Genetics and molecular biology of melorheostosis

骨髓变性的遗传学和分子生物学

• 批准号: 10266555
• 负责人: Joan C Marini
• 金额: $ 60.5万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10266555
• 关键词: Affect; Appearance; BMP2 gene; Biopsy; Blood Vessels; Cells; Cellularity; Child; Clinical; Clinical Trials; Collaborations; Contracture; Contralateral; Diagnostic; Diagnostic radiologic examination; Extracellular Matrix; Functional disorder; Genes; Genetic; Goals; Hardness; Histologic; Immune response; Impairment; In Vitro; Institutes; Joints; Lesion; Leukocytes; MADH3 gene; MAP Kinase Gene; MAP2K1 gene; MAPK3 gene; Melorheostosis; Metabolism; Molecular; Molecular Biology; Mosaicism; Mutation; National Institute of Arthritis and Musculoskeletal and Skin Diseases; National Institute of Child Health and Human Development; Osteoblasts; Osteogenesis; Osteoid; Pain; Parents; Pathway interactions; Patients; Pattern; Physiologic Ossification; Physiologic calcification; Process; Research Personnel; Role; Signal Transduction; Skeleton; Skin; Somatic Mutation; Surface; Testing; Thick; Time; Tissues; Transcript; Waxes; angiogenesis; base; bone; causal variant; cell growth; cortical bone; functional disability; gain of function; inhibitor/antagonist; insight; mineralization; mouse model; mutant; osteoblast differentiation; osteoblast proliferation; skeletal; soft tissue; transcriptome; transcriptome sequencing; transmission process

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.

骨质疏松症是一种散发性的骨发育不全，其特征是不对称的骨过度生长和功能障碍。经典形式的melorheostosis有放射线照片的特点是滴蜡外观。在我们的项目之前，melorheostosis的遗传原因是未知的;体细胞嵌合体被假定，因为没有父母到孩子的传播和突变没有检测到白细胞。NICHD、NIAMS和维也纳骨学研究所的研究人员合作，共同研究了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melorheostosis患者接受了受累骨和对侧未受累骨的活检，以便可以检测到受累组织中的低百分比镶嵌。我们分别在经典型和骨内膜骨质疏松症中发现了MAP 2K 1和SMAD 3突变的体细胞嵌合现象。 15名活组织检查患者中的8名，所有患者在X光照片上都有滴落的蜡烛蜡图案，在NRD中的MAP 2K 1 p.K57或p.Q56处具有突变的体细胞嵌合体（Kang，Jha，Deng等人，2018年）。MEK 1取代激活ERK 1/2 MAPK，增强成骨细胞增殖，延迟成骨细胞分化和ECM矿化。这些发现是受影响骨较深区域组织学检查发现的类骨质积聚增加、细胞构成增加和重塑的基础。蜡样病变的外区由骨膜附着形成的多层初级骨板组成，其赋予过度生长的骨硬度。受影响的骨和覆盖的皮肤中存在血管分布增加：皮质骨血管分布增加;覆盖的皮肤是肿胀的，血管壁厚度增加，与血管生成相关的转录物表达增加。在许多情况下，上覆皮肤中的MAP 2K 1嵌合体是诊断性的。 15名活组织检查的患者中有4名在X线照片上都有骨内膜过度生长，在受影响但未受影响的骨中有SMAD 3（p.S264）的体细胞突变（Kang，Jha，Ivovic等人，2020年）。qBEI显示受累骨的基质矿化程度高于未受累骨。SMAD 3突变增加了典型的TGF-/SMAD信号传导并抑制了melorheostotic成骨细胞增殖，表明功能获得。SMAD 3突变刺激受影响细胞中的成骨细胞分化和ECM矿化;当在BMP 2存在下驱动骨生成时，这些途径被抑制。基于RNA-Seq的转录组分析证实SMAD 3突变显著影响TGF-途径和骨化相关过程，并揭示了对病理生理学的见解，包括ECM组织，细胞生长和免疫应答相关途径。 总体而言，我们证明，melorheostosis是遗传异质性的，在两个基因的体细胞突变负责通过不同的分子和细胞机制不同的放射学和临床模式。