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. 2017 Aug 29;114(35):E7341-E7347.
doi: 10.1073/pnas.1709255114. Epub 2017 Aug 14.

De novo mutations in inhibitors of Wnt, BMP, and Ras/ERK signaling pathways in non-syndromic midline craniosynostosis

Andrew T Timberlake  1   2 , Charuta G Furey  1   3 , Jungmin Choi  1 , Carol Nelson-Williams  1 ; Yale Center for Genome Analysis; Erin Loring  1 , Amy Galm  4 , Kristopher T Kahle  3 , Derek M Steinbacher  2 , Dawid Larysz  5 , John A Persing  2 , Richard P Lifton  6   7
Collaborators, Affiliations

De novo mutations in inhibitors of Wnt, BMP, and Ras/ERK signaling pathways in non-syndromic midline craniosynostosis

Andrew T Timberlake et al. Proc Natl Acad Sci U S A. .

Abstract

Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent-offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10-11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

Keywords: BMP signaling; Ras/ERK signaling; Wnt signaling; craniosynostosis; de novo mutation.

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Conflict of interest statement

Conflict of interest statement: J.S. and R.P.L. were among 42 coauthors on a 2015 review article.

Figures

Fig. S1.
Fig. S1.
Number of de novo mutations identified per proband in 291 craniosynostosis trios. The blue bars represent the observed distributions of de novo mutations identified per proband in craniosynostosis kindreds, and the red bars represents the expected number from the Poisson distribution. The observed distribution closely approximates expectation.
Fig. 1.
Fig. 1.
De novo mutations in craniosynostosis probands identified in negative regulators of Wnt, BMP, and Ras-ERK signal transduction. Schematic of signaling in Wnt (A), BMP (B), and RAS/ERK (C) pathways are shown. All contribute to osteoblast differentiation and bone formation via common transcriptional targets. Genes in italics are mutated in syndromic craniosynostosis. Genes in red font are negative regulators of signaling that have damaging de novo mutations in probands. Genes noted with yellow stars are known syndromic genes found mutated in kindreds with midline NSC.
Fig. 2.
Fig. 2.
Quantile-quantile plot of P values for LOF alleles in all protein-coding genes in 384 craniosynostosis probands. Rare (ExAC frequency < 2 × 10−5) LOF alleles were identified in probands. The probability of the observed number of variants in each gene occurring by chance was calculated from the total number of observed variants and the proportion of the coding length of the exome comprising each gene using the binomial test. (A) The observed distribution of P values matches the expected binomial distribution with the exception of SMAD6, in which 12 LOF alleles are observed compared with the expected 0.10. (120-fold enrichment; P = 2.28 × 10−21). (B) Distribution of all damaging mutations in SMAD6.
Fig. S2.
Fig. S2.
SMAD6 and BMP2 genotypes in probands with sagittal or metopic craniosynostosis. Pedigrees harboring de novo (denoted by stars within pedigree symbols) or rare transmitted variants in SMAD6. Filled and unfilled symbols denote individuals with and without craniosynostosis, respectively. The SMAD6 mutation identified in each kindred is noted above each pedigree. Below each symbol, genotypes are shown first for SMAD6 (with “D” denoting the damaging allele) and for rs1884302 risk locus downstream of BMP2, (with “T” conferring protection from and “C” conferring increased risk of sagittal craniosynostosis). Maximum LOD scores under two-locus and single-locus linkage models for each kindred are shown in Tables S8 and S9.
Fig. S3.
Fig. S3.
Pedigrees of kindreds with atypical presentations resulting from mutations in known craniosynostosis genes. Four kindreds with dominant transmission of craniosynostosis and novel alleles in genes known to cause syndromic craniosynostosis. Filled and unfilled circles represent the presence or absence of craniosynostosis, with “D” representing the mutation specified above the pedigree, and “+” representing a wild-type allele. In each kindred, all affected individuals share the mutation found in the proband. Sanger sequence electropherograms demonstrating the mutation in each pedigree are shown. None of the affected individuals display the traits typical of the syndrome associated with each gene.
Fig. S4.
Fig. S4.
Estimation of the number of craniosynostosis risk genes by simulation. The likelihood of the model under the assumption of there being 0–1,000 risk genes is plotted, with maximum likelihood observed at 190 genes (Methods).
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