AN INVESTIGATION OF RECENT AND NOVEL GENETIC VARIANTS THAT ARE ASSOCIATED WITH THE PATHOGENESIS OF AMYOTROPHIC LATERAL SCLEROSIS AND THEIR IMPLICATIONS ON PHENOTYPES OF THE DISEASE Kwok, Chun Tak In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Division of Brain Sciences Department of Medicine Imperial College London 2014 1 ABSTRACT Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by loss of motor neurons in the spinal cord, brain stem and cerebral cortex. ALS is characterized by both upper and lower motor neuron symptoms and death usually occurs 3-5 years after onset. Familial histories are found in 5-10% of ALS cases while the rest are sporadic. This study is focused on analysing known and novel candidate genes in ALS and the aims of study are to characterize causal genes and risk factors for Familial ALS (FALS) and Sporadic ALS (SALS) in the Imperial Cohorts, in which genetic causes have been assigned for 64% of FALS cases. Three strategies were pursued and genes involved in proteostasis pathways were emphasized in this study. Firstly, we sequenced known candidate genes in our FALS cases excluded for known mutations. VCP and SQSTM1 genes were sequenced. We did not identify any coding changes in VCP but report a 5’ hexanucleotide expansion exclusively found in ALS. Known and novel SQSTM1 mutations, P392L and E155K, were identified in FALS kindred presenting with a history of Paget’s disease of bone. Secondly, we carried out association studies for two candidate genes on Chromosome 17, P4HB and NPLOC4, and showed that they were risk factors for FALS and SALS respectively. The association of P4HB SNPs with FALS survival time indicates that it is a modifier gene. Thirdly, to explore novel genes in ALS, we investigated Variable number tandem repeats (VNTR) from top candidate genes selected based on association signals from previous Genome wide association (GWA) studies and protein functions. VNTRs in NIPA1 and HSPB8 gene were associated with FALS and SALS respectively. Finally, we characterized the size of the reported hexanucleotide GGGGCC expansion in the C9orf72 gene using Southern blot analysis in our FALS cohort and interim results are presented. 2 DECLARATION I declare that this thesis is my own composition and data present herein is my own work unless otherwise stated. This thesis has not been submitted in any form for another degree or diploma at any university or institute. The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to Copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Kwok, Chun Tak 3 ACKNOWLEDGEMENTS First of all I would like to thank my supervisor Professor Jackie de Belleroche for giving me the precious opportunity to study Neurogenetics, an area that always interests me. During my study, Jackie experiencedly guided me through all PhD milestones and encouraged me writing for publications. I would like to thank Dr. Alex Morris for his daily guidance. Alex patiently took me through all practical steps in my research projects from pipetting to statistics with his broad knowledge in genetics and basic sciences. I would also like to acknowledge people who carried out the experiments for some results that are presented in this thesis. Ms Jennifer Frampton carried out an initial association study of the P4HB gene in her MSc Project (2008-2009). Ms Hsiang-Ya Wang and Dr. Alex Morris carried out the sequencing of 5’ and 3’UTR, Exon 1, 13 and 17 of the VCP gene and the screening of the hexanucleotide repeat in ALS cases and Controls in Wang’s MSc Project (2010-2011). Dr. Alex Morris also carried out fragment length analysis of the NPLOC4 TG repeat. Finally I would like to thank my family for unconditionally supporting my study. 4 DEDICATION This thesis is dedicated to my parents, Kin Lun Kwok and Siu Yin Kwok. 5 CONTENTS Chapter 1 Introduction to ALS Chapter 2 Methodology and Materials Chapter 3 Sequence Analysis of VCP Gene in the Imperial College ALS Cohort Chapter 4 Sequence Analysis of SQSTM1 Gene in the Imperial College ALS Cohort Chapter 5 Association Studies of Candidate Genes in ALS Chapter 6 VNTR analysis in ALS Chapter 7 Characterization of C9orf72 GGGGCC expansion Chapter 8 Discussion 6 DETAILED CONTENTS ABSTRACT 2 DECLARATION 3 ACKNOWLEDGEMENTS 4 DEDICATION 5 CONTENTS 6 DETAILED CONTENTS 7 LIST OF FIGURES 12 LIST OF TABLES 14 ABBREVIATIONS 16 CHAPTER 1 INTRODUCTION TO ALS 19 1.1 Clinical features of ALS: Symptoms begin in various sites and progress throughout the body 20 1.2 The diagnosis of ALS is based on clinical and electrophysiological findings 21 1.3 Current treatments for ALS are limited 26 1.4 Pathological findings in ALS 26 1.5 Genetic studies have led to the understanding of underlying pathogenesis of ALS 27 1.6 Mutant SOD1 causes oxidative stress, a common feature in ALS 35 1.7 Mutant SOD1 proteins are prone to aggregate 38 1.8 ER Stress, Unfolded Protein Response (UPR) and VAPB Mutations (ALS8) 39 1.9 Mitochondrial dysfunction 42 1.10 Neurofilaments, impaired axonal transport and DCTN1 mutations 43 1.11 Excitotoxicity and DAO mutations 44 1.12 Apotosis: a possible route to neuronal death 45 1.13 Other forms of protein aggregates, FUS (ALS6) and TARDBP (ALS10) mutations 46 1.14 The Ubiquitin-Proteasome System (UPS), autophagy and VCP mutations (ALS14) 49 1.15 The NF-kB pathway: TDP-43 aggregates, SQSTM1 and OPTN mutations 52 1.16 ALS-FTD and C9orf72 mutations 53 1.17 Other ALS Loci 55 1.17.1 Alsin (ALS2) mutations 55 7 1.17.2 18q21 (ALS3) 56 1.17.3 SETX (Senataxin, ALS4) 56 1.17.4 SPG11, Spatacsin (ALS5) 57 1.17.5 20p13 (ALS7) 58 1.17.6 ALS9: Angiogenin (ANG) 58 1.17.7 FIG4 (ALS11) 60 1.17.8 OPTN (ALS12) 61 1.17.9 ATXN2 (Ataxin-2, ALS13) 62 1.17.10 UBQLN2 (ALS15) 63 1.17.11 SIGMAR1 (ALS16) 64 1.17.12 CHMP2B (ALS17) 65 1.17.13 PFN-1 (ALS18) 65 1.18 Sequence analysis and the importance of studying known candidate genes in extended cohorts in ALS 66 1.19 Aims of study 69 1.19.1 Characterizing known genes in the IC-FALS cohort: Sequence analysis and Southern’s Blot 69 1.19.2 Association studies 70 1.19.3 Characterization of VNTR length in candidate genes for ALS 70 CHAPTER 2 METHODOLOGY AND MATERIALS 72 2.1 Subjects 72 2.2 DNA Extraction 74 2.2.1 DNA extraction from whole blood or the buffy coat layer 74 2.2.2 DNA purification from agarose gels 74 2.2.3 Total RNA extraction from blood 75 2.3 Polymerase chained reaction (PCR) 75 2.4 cDNA Synthesis and Endpoint RT-PCR 77 2.5 DNA Sequencing 77 2.5.1 DNA Purification 77 2.5.2 DNA Sequencing 78 2.6 Restriction digest 78 2.7 Electrophoresis 79 2.7.1 Agarose gel electrophoresis 79 2.7.2 Denaturing Polyacrylamide Gel Electrophoresis (PAGE) 80 2.8 SNP Genotyping using Kompetitive Allele Specific PCR (KASPTM) assay 84 2.9 Fragment analysis 84 2.10 Southern Blot 84 2.10.1 Sample preparation and electrophoresis 85 2.10.2 Preparation for DNA transfer 86 2.10.3 Assembling the blot 86 2.10.4 Dissembling the blot and preparation for hybridization 88 2.10.5 Hybridization using Digoxigenin (DIG) labelled probes 88 8 2.10.6 Chemiluminesence detection 90 2.10.7 Dot Blot 91 2.11 Statistics 92 2.12 Stock solutions 94 CHAPTER 3 SEQUENCE ANALYSIS OF VCP GENE IN THE IC ALS COHORT 96 3.1 Introduction 96 3.2 Method 97 3.2.1 Sample collection and DNA extraction 97 3.2.2 DNA Sequencing 97 3.2.3 Gel electrophoresis 98 3.2.4 Total RNA extraction and RT-PCR 98 3.2.5 Statistics 98 3.3 Results 100 3.3.1 Sequence analysis of the VCP gene 100 3.3.2 Screening for the hexanucleotide expansion in additional ALS cohorts 107 3.3.3 SNPs identified in the VCP gene 107 3.4 Discussion 111 CHAPTER 4 SEQUENCE ANALYSIS OF SQSTM1 GENE IN THE IC ALS COHORT 116 4.1 Introduction 116 4.2 Methods 117 4.2.1 Sample collection 117 4.2.2 Genotyping 117 4.2.3 Data analysis 118 4.3 Results 120 4.3.1 Identification of SQSTM1 sequence variants in a UK-FALS cohort 120 4.3.2 Founder haplotype of P392L SQSTM1 kindred 130 4.4 Discussion 134 CHAPTER 5 ASSOCIATION STUDIES OF CANDIDATE GENES IN ALS 136 5.1 General introduction: Association study and its use for identifying susceptibility genetic variants in ALS 136 5.2 Association studies indicate that P4HB is a risk factor in Amyotrophic lateral sclerosis 140 5.2.1 Introduction 140 5.2.2 Methods for P4HB association study 142 5.2.2.1 Sample collection 142 5.2.2.2 Identification and genotyping of SNPs 142 5.2.2.3 DNA Sequencing 143 5.2.2.4 Statistics 143 5.2.3 Results 144 5.2.3.1 Genotype Analysis of P4HB yielded Significant Genotypic Associations with ALS 147 9 5.2.3.2 Rare Haplotypes are associated with FALS and SALS 149 5.2.3.3 Genotypes and Haplotypes of P4HB affect the Survival of ALS 155 5.2.4 Discussion: P4HB 157 5.3 Association study of the NPLOC4 Gene: 161 5.3.1 Introduction 161 5.3.2 Methods for NPLOC4 association study 162 5.3.2.1 Subjects 162 5.3.2.2 Fragment analysis 164 5.3.2.3 Statistics 164 5.3.3 Results 165 5.3.3.1 GWAS SNPs tagged for independent haplotype blocks in the NPLOC4 gene 165 5.3.3.2 SNPs in NPLOC4 gene are significantly associated with Sporadic ALS 166 5.3.3.3 Linkage disequilibrium and Haplotype Analysis 170 5.3.3.4 Frequencies of [TG]n repeats 172 5.3.3.5 Gender and age of onset effects of NPLOC4 SNPs 173 5.3.4 Discussion: NPLOC4 174 CHAPTER 6 VNTR ANALYSIS IN ALS 178 6.1 Introduction 178 6.2 Backrgounds for candidate genes 180 6.2.1 SSBP3 180 6.2.2 HSPB8 180 6.2.3 EIF2AK2 181 6.2.4 YWHAQ 181 6.2.5 GPX1 182 6.2.6 SLC12A2 183 6.2.7 RBM23 183 6.2.8 NIPA1 183 6.2.9 CAPNS1 183 6.2.10 UBQLN3 184 6.2.11 TMEM158 184 6.2.12 IRX2, IRX3 and IRX4 185 6.2.13 CTNND2 185 6.2.14 ID4 185 6.2.15 TXNDC5 186 6.2.16 RXRB 186 6.2.17 FAM120C 187 6.3 Methodology 187 6.3.1 Subjects 187 6.3.2 Genotyping and DNA sequencing 188 6.3.3 Data analysis and Statistics.