Charlotte A. Haaxma, Heather Olson, Laufey Yr. Sigurdardottir, Georgianne L. Arnold, Erica H. Gerkes, M. Boon, M. Rebecca Heiner-Fokkema, Sandra Noble, Marjolein Bosma, Judith Jans, David A. Koolen, Erik-Jan Kamsteeg, Britt Drögemöller, Colin J. Ross, Jacek Majewski, Megan T. Cho, Amber Begtrup, Wyeth W. Wasserman, Tuan Bui, Elise Brimble, Sara Violante, Sander M. Houten, Ron A. Wevers, Martijn van Faassen, Ido P. Kema, Nathalie Lepage, Care4Rare Canada Consortium, Matthew A. Lines, David A. Dyment, Ronald J.A. Wanders, Nanda Verhoeven-Duif, Marc Ekker, Kym M. Boycott, Jan M. Friedman, Izabella A. Pena, and Clara D.M. van Karnebeek. (2019). PLPHP deficiency: Clinical, genetic, biochemical, and mechanistic insights. Brain: A Journal of Neurology, 142(3), 542–559. (* joint first authors). I performed recruitment of index patients (Patients 1, 2, 8 and 9), SNP homozygosity mapping, analysis of exome candidate variant list and discovery of the causative gene (PLPBP, novel at the vi time of discovery). Bioinformatics analysis of exome data and generation of the candidate variant lists for Patients 1 and 2 were performed by M. Tarailo-Graovac. Sanger validation of exome variants and SNP microarray experiment in Patients 1, 2, 8 and 9 was performed by C. Ross and B. Drögemöller. Patient 5 was recruited by M. Lines and D. Dyment. Remaining patients were recruited by C. van Karnebeek. Clinical patient descriptions were originally written by their referring clinicians (K. Al-Thihli and R. Abdelrahim for Patients 1, 2, 8 and 9; E. Gerkes and M. Boon for Patient 3; C. Haaxma and E. Kamsteeg for Patient 4; M. Lines and D. Dyment for Patient 5; H. Olson for Patient 6; A. Ivy for Patient 7; S. Demarest for Patients 10 and 11; L. Sigurdardottir and G. Arnold for Patient 12). I assembled the patient clinical reports, structured them for the manuscript and filled in missing patients’ clinical and biochemical data by discussing them with the collaborative clinicians. I performed PLPBP variant annotations and in silico variant effect predictions (SIFT, Polyphen2, MutationTaster, MutationAssessor, FATHMM MKL, PROVEAN) for all patients. The genotype-phenotype correlation based on variant location relative to PLP binding pocket in PLPHP protein was first discovered by me. Clinical severity score assessment and related methods section and Table 4 were prepared by D. Johnstone. Analysis of patients’ MRIs and related Figure 1 and Table 2 were prepared by N. Wolf. Table 1, Table 3 (with some contribution from D. Johnstone and I. Pena) and Figure 2A were prepared by me. Results sections 2.3.1 and 2.3.3 were jointly written by me and D. Johnstone. Results section 2.3.4 was written by me with contribution from D. Johnstone. Structural modelling of human PLPHP and related methods and figures in the manuscript (Figure 2B, C, D and Figure 4) were prepared by I. Pena. Protein analysis in Patient 5 fibroblasts and its related methods and immunoblot figure were prepared by D. Johnstone. Mitochondrial localization of PLPHP in HeLa cells and related methods section and figures were prepared by S. Houten. PLPBP studies in yeast and HEK293 cells and vii related methods section and figures were prepared by C. van Roermund and R. Wanders. Seahorse assay in Patient’s 5 fibroblasts was performed by D. Johnstone. Generation and characterization of plpbp-KO zebrafish and related sections and figures in the manuscript were prepared by I. Pena and D. Johnstone. Vitamin B6 vitamer analysis in patient samples, PLPHP-deficient HEK293 cells and plpbp-KO zebrafish and related methods section were prepared by J. Ciapaite, M. Bosma and N. Verhoeven-Duif. Introduction and Discussion sections were written
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