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Publication

Molecular and functional study of two frequent syndromic Autism Spectrum Disorders

Book - Dissertation

Subtitle:Helsmoortel-Van der Aa Syndrome and Fragile X Syndrome
Neurodevelopmental disorders enclose a heterogeneous group of conditions implicating central nervous system development. The majority of cases are caused by genetic defects. Mutations in the genes encoding Activity-Dependent Neuroprotective Protein (ADNP) and Fragile X Mental Retardation Protein (FMRP) make up the two common genetic causes of ASD combined with ID. Although both are ASD/ID syndromes, their genetic mechanism is completely different and the focus of this thesis. In a first part of this thesis, we elaborate on the impact of ADNP mutations. A first objective was finding a reliable ADNP detection method to evaluate its stability and functioning upon mutation. We tested several ADNP antibodies. The blocking peptide strategy enabled us to differentiate between specific and non-specific signals in different specimens. With a GFP-ADNP expression vector, we detected a shorter, mutant ADNP protein. Our results verify the presence of mutant forms of ADNP, caused by Helsmoortel-Van der Aa-like mutations. In a second objective, we studied correlations between the distinct ADNP mutations and their impact on the protein, by examining the expression and subcellular localization of GFP-tagged mutant transcripts in transfected HEK293T cells. We found mutations presenting a pattern based on the genetic position. Mutations affecting the nuclear localization signal caused mislocalization of mutant protein in the cytoplasm. N-terminal truncated ADNP mutants are routed towards cytosolic proteasomal degradation and rescued with the proteasome inhibitor MG132. C-terminal mutants presented wildtype-like nuclear localization, but those with an arrested HP1α interaction motif showed decreased affinity for heterochromatin interaction. Our results suggest a correlation between the position of the mutations, protein stability and subcellular localization. The third objective was to generate and validate a “humanized” (carrying a human mutation) mouse model to further study HVDAS-related mutations on a functional level. Using Crispr/Cas9, we introduced a frameshift mutation near a clustering site of known patient mutations. We assigned the mutant mouse model to a standardized behavioral test battery. Our model presented similarities to HVDAS patients, including impaired cognitive functioning, increased anxiety and repetitive behavior. The second part of this thesis focuses on kinome analysis in Fmr1 knockout mice, a model for Fragile X Syndrome (FXS), using the PamGene platform. We observed a general upregulation of kinase activity in an Fmr1 knockout mice. Through upstream kinase analysis, we observed dysregulation in glycogen and immune pathways. Furthermore, we found statistical evidence for the dysregulation of 46 proteins. These candidates have to be validated for biomarker potency for future drug testing.
Number of pages: 1
Publication year:2021
Keywords:Doctoral thesis
Accessibility:Closed