Master thesis defense: Cecilie Keller

Functional characterization of a novel candidate disease gene RRP7A in primary microcephaly

Supervisors
Prof. Søren Tvorup Christensen, Section for Cell Biology and Physiology, BIO-UCPH
Prof. Lars Allan Larsen, Department of Cellular and Molecular Medicine, Panum Institute, UCPH

External examiner
Prof. Steen Gammeltoft

Abstract
Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder that is characterized by microcephaly present at birth and varying degree of mental retardation. MCPH is genetically heterogeneous, and at least 14 genes involved in MCPH have been identified. These genes encode proteins that have been implicated in centrosome function and biogenesis, which may impact on cell cycle progression of neuronal progenitors in the developing neocortex. Here, we describe the functional characterization of a novel MCPH gene, which was identified in a consanguineous Pakistani family presenting with autosomal recessive microcephaly in 10 affected individuals. Homozygosity mapping and exome sequencing led to discovery of a homozygous missense mutation (p.Trp155Cys) in the gene encoding Ribosomal RNA-processing Protein, RRP7A. Analysis of human fetal brain sections showed that RRP7A localizes to cilia and is predominantly expressed in radial glia cells in the ventricular zone of the developing forebrain. Analysis of zebrafish with loss of function mutation in rrp7a confirmed that depletion of RRP7A leads to reduced brain size. In order to study the function of RRP7A in more detail, we set out to investigate the role of RRP7A in in vitro neurogenesis and in regulation of cell cycle control, including resorption of primary cilia, which is a prerequisite for proper cell cycle entry and progression through the cell cycle. We initially show that CRISPR/Cas9-mediated deletion of RRP7A leads to dysfunction in neurogenesis and that stem cells mutated in RRP7A display reduced proliferation rates. Secondly, experiments with human dermal fibroblast cells show that RRP7A localizes to the cilia-centrosome axis as well as to nucleoli, and that mutation in RRP7A leads to cell cycle retardation, which is partly characterized by defects in timely resorption of primary cilia. These findings provide novel insight into the cellular and molecular mechanisms underlying MCPH.