MOLECULAR BASIS OF OSTEOGENESIS IMPERFECTA

描述(改编自申请者摘要)：成骨 不完美是一种临床上的异质性疾病，影响的不止是 每20,000人中有1人。表型范围从围产期的致死性 期间，骨折频率略有增加。90%以上的受影响用户 个体中编码I型链的基因发生突变 胶原蛋白，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扰乱 分子组装、细胞内运输、分泌和细胞外 处理和；确定通过哪些机制和细胞内 细胞识别异常蛋白质并启动的位置 防止它们分泌的策略。酵母双杂交系统将是 用于识别与I型部分相互作用的新蛋白质 前胶原分子，以及这些基因的分离和鉴定。 针对新发现的蛋白质的抗体，以及针对I型的抗体 前胶原、HSP47、GRP78和GRP94，以及Pro-羟基酶(PDI) 用来确定哪些蛋白质与异常分子相互作用 由不同类别突变的患者细胞合成的 COLIA1和COLIA2基因。将确定更多的突变 发生在编码三重甘氨酸残基的区域的CpG位点 用限制性内切酶分析I型前胶原链的螺旋 这些已知位点的内切酶(COLIAI基因36个，COLIAI基因27个 COLIA2)。在大家族中发生进一步的额外突变 将寻找变量表达式以及父变量为镶嵌的变量表达式 阐明可变表达的基础。最后，动力学研究表明， 影响剪接的突变的mRNA处理将被识别。 这些研究的目的是为了提高理解 突变的表型效应，并识别加工过程中的位置 干预可改善其表型效应的途径 胶原蛋白基因的突变。

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.