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Cellular and Molecular Investigations of Undiagnosed Neurometabolic

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Cellular and Molecular Investigations of Undiagnosed Neurometabolic CELLULAR AND MOLECULAR INVESTIGATIONS OF UNDIAGNOSED NEUROMETABOLIC DISORDERS Submitted in application for award of Doctor of Philosophy (PhD) Emma Reid Centre for Translational Omics Genetics and Genomic Medicine Institute of Child Health University College London August 2016 DECLARATION I, Emma Reid, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Where experimental or analytical work has been completed by others, this has been stated in the relevant section of this thesis. However, these instances are also listed below: • Alignment of sequencing reads to the reference genome, variant calling (Section 2.4.11) and ExomeDepth analysis (Section 3.3.3) of the gene panel sequencing data was performed by Dr Chris Boustred (NE Thames Regional Genetics Service, GOSH, UK). • Whole exome sequencing of patients X, 1, 4 and 5 was outsourced to BGI Genomics Hong Kong and the resulting raw data files were processed and aligned by Dr Chela James (GOSgene, ICH, UK) (Section 2.5). • Whole exome sequencing of patient Y and processing of the resulting raw data was performed by Dr Olaf Bodamer (University of Miami, USA) (Section 6.4.1). • Homozygosity mapping and whole exome sequencing of index PROSC family was performed by Dr Niklas Darin (The Queen Silvia Children’s Hospital, Sweden) (Section 7.1.4). • Sanger sequencing of additional PROSC and PNPO-deficient patients was performed by Dr Philippa Mills (ICH, UK) (Section 7.2). • Sample processing for electron microscopy analysis was carried out by Elizabeth Latimer- Bowman (Histopathology Department, GOSH, UK) and imaging was performed by Glenn Anderson (Histopathology Department, GOSH, UK) (Section 2.14.2). • Immunofluorescence staining of α-dystroglycan in a muscle sample from patient 23 was performed by the Dubowitz Neuromuscular Centre, ICH, UK (Section 3.3.6.1). • Amino acid analyses of plasma, urine and CSF listed throughout Chapters4,5 and6 were performed on a clinical basis by the Chemical Pathology Department, GOSH, UK. London, August 2016 Emma Sarah Reid 2 ABSTRACT Inborn errors of metabolism (IEM) affect 1 in 500 newborns causing significant disease-burden and mortality throughout childhood. However, despite extensive genetic and biochemical investigations the cause of disease remains unknown in up to 50% of patients with neurological symptoms; so-called neurometabolic disorders (NMD). The overarching aim of this thesis was to determine the cellular and molecular aetiologies for the clinical phenotypes seen in patients with undiagnosed NMD. In order to improve the diagnosis of these disorders in clinical practice, a comprehensive targeted gene panel of 614 genes known to cause IEM was designed and a cohort of 44 patients was analysed. A definitive or probable genetic diagnosis was achieved in 53% of patients without a prior genetic diagnosis. Method optimisation and validation, comparison to other diagnostic strategies and the advantages and disadvantages of targeted sequencing are reviewed. Case reports, novel mutations/phenotypes and their contribution to the expansion of the literature are described. Whole exome sequencing and functional characterisation was also undertaken for patients who had been extensively clinically investigated previously. Five patients identified with mutations in the mitochondrial glutamate transporter, SLC25A22, presenting with novel biochemical phenotypes are described and novel transporter functions are postulated. One patient diagnosed with a potassium channelopathy with biochemical abnormalities and anticonvulsant responses suggestive of an inborn error of vitmain B6 metabolism is documented and the mechanisms underlying the generalised anticonvulsant effects of vitamin B6 are postulated. Characterisation of a possible novel inborn error of lysine metabolism in a patient presenting with hyperlysinaemia and motor neuron disease is also discussed. These studies also demonstrate the complexity of unravelling the relationship between genotype and phenotype and highlight the need for novel functional assays to assess the pathogenicity of sequence variants. Mass spectrometry-based assays were developed to enable characterisation of disorders affecting vitamin B6 homeostasis, including pyridox(am)ine 5’-phosphate oxidase (PNPO), antiquitin and PROSC deficiency, the latter being a novel disorder. The differences between pyridoxine- and pyridoxal phosphate-responsive PNPO deficiency and fibroblast vitamer profiles in all patients were all investigated. Finally, multiple methodologies were employed with the aim of understanding the biological function of PROSC. 3 PUBLICATIONS Some ideas, figures and methodologies have appeared previously in the following publications: 1. Reid ES, Williams H, Stabej PL, James C, Ocaka L, Bacchelli C, Footitt EJ, Boyd S, Cleary MA, Mills PB, Clayton PT (2015) Seizures Due to a KCNQ2 Mutation: Treatment with Vitamin B6. JIMD Reports Oct 8. [epub ahead of print] 2. Oppici E, Fargue S, Reid ES, Mills PB, Clayton PT, Danpure CJ, Cellini B (2015) Pyridoxamine and pyridoxal are more effective than pyridoxine in rescuing folding-defective variants of human alanine:glyoxylate aminotransferase causing primary hyperoxaluria type I. Human Molecular Genetics 24(19):5500-11. 3. Reid ES, Papandreou A, Drury S, Boustred C, Yue WW, Wedatilake Y, Beesley C, Jacques TS, Anderson G, Abulhoul L, Broomfield A, Cleary M, Grunewald S, Varadkar SM, Lench N, Rahman S, Gissen P, Clayton PT, Mills PB (2016) Advantages and pitfalls of an extended gene panel for investigating complex neurometabolic phenotypes. Brain [in press]. 4 ACKNOWLEDGEMENTS Above all I would like to thank the patients, carers, relatives and other individuals who donated samples and their time, freely and without necessarily expecting benefit to themselves. I am very grateful to the organisations that funded this work: Great Ormond Street Hospital Children’s Charity and UCL Impact. I am indebted to my supervisors: Philippa Mills for her continual support both inside and outside the lab, not only helping me to grow as a scientist, but also in confidence as a person. Without her this work would not have been possible and I am very grateful to have had the opportunity to work in her lab. Paul Gissen I must thank for always playing the devil’s advocate and pushing me to aim higher than I thought was possible. I also owe a great deal to Peter Clayton for sharing merely a slice of his brilliance with me whilst never failing to bamboozle me with hand-drawn biochemistry. There are many other people that I have met during the course of my PhD who have been of enormous help to me. Firstly, I would like to thank Nick Lench for giving me the opportunity to learn in such a unique diagnostic laboratory, along with Suzie Drury and Chris Boustred for helping to make our panel a reality and always having time for my never-ending questions. I also thank the members of GOSgene for their generous collaboration and assistance with interpretation of the whole exome sequencing data. Thanks to Kevin Mills for introducing me to the wonders (and woes!) of mass spectrometry. I am also eternally grateful to the GOSH Metabolic Team for their help recruiting patients, enthusiasm in the project and for rekindling my passion for medicine. Many, many thanks to my colleagues and friends at the Institute of Child Health and particularly within the Biological Mass Spectrometry Centre, for their readiness to help and making each day in the lab a fun day. Last but not least, I thank my family for their continual support and encouragement, and Malika for keeping me chained to that desk, no matter how much I protested! 5 CONTENTS 1 introduction 24 1.1 Inborn errors of metabolism (IEM) . 24 1.1.1 First description of IEM . 24 1.1.2 Pathogenic mechanisms underlying IEM . 25 1.1.3 Classes of IEM . 26 1.1.4 The importance of early diagnosis . 32 1.2 Neurometabolic disorders (NMD) . 36 1.3 Current methods for the diagnosis of IEM/NMD . 37 1.3.1 Profiling of analytes in biofluids . 37 1.3.2 Specialised biochemical tests . 40 1.3.3 Single-gene Sanger sequencing . 42 1.3.4 Gene panels targeting multiple disease genes . 43 1.3.5 Whole exome (WES) and genome (WGS) sequencing . 47 1.4 The importance of functional confirmation and characterisation of identified variants . 49 1.5 Aims of this thesis . 50 2 materials and methods 51 2.1 Materials . 51 2.2 Ethics statement . 54 2.3 DNA extraction and Sanger sequencing . 54 2.3.0.1 Automated DNA extraction . 54 2.3.0.2 Manual DNA extraction . 54 2.3.1 Primer design . 54 2.3.2 Amplification of target genes from genomic DNA using Polymerase Chain Reaction (PCR) . 55 2.3.2.1 PCR conditions . 55 2.3.2.2 Visualisation of PCR products by agarose gel electrophoresis . 56 2.3.3 Sanger sequencing . 57 2.3.3.1 Purification of PCR products using ExoSAP . 57 2.3.3.2 Sanger sequencing . 57 2.3.3.3 DNA precipitation . 57 6 2.4 Gene panel sequencing . 58 2.4.1 Qubit quantitation . 58 2.4.2 Target capture . 58 2.4.3 Restriction digestion validation . 59 2.4.4 Target enrichment and sample indexing . 60 2.4.5 Target DNA capture, ligation and elution . 60 2.4.6 PCR amplification of target libraries . 61 2.4.7 Purification of target libraries . 62 2.4.8 Validation of DNA enrichment . 62 2.4.9 Library preparation for sequencing . 63 2.4.10 Next-generation sequencing . 63 2.4.11 Data analysis . 64 2.5 Whole exome sequencing (WES) . 64 2.6 Biological interpretation using bioinformatics tools . 65 2.7 Cell culture . 65 2.7.1 Fibroblast cell culture conditions . 65 2.7.2 Counting cells . 66 2.7.3 Fibroblast cell storage . 66 2.7.4 Protein assay . 66 2.8 cDNA analysis . 67 2.8.1 Isolation and purification of total RNA from fibroblasts .
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