Germline-Activating RRAS2 Mutations Cause Noonan Syndrome

Abstract

Noonan syndrome (NS) is characterized by distinctive craniofacial appearance, short stature, and congenital heart disease. Approximately 80% of individuals with NS harbor mutations in genes whose products are involved in the RAS/mitogen-activating protein kinase (MAPK) pathway. However, the underlying genetic causes in nearly 20% of individuals with NS phenotype remain unexplained. Here, we report four de novo RRAS2 variants in three individuals with NS. RRAS2 is a member of the RAS subfamily and is ubiquitously expressed. Three variants, c.70_78dup (p.Gly24_Gly26dup), c.216A>T (p.Gln72His), and c.215A>T (p.Gln72Leu), have been found in cancers; our functional analyses showed that these three changes induced elevated association of RAF1 and that they activated ERK1/2 and ELK1. Notably, prominent activation of ERK1/2 and ELK1 by p.Gln72Leu associates with the severe phenotype of the individual harboring this change. To examine variant pathogenicity in vivo, we generated zebrafish models. Larvae overexpressing c.70_78dup (p.Gly24_Gly26dup) or c.216A>T (p.Gln72His) variants, but not wild-type RRAS2 RNAs, showed craniofacial defects and macrocephaly. The same dose injection of mRNA encoding c.215A>T (p.Gln72Leu) caused severe developmental impairments and low dose overexpression of this variant induced craniofacial defects. In contrast, the RRAS2 c.224T>G (p.Phe75Cys) change, located on the same allele with p.Gln72His in an individual with NS, resulted in no aberrant in vitro or in vivo phenotypes by itself. Together, our findings suggest that activating RRAS2 mutations can cause NS and expand the involvement of RRAS2 proto-oncogene to rare germline disorders.

Keywords: Noonan syndrome; RAS/MAPK; RASopathies; RRAS2; exome sequencing; functional profiling; macrocephaly; zebrafish.

Figure 1

RRAS2 Mutations Identified in Individuals with Noonan Syndrome (A) Exon-intron structure of RRAS2 (upper) and functional domains of RRAS proteins (lower). RRAS2 variants in individuals with Noonan syndrome (NS) were located in phosphate-binding loop or switch II region. (B) Crystal structure of human RRAS2. Phosphate-binding loop, switch I, switch II, GDP, and residues mutated in individuals with Noonan syndrome (NS) are highlighted in pink, purple, blue, yellow, and red, respectively. (C) Partial amino acid sequence alignment of human RRAS2, RRAS, MRAS, KRAS, HRAS, and NRAS. High conservation of residues of Gly24-Gly26 and Gln72 through all paralogs are shown. Conservation of residues at Phe75 was limited in the RRAS subfamily. Red circles indicate hotspot residues mutated in various cancers in KRAS, HRAS, and NRAS. (D and E) Photos of individual NS833. She had the definitive facial appearance of NS at 4 years of age. (F–J) Photos of individual HU1 at (F) neonatal period, (G) 7 months, (H) 22 months, and (I and J) 3 years of age. (K) Axial slice of brain magnetic resonance image of HU1 taken at 6 months of age shows ventriculomegaly and widened subarachnoid spaces in the frontal and temporal lobes.

Figure 2

Functional Assays of RRAS2 Variants (A) Representative immunoblots of three independent experiments. HEK293 cells transfected with WT or mutant RRAS2 constructs or empty vector (Mock) were used for pull-down assays and immunoblotting. RRAS2-guanosine triphosphate (GTP) which was pulled down using RAF1-RBD agarose, total RRAS2, phospho-MEK1/2, total MEK1/2, phospho-ERK1/2, total ERK1/2, and β-actin as a loading control were shown. WT, wild-type. (B) Stimulation of ELK transcription by RRAS2 mutants. ELK-GAL4 and GAL4-luciferase trans-reporter vectors were transiently co-transfected with RRAS2 constructs into unstimulated HEK293 cells. Relative luciferase activity (RLA) was normalized to the activity of a co-transfected control vector (phRLnull-luc) expressing Renilla reniformis luciferase. Folds under each bar were calculated as a multiple of WT. Data are presented as mean ± SD; n = 3 per group. WT, wild-type. ^∗∗∗p < 0.001 compared with WT.

Figure 3

Morphology of Zebrafish Larvae Injected with Wild-Type (WT) or Mutant RRAS2 mRNA at 3 Days Post Fertilization (dpf) (A) Tg(−1.4col1a1:egfp) transgenic embryos, in which cartilage cells are marked by EGFP, were injected at the 1- to 4-cell stage with RNA encoding RRAS2-WT or variants identified in affected individuals. The angle of the ceratohyal cartilage was measured at 3 dpf. Representative images of an uninjected control (UI) and RRAS2-variant mRNA injected larvae are depicted. Scale bar: 200 μm. (B) Quantification of the ceratohyal angle. Left: we injected 25 pg mRNA encoding each indicated RRAS2 condition. n = 61–80 embryos per batch. n.s., not significant; ^∗∗p < 0.001 by Dunnett’s test. Right: 5 pg of p.Gln72Leu endoding mRNA was injected. n = 39–56 embryos per batch. ^∗p < 0.01 by Student’s t test. The thick line in the box represents median value; the bottom and top lines of the box represent first and third quartiles, respectively; the whiskers extend from the hinge to the lowest or highest value that is within 1.5-fold of interquartile range from the hinge; the filled circles are outliers. (C) The body (lower arrow) and head (upper arrow) length were measured at 3 dpf. Representative images of an uninjected control (UI) and RRAS2-mRNA injected larvae are shown. Scale bar: 500 μm. (D and E) Quantification of body length (D) and relative head size, which was the value of the head length divided by the value of the body length (E). Left: we injected 25 pg of each RRAS2 mRNA. n = 61–82 embryos per batch. n.s., not significant; ^∗∗p < 0.001 by Dunnett’s test. Right: 5 pg of p.Gln72Leu encoding RNA was injected. n = 36–53 embryos per batch. n.s., not significant by Student’s t test. The thick line in the box represents median value; the bottom and top lines of the box represent first and third quartiles, respectively; the whiskers extend from the hinge to the lowest or highest value that is within 1.5-fold of interquartile range from the hinge; the filled circles are outliers.