Molecular Etiology of Spondyloepimetaphyseal Dysplasia with Joint Laxity - Leptodactylic Type

项目摘要 骨骼发育不良是骨骼发育障碍，发生率约为1/5000 出生DNA测序能够识别这些疾病的致病基因，但机制研究 需要了解已确定的突变如何导致骨骼异常生长。该提案针对 脊柱干骺端发育不良伴关节松弛的机制性知识缺口-指端型 （SEMDJL 2），一种以软骨内骨形成缺陷为特征的骨骼发育不良。使役显性 已经在SEMDJL 2患者中鉴定了基因KIF 22中的突变。然而，KIF 22，一种驱动蛋白， 马达蛋白，在骨发育和KIF 22中的单个氨基酸变化对这一过程的影响 不知道。软骨内骨生长需要间充质干细胞分化， 软骨细胞和成骨细胞。软骨细胞增殖并为骨骼产生钙化软骨模板 增长在软骨细胞肥大时，成骨细胞被募集到钙化的软骨和存款骨 矩阵我们假设KIF 22的突变通过细胞分裂缺陷导致这一过程的中断， 增殖、祖细胞向软骨细胞和成骨细胞分化的缺陷、或两者的缺陷 分化和分化。为了检测KIF 22在造血祖细胞分裂和增殖中的作用， 细胞水平，我们将使用固定和活细胞成像的组合来评估KIF 22与SEMDJL 2- 衍生的突变，以产生有丝分裂细胞中染色体臂运动的力。接触细胞 将评估增殖的周期停滞或减少。为了确定KIF 22的突变是否会导致 SEMDJL 2病理由于多能间充质干细胞分化缺陷，我们将诱导 在体外细胞向软骨细胞和成骨细胞的分化。将评估分化和成熟 在这些系统中，通过组织学染色进行基质产生和矿化，以及表达水平 软骨细胞、成骨细胞或骨细胞的标志物。总之，这些检测将使我们能够 确定KIF 22突变影响软骨内骨生长的机制， 在控制骨骼发育方面有专长。

PROJECT SUMMARY Skeletal dysplasias are disorders of skeletal development and occur at a rate of approximately 1 in 5000 births. DNA sequencing is capable of identifying causative genes for these disorders, but mechanistic studies are needed to understand how identified mutations cause abnormal bone growth. This proposal addresses the mechanistic gap in knowledge for spondyloepimetaphyseal dysplasia with joint laxity- leptodactylic type (SEMDJL2), a skeletal dysplasia characterized by defects in endochondral bone formation. Causative dominant mutations in the gene KIF22 have been identified in SEMDJL2 patients. However, the function of KIF22, a kinesin motor protein, in bone development and the consequences of single amino acid changes in KIF22 on this process are not known. Endochondral bone growth requires the differentiation of mesenchymal stem cells to chondrocytes and osteoblasts. Chondrocytes proliferate and produce a calcified cartilage template for bone growth. Upon chondrocyte hypertrophy, osteoblasts are recruited to the calcified cartilage and deposit bone matrix. We hypothesize that mutations in KIF22 result in disruption of this process via defects in cell division and proliferation, defects in the differentiation of progenitors to chondroctyes and osteoblasts, or defects in both division and differentiation. To test the function of KIF22 in division and proliferation of progenitor cells at the cellular level, we will use a combination of fixed and live cell imaging to assess the ability of KIF22 with SEMDJL2- derived mutations to generate forces for the movement of chromosome arms in mitotic cells. Consequent cell cycle arrest or reduction in proliferation will be assessed. To determine whether mutations in KIF22 result in SEMDJL2 pathology due to defects in pluripotent mesenchymal stem cell differentiation, we will induce differentiation of cells to chondrocytes and osteoblasts in vitro. Differentiation and maturation will be assessed in these systems by histological staining for matrix production and mineralization, as well as expression levels of markers indicative of chondrocytes, osteoblasts, or osteocytes. Together, these assays will allow us to determine the mechanism by which mutations in KIF22 affect endochondral bone growth by combining expertise in cell division with expertise in the control of bone development.