MOLECULAR GENETICS OF HUMAN BMP-4 IN FOP

FOP 中人类 BMP-4 的分子遗传学

• 批准号: 6016880
• 负责人: FREDERICK Samuel KAPLAN
• 金额: $ 30.28万
• 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/6016880
• 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 发展事件依赖于成功诱导 软骨内成骨 然而，人们对它知之甚少。 骨诱导的遗传控制。 骨形态发生蛋白 骨形成蛋白（BMPs）是一个高度保守的分子家族的成员， 参与胚胎模式形成的调节， 成骨 重组人BMP-4可以诱导整个 异位软骨内成骨的发育程序 位点的方式与常染色体显性遗传中所见相同 进行性骨化性纤维发育不良（FOP）。FOP 是一种逐渐致残的疾病， 脚趾畸形和时空紊乱 在异位部位诱导软骨内成骨。 BMP-4 信使RNA和蛋白质独特地过度表达 FOP患者的淋巴细胞中。 这些数据表明 BMP-4的过度表达，一种有效的骨诱导形态原， 与人类异位成骨的失能有关。 相关假设为我们的长期目标提供了焦点;首先， 人BMP-4基因的分子结构和功能 提供了对遗传调控的基本见解， 人类的软骨内骨诱导和模式形成; 第二，BMP-4是FOP的主要候选基因; 该基因分子结构和/或调节控制被 在FOP患者中异常。为了解决这些假设， 我们打算：定义人BMP-4的调控区 基因通过报告基因表达测定。 2.鉴定DNA结合 通过凝胶电泳分析正常和FOP细胞中BMP-4基因的蛋白质 使用核蛋白提取物的迁移率变化和足迹分析 从FOP和非FOP细胞。 3.检测BMP-4的表达率 转录起始和mRNA稳定性 通过核启动和转录抑制控制细胞 实验4.检测I型和II型BMP的表达 通过RT-PCR和免疫组织化学方法检测FOP细胞中受体的表达。5. 识别与BMP-4基因紧密相关的遗传标记 基因座，并利用这些标记来建立或排除遗传 BMP-4基因与FOP的连锁。监管分析 人类BPM-4基因的控制将促进这一长期目标， 阐明正常和紊乱骨骼的基本机制 诱导，设计合理的分子诊断和 一系列发育障碍的治疗策略， 人类的骨架