Sant David W, Margraf Rebecca L, Stevenson David A, Grossmann Allie H, Viskochil David H, Hanson Heather, Everitt Melanie D, Rios Jonathan J, Elefteriou Florent, Hennessey Theresa, Mao Rong
ARUP Laboratories, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA.
Department of Pediatrics, Division of Medical Genetics, Stanford University, Stanford, California, USA Departments of Pediatrics, Division of Medical Genetics, University of Utah, School of Medicine, Salt Lake City, Utah, USA Shriners Hospital for Children Salt Lake City, Salt Lake City, Utah, USA.
J Med Genet. 2015 Apr;52(4):256-61. doi: 10.1136/jmedgenet-2014-102815. Epub 2015 Jan 22.
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.
Exome sequencing of different sections of surgically resected NF1 tibial pseudarthrosis tissue was performed and compared to germline (peripheral blood).
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.
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.
胫骨假关节与1型神经纤维瘤病（NF1）相关，且NF1患者的胫骨发育异常存在广泛的临床变异性，提示可能存在基因修饰因子。据推测，NF1的双等位基因失活是胫骨假关节发生所必需的，但“第二次打击”突变的组织或细胞起源仍不清楚。
对手术切除的NF1胫骨假关节组织的不同部分进行外显子组测序，并与种系（外周血）进行比较。
从外周血提取的DNA中鉴定出一个种系NF1剪接位点突变（c.61-2A>T，p.L21 M68del）。对胫骨假关节手术中切除的组织提取的DNA进行外显子组测序，结果显示正常种系等位基因中存在NF1的体细胞突变（c.3574G>T，p.E1192*）。对胫骨假关节样本不同区域的进一步分析显示，假关节部位软组织中体细胞突变富集，而皮质骨中不存在体细胞突变。此外，还鉴定出PTPN11基因（c.1658C>T，p.T553M）的一个种系变异，该基因参与RAS信号转导途径，但其临床意义尚不清楚。
鉴于NF1体细胞突变主要在假关节部位的增殖性软组织中检测到，第二次打击可能发生在骨膜的间充质祖细胞中。这些结果与分化缺陷一致，这可能解释了为什么突变存在于增殖细胞中，而不存在于皮质骨组织中，因为根据定义，后者主要包含成熟的分化成骨细胞和骨细胞。
