MOLECULAR BASIS OF OSTEOGENESIS IMPERFECTA

成骨细胞（OI）是一个临床，遗传和生化 以骨脆性和其他结缔组织为特征的异质性疾病 组织异常 在绝大多数受影响的个体中， 临床状况是由两种基因（COL 1A 1和COL 1A 2）的突变引起的。 COL 1A 2），其编码I型前胶原的链。 的目标 本申请中提出的研究是表征突变 在引起不同形式的OI的胶原基因中， 突变的性质、突变所处的基因之间的关系 定位和临床表型，以确定突变如何改变 细胞内和细胞外行为的分子，含有异常 链，以确定新的显性突变的起源亲本，并 确定胶原基因突变的嵌合程度， 首先受影响的人的父母。 对于改变链的突变 结构，突变位点将近似于肽 映射，突变的精确位置将通过以下方式确定： 单碱基错配化学切割或单链构象 多态性，来自成纤维细胞或基因组的cDNA的适当区域， 将扩增DNA并直接测序或克隆到M13中， 对选定的克隆进行测序。 对于改变表达的突变 在胶原蛋白基因中，异常等位基因将通过缺乏 从该基因合成的mRNA表达多态性标记（在 杂合子）和突变的结构鉴定， 在使用碱基错配化学物质搜索基因后， 切割单链构象多态性以检测区域 的差异。 Ⅰ型前胶原异常链的作用 分子将通过检查分泌动力学来确定， RER结合蛋白对分泌的影响， 具有一个或多个异常链的分子的分泌， 效应-这些分子在EM水平上对分子结构的影响， 分子的热稳定性，以及分子参与 在纤维形成中。 最后，镶嵌的程度将由以下因素决定： 检查来自父母的组织并测量 通过用适当的限制性内切酶进行双杂交或通过 等位基因特异性寡核苷酸杂交。 这些研究应该有助于 了解OI的分子基础，以及突变是如何翻译的， to phenotype表型. 最后，它们对更多疾病的发病机制有影响。 骨形成的常见疾病。

Osteogenesis imperfecta (OI) is a clinically, genetically and biochemically heterogeneous disorder characterized by bone fragility and other connective tissue abnormalities. In the vast majority of affected individuals the clinical condition results from mutations in the two genes (COL1A1 and COL1A2) that encode the chains of type I procoliagen. The objectives of the studies proposed in this application are to characterize the mutations in the collagen genes that give rise to different forms of OI, to determine the relationship among the nature of the mutation, the gene in which it is located, and the clinical phenotype, to determine how mutations alter the intracellular and extracellular behavior of molecules that contain abnormal chains, to identify the parent of origin of new dominant mutations, and to determine the extent of mosaicism for mutations in collagen genes among parents of first affected individuals. For mutations that alter chain structure, the site of the mutation will be approximated by peptide mapping, the precise location of the mutation will be determined by single-base mismatch chemical cleavage or single stranded conformational polymorphisms, the appropriate region of cDNA from fibroblasts or genomic DNA will be amplified and either sequenced directly or cloned into M13 and selected clones will be sequenced. For mutations that alter the expression of collagen genes, the abnormal allele will be identified by the absence of expressed polymorphic markers from the mRNA synthesized from that gene (in heterozygotes) and the structure of the mutation identified and characterized following search of the gene using base mismatch chemical cleavage of single stranded conformational polymorphisms to detect regions of difference. The effects of abnormal chains in type I procollagen molecules will be determined by examinining the kinetics of secretion and the effects of RER binding proteins on secretion, the efficiency of secretion of molecules with one or more than one abnormal chain, the effect-,of these molecules on molecular structure at the EM level, the thermal stability of molecules, and the ability of molecules to participate in fibrillogenesis. Finally, the extent of mosaicism will be determined by examination of tissues from parents and measuring the presence of the mutant allele by digestions with appropriate restriction enzymes or by allele-specific oligonucleotide hybridization. These studies should help to understand the molecular basis of OI, and how mutations are translated to phenotype. Finally, they have implications for the pathogenesis of more common disorders of bone formation.