Regulatory role of RASIP1 in cardiovascular cell differentiation

Abstract

Noonan syndrome (NS) an autosomal dominant and genetically heterogeneous disorder is characterized by short stature developmental delay and congenital heart diseases Previously we identified candidate NS-associated variants in RASIP1 through exome sequencing Patients carrying RASIP1 variants showed atypical cardiac phenotypes for NS such as dilation of aortic root coarctation of aorta and aortic aneurysms which raise the possibility that these cardiac phenotypes occurred specifically to NS patients carrying RASIP1 To investigate the molecular mechanisms through which RASIP1 mutations lead to NS we aim to generate cell models that carry patient-specific RASIP1 mutations through genome editing in the induced pluripotent stem cells (iPSCs) Because iPSCs have the ability to differentiate into many cell lineages of cardiovascular cells we can study the impact of mutations on the function of cardiovascular cells Previous studies have successfully utilized CRISPR system in iPSCs however the efficiency of CRISPR/Cas9 mediated knock-in was only 2-3% in iPSCs In this study I aim to improve the homology-direct repair (HDR) rates in iPSCs and generate iPSCs carrying patient-specific mutations I used RNA-sequencing (RNA-seq) to establish double strand break-related gene profile in iPSCs I also generated a reporter system in iPSCs In addition I compared the HDR rate in cells supplied with long dsDNA donor and that with single strand oligodeoxy nucleotides Moreover I differentiated RASIP1 knock-out (RKO) iPSCs into cardiac cell lineages and found that the differentiation ability of RKO iPSCs was decreased The ability of forming plexus like vascular structures was also decrease Furthermore results of RNA-seq suggested that several signaling pathways were altered in RKO cells Lastly I found that phosphorylated-GSK3β level was altered in RKO cells Taken together these results revealed the possible mechanisms through which RASIP1 mutation affect the cardiovascular cell differentiation and function

Date of Award	2018 Sept 4
Original language	English
Supervisor	Peng-Chieh Chen (Supervisor)