Specialeforsvar: Vivi Søgaard Nielsen

Functional characterization of novel candidate disease genes in primary microcephaly

Vejledere: Søren Tvorup Christensen og Lars Allan Larsen

Censor: Overlæge dr. med. Steen Gammeltoft

Abstract
Primary microcephaly (MCPH) is a rare autosomal recessive neurodevelopmental disorder in which the cerebral cortex is significantly reduced in size leading to mild to severe learning disabilities in the affected subjects. In a recent study using samples from 26 consanguineous Pakistani MCPH families, Dr. Muhammad Farooq identified a homozygous missense mutation affecting a highly conserved amino acid residue in the Ribosomal RNA-processing protein 7 homolog A (RRP7A). The mutation is not present in the exome variant database or in 180 healthy controls of Pakistani origin and has not previously been characterized, suggesting that the variant may be subject to negative selection. This master thesis was performed as a part of larger project to characterize the function of RRP7A. For our part of the study, we used CRISPR/Cas9-mediated protein depletion in P19.CL6 cells, a mouse embryonic teratocarcinoma cell line as an easily managed culture system with the capability for in vitro neurogenesis. Furthermore, we used RNAi-mediated knockdown in human RPE cells in order to study the role of RRP7A in neurogenesis, centriole duplication, cell cycle control and migration as well as various aspects associated with primary cilia, including ciliogenesis, ciliary length and ciliary disassembly. The first results include immunohistochemical analysis on the localization of RRP7A in mouse embryonic tissue sections, including the brain. We were further able to construct a stable RRP7A CRISPR/Cas9 knock-out clone, followed by work on the role of RRP7A in in vitro neurogenesis. The third and final part of the Results section presents results on the role of RRP7A in centrosome duplication, cell cycle control and migration as well as formation, length and disassembly of primary cilia in in RPE cells subjected to RNAi-mediated depletion of RRP7A. Our preliminary findings on the neuronal differentiation of P19.CL6 CRISPR/Cas9 knockout clone suggest a change of rate in which stem cells differentiate. Furthermore, we were able to efficiently deplete RRP7A in RPE cells and show a tight connection between RRP7A’s various cellular functions connected to regulatory functions of primary cilia. Consequently, by depleting RRP7A, we initially show an elongation of primary cilia as well as a lack in formation of primary cilia. Additionally, by immunofluorescent microscopy analysis, we show entry and exiting of cell cycle disturbance in RRP7A depleted cells. Lastly, we found disorientation in migrating cells using a scratch assay. All these functions are key factors in neurogenesis and therefore in the development of the cerebral cortex.