A restricted spectrum of mutations in the SMAD4 tumor-suppressor gene underlies Myhre syndrome

Abstract

Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor β and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth.

Figure 1

A Restricted Spectrum of Germline Missense Mutations in SMAD4 Underlies Myhre Syndrome (A) Clinical features of representative subjects (from left, OBG_01, TO_01, and TO_02) carrying the heterozygous SMAD4 mutations. Pictures illustrate the distinctive facial features, muscular build, joint stiffness, camptodactyly, and brachydactyly. Radiographs show the thickening of the skull bones, vertebrae with large pedicles and irregular endplates, wide ribs, and hypoplastic iliac wings. (B) The location of Ile500 is shown below the SMAD4 domain structure scheme. SMAD4 comprises an N-terminal MAD homology 1 domain (MH1) connected by a linker to the SMAD4 activation domain (SAD) and the C-terminal MAD homology 2 domain (MH2). Numbers below the domain structure indicate the amino acid boundaries of those domains. Nonsense (red circle) and truncating (pink triangle) mutations reported in juvenile polyposis syndrome (JPS) and JPS-hereditary hemorrhagic telangiectasia are shown below the illustration. The distribution of somatic SMAD4 mutations occurring in cancer (blue-filled peaks) is shown above the illustration. The location of Lys519, which is the residue subject to ubiquination, is also shown.

Figure 2

Location of Ile500 in the Spatial Structure of the SMAD3/SMAD4 MH2 Domain Complex (A) Solvent accessible surface representation of the SMAD4/SMAD3 MH2 domain complex (Protein Data Bank [PDB] ID codes 1U7F). The SMAD4 monomer is shown in cyan, whereas the two SMAD3 subunits are colored in yellow and white. The solvent accessible surface of residues affected by somatic (blue) and germline (magenta) mutations and lesions occurring as both germline and somatic events (green) are shown. The residue mutated in Myhre syndrome, Ile500, is shown in red. The red and blue ovals indicate the location of the loop/helix and helix bundle SMAD4 subdomains, respectively. Orange ovals indicate the position of the serine residues that are phosphorylated in activated SMAD3. (B) Cα ribbon trace (PDB ID codes 1U7F) of SMAD4 MH2 domain (cyan) and those of the two interacting SMAD3 subunits (orange and gray). Ile500 is represented in space-filling mode (red). Residues interacting with Ile500 (Arg496, Arg497, Arg502, Glu526, and His528) are shown as blue sticks. Asp493 (green), which is important for interdomain interactions, and Lys519 (violet), which is subject to ubiquination, are also shown. SMAD3 residues directly contributing to interactions with the evidenced SMAD4 residues (Ser275, Arg279, Glu284, and Arg287) are shown as yellow sticks, together with the phosphorylated SMAD3 serine residues (brown). The anion binding site located in proximity of Ile500, as indicated by the structure of the SMAD4 homotrimer (pdb code 1DD1), is shown by a yellow sphere. Visualization and analysis of the molecular structure was performed with the program UCSF Chimera.