Spatial regulation of gene expression in nonsyndromic sagittal craniosynostosis

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OBJECTIVE

Cranial suture patterning and development are highly regulated processes that are not entirely understood. While studies have investigated the differential gene expression for different sutures, little is known about gene expression changes during suture fusion. The aim of this study was to examine gene expression in patent, fusing, and fused regions along sagittal suture specimens in nonsyndromic craniosynostosis patients.

METHODS

Sagittal sutures were collected from 7 patients (average age 4.5 months) who underwent minimally invasive craniotomies at the Children’s Hospital of Richmond at VCU under IRB approval. The sutures were analyzed using micro-CT to evaluate patency. The areas were classified as open, fusing, or fused and were harvested, and mRNA was isolated. Gene expression for bone-related proteins, osteogenic and angiogenic factors, transforming growth factor–β (TGF-β) superfamily, and Wnt signaling was analyzed using quantitative polymerase chain reaction and compared with normal sutures collected from fetal demise tissue (control).

RESULTS

Micro-CT demonstrated that there are variable areas of closure along the length of the sagittal suture. When comparing control samples to surgical samples, there was a significant difference in genes for Wnt signaling, TGF-β, angiogenic and osteogenic factors, bone remodeling, and nuclear rigidity in mRNA isolated from the fusing and fused areas of the sagittal suture compared with patent areas (p < 0.05).

CONCLUSIONS

In nonsyndromic sagittal craniosynostosis, the affected suture has variable areas of being open, fusing, and fused. These specific areas have different mRNA expression. The results suggest that BMP-2, FGFR3, and several other signaling pathways play a significant role in the regulation of suture fusion as well as in the maintenance of patency in the normal suture.

ABBREVIATIONS MSC = mesenchymal stem cell; qPCR = quantitative polymerase chain reaction; TGF-β = transforming growth factor–β.

Article Information

Correspondence René Olivares-Navarrete: VCU School of Engineering, Virginia Commonwealth University, Richmond, VA. ronavarrete@vcu.edu.

INCLUDE WHEN CITING Published online September 14, 2018; DOI: 10.3171/2018.6.PEDS18229.

G.N.C. and J.W.O. contributed equally to this work.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

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Figures

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    Reconstruction of sagittal suturectomy tissue samples using micro-CT. Samples were collected by minimally invasive suturectomy. A: Regions of suture from synostotic samples (n = 7) were classified as patent (green) if there was a continuous space from the internal to external face of the suture, fusing (magenta) if the internal face was fused but the external was not, or fused (blue) if the region was fully fused from the internal to external face. B: Control samples, collected from deceased fetuses (n = 4), were all patent. Figure is available in color online only.

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    Differential expression of genes associated with calvarial bone formation and homeostasis. Gene expression was analyzed using PCR array. A: Heat map characterizing the magnitude of upregulation of 90 genes with respect to fetal demise controls. B–D: Volcano plots demonstrating the pattern of upregulation of genes in patent (B), fusing (C), and fused (D) craniosynostosis patient samples compared with control; dotted lines indicate threshold of statistical significance. Data are presented as log2(fold change). Figure is available in color online only.

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    Upregulation of bone-associated and TGF-β superfamily genes in patent, fusing, and fused craniosynostotic samples. Gene expression was evaluated by PCR. Data are presented as fold change (2−ΔΔCt) with respect to control samples and to respective sample housekeeping gene expression. Figure is available in color online only.

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    Differential regulation of Wnt signaling in patent, fusing, and fused craniosynostotic suture. Gene expression was evaluated by PCR. Data are presented as fold change (2−ΔΔCt) with respect to control samples and to respective sample housekeeping gene expression. Figure is available in color online only.

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    Upregulation of genes associated with angiogenesis and mechanotransduction in craniosynostotic sagittal suture. Gene expression was evaluated by PCR. Data are presented as fold change (2−ΔΔCt) with respect to control samples and to respective sample housekeeping gene expression. Figure is available in color online only.

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