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Potential Candidate Genes for Neuropsychiatric Disorders: MLC1

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Potential Candidate Genes for Neuropsychiatric Disorders: MLC1 Potential candidate genes for neuropsychiatric disorders: MLC1, C15orf53 and OXTR Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) durch den Fachbereich I – Psychobiologie der Universität Trier vorgelegt von Thorsten Manfred Kranz Betreuer: Prof. Dr. Jobst Meyer Prof. Dr. Claude P. Muller Trier, 2012 Der Nutzen von Information liegt eindeutig in der Auswahl, nicht in der Fülle, in ihrer Relevanz, nicht im Übertragungstempo. Die Kunst vernetzt zu denken Frederic Vester (1925 – 2003), deutscher Biochemiker und Biokybernetiker Table of Contents Table of Contents Table of Contents ...................................................................................................................... I Index of Figures .................................................................................................................... VII Index of Tables ....................................................................................................................... IX List of Abbreviations .............................................................................................................. XI 1 Introduction ....................................................................................................................... 1 1.1 The short outline on the history of psychiatry from ancient times till present ............ 1 1.2 Main characteristics of schizophrenia, bipolar disorder, periodic catatonia (SCZD10) and autism .................................................................... 4 1.3 Neurodevelopmental hypothesis of mental disorders .................................................. 5 1.4 Biopsychosocial factors trigger the onset of mental disorders: gene – environment interaction .................................................. 6 1.5 Chronic stress deregulates the hypothalamus-pituitary-adrenal gland (HPA) axis and triggers the onset of psychiatric disorders ............................................................ 9 1.6 Mental disorders follow a complex inheritance mode but in rare cases they appear to accumulate in multiplex families ............................ 13 1.7 Multiplex families segregating periodic catatonia (SCZD10, OMIM: #605419) reveal susceptibility loci on chromosome 15q and 22q ............................................. 14 1.8 MLC1 mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC) .................................................................................... 16 1.9 SCZD10 (periodic catatonia): an entity on its own in psychiatric classification? ..... 17 1.10 Candidate genes for SCZD10, BD and ASD: C15orf53 (Chr. 15q14), oxytocin receptor ( OXTR , Chr. 3p25) and MLC1 (Chr. 22q13.33). ......................................... 17 1.11 Non-coding RNAs and their influence in pathophysiology: regulation of microRNAs (miRNAs) in psychiatric disorders ........................................................ 27 1.12 Transgenic mouse models are a suitable approach to investigate the etiological mechanisms of mental disorders ................................................................................ 32 1.13 Research objective ..................................................................................................... 38 I Table of Contents 2 Material & Methods ....................................................................................................... 40 2.1 Psychiatric disorder sample collections ..................................................................... 40 2.2 Individual patient samples (non-related) ................................................................... 40 2.2.1 Bipolar disorder (BD) patients ........................................................................... 40 2.2.2 Periodic catatonia (SCZD10) patients ................................................................ 41 2.3 Parents-offspring trios and multiplex families (related) ............................................ 41 2.3.1 Parents-offspring trios with autism spectrum disorder (ASD) ........................... 41 2.3.2 Multiplex families segregating SCZD10 ............................................................ 42 2.4 Cell lines .................................................................................................................... 42 2.4.1 Cell lines used for expression analysis and knock down experiments of MLC1 .................................................................... 42 2.4.2 Cell lines cDNA used for gene expression analysis of C15orf53 ...................... 43 2.5 Bacterial strains & transformation of DNA ............................................................... 44 2.6 Bacterial DNA vectors ............................................................................................... 45 2.7 Human brain tissue .................................................................................................... 45 2.8 Genomic murine DNA & murine embryonic stem cells (ES) ................................... 45 2.9 Buffers, media and reagents ...................................................................................... 46 2.9.1 Used buffers and media ...................................................................................... 46 2.10 Reagents ..................................................................................................................... 48 2.11 Oligonucleotides ........................................................................................................ 51 2.11.1 Oligonucleotides (“primers”) used for PCR reactions ....................................... 51 2.11.2 Oligonucleotides (“primers”) used for SNP detection ....................................... 54 2.11.3 Oligonucleotides (“primers”) used for sequencing reactions ............................. 54 2.12 Precursor sequences of miRNAs and applied antibodies .......................................... 55 2.13 Commercial kits ......................................................................................................... 56 2.14 Equipment, bioinformatic tools & databases ............................................................. 57 2.14.1 Equipment .......................................................................................................... 57 2.14.2 Bioinformatic tools & databases ........................................................................ 58 2.15 Methods ..................................................................................................................... 59 2.15.1 Non-conditional murine Mlc1 knockout mouse procedure ................................ 59 2.16 PCR reactions on gDNA of ES .................................................................................. 62 2.16.1 Estimation of ES gDNA amount by PCR on Serpina6 ...................................... 62 2.16.2 Detection of aberrant Mlc1 gDNA sequence into ES genome ........................... 63 II Table of Contents 2.16.3 Detection of homologous recombination of aberrant Mlc1 into ES genome ..... 63 2.16.4 In silico predicition of miRNA target sites in 3’-UTRs of MLC1 and NR3C1 .......................................................................................... 64 2.16.5 Heat inactivation of fetal calf (FCS) and horse serum (HS) .............................. 65 2.16.6 Depletion of steroids in FCS and HS by charcoal filtering ................................ 65 2.17 Cell culture experiments with miR-137, miR-22 and miR-124 ................................ 66 2.17.1 Immunoblot analysis of miR-137, miR-22 and miR-124 effects on NR3C1 expression ......................................................................................... 68 2.17.2 Amplification of NR3C1 3’-UTR fragment ....................................................... 68 2.17.3 Verification of NR3C1 3’-UTR fragment by enzymatic restriction ................... 69 2.17.4 Cloning of NR3C1 3’-UTR fragment into pMIR-REPORT vector ................... 70 2.17.5 Transformation of pMIR_REPORT_ NR3C1 construct into bacterial strains TOP10 & XL10-Gold .............................................................. 71 2.17.6 Inoculated agar plates & liquid bacterial cultures .............................................. 72 2.17.7 Colony PCR on selected bacterial colonies ........................................................ 72 2.17.8 Quantification of immunoblot signals ................................................................ 73 2.18 Investigation of C15orf53 in BD and SCZD10 patients ............................................ 73 2.18.1 Mutational analysis of C15orf53 ........................................................................ 74 2.18.2 Genotyping of C15orf53 in SCZD10 and BD individual samples and SCZD10 families ......................................................................................... 74 2.18.3 Case-control association analysis of C15orf53 with BD and SCZD10 ............. 75 2.18.4 C15orf53 gene expression analysis in human post-mortem brain tissue and leukocyte subpopulations ........................................................................... 75 2.19 Association study of the human oxytocin receptor ( OXTR ) with ASD ..................... 76 2.19.1 PCRs of SNP-encompassing genomic regions in OXTR ................................... 76 2.19.2 Exo-SAP -procedure ............................................................................................ 78 2.19.3 Single base extension procedure (“SNP reaction”) ...........................................
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