RETA LILA WESTON INSTITUTE OF NEUROLOGICAL STUDIES INSTITUTE OF NEUROLOGY UNIVERSITY COLLEGE LONDON DEFINING THE GENETIC BASIS OF THREE HEREDITARY NEUROLOGICAL CONDITIONS IN FAMILIES FROM THE INDIAN SUBCONTINENT Dr Vafa Alakbarzade PhD Thesis 2016 1 DEFINING THE GENETIC BASIS OF THREE HEREDITARY NEUROLOGICAL CONDITIONS IN FAMILIES FROM THE INDIAN SUBCONTINENT Submitted by Dr Vafa Alakbarzade, MBBS, MRCP (UK), MSc University College London Student Number: 1028294 to University College London as a thesis for the degree of Doctor of Philosophy, January 2016 This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement I confirm that the work presented in this thesis is my own and information derived from other sources has been indicated in the thesis (Signature) …………………………………………………… 2 ACKNOWLEDGEMENTS Foremost I would like to thank the families who took part in these studies. I am sincerely grateful to Professor Tom Warner and Professor Andrew Crosby, without whom I would never have had all the wonderful experiences this PhD brought me. They have always supported and encouraged me in whatever scientific endeavours I have followed. Dr. Barry Chioza and Dr. Sreekantan-Nair Ajith provided invaluable support and advice throughout my PhD; I am hugely appreciative of their guidance and encouragement. None of the work in this thesis would have been possible without guidance of Dr. Barry Chioza. I would specifically like to appreciate contribution of the team of Prof. David Silver and Dr. Kulkarni Abhijit who provided functional follow up of our genetic findings and Dr. Iype Thomas and Dr. Arshia Ahmad who provided clinical information of the families under investigation. David Newman has shaped out my thesis for which I am deeply grateful for. I would also like to thank all the members of the Crosby group for their help. On a personal level I would like to sincerely thank my beloved mother who has always been there for me. 3 ABSTRACT Neurogenetic studies have revolutionised our understanding of the genetic and molecular basis of inherited neurological disorders, primarily as a result of the identification of single disease-causing genes. The incidence of such disorders is increased amongst populations with common shared ancestry or a high rate of consanguinity. Hence, the investigation of inherited neurological conditions in genetic isolates provides a robust opportunity to define the molecular pathogenic basis of these conditions. Neurological and neurodevelopmental disorders present important public health issues in the developing countries in the Indian subcontinent. The global burden of these disorders is worsened by the lack of targeted research funding and relevant in-country research capacity. This project, undertaken as part of a wider research study investigating inherited disorders in the Indian subcontinent, aimed to define the molecular genetic bases of three extended families with distinct neurological and neurodevelopmental disorders. In the first family with multiple individuals affected by a severe autosomal recessive form of neurodevelopmental delay with microcephaly, genetic studies identified mutation in a gene (MFSD2A), not previously associated with inherited disease, which led to a reduction of fatty acid transportation in patients homozygous for the disease-causing mutation. In the second family, genotyping identified a complex chromosomal rearrangement associated with diverse clinical outcomes including Wolf Hirschorn-, 3p deletion-, and 4p duplication syndrome, among ten 4 chromosomally-imbalanced affected individuals. In the third family, a duplication event on chromosome 15q24 encompassing the LINGO1 gene was identified as a likely cause of dystonic tremor in affected individuals. Together these molecular discoveries provide fundamentally important biological insight into the pathogenic basis of abnormal brain growth and control of movement with the potential diagnostic and treatment applications. 5 CONTENTS 1 GENERAL INTRODUCTION ................................................................ 15 1.1 Abnormal brain development: embryological aspects, definitions and underlying causes ............................................................................. 15 1.1.1 Structural development of the brain ................................................... 15 1.1.2 Microcephaly ..................................................................................... 22 1.2 Neurodevelopmental disorders and congenital malformations: definitions .......................................................................................... 28 1.3 Dystonic tremor ................................................................................. 34 1.3.1 Definitions, classifications and misdiagnosis ..................................... 34 1.3.2 Pathophysiology ................................................................................ 39 1.3.3 Autosomal dominant dystonic tremors .............................................. 41 1.4 Basic principles of neurogenetic studies ........................................... 45 1.4.1 Analysis of structural chromosome variants ...................................... 45 1.4.1.a Structural chromosome variants ............................................................................... 45 1.4.1.b Genetic characterisation of structural variation ......................................................... 50 1.4.2 Systematic elucidation of single gene disorders ................................ 57 1.4.2.a Single gene disorders ................................................................................................ 57 1.4.2.b Linkage mapping and whole exome sequencing ...................................................... 58 1.5 Applying Genomic Technologies to address Clinical Challenges in the Indian subcontinent study .................................................................. 63 1.5.1 Indian subpopulations and founder mutations ................................... 63 1.5.2 ‘Applying Genomic Technologies to address Clinical Challenges in the Indian Subcontinent’ (AGTC-India) .................................................... 66 1.6 Aims .................................................................................................. 68 2 METHODS AND MATERIALS ............................................................. 70 2.1 Subjects and samples ....................................................................... 70 2.2 Molecular methods ............................................................................ 72 2.2.1 RNA and DNA extraction ................................................................... 72 2.2.2 Reverse transcriptase PCR reaction ................................................. 72 2.2.3 Quantitative real-time PCR ................................................................ 73 2.2.4 Genotyping, linkage & CNV analysis ................................................. 74 2.2.5 Whole exome sequencing data analysis ........................................... 76 2.2.6 Primer design .................................................................................... 77 2.2.7 Polymerase chain reaction (PCR) ..................................................... 77 2.2.1 Cycling conditions for PCR ................................................................ 78 2.2.2 Agarose gel electrophoresis .............................................................. 80 6 2.2.3 PCR purification ................................................................................ 80 2.2.4 DNA sequencing and analysis ........................................................... 81 2.2.5 Co-segregation analysis .................................................................... 82 2.2.6 Genomic library preparation .............................................................. 84 2.2.7 MFSD2A p.Ser339Leu mutation analysis .......................................... 84 3 A PARTIALLY INACTIVATING MUTATION IN THE SODIUM- DEPENDENT LYSOPHOSPHATIDYLCHOLINE TRANSPORTER MFSD2A CAUSES A NON-LETHAL MICROCEPHALY SYNDROME 86 3.1 Introduction ........................................................................................ 86 3.2 Results .............................................................................................. 93 3.2.1 Clinical features ................................................................................. 93 3.2.2 330K SNP analysis ............................................................................ 99 3.2.3 Genotyping and whole exome sequencing data analysis ................ 101 3.2.4 MFSD2A 40433304C>T variant ...................................................... 105 3.2.5 p.Ser339Leu functional outcomes ................................................... 108 3.3 Discussion and future work ............................................................. 113 4 A COMPLEX CHROMOSOMAL REARRANGEMENT IN AN INDIAN FAMILY WITH NEURODEVELOPMENTAL DELAY ......................... 121 4.1 Introduction ...................................................................................... 121 4.2 Results ............................................................................................ 124 4.2.1 Clinical report .................................................................................. 124 4.2.2 Microarray and fluorescence in situ hybridization (FISH) analysis .. 133 4.3 Discussion ......................................................................................