(''%'#($ )/)# ""($)%" (#(''%'# $ %)%'"'%'## $ ' $$ $ *").% $ $ +'( ).%"( $! $"$ %&'($), ))&'# (( %$%) *").% $%)$ +'( ). %"( $! %'&*" -# $) %$'#%)".'%#")*'"" %# *#%$) %.)%0"%! "( $! $"$ Supervised by Associate Professor Henna Tyynismaa, Ph.D Research Programs Unit, Faculty of Medicine University of Helsinki Docent Emil Ylikallio, MD, Ph.D Research Programs Unit, Faculty of Medicine University of Helsinki Reviewed by Professor Johanna Uusimaa, MD, Ph.D Faculty of Medicine University of Oulu Doctor Vihandha Wickramasinghe, Ph.D School of Biomedical Sciences University of Melbourne Opponent Professor Marina Kennerson, Ph.D Faculty of Health and Medicine University of Sydney Custos Associate Professor Henna Tyynismaa, Ph.D Research Programs Unit, Faculty of Medicine University of Helsinki The Faculty of Medicine uses the Urkund system (plagiarism recognition) to examine all doctoral dissertations. Cover image: Human neurons differentiated from induced pluripotent stem cells expressing MAP2 (green) and GANP (red). ISBN 978-951-51-7217-4 (print) ISBN 978-951-51-7218-1 (online) ISSN 2342-3161 (print) ISSN 2342-317X (online) Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis Unigrafia Helsinki 2021 To my husband Dr. Michael Woldegebriel. Rare diseases are a heterogenous group of disorders, which together affect a large number of people. These diseases typically have a genetic cause, which affects various cellular processes and pathways. One of the key processes in the cell is RNA metabolism, which is crucial for gene expression. Defects in RNA processing steps contribute to a variety of human diseases ranging from motor neuron diseases to cancer. Most rare diseases currently have no cure. In this dissertation, biallelic variants in Minichromosome maintenance complex 3 associated protein (MCM3AP) gene were found to underlie a neurological syndrome: early-onset peripheral neuropathy with/without intellectual disability. Through international collaboration, patients from multiple families and the disease variants in MCM3AP were identified. Germinal center associated nuclear protein (GANP), encoded by MCM3AP, is a large scaffold protein in the Transcription and Export 2 (TREX-2) complex, which is responsible for the transport of mRNAs from nucleus to the cytoplasm. The effects of different disease-causing variants on GANP abundance on the nuclear envelope were determined, which led to establishment of a genotype/phenotype correlation regarding the severity of the motor phenotype. This research showed that GANP regulates gene expression in a cell type specific manner. In patient fibroblasts, GANP was found to dysregulate genes depending on intron content. In addition, stem cell-based technology and genome editing was utilized to allow modeling of GANP defects in human cultured motor neurons. We found that GANP is a major regulator of gene expression in developing motor neurons, with GANP defects altering the expression of genes related to synaptic functions and resulting in compensatory induction of protein synthesis and mitochondrial pathways. Understanding of the causes and mechanisms of rare diseases is needed for improving diagnostics, therapeutic development and personalized treatments. In this dissertation, the foundation for understanding the mechanisms of MCM3AP-linked neurological syndrome has been established through identification of the causative gene and description of the disease phenotype. The different cellular models including patient-specific motor neurons used in this study have revealed molecular pathways that may be targets for treatment in preclinical trials. 4 Abstract .................................................................................................. 4 Contents ................................................................................................. 5 List of original publications .................................................................. 9 Abbreviations ...................................................................................... 10 1 Introduction ............................................................................ 14 2 Review of the literature ........................................................... 16 2.1 Introduction to human genetics .......................................... 16 2.1.1 DNA ............................................................................... 16 2.1.2 Human genome organization ........................................ 16 2.1.3 Genes and other elements of the genome ..................... 16 2.1.4 Genetic variation ........................................................... 17 2.1.5 Mendelian inheritance of traits ..................................... 18 2.2 mRNA export is a key step in gene expression .................... 19 2.2.1 Gene expression ............................................................ 19 2.2.2 mRNAs are exported through nuclear pores ............... 20 2.2.3 Pre-mRNA processing and splicing .............................. 21 2.2.4 Macromolecular complexes in gene expression and export 22 2.2.5 mRNA export factor GANP .......................................... 24 2.2.6 Selectivity of mRNA export ........................................... 25 2.2.7 Export and expression of intronless genes .................. 26 2.2.8 Nucleocytoplasmic transport in neurological diseases 27 2.3 Charcot- Marie Tooth disease: from phenotypes to genetics and variant detection .......................................................................... 28 5 2.3.1 Introduction and etiology ............................................. 28 2.3.2 Modes of inheritance .................................................... 28 2.3.3 Classification ................................................................. 29 2.3.4 Clinical features ............................................................ 30 2.3.5 Genetic causes and molecular mechanisms .................. 31 2.3.6 Genetic testing .............................................................. 34 2.3.7 Advances in the identification of disease-causing variants by next generation sequencing ......................................... 35 2.4 Stem cell-based modeling of diseases affecting motor neurons 36 2.4.1 Introduction and stem cell types .................................. 36 2.4.2 Induced pluripotent stem cells ...................................... 37 2.4.3 Modeling neuronal disease in human neurons ............ 38 2.4.4 Assessing local translation in neurons ......................... 39 2.4.5 Editing the genome with CRISPR-Cas9 for disease modeling 40 3 Aims of the study .................................................................... 42 4 Materials and methods ........................................................... 43 4.1 Collection of clinical data (I, II) .......................................... 43 4.2 Ethical permissions (I, II, III) ............................................. 43 4.3 Affected individual and control cells (I, II, III) .................. 43 4.4 Whole exome and genome sequencing (I, II) ..................... 44 4.5 Fibroblast cell culture (I, II) ................................................ 44 4.6 RNA isolation and cDNA synthesis (I, II, III) .................... 44 4.7 Quantitative reverse transcription polymerase chain reaction (qRT-PCR) (I, II, III) ........................................................................... 45 6 4.8 Minigene assay to detect altered mRNA splicing in fibroblasts (I) ........................................................................................ 45 4.9 Mixed neuronal cultures (I) ................................................. 45 4.10 DNA repair assay (I) ............................................................ 45 4.11 Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and western blotting (I, II, III) ..................................... 46 4.12 Immunocytochemistry (I, II, III) ........................................ 46 4.13 Microscopy (I, II, III) .......................................................... 46 4.14 RNA in situ hybridization (II, III) ....................................... 47 4.15 Transmission electron microscopy (II) ............................... 47 4.16 Molecular modeling of Sac3 region of GANP (II) ............... 47 4.17 RNA sequencing and bioinformatics (II, III) ...................... 47 4.18 Culture of human induced pluripotent stem cells (hiPSC) (I, III) 48 4.19 Motor neuron differentiation (III) ...................................... 48 4.20 Axon isolation for RNA and protein (III) ........................ 49 4.21 Nuclei isolation for RNA and protein (III) ......................... 49 4.22 CRISPR-Cas9 gene editing (III) .......................................... 50 4.23 Statistics (I, II, III) .............................................................. 50 5 Results and discussion ............................................................ 51 5.1 MCM3AP variants underlie an early-onset neurological disease spectrum (I, II) ........................................................................ 51 5.1.1 Clinical features ............................................................. 51 5.1.2 Genetic features ............................................................. 53 5.2 Distinct effects on GANP by variant location (I-III) ........... 54 5.2.1 Defects in GANP correlate with severity of disease phenotype 54 7 5.3 MCM3AP variants have different effects