Neurofibromin deficiency-associated transcriptional dysregulation suggests a novel therapy for tibial pseudoarthrosis in NF1

Abstract

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in NF1. Among the earliest manifestations is tibial pseudoarthrosis and persistent nonunion after fracture. To further understand the pathogenesis of pseudoarthrosis and the underlying bone remodeling defect, pseudoarthrosis tissue and cells cultured from surgically resected pseudoarthrosis tissue from NF1 individuals were analyzed using whole-exome and whole-transcriptome sequencing as well as genomewide microarray analysis. Genomewide analysis identified multiple genetic mechanisms resulting in somatic biallelic NF1 inactivation; no other genes with recurring somatic mutations were identified. Gene expression profiling identified dysregulated pathways associated with neurofibromin deficiency, including phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Unlike aggressive NF1-associated malignancies, tibial pseudoarthrosis tissue does not harbor a high frequency of somatic mutations in oncogenes or other tumor-suppressor genes, such as p53. However, gene expression profiling indicates that pseudoarthrosis tissue has a tumor-promoting transcriptional pattern, despite lacking tumorigenic somatic mutations. Significant overexpression of specific cancer-associated genes in pseudoarthrosis highlights a potential for receptor tyrosine kinase inhibitors to target neurofibromin-deficient pseudoarthrosis and promote proper bone remodeling and fracture healing.

Keywords: CELL/TISSUE SIGNALING-TRANSCRIPTION FACTORS; DISEASES AND DISORDERS RELATED TO BONE-OTHER; HUMAN ASSOCIATION STUDIES; MOLECULAR PATHWAYS-DEVELOPMENT; TUMOR-INDUCED BONE DISEASE.

Figure 1. Genomic analysis in tibial pseudoarthrosis

(A) Homozygosity plots from microarray SNP genotyping of DNA from cells cultured from tibial pseudoarthosis (top) and blood (bottom) from one individual (NF#7) are shown. Somatically acquired homozygosity of the entire q-arm of chromosome 17 was evident and does not result from chromosomal deletion (data not shown). The split signal is consistent with a mix of NF1-haploinsufficient and pseudoarthrosis cells. Red asterisk shows the location of NF1. (B) RNA-sequencing from cells cultured from pseudoarthrosis (top) and iliac crest (bottom) from the same individual (NF#7). The constitutional c.4537C>T (p.Arg1513*) mutation is shown, with the proportion of reads shown above for normal (blue) and mutant (red) alleles. While the mutant allele was expressed less compared to normal in the iliac crest cells, the mutant allele was expressed higher than the normal allele in the pseudoarthrosis sample, suggesting homozygosity of the mutant allele in pseudoarthrosis.

Figure 2. Expression profiling in tibial pseudoarthrosis

(A) Hierarchical clustering of 258 differentially expressed genes associated with neurofibromin deficiency identified 76 genes upregulated and 182 genes downregulated in pseudoarthrosis samples. Expression is shown as a heatmap with low (blue) to high (red) relative expression. Sample groups are shown on the left, including control (black), iliac crest (yellow) and pseudoarthrosis (green). (B) Venn diagram of genes significantly upregulated in pseudoarthrosis samples and annotated in the PI3K-Akt Signaling, Cancer and/or MAPK Signaling pathways.

Figure 2. Expression profiling in tibial pseudoarthrosis

(A) Hierarchical clustering of 258 differentially expressed genes associated with neurofibromin deficiency identified 76 genes upregulated and 182 genes downregulated in pseudoarthrosis samples. Expression is shown as a heatmap with low (blue) to high (red) relative expression. Sample groups are shown on the left, including control (black), iliac crest (yellow) and pseudoarthrosis (green). (B) Venn diagram of genes significantly upregulated in pseudoarthrosis samples and annotated in the PI3K-Akt Signaling, Cancer and/or MAPK Signaling pathways.