Abstract
Noonan syndrome is an autosomal dominant developmental disorder. Although it is relatively common, and its phenotypical variability is well documented, its pathophysiology is not fully understood. Previously, with the aim of revealing the pathogenesis of genetic disorders, we reported the induction of cleidocranial dysplasia-specific human-induced pluripotent stem cells (hiPSCs) from patient’s dental pulp cells (DPCs) under serum-free, feeder-free, and integration-free conditions. Notably, these cells showed potential for application to genetic disorder disease models. Furthermore, using similar procedures, we reported the induction of hiPSCs derived from peripheral blood mononuclear cells (PBMCs) of healthy volunteers. These methods are beneficial, because they are carried out without invasive and painful biopsies. Using those procedures, we reprogrammed DPCs and PBMCs that were derived from a patient with Noonan syndrome (NS) to establish NS-specific hiPSCs (NS-DPC-hiPSCs and NS-PBMC-hiPSCs, respectively). The induction efficiency of NS-hiPSCs was higher than that of WT-hiPSCs. We hypothesize that this was caused by high NANOG expression. Here, we describe the experimental results and findings related to NS-hiPSCs. This is the first report on the establishment of NS-hiPSCs and their disease modeling.
Highlights
Noonan syndrome (NS:MIM 163950) is an autosomal dominant inherited disorder of the RAS/MAPK signaling pathway, and it is generally characterized by peculiar facial features, Supplementary Information The online version of this article contains supplementary material, which is available to authorized users.(AIST), Tsukuba, Ibaraki, Japan 5 Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan short stature, congenital heart disease, mental retardation, and other characteristics (Noonan, 2006)
DNA isolation and short tandem repeat (STR) analysis The Missense mutation next-generation sequencing (NGS) analysis revealed that the patient patient’s genomic DNA was isolated from patient-derived exhibited a missense mutation in the protein coding sequence of KRAS (456 A > T) indicating substitution of Val for Asp at amino acid position 153 (D153V). This mutation was revealed by MiSeq using a TruSight One Panel and was further confirmed by Sanger sequencing (Fig. 1)
High expression of NANOG in NS-peripheral blood mononuclear cells (PBMCs) The NS-PBMCs cultured in RD6F serum-free medium supplemented with IL-2 showed 9-fold higher expression of NANOG than that of WT-PBMCs
Summary
Congenital mutations in genes such as PTPN 11, SOS 1, RAF 1, KRAS, and BRAF are involved in causing RAS/MAPK pathway abnormalities in NS (Roberts et al 2013). That is why NS is considered a part of the group of so-called RASopathies, which are caused by mutations in genes that encode the ERK/MAPK signaling pathway. The ERK/MAPK signaling pathway is downstream of the fibroblast growth factor receptor. Disruption of the ERK/MAPK signaling pathway during embryogenesis impedes neural crest cell development and causes defects in the structure of the cardiac, craniofacial, and central nervous systems (Makishima et al 2009)
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