Genetics and molecular biology of melorheostosis

骨质疏松症是一种以非对称性骨过度生长和功能障碍为特征的散发性骨质疏松症。典型的硬化性骨质疏松症的X线片特征是有滴漏的烛蜡外观。在我们的项目之前，骨质疏松症的遗传原因尚不清楚；体细胞嵌合性是假设的，因为没有亲子传播，也没有在白细胞中检测到突变。NICHD、NIAMS和维也纳骨科研究所的研究人员共同合作，研究了马蹄内骨质疏松症的遗传学。为了提高WES测序检测致病突变的能力，15名骨质疏松症患者接受了患侧和对侧健侧骨骼的活组织检查，以便能够检测到患侧组织中的低百分比嵌合体。我们发现MAP2K1和Smad3突变的体细胞嵌合体分别存在于经典型骨质疏松症和骨内膜骨质疏松症。 在15名活检患者中，有8名患者在X线片上都有滴落的烛蜡图案，在NRD的MAP2K1 p.K57或p.Q56处有体细胞嵌合体突变(Kang，Jha，邓等人，2018年)。MEK1替换激活了ERK1/2MAPK，促进了成骨细胞的增殖，延迟了成骨细胞的分化和ECM的矿化。这些发现是在受影响骨的深层组织学检查中发现的类骨积聚增加、细胞性增加和重塑的基础。烛蜡病变的外层由骨膜贴合形成的多层原始板组成，使过度生长的骨变得坚硬。受累骨骼和上覆皮肤血管增多：皮质骨血管增多；上覆皮肤红斑，血管壁厚度增加，与血管生成相关的转录表达增加。在许多情况下，皮肤上的MAP2K1嵌合体具有诊断性。 在15名接受活组织检查的患者中，有4名患者的SMAD3基因有体细胞突变(第S264页)，这些患者的X线片上都有骨内膜过度生长，但并非未受影响的骨骼(Kang，Jha，Ivovic等人，2020)。QBEI显示，患骨的基质矿化程度高于非患骨。SMAD3突变增加了典型的转化生长因子-/SMAD信号，并抑制了成骨细胞的增殖，表明功能获得。Smad3突变刺激了受影响细胞中的成骨细胞分化和ECM矿化；当在BMP2存在的情况下驱动成骨时，这些途径被抑制。基于RNA-Seq的转录组分析证实，Smad3突变显著影响了转化生长因子途径和骨化相关过程，并揭示了病理生理学，包括细胞外基质组织、细胞生长和免疫反应相关途径。 总体而言，我们证明了骨质疏松症在遗传上是异质性的，两个基因的体细胞突变通过不同的分子和细胞机制导致了不同的放射学和临床模式。

Melorheostosis is a sporadic dysostosis characterized by asymmetric bone overgrowth and functional impairment. The classic form of melorheostosis has radiographs characterized by a dripping candle wax appearance. Prior to our project the genetic cause of melorheostosis was unknown; somatic mosaicism was postulated because there was no parent to child transmission and mutations were not detected in leukocytes. A collaboration of investigators in NICHD, NIAMS and the Institute of Osteology in Vienna came together to investigate the genetics of melorheostosis. To increase the ability of WES sequencing to detect causative mutations, 15 melorheostosis patients underwent biopsies of both affected and contralateral unaffected bone, so that low percent mosaicism in the affected tissue could be detected. We identified somatic mosaicism for MAP2K1 and SMAD3 mutations in classical and endosteal melorheostosis, respectively. Eight of 15 biopsied patients, all of whom had dripping candle wax pattern on radiographs, had somatic mosaicism for mutations at MAP2K1 p.K57 or p.Q56 in the NRD (Kang, Jha, Deng et al., 2018). The MEK1 substitutions activated ERK1/2 MAPK, enhanced osteoblast proliferation and delayed osteoblast differentiation and ECM mineralization. These findings underlie the increased osteoid accumulation, increased cellularity and remodelling identified on histologic examination in deeper regions of affected bone. The outer zone of the candle wax lesion is composed of multi-layered primary lamellae formed by periosteal apposition, which confers hardness to the overgrowth bone. Increased vascularity was present in affected bone and overlying skin: cortical bone vascularity was increased; overlying skin was erythematous, with increased thickness of vessel walls and expression of transcripts related to angiogenesis. MAP2K1 mosaicism in overlying skin is diagnostic in many cases. Four of 15 biopsied patients, all of whom had endosteal overgrowth on radiographs, had somatic mutations in SMAD3 (p.S264) in affected, but not unaffected, bone (Kang, Jha, Ivovic et al., 2020). qBEI revealed higher matrix mineralization in affected than unaffected bone. The SMAD3 mutations increased canonical TGF-/SMAD signaling and inhibited melorheostotic osteoblast proliferation, indicating gain-of-function. The SMAD3 mutations stimulated osteoblast differentiation and ECM mineralization in affected cells; these pathways were inhibited when osteogenesis was driven in the presence of BMP2. RNA-Seq-based transcriptome profiling confirmed the SMAD3 mutation significantly influenced TGF- pathway and ossification-related processes, and uncovered insights into pathophysiology, including ECM organization, cell growth, and immune response-related pathways. Overall, we demonstrated that melorheostosis is genetically heterogeneous, with somatic mutations in two genes responsible for distinct radiographic and clinical patterns through different molecular and cellular mechanisms.