MOLECULAR GENETICS OF PROGRESSIVE OSSEOUS HETEROPLASIA

进行性骨异质发育的分子遗传学

• 批准号: 6090796
• 负责人: EILEEN M SHORE
• 金额: $ 24.89万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6090796
• 关键词: autosomal dominant trait; cell differentiation; clinical research; connective tissue disorder; gene expression; gene mutation; genetic screening; human genetic material tag; human subject; immunocytochemistry; mesenchyme; molecular pathology; muscle disorders; northern blottings; nucleic acid sequence; osteogenesis; pathologic ossification; polymerase chain reaction; skeletal disorder; skin disorder; striated muscles; tendons; western blottings

DESCRIPTION (Adapted from the Applicant's Abstract): During embryogenesis, the skeleton develops according to a genetic plan that controls its precise temporal and spatial formation. Heterotopic ossification results from alterations in the normal regulation of osteogenesis. Progressive osseous heteroplasia (POH) is one of the most disabling disorders of heterotopic ossification in humans. POH is a distinct autosomal dominant genetic disorder of connective tissue characterized by heterotopic ossification of skin and subcutaneous tissue during childhood with progressive and severe ossification of deep connective tissues, including tendons, ligaments and skeletal muscles, throughout life. The genetic defect and pathophysiology are unknown. Three hypotheses provide the focus for this research proposal: (1) POH is related to Albright hereditary osteodystrophy (AHO), a genetic disorder associated with minor and superficial heterotopic ossification caused by inactivating mutations in the GNAS1 gene; (2) the human GNAS1 gene is mutated in POH, resulting in reduced levels and/or activity of GNAS1 mRNA and/or protein; (3) maternal/paternal inheritance of the GNAS1 gene influences the osteogenic phenotypic of inactivating GNAS1 mutations in POH. To address these hypotheses, the applicants intend to: (1) screen genomic DNA for mutations in the GNAS1 gene in POH patients by PCR amplification and DNA sequencing (a preliminary analysis has already detected such mutations) and compare GNAS1 mutations in POH patients to those that have been found in patients with AHO; (2) examine expression of the GNAS1 gene in POH patients by quantitation of GNAS1 mRNA by RT-PCR, and Gsalpha protein by immunoblot analysis; (3) perform functional analysis of Gsalpha in POH patients by G-protein-mediated cAMP activity assays; (4) evaluate the effects of maternal/paternal inheritance of the GNAS1 gene in families with inheritance of POH; and (5) investigate Gsalpha protein expression in POH lesional tissue by immunohistochemical analysis. Analysis of the molecular genetics of POH is intended to contribute to the applicants' long-term goals of (a) understanding the molecular and cellular pathways of normal and disordered bone induction, and (b) designing rational molecular diagnostic and treatment strategies for a wide range of developmental disorders of the skeleton in humans. Accomplishment of these Specific Aims will provide the foundation for future work that will examine the pathways through which GNAS1 expression leads to regulation of normal and ectopic bone formation.

描述（改编自申请人的摘要）：在胚胎发生期间， 骨骼是按照基因计划发育的， 时间和空间结构。异位骨化是由 骨生成正常调节的改变。进行性骨性 子宫内膜异位症（POH）是异位性子宫内膜异位症中最致残的疾病之一， 人类的骨化POH是一种独特的常染色体显性遗传病 以皮肤异位骨化为特征的结缔组织， 儿童期皮下组织进行性和严重骨化 深层结缔组织，包括肌腱、韧带和骨骼肌， 在生活中。遗传缺陷和病理生理学尚不清楚。三 假设提供了本研究建议的重点：（1）POH与 奥尔布赖特遗传性骨营养不良（AHO），一种与以下疾病相关的遗传性疾病： 失活突变引起的轻微和浅表异位骨化 GNAS 1基因中;（2）人类GNAS 1基因在POH中发生突变，导致 降低的GNAS 1 mRNA和/或蛋白质的水平和/或活性;（3） GNAS 1基因的母系/父系遗传影响成骨细胞的生长， POH中失活GNAS 1突变的表型。为了解决这些假设， 申请人打算：（1）筛选基因组DNA中的GNAS 1突变， 通过PCR扩增和DNA测序，对POH患者的基因进行了初步分析， 分析已经检测到这样的突变），并比较 POH患者与AHO患者中发现的患者比较;（2）检查 POH患者中GNAS 1基因的表达， RT-PCR和免疫印迹分析Gsalpha蛋白;（3）进行功能鉴定 通过G蛋白介导的cAMP活性测定分析POH患者中的Gsalpha; (4)评估GNAS 1基因的母系/父系遗传对 POH家系;（5）研究Gsalpha蛋白 免疫组化分析POH病变组织中的表达。分析 POH的分子遗传学旨在有助于申请人 长期目标：（a）了解 正常和紊乱的骨诱导，和（B）设计合理的分子 广泛的发育障碍的诊断和治疗策略 人类骨骼的最后一部分这些具体目标的实现将提供 未来工作的基础，将研究通过哪些途径， GNAS 1表达导致正常和异位骨形成的调节。