MOLECULAR GENETICS OF HUMAN BMP-4 IN FOP

FOP 中人类 BMP-4 的分子遗传学

• 批准号: 2712450
• 负责人: FREDERICK Samuel KAPLAN
• 金额: $ 29.47万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2712450
• 关键词: DNA binding protein; DNA footprinting; blood chemistry; bone development disorder; clinical research; developmental genetics; gel mobility shift assay; gene expression; gene mutation; genetic markers; genetic promoter element; genetic regulatory element; human genetic material tag; human subject; immunocytochemistry; linkage mapping; messenger RNA; nuclear runoff assay; osteogenesis; pathologic ossification; polymerase chain reaction; progressive myositis ossificans; protein structure function; regulatory gene

The embryogenesis and regeneration of the skeleton are complex developmental events dependent on the successful induction of endochondral osteogenesis. Little is known, however, about the genetic control of bone induction. Bone morphogenetic proteins (BMPs) are members of a highly conserved family of molecules involved in the regulation of embryonic pattern formation and osteogenesis. Recombinant human BMP-4 can induce the entire developmental program of endochondral osteogenesis in an ectopic site in a manner identical to that seen in the autosomal dominant genetic disorder, fibrodysplasia ossificans progressive (FOP). FOP is a progressively disabling condition characterized by congenital malformations of the toes and disordered temporal and spatial induction of endochondral osteogenesis at ectopic sites. BMP-4 messenger RNA and proteins uniquely over-expressed inlymphocytes from patients who have FOP. These data indicate that over expression of BMP-4, a potent bone-inducing morphogen, is associated with disabling ectopic osteogenesis in man. Tow related hypotheses provide the focus for our longterm goals; First, the molecular structure and function of the human BMP-4 gene provides fundamental insight into the genetic regulation of endochondral bone induction and pattern formation in humans; second, BMP-4 is the prime candidate gene for FOP; and the molecular structure and/or regulatory control of that gene is abnormal in patients who have FOP. To address these hypothesis, we intend to: 1. Define the regulatory regions of the human BMP-4 gene by reporter gene expression assays. 2. Identify DNA-binding proteins for the BMP-4 gene in normal and FOP cells by gel mobility shift and footprinting assays using nuclear protein extracts from FOP and non-FOP cells. 3. Examine the rate of BMP-4 transcript initiation and mRNA stability in FOP cells relative to control cells by nuclear run-on and transcription inhibition experiments. 4. Examine the expression of BMP type I and type II receptors in FOP cells by RT-PCR and immunohistochemistry. 5. Identify genetic markers that are closely linked to the BMP-4 gene locus and utilize these markers to establish or exclude genetic linkage of the BMP-4 gene with FOP. Analysis of the regulatory control of the human BPM-4 gene will foster that longterm goal of elucidating basic mechanisms of normal and disordered bone induction, and of designing rational molecular diagnostic and treatment strategies for a wide range of developmental disorders of the skeleton in humans.

骨骼的胚胎发生和再生是复杂的 发育事件依赖于成功地诱导 软骨内成骨。然而，人们对此知之甚少 骨诱导的遗传控制。骨形态发生蛋白 (BMP)是高度保守的分子家族的成员 参与调节胚胎模式的形成和 成骨作用。重组人骨形态发生蛋白-4可诱导 异位软骨内成骨的发育程序 以与常染色体显性遗传相同的方式定位 遗传性疾病，进行性骨化纤维发育不良(FOP)。FOP 是一种渐进性致残状态，其特征是先天性 足趾畸形与时空紊乱 异位部位软骨内成骨的诱导。BMP-4 信使RNA和蛋白质独特的过度表达 有FOP患者的淋巴细胞。这些数据表明 骨形态发生蛋白-4的过度表达，一种强有力的骨诱导形态原， 与人的异位成骨功能障碍有关。拖车 相关假设为我们的长期目标提供了重点；第一， 人骨形成蛋白-4基因的分子结构和功能 提供了对基因调控的基本见解 人软骨内骨诱导和形态形成； 第二，BMP-4是FOP的首选候选基因； 该基因的分子结构和/或调控是 在有FOP的患者中异常。为了解决这些假设， 我们的目标是：1.确定人骨形成蛋白-4的调控区域 基因与报告基因表达分析。2.识别DNA绑定 正常细胞和FOP细胞中BMP-4基因蛋白的凝胶表达 利用核蛋白提取物进行迁移率变化和足迹分析 来自FOP和非FOP单元格。3.检测BMP-4的比率 FOP细胞转录起始与mRNA稳定性的关系 通过核跑动和转录抑制控制细胞 实验。4.检测BMP I型和II型的表达 逆转录-聚合酶链式反应和免疫组织化学检测FOP细胞的受体。5. 识别与BMP-4基因紧密连锁的遗传标记 定位并利用这些标记建立或排除遗传 骨形态发生蛋白-4基因与FOP的连锁。对监管的分析 对人类BPM-4基因的控制将促进实现这一长期目标 阐明正常和紊乱骨的基本机制 诱导，并设计合理的分子诊断和 多种发育障碍的治疗策略 人类的骨骼。