MOLECULAR BASIS OF OSTEOGENESIS IMPERFECTA

描述（改编自申请人摘要）：成骨 多发性硬化症是一种临床异质性疾病， 1/20，000人。 表型范围从围产期致死 期间，骨折频率略有增加。 超过90%的受影响 个体在编码I型链的基因中有突变 胶原蛋白是骨骼的主要结构蛋白。 研究的目标 在本申请中提出的是确定这些突变如何干扰 分子组装、细胞内转运、分泌和细胞外 处理和;确定的机制，和细胞内 细胞识别异常蛋白质并启动 防止其分泌的策略。 酵母双杂交系统将是 用于鉴定与I型蛋白质相互作用的新蛋白质 前胶原分子，以及分离和表征的那些基因。 新发现的蛋白质的抗体，以及I型抗体 前胶原、HSP 47、GRP 78和GRP 94以及脯氨酰羟化酶（PDI）将被 用于确定哪些蛋白质与异常分子相互作用 由来自不同类型突变的患者的细胞合成， COLIA 1和COLIA 2基因。 将鉴定另外的突变， 发生在编码三联体中甘氨酸残基的区域中的CpG位点 Ⅰ型前胶原双链螺旋的限制性分析 这些已知位点的核酸内切酶（36个在COLIAI基因中，27个在 COLIA2）。 在大家族中发生的其他突变， 变量表达式，以及父级为马赛克的变量表达式，将被查找 阐明变量表达的基础。 最后， 将鉴定影响剪接的突变的mRNA加工。 这些研究的目的是提高理解的能力， 表型效应的突变，并确定网站沿着处理 干预可以改善表型效应的途径 胶原基因的突变。

DESCRIPTION (Adapted from the Applicant's Abstract): Osteogenesis imperfecta is a clinically heterogeneous disorder that affects more than 1/20,000 individuals. The phenotypic range is from lethal in the perinatal period, to a mild increase in fracture frequency. More than 90% of affected individuals have mutations in the genes that encode the chains of type I collagen, the major structural protein of bone. The objectives of studies proposed in this application are to determine how these mutations perturb molecular assembly, intracellular transport, secretion, and extracellular processing and; to identify the mechanisms by which, and the intracellular locations at which, cells recognize abnormal proteins and initiate strategies to prevent their secretion. The yeast two-hybrid system will be used to identify novel proteins that interact with portions of the type I procollagen molecule, and those genes isolated and characterized. Antibodies to the newly identified proteins, as well as antibodies to type I procollagen, HSP47, GRP78 and GRP94, and prolyl-hydroxylase (PDI) will be used to determine which proteins interact with the abnormal molecules synthesized by cells from patients with different classes of mutations in the COLIAI and COLIA2 genes. Additional mutations will be identified that occur at CpG sites in the regions that encode glycine residues in the triple helix of both chains of type I procollagen, by analysis with restriction endonucleases of those known sites (36 in the COLIAI gene and 27 in the COLIA2). Further additional mutations that occur in large families with variable expression, and those for which a parent is mosaic, will be sought to elucidate the basis for variable expression. Finally, the kinetics of MRNA processing for mutations that affect splicing will be identified. These studies are intended to increase the ability to understand the phenotypic effects of mutations, and identify sites along the processing pathway where intervention could ameliorate the phenotypic effects of mutations in collagen genes.