STUDY OF TWO BIPOLAR SUSCEPTIBILITY GENES: SLYNAR AND IGF1 Ana Catarina Parente Pereira University College London PhD 2009 1 I, Ana Catarina Parente Pereira 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. 2 AKNOWLEDGMENTS I want to thank Professor Hugh Gurling and Dr Andrew McQuillin for all the through out my PhD. I want to thank Fundacao para a Ciencia e Tecnologia (FCT) for the funding that allowed me to pursue this PhD. I want to thank the Molecular Psychiatry Lab Team for all the help and support as a team and for the good moments we had as friends. I want to thank my friends and my family for the constant support, especially mother, my aunty Teresa and Vinay. I want this PhD to be dedicated to my father. He always has been a pillar of my life. His life has inspired me and now I want to give him this PhD as a proof of my love. 3 ABSTRACT Linkage studies have implicated the 12q22-24 region in susceptibility to bipolar disorder. In this region alleles at the “Slynar” and Insulin Like Growth Factor 1 (IGF1) genes showed association with bipolar disorder. The Slynar gene is contained within a region of 278 kb on chromosome 12q24 and expresses the sequence AY070435 in the human brain. AY070435 has no known function. A Macaque brain expressed cDNA which is highly homologous to human AY070435 has been cloned and sequenced. To further characterise the human Slynar gene and expressed mRNA transcript studies were carried out to identify Slynar in the mouse and in human neuroblastoma cell lines. Exhaustive efforts were taken to find a mouse homologue but these proved negative. Slynar shared no homology, or partial homology with any other gene in the human genome. The other 12q24 bipolar susceptibility gene IGF1 is highly expressed in the human brain and a well known for its neuromodulatory functions. IGF1 protein has been shown to have an antidepressant and anxiolytic-like effect in the mouse brain. On a genome wide association study (GWAS) with the UCL case control sample, IGF1 was found to be associated to disease with 5 SNPs showing association within the gene. In order to further implicate IGF1 and find the aetiological base pair changes responsible for disease, IGF1 was sequenced. New three new non database SNPs, three previously characterised polymorphisms and a CA repeat were found and genotyped in an extended UCL sample of 1,000 cases and 1,000 controls. One of the novel SNPs and the CA repeat, both located in the promoter region, were associated with bipolar disorder. Haplotype analysis of the GWAS and new markers data confirmed association to bipolar disorder. 4 TABLE OF CONTENTS STUDY OF TWO BIPOLAR SUSCEPTIBILITY GENES: SLYNAR AND IGF1 1 AKNOWLEDGMENTS 3 ABSTRACT 4 TABLE OF CONTENTS 5 TABLE OF TABLES 9 TABLE OF FIGURES 10 1 INTRODUCTION 11 1.1 BIPOLAR DISORDER 11 1.1.1 HISTORICAL BACKGROUND 11 1.1.2 BIPOLAR DISORDER PHENOMENOLOGY AND CLASSIFICATION 15 1.1.2.1 Phenomenology 15 1.1.2.2 Classification of Bipolar Disorder 16 1.1.3 DIAGNOSIS AND TREATMENT 17 1.1.3.1 Diagnosis 17 1.1.3.2 Treatment 18 1.1.3.3 Hospitalization 20 1.1.4 IMPACT OF BIPOLAR ON SOCIETY 21 1.2 NEUROBIOLOGY OF BIPOLAR DISORDER 23 1.2.1 NEUROTRANSMITTER SYSTEMS 23 1.2.1.1 Noradrenergic System 23 1.2.1.2 Dopaminergic System 24 1.2.1.3 Serotonergic System 24 1.2.1.4 Cholinergic System 25 1.2.1.5 GABAergic System 26 1.2.1.6 Glutamergic System 26 1.2.2 NEUROENDOCRINE SYSTEMS 28 1.2.3 SIGNALLING NETWORKS 28 1.2.3.1 Gαs/cAMP-generating signalling pathway 29 1.2.3.2 Phosphoinositide/Protein Kinase C Signalling Pathway 30 1.2.3.3 GSK-3 Signalling Pathway 31 1.2.3.4 Neurotrophic Signalling Cascades 32 1.2.4 HISTONE DEACETYLASE 33 1.2.5 NEUROANATOMY STUDIES 33 1.3 GENETICS AND BIPOLAR DISORDER 35 1.3.1 FAMILY STUDIES 35 1.3.2 SEGREGATION ANALYSIS 37 1.3.3 TWIN STUDIES 38 1.3.4 ADOPTION STUDIES 40 1.4 MAPPING BIPOLAR DISORDER 42 1.4.1 GENETIC AND PHENOTYPIC HETEROGENEITY 42 5 1.4.2 POWER ANALYSIS 43 1.4.3 GENETIC MARKERS 43 1.4.4 LINKAGE ANALYSIS 45 1.4.4.1 Parametric Linkage Analysis 46 1.4.4.1.1 Two-Point Analysis 47 1.4.4.2 Non-Parametric Models 49 1.4.4.3 Two-Locus Linkage 50 1.4.5 ALLELIC AND HAPLOTYPIC ASSOCIATION STUDIES 51 1.4.5.1 Measures of Allelic Association 52 1.4.5.2 Tests for Association 53 1.4.5.2.1 Case-control Test 53 1.4.5.3 Haplotype Analysis 55 1.5 LINKAGE AND ASSOCIATION RESULTS IN BIPOLAR DISORDER 56 1.5.1 LINKAGE 56 1.5.2 ASSOCIATION 58 1.5.2.1 Association studies on Chromosome 12 62 1.5.3 GENOME WIDE ASSOCIATION STUDIES (GWAS) 63 1.5.4 COPY NUMBER VARIATIONS 69 1.6 MOUSE AS AN ANIMAL MODEL 71 1.7 SLYNAR AND IGF1 74 1.7.1 SLYNAR 74 1.7.2 IGF1 77 1.7.2.1 IGF1 GWAS results 79 2 AIMS OF THE PROJECT 81 3 METHODOLOGY 83 3.1.1 DNA EXTRACTION 83 3.1.1.1 DNA Extraction from Whole Blood Cells 83 3.1.1.2 DNA Extraction from Saliva Samples 85 3.1.2 DNA QUANTIFICATION 86 3.1.2.1 Quantification of Saliva and Whole Blood DNA Samples 86 3.1.2.2 Quantification of cDNA and plasmid DNA 87 3.1.3 RNA EXTRACTION 88 3.1.3.1 RNA Extraction from Tissue 88 3.1.3.2 RNA Extraction from Cells 89 3.1.3.3 Total and PolyA+ RNA Purification 89 3.1.4 RNA QUANTIFICATION AND QUALITY ASSESSMENT 90 3.1.5 CDNA SYNTHESIS 91 3.1.6 PRIMER DESIGN 92 3.1.7 POLYMERASE CHAIN REACTION (PCR) 94 3.1.7.1 Mastermix for General Optimization and Amplification 94 3.1.7.2 PCR Cycling Conditions 95 3.1.8 ELECTROPHORESIS 97 3.1.8.1 Agarose Gels 97 3.1.8.2 Polyacrylamide Gels 98 3.1.9 PCR PRODUCT CLEANING METHOD 100 3.1.10 SEQUENCING 101 3.1.10.1 Choice of Samples to Sequence 101 3.1.10.2 Chain Termination Sequencing Method (Sanger-Coulson) 103 3.1.10.3 Big Dye Terminator Method 105 3.1.11 GENOTYPING 108 6 3.1.11.1 KASPar Method 108 3.1.11.2 High Resolution Melting Curve Method 111 3.1.11.3 Microsatellites 113 3.1.11.4 Data analysis 115 3.1.12 CLONING 116 3.1.12.1 Cloning Process 117 3.1.12.2 Transformation 118 3.1.12.3 Colony Selection 119 3.1.12.4 Plasmid Extraction and Purification 120 3.1.13 NORTHERN BLOT 120 3.1.13.1 Formaldehyde/Formamide denaturing agarose gel 121 3.1.13.2 RNA Transfer to the Nylon Membrane 121 3.1.13.3 Fixation of RNA to Membrane 123 3.1.13.4 Pre-Made Membranes 123 3.1.13.5 Riboprobe Synthesis 124 3.1.13.6 Hybridisation 126 3.1.13.7 Exposure and Development of the Film 127 3.1.13.8 Probe Removal 127 3.1.14 QUANTITATIVE REAL TIME PCR (QRT-PCR) 128 3.1.14.1 Detection Systems Used 129 3.1.14.2 Experimental Design 132 3.1.14.3 qRT-PCR Normalization 134 3.1.14.4 Running Programs 136 3.1.14.5 Interpretation of Results 138 3.1.15 CDNA LIBRARY SCREENING 140 3.1.16 RAPID AMPLIFICATION OF CDNA ENDS (RACE) 143 3.1.16.1 Principles of RACE 143 3.1.16.2 5’RACE 144 3.1.16.3 3’ RACE 146 3.1.17 ANIMAL CELL CULTURE 147 3.1.17.1 Cell Lines 147 3.1.17.2 Growth Conditions 148 3.1.17.3 Harvesting of Cells 148 3.1.17.4 Cryogenic Storage of Animal Cell Lines 149 3.1.17.5 SH-SY5Y Neuroblastoma Cells Differentiation 150 3.1.1 SIRNA 150 3.1.1.1 Day 1: Seeding the cells 152 3.1.1.2 Day 2: Transfection 152 3.1.1.3 Day 3: RNA extraction and quantification by qPCR 153 4 SLYNAR RESULTS 154 4.1 SLYNAR STUDIES IN THE MOUSE 154 4.1.1 SLYNAR MOUSE HOMOLOGUE 154 4.1.2 FINDING SLYNAR HOMOLOGUE TRANSCRIPTS 155 4.1.3 SLYNAR’S ALTERNATIVE TRANSCRIPTION 157 4.1.4 THE SEARCH FOR THE DETECTED TRANSCRIPTS – GETTING THE CDNA CLONE 159 4.1.5 SLYNAR AND LITHIUM 160 4.1.6 DISCUSSION / CONCLUSION 161 4.2 SLYNAR STUDIES IN THE HUMAN 164 4.2.1 SLYNAR IN THE HUMAN 164 4.2.2 DETECTING SLYNAR IN CELL LINES 164 4.2.3 HUMAN SLYNAR’S CLONE 167 4.2.4 DETECTION OF SLYNAR BY QPCR 169 7 4.2.5 SILENCING SLYNAR 170 4.2.6 DISCUSSION/CONCLUSION 173 5 IGF1 RESULTS 177 5.1 IGF1 SEQUENCING 177 5.2 IGF1 HAPLOTYPIC ASSOCIATION 180 5.3 DISCUSSION/CONCLUSION 183 6 FUTURE WORK 189 6.1 SLYNAR 189 6.2 IGF1 190 7 BIBLIOGRAPHY 193 8 APPENDIX 264 8.1 PRIMERS AND PROBES SEQUENCES FOR SLYNAR EXPERIMENTS 264 8.1.1 RT-PCR 264 8.1.2 CDNA LIBRARY SCREENING 265 8.1.3 RACE 265 8.1.4 QPCR 267 8.1.5 NORTHERN BLOT 268 8.1.6 SIRNA 269 8.2 IGF1 SEQUENCING PRIMERS 269 8.3 IGF1 ASSOCIATION TABLE 270 8.4 CLUMP TEST RESULTS FOR CA THE REPEAT 273 8 TABLE OF TABLES Table 1.1 2x2 Contingency table for case/control analysis....................................................54 Table 1.2 Summary of the main studies done for linkage analysis on bipolar disorder.