Whole Genome Expression Profiling of Semitendinosus Tendons from Children with Diplegic and Tetraplegic Cerebral Palsy

Abstract

Cerebral palsy (CP) is the most common movement disorder in children, with a prevalence ranging from 1.5 to 4 per 1000 live births. CP is caused by a non-progressive lesion of the developing brain, leading to progressive alterations of the musculoskeletal system, including spasticity, often leading to the development of fixed contractures, necessitating tendon lengthening surgery. Total RNA-sequencing analysis was performed on semitendinosus tendons from diplegic and tetraplegic CP patients subjected to tendon lengthening surgery compared to control patients undergoing anterior cruciate ligament reconstructive surgery. Tetraplegic CP patients showed increased expression of genes implicated in collagen synthesis and extracellular matrix (ECM) turnover, while only minor changes were observed in diplegic CP patients. In addition, tendons from tetraplegic CP patients showed an enrichment for upregulated genes involved in vesicle-mediated transport and downregulated genes involved in cytokine and apoptotic signaling. Overall, our results indicate increased ECM turnover with increased net synthesis of collagen in tetraplegic CP patients without activation of inflammatory and apoptotic pathways, similar to observations in athletes where ECM remodeling results in increased tendon stiffness and tensile strength. Nevertheless, the resulting increased tendon stiffness is an important issue in clinical practice, where surgery is often required to restore joint mobility.

Keywords: RNA-sequencing; cerebral palsy; extracellular matrix; gene expression; tendons.

Figure 1

RNA-Seq analysis on semitendinosus tendons from 12 diplegic CP (D), 12 tetraplegic CP (T), and 5 control (C) patients. (A) Principal components analysis (PCA) based on transcriptional profiles with ellipses of confidence intervals set at 0.9 for each group. (B) Heat map of unsupervised hierarchical clustering of 1223 differentially expressed protein-coding genes (FDR ≤ 0.1; |log2FC| ≥ 0.4). (C) Venn diagram of the pairwise differential expression (DE) analysis (FDR ≤ 0.1; |log2FC| ≥ 0.4).

Figure 2

EnrichR analysis for upregulated and downregulated genes in semitendinosus tendons from tetraplegic CP (T) patients vs. control (C) patients (FDR ≤ 0.1; |log2FC| ≥ 0.4). (A) Gene ontology (GO) analysis for biological processes, and (B) Reactome analysis (adjusted p value ≤ 0.05). For upregulated genes, the 20 most significantly enriched GO terms and pathways are shown.

Figure 3

Differentially expressed genes in semitendinosus tendons from tetraplegic (T) and diplegic (D) CP patients vs. control (C) patients involved in ECM organization (A), membrane trafficking (B), cytokine signaling (C), and apoptotic processes (D) (FDR ≤ 0.1; |log2FC| ≥ 0.4).

Figure 4

qRT-PCR analysis for selected differentially expressed genes on tendon RNA from tetraplegic (T) and diplegic (D) CP patients vs. control (C) patients. Data are represented as mean ± standard error of the mean (SEM) (n = 5–7 biological replicates and 3 technical replicates per group). Data were normalized to GAPDH. * p < 0.05; ** p < 0.01; *** p < 0.001; one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.