Next-generation sequencing identifies rare variants associated with Noonan syndrome

Abstract

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.

Keywords: PTPN11; RAS; developmental diseases; human genetics; whole exome sequencing.

Fig. 1.

NS-associated NF1 variants are loss-of-function alleles. (A) Variants found in NS patients: RAS-GAP, RAS-GTPase activating protein-related; SEC, Sec14p-like lipid-binding domain. (B) NS-associated residue (pink) maps to RAS-GAP domain. RAS binding sites are in yellow. (C) WT and mutant NF1-GRD constructs express similar mRNA levels following transient transfection. Real-time qPCRs were performed using the indicated primers (arrows). (D) NS-associated NF1 variant encodes unstable protein. HEK293T cells were cotransfected with expression constructs for HA-ERK1 and WT NF1-GRD or NF1-GRD^L1361R, starved, and restimulated with EGF. Lysates were immunoblotted, as indicated. Note decreased level of the mutant and its inability to inhibit EGF-evoked ERK activation. Triangles show HA-ERK1. A representative result from three biological replicates is shown.

Fig. 2.

NS-associated MAP2K1 variant is gain-of-function allele. (A) Position of MAP2K1 variant: ED, ERK docking; NE, nuclear export; NR, negative regulation; PKc, protein kinase catalytic; PR, proline-rich domains. (B) Increased activity of variant. HA-ERK1 and FL-tagged WT MEK1 or NS-associated MEK1^D67N were transiently cotransfected into HEK293T cells. Transfected cells were starved and restimulated with EGF as indicated, and cell lysates were subjected to immunoblotting. Note that the MEK1 variant evokes constitutive ERK activation. Triangles indicate HA-ERK1. A representative result from three biological replicates is shown.

Fig. 3.

NS-associated RIT1 mutants activate RAS-ERK pathway. (A) Positions of variants are shown. Like all RAS family members, RIT1 contains five conserved functional domains: phosphate binding (G1 and G3), GTP binding and hydrolysis (G4 and G5), and effector protein binding (G2). Switch II indicates region homologous to switch II domain in RAS. (B) Representative immunoblots of lysates from cell lines expressing FL-tagged WT RIT1 or RIT variants, which were either nonsynchronized (NS) or starved (0) and then restimulated with EGF as indicated, lysed, and immunoblotted. Note increased basal and/or stimulated ERK1/2 phosphorylation in RIT1 mutant (AG, AP, FV, and GA) compared with WT RIT1-expressing cells. Lysates from cells expressing dominant-negative RIT1 (SN) or known activating mutant (QL) are also shown. Representative result from three independent biological replicates. (C) Representative immunoblots of lysates from cell lines expressing FL-tagged WT RIT1 or RIT variants, treated with cycloheximide, and then harvested, as indicated. Lysates from 1.5 × 10⁵ (AG, AP, FV, and QL) or 3 × 10⁵ (WT and SN) cell equivalents were immunoblotted with anti-FLAG antibodies. Note the increased stability of RIT1 mutants compared with WT or SN RIT. Also see Fig. S7.

Fig. 4.

NS-associated RASA2 variants are loss-of-function alleles. (A) Positions of variants: C2A, C2 domain first repeat; C2B, C2 domain second repeat; RAS-GAP, RAS-GTPase activating protein; PH, Pleckstrin homology-like; BTK, Bruton’s tyrosine kinase cysteine-rich motif. (B) NS-associated residues (pink) map to RAS-GAP domain. Note that R511 is a putative RAS binding site (yellow). (C) RASA2 depletion enhances ERK1/2 activation. HEK293T cells transfected with either of two siRNAs for 48 h were lysed and immunoblotted. Note that increased ERK1/2 phosphorylation in RASA2-depleted cells correlates inversely with RASA2 level, with ∼50% reduction in RASA2 increasing ERK activation. (D) NS-associated RASA2 variants have defective GAP activity. Activated RAS levels in HEK293T cells transiently transfected with MYC-tagged WT RASA2 or NS-associated RASA2 variant (YC, YN, and RC) constructs were measured by RAF-RBD binding. Note that WT RASA2 suppresses, whereas NS-associated RASA2 variants enhance, RAS activation compared with controls. (E) Cell lines expressing Flag-tagged WT RASA2 or the indicated RASA2 variants (YC, YN, and RC) were starved and then restimulated with EGF. Lysates were immunoblotted, as indicated. A representative result from three biological replicates is shown.