The utility of chorionic villus sample cells for functionally assessing noonan syndrome variants

Abstract Noonan syndrome (NS) is a congenital disorder that causes pleiotropic developmental problems of varying severity. Its main cause is gain-of-function mutations in genes of the essential Ras-mitogen-activated protein kinase (Ras/MAPK) pathway. There are currently no routine functional tests available for prenatal cases with a variant of uncertain significance (VUS) in an NS gene, hence we aimed to investigate chorionic villus sampling (CVS) cells taken during the first trimester of pregnancy as a system for direct, patient cell-based functional analysis. We tested Ras/MAPK pathway functional readouts based on established assays in immortalized cell lines, as well as a novel kinase profiling assay and transcriptomic analysis. We found no significant differences between CVS harbouring NS variants and CVS controls when assessing ERK1/2 activation via Western blot, RAS activation via ELISA, or analysis via the PamGene® Serine/Threonine kinase assay. This suggests a potential compensatory mechanism in CVS cells to maintain Ras/MAPK homeostasis within a cell type vital to foetal development. Nonetheless, RNA sequencing of CVS cells displayed discrete differences between the transcriptomic profiles of NS cases compared to controls, including factors related to Ras/MAPK regulation such as VEGFA, providing promising leads for the development of a direct functional readout.