Next-generation sequencing identifies rare variants associated with Noonan syndrome

Significance Noonan syndrome (NS) is one of several RASopathies, which are developmental disorders caused by mutations in genes encoding RAS-ERK pathway components. The cause of 20–30% of NS cases remains unknown, and distinguishing NS from other RASopathies and related disorders can be difficult. We used next-generation sequencing (NGS) to identify causative or candidate genes for 13 of 27 NS patients lacking known NS-associated mutations. Other patients harbor single variants in potential RAS-ERK pathway genes, suggesting rare private variants or other genetic mechanisms of NS pathogenesis. We also found mutations in causative genes for other developmental syndromes, which together with clinical reevaluation, prompted revision of the diagnosis. NGS can aid in the challenging diagnosis of young patients with developmental syndromes. Noonan syndrome (NS) is a relatively common genetic disorder, characterized by typical facies, short stature, developmental delay, and cardiac abnormalities. Known causative genes account for 70–80% of clinically diagnosed NS patients, but the genetic basis for the remaining 20–30% of cases is unknown. We performed next-generation sequencing on germ-line DNA from 27 NS patients lacking a mutation in the known NS genes. We identified gain-of-function alleles in Ras-like without CAAX 1 (RIT1) and mitogen-activated protein kinase kinase 1 (MAP2K1) and previously unseen loss-of-function variants in RAS p21 protein activator 2 (RASA2) that are likely to cause NS in these patients. Expression of the mutant RASA2, MAP2K1, or RIT1 alleles in heterologous cells increased RAS-ERK pathway activation, supporting a causative role in NS pathogenesis. Two patients had more than one disease-associated variant. Moreover, the diagnosis of an individual initially thought to have NS was revised to neurofibromatosis type 1 based on an NF1 nonsense mutation detected in this patient. Another patient harbored a missense mutation in NF1 that resulted in decreased protein stability and impaired ability to suppress RAS-ERK activation; however, this patient continues to exhibit a NS-like phenotype. In addition, a nonsense mutation in RPS6KA3 was found in one patient initially diagnosed with NS whose diagnosis was later revised to Coffin–Lowry syndrome. Finally, we identified other potential candidates for new NS genes, as well as potential carrier alleles for unrelated syndromes. Taken together, our data suggest that next-generation sequencing can provide a useful adjunct to RASopathy diagnosis and emphasize that the standard clinical categories for RASopathies might not be adequate to describe all patients.