Germline missense mutations affecting KRAS Isoform B are associated with a severe Noonan syndrome phenotype

Abstract

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring-binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor.

Figure 1.

Heterozygous KRAS missense mutations causing a severe Noonan syndrome phenotype with features overlapping those of CFC and CS. A, Schematic representation of the structural and functional domains defined within RAS proteins. The conserved domain (G domain) is indicated, together with the motifs required for signaling function. The hypervariable region is shown (gray), as is the C-terminal motifs that direct posttranslational processing and plasma membrane anchoring (dark gray). The arrow indicates the location of affected residues. B, KRAS gene organization and transcript processing that produces the alternative KRAS isoforms A and B (Gene accession numbers NC_000012, NM_004985, and NM_033360). The numbered black and gray boxes indicate the invariant coding exons and the exons undergoing alternative splicing, respectively. KRASB mRNA results from exon 5 skipping. In KRASA mRNA, exon 6 encodes the 3′ UTR. The arrows indicate locations of mutations. C, DHPLC elution profiles (left) and electropherograms (right) of KRAS exon 6 PCR products showing the de novo heterozygous 455T→G (above) and 458A→T (below) changes. D, Dysmorphic facial features of individual 2 (with Asp153Val).

Figure 2.

Structural analyses. A, Superimposition of the crystallographic structures of HRAS (yellow) and RRAS (light blue), in the region encompassing the β6 strand and α5 helix. The side chain of HRAS residues Arg¹⁴⁹ (β6-α5 loop) and Glu¹⁵³ (α5) and the corresponding RRAS residues Arg¹⁷⁶ and Asp¹⁸⁰ are shown as a stick model. B, Crystallographic structure of GTP-HRAS showing the interactions of residues 152 and 153. The side chain of Val¹⁵² (red surface) participates in a hydrophobic core involving residues Leu¹⁹, Gln²², and Leu²³ (gray surfaces); Ala¹⁴⁶ (pink surface); and Arg¹⁴⁹ (semitransparent yellow surface). The side chains of residues 149 and 153, forming a salt bridge, are shown as a stick model. The yellow dashed line indicates the hydrogen bond formed by the N-H of the Ala¹⁴⁶ backbone and the O6 atom (red stick) of the GTP molecule (blue sticks). C, Superimposition of the crystallographic structures of GTP-HRAS (light blue) and GDP-HRAS (yellow) complexes. A ribbon representation is used for the protein backbone, whereas GDP and GTP are shown as a stick model. The positions of the mutated residues (N-terminus of the α5 helix) and the G-5 motif are shown in red and pink, respectively. Regions assuming a different conformation in the two complexes (Switch I [residues 32–38] and Switch II [residues 59–67]) are shown in orange (in GDP-HRAS) and blue (in GTP-HRAS).