Background: Tibial pseudarthrosis is associated with neurofibromatosis type 1 (NF1) and there is wide clinical variability of the tibial dysplasia in NF1, suggesting the possibility of genetic modifiers. Double inactivation of NF1 is postulated to be necessary for the development of tibial pseudarthrosis, but tissue or cell of origin of the 'second hit' mutation remains unclear. Methods: Exome sequencing of different sections of surgically resected NF1 tibial pseudarthrosis tissue was performed and compared to germline ( peripheral blood). Results: A germline NF1 splice site mutation (c.61- 2A>T, p.L21 M68del) was identified from DNA extracted from peripheral blood. Exome sequencing of DNA extracted from tissue removed during surgery of the tibial pseudarthrosis showed a somatic mutation of NF1 (c.3574G>T, p.E1192*) in the normal germline allele. Further analysis of different regions of the tibial pseudarthrosis sample showed enrichment of the somatic mutation in the soft tissue within the pseudarthrosis site and absence of the somatic mutation in cortical bone. In addition, a germline variant in PTPN11 (c.1658C>T, p.T553M), a gene involved in the RAS signal transduction pathway was identified, although the clinical significance is unknown. Conclusions: Given that the NF1 somatic mutation was primarily detected in the proliferative soft tissue at the pseudarthrosis site, it is likely that the second hit occurred in mesenchymal progenitors from the periosteum. These results are consistent with a defect of differentiation, which may explain why the mutation is found in proliferative cells and not within cortical bone tissue, as the latter by definition contains mostly mature differentiated osteoblasts and osteocytes.
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