Körkkö J, Ala-Kokko L, De Paepe A, Nuytinck L, Earley J, Prockop D J
Center for Gene Therapy, Allegheny University of the Health Sciences, MCP-Hahnemann School of Medicine, Philadelphia, PA 19102-1192, USA.
Am J Hum Genet. 1998 Jan;62(1):98-110. doi: 10.1086/301689.
Although >90% of patients with osteogenesis imperfecta (OI) have been estimated to have mutations in the COL1A1 and COL1A2 genes for type I procollagen, mutations have been difficult to detect in all patients with the mildest forms of the disease (i.e., type I). In this study, we first searched for mutations in type I procollagen by analyses of protein and mRNA in fibroblasts from 10 patients with mild OI; no evidence of a mutation was found in 2 of the patients by the protein analyses, and no evidence of a mutation was found in 5 of the patients by the RNA analyses. We then searched for mutations in the original 10 patients and in 5 additional patients with mild OI, by analysis of genomic DNA. To assay the genomic DNA, we established a consensus sequence for the first 12 kb of the COL1A1 gene and for 30 kb of new sequences of the 38-kb COL1A2 gene. The sequences were then used to develop primers for PCR for the 103 exons and exon boundaries of the two genes. The PCR products were first scanned for heteroduplexes by conformation-sensitive gel electrophoresis, and then products containing heteroduplexes were sequenced. The results detected disease-causing mutations in 13 of the 15 patients and detected two additional probable disease-causing mutations in the remaining 2 patients. Analysis of the data developed in this study and elsewhere revealed common sequences for mutations causing null alleles.
尽管据估计超过90%的成骨不全症（OI）患者在I型前胶原的COL1A1和COL1A2基因中存在突变，但在所有患有最轻微形式疾病（即I型）的患者中，突变一直难以检测到。在本研究中，我们首先通过分析10例轻度OI患者成纤维细胞中的蛋白质和mRNA来寻找I型前胶原中的突变；蛋白质分析在2例患者中未发现突变证据，RNA分析在5例患者中未发现突变证据。然后，我们通过分析基因组DNA，在最初的10例患者和另外5例轻度OI患者中寻找突变。为了检测基因组DNA，我们确定了COL1A1基因前12 kb和COL1A2基因38 kb新序列中30 kb的共有序列。然后使用这些序列设计用于这两个基因103个外显子和外显子边界的PCR引物。PCR产物首先通过构象敏感凝胶电泳扫描异源双链体，然后对含有异源双链体的产物进行测序。结果在15例患者中的13例中检测到致病突变，在其余2例患者中检测到另外两个可能的致病突变。对本研究及其他地方得出的数据进行分析，揭示了导致无效等位基因的突变的共有序列。
