The ASD-associated CNV 16p11.2: Functional study of the candidate gene QPRT Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich 15 der Johann Wolfgang Goethe-Universität in Frankfurt am Main von Denise Haslinger aus Braunau am Inn, Österreich Frankfurt 2018 D 30 vom Fachbereich 15 der Johann Wolfgang Goethe-Universität als Dissertation angenommen. Dekan: Prof. Dr. Sven Klimpel 1. Gutachterin: Prof. Dr. Amparo Acker-Palmer 2. Gutachterin: Prof. Dr. Christine M. Freitag Datum der Disputation: ………………………………………………. Dissertation Denise Haslinger Index Index Index ............................................................................................................................................................................ I List of figures ............................................................................................................................................................. IV List of tables .............................................................................................................................................................. IV List of abbreviations used in this study ....................................................................................................................... V List of genes discussed in this study ......................................................................................................................... VII Vorwort ..................................................................................................................................................................... XI Ehrenwörtliche Erklärung ........................................................................................................................................ XIII Zusammenfassung .................................................................................................................................................... XV 1 Abstract .............................................................................................................................................................. 1 2 Introduction ........................................................................................................................................................ 3 2.1 Autism spectrum disorder (ASD) ....................................................................................................................... 3 2.1.1 Phenotype and diagnostics ........................................................................................................................... 3 2.1.2 Epidemiology ................................................................................................................................................ 4 2.1.3 Aetiology and heritability ............................................................................................................................. 5 2.1.4 Molecular genetic findings ........................................................................................................................... 6 2.2 Neurobiology and current animal or cell models of ASD ................................................................................ 16 2.2.1 Neuropathology .......................................................................................................................................... 17 2.2.2 Neuroanatomy ........................................................................................................................................... 17 2.2.3 Genetic basis of altered neurobiology ........................................................................................................ 17 2.2.4 Current animal and cell models .................................................................................................................. 18 2.3 ASD-associated region 16p11.2 ...................................................................................................................... 23 2.3.1 Penetrance and phenotype of 16p11.2 CNVs ............................................................................................ 23 2.3.2 Functional studies of the whole CNV or single genes located in 16p11.2 .................................................. 24 2.4 Summary and aims ......................................................................................................................................... 33 3 Material and methods ...................................................................................................................................... 35 3.1 Genetic methods ............................................................................................................................................. 35 3.1.1 DNA extraction ........................................................................................................................................... 35 3.1.2 Real-time PCR ............................................................................................................................................. 35 3.2 RNA methods .................................................................................................................................................. 36 3.2.1 RNA extraction............................................................................................................................................ 36 3.2.2 Real-time RT-PCR ........................................................................................................................................ 36 3.2.3 Whole transcriptome analyses ................................................................................................................... 37 3.3 Protein methods .............................................................................................................................................. 38 I Dissertation Denise Haslinger Index 3.3.1 Protein extraction, gel electrophoresis and Western blot .......................................................................... 38 3.4 Cell culture methods ........................................................................................................................................ 40 3.4.1 Cell lines and growth conditions ................................................................................................................. 40 3.4.2 Cell culture assays ....................................................................................................................................... 43 3.5 Statistics........................................................................................................................................................... 47 3.5.1 Group differences ....................................................................................................................................... 47 3.5.2 Correlations ................................................................................................................................................. 47 3.5.3 Targeted mRNA expression ......................................................................................................................... 47 3.5.4 Transcriptome analyses............................................................................................................................... 48 3.5.5 Gene list enrichment test in gene networks of brain development ........................................................... 49 4 Results .............................................................................................................................................................. 51 4.1 Characterization of the SH-SY5Y cell model ..................................................................................................... 51 4.1.1 Confirmation and evaluation of neuronal differentiation........................................................................... 51 4.1.2 Identification of genes implicated in neuronal differentiation ................................................................... 54 4.1.3 Co-expression network analysis .................................................................................................................. 54 4.2 QPRT as candidate gene for functional validation .......................................................................................... 55 4.2.1 QPRT is differentially regulated during neuronal differentiation................................................................ 55 4.2.2 QPRT correlates with neuronal morphology during neuronal differentiation ............................................ 56 4.2.3 Validation of 16p11.2 CNVs ......................................................................................................................... 57 4.2.4 Validation of QPRT expression in 16p11.2 deletion carrier ........................................................................ 58 4.3 Functional validation of QPRT in SH-SY5Y ....................................................................................................... 59 4.3.1 siRNA mediated knock down (KD) of QPRT ................................................................................................. 59 4.3.2 Chemical inhibition of QPRT ........................................................................................................................ 61 4.3.3 CRISPR/Cas9 mediated knock out (KO) of QPRT ........................................................................................