MOLECULAR GENETICS OF PROGRESSIVE OSSEOUS HETEROPLASIA

描述(改编自申请人的摘要)：在胚胎发育过程中， 骨骼根据控制其精确度的遗传计划发育 时间和空间形态。异位骨化的原因是 成骨的正常调节的改变。进行性骨性 异位症(POH)是异位致残最严重的疾病之一。 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 儿童期进行性严重骨化的皮下组织 深结缔组织，包括肌腱、韧带和骨骼肌， 在一生中。遗传缺陷和病理生理学尚不清楚。三 假设为本研究提供了重点：(1)POH与 奥尔布赖特遗传性骨营养不良症(Aho)，一种与 失活突变引起的轻度和浅表性异位骨化 在GNAS1基因中；(2)人GNAS1基因在POH中突变，导致 GNAS1mRNA和/或蛋白水平和/或活性降低；(3) GNAS1基因的母系/父系遗传影响成骨 POH中GNAS1基因突变失活的表型。为了解决这些假设， 申请者打算：(1)筛选基因组DNA中的GNAS1突变 应用聚合酶链式反应和DNA测序技术检测POH患者的基因 分析已经检测到这种突变)，并将GNAS1突变与 对POH患者中已发现的AHO患者进行检查； 应用免疫组织化学方法定量检测POH患者GNAS1基因的表达 RT-PCR，免疫印迹分析Gsalpha蛋白；(3)功能 G蛋白介导的cAMP活性测定分析POH患者的Gsalpha； (4)评价GNAS1基因母本/父本遗传对儿童的影响 POH遗传性家系；(5)Gsalpha蛋白研究 免疫组织化学方法检测其在POH皮损中的表达。分析 POH的分子遗传学是为了帮助申请者 (A)了解分子和细胞途径的长期目标 正常和无序的骨诱导，以及(B)设计合理的分子 针对多种发育障碍的诊断和治疗策略 在人类的骨骼上。这些具体目标的实现将提供 为今后的工作奠定基础，审查通过哪些途径 GNAS1的表达导致正常和异位骨形成的调节。

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.