Differential expression of extracellular matrix-mediated pathways in single-suture craniosynostosis

Abstract

Craniosynostosis is a disease defined by premature fusion of one or more cranial sutures. The mechanistic pathology of single-suture craniosynostosis is complex and while a number of genetic biomarkers and environmental predispositions have been identified, in many cases the causes remain controversial and inconclusive. In this study, gene expression data from 199 patients with isolated sagittal (n = 100), unilateral coronal (n = 50), and metopic (n = 49) synostosis are compared against both a control population (n = 50), as well as each other. After controlling for variables contributing to potential bias, FGF7, SFRP4, and VCAM1 emerged as genes associated with single-suture craniosynostosis due to their significantly large changes in gene expression compared to the control population. Pathway analysis implicated focal adhesion and extracellular matrix (ECM)-receptor interaction as differentially regulated gene networks when comparing all cases of single-suture synostosis and controls. Lastly, overall gene expression was found to be highly conserved between coronal and metopic cases, as evidenced by the fact that WNT2 and IGFBP2 were the only genes differentially regulated to a significantly large extent in a direct comparison. The identification of genes and gene networks associated with Fgf/Igf/Wnt signaling and ECM-mediated focal adhesion not only support the involvement of biomarkers previously reported to be related to craniosynostosis, but also introduce novel transcripts and pathways that may play critical roles in its pathogenesis.

Figure 1. Comparison of Gene Expression patterns between osteoblasts derived from cases of synostosis and control lines.

Heatmaps with 2-dimensional hierarchical clustering were generated for the 2000 genes with the highest correlation scores for probe expression (A), and enriched subsets of this gene set where expression levels were considered significant (p<0.05) compared to controls (736 genes) (B), or both significant (p<0.05) and large (|% change| >50) compared to controls (49 genes) (C).

Figure 2. MA-plots highlighting differential gene expression between osteoblasts derived from cases of synostosis and control lines.

Genes whose expression was considered to be significant (p<0.05) and large (|% change| >50) are represented by a red “X”, whereas genes whose expression did not meet threshold values are represented by black dots. Comparisons were made between coronal cases and control populations (A), metopic cases and control populations (B), and sagittal cases and control populations (C).

Figure 3. Venn diagram highlighting unique or shared gene sets among different forms of single-suture craniosynostosis.

Venn region m1 contains genes shared among all three cases of single-suture synostosis, genes shared between two cases are contained in Venn regions m2, m3, and m4, and genes unique to a specific case are contained in Venn regions m5 (coronal), m6 (metopic), and m7 (sagittal).

Figure 4. MA plots highlighting differential gene expression by directly comparing osteoblasts derived from cases of synostosis.

Genes whose expression was considered to be significant (p<0.05) and large (|% change| >50) are represented by a red “X”, whereas genes whose expression did not meet threshold values are represented by black dots. Comparisons were made between coronal and metopic cases (A), coronal and sagittal cases (B), and metopic and sagittal cases (C).

Figure 5. Differential expression of genes related to extracellular matrix-mediated focal adhesion in synostosis cases.

Changes in gene expression that were robustly expressed across the population of samples were uploaded into DAVID to identify enriched KEGG pathways potentially affected in craniosynostosis. Genes with significant changes in expression between cases and controls that were related to either focal adhesion or ECM-receptor interactions are mapped in this modified KEGG pathway. Differentially upregulated genes are boxed in red, differentially downregulated genes are boxed in blue, and when up- and downregulated isoforms of the same gene family were observed, mixed expression was assigned (boxed in gray).