Calvarial osteoblast gene expression in patients with craniosynostosis leads to novel polygenic mouse model

Abstract

Craniosynostosis is the premature fusion of the sutures of the calvaria and is principally designated as being either syndromic (demonstrating characteristic extracranial malformations) or non-syndromic. While many forms of syndromic craniosynostosis are known to be caused by specific mutations, the genetic etiology of non-syndromic, single-suture craniosynostosis (SSC) is poorly understood. Based on the low recurrence rate (4-7%) and the fact that recurrent mutations have not been identified for most cases of SSC, we propose that some cases of isolated, single suture craniosynostosis may be polygenic. Previous work in our lab identified a disproportionately high number of rare and novel gain-of-function IGF1R variants in patients with SSC as compared to controls. Building upon this result, we used expression array data from calvarial osteoblasts isolated from infants with and without SSC to ascertain correlations between high IGF1 expression and expression of other osteogenic genes of interest. We identified a positive correlation between increased expression of IGF1 and RUNX2, a gene known to cause SSC with increased gene dosage. Subsequent phosphorylation assays revealed that osteoblast cell lines from cases with high IGF1 expression demonstrated inhibition of GSK3β, a serine/threonine kinase known to inhibit RUNX2, thus activating osteogenesis through the IRS1-mediated Akt pathway. With these findings, we have utilized established mouse strains to examine a novel model of polygenic inheritance (a phenotype influenced by more than one gene) of SSC. Compound heterozygous mice with selective disinhibition of RUNX2 and either overexpression of IGF1 or loss of function of GSK3β demonstrated an increase in the frequency and severity of synostosis as compared to mice with the RUNX2 disinhibition alone. These polygenic mouse models reinforce, in-vivo, that the combination of activation of the IGF1 pathway and disinhibition of the RUNX2 pathway leads to an increased risk of developing craniosynostosis and serves as a model of human SSC.

Fig 1. Heat map representation of RNA expression of genes involved with bone biology.

The candidate genes of interest depicted in this heat map have a statistically significant difference in expression between (A) 23 cases with the highest IGF1 expression levels and unaffected controls and (B) 23 cases with the highest IGF1 expression levels and 188 remaining cases (affected controls). Affected controls represent SSC cases with lower IGF1 expression. IGF1 expression is shown in the far-left column (arrow) and sorted high to low. The expression of each transcript is represented with conditional formatting with green indicating highest and red representing lowest expression among individuals. Candidate gene expression is ordered left to right by the ratio of the mean expression value of the top 23 (high IGF1 cases) to the mean of unaffected controls (A) and affected control cases (B). See S1 Table for the complete gene list and statistical significance.

Fig 2. MicroCT images of representative P28 skulls.

(A) wildtype C57BL/6J mouse with normal calvarial suture pattern, (B) Twist1^(+/-) mouse with right coronal craniosynostosis, (C) Twist1 ^(+/-)/Gsk3β^(+/-) mouse with partial bilateral coronal craniosynostosis, and (D) Twist1^(+/-)/Igf1^(+/tg) mouse with right coronal craniosynostosis. Sutures are labelled as follows: M (metopic or interfrontal), C (coronal), S (sagittal), and L (lambdoid). Arrows designate areas of premature suture fusion.

Fig 3. Percentage of mice with craniosynostosis by genotype.

Mice that harbored mutations in both Twist1 ^(+/-) and Gsk3β^(+/-) showed a significant increase in rate of craniosynostosis as compared to the expected additive outcome of Twist1 ^(+/-) and Gsk3β^(+/-) independently (based on their individual rates of craniosynostosis). Mice that harbored mutations in both Twist1 ^(+/-) and Igf1^(+/tg) showed a near-significant increase in instances of bilateral craniosynostosis as compared to the expected additive outcome of bilateral craniosynostosis in Twist1 ^(+/-) and Igf1^(+/tg) (or Igf1^(tg/tg)) independently.

Fig 4. Schematic representation of intersection of pathways of interest.

(Left Panel) In the absence of IGF1/IGF1R binding, the Akt pathway is inactive and GSK3β Ser9 is unphosphorylated resulting in inhibition of RUNX2 (through phosphorylation of S369, S373, S377) and β-catenin. In parallel, TWIST1 suppresses RUNX2 function via direct interaction of RUNX2 with the twist-box domain of TWIST1. (Right Panel) In our experimental system we tested the hypothesis that dysregulation of both the TWIST1/RUNX2 and IGF1/Akt pathways would result in an increased incidence of craniosynostosis. This schematic demonstrates our hypothesis that the combination of a twist-box mutation of Twist1 and activation of the IGF1/AKT pathway would result in activation of osteogenic transcripts and manifest as craniosynostosis. The pathways shown are derived from previously described data [19, 41, 43, 44].