Gene analysis of congenital connective tissue disorders using human collagen gene probes

使用人类胶原蛋白基因探针对先天性结缔组织疾病进行基因分析

• 批准号: 62570445
• 负责人: KONOMI Hiroshi
• 金额: $ 1.54万
• 依托单位: National Institute of Neuroscience, NCNP
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570445/
• 关键词: Marfan syndrome; osteogenesis imperfecta; collagen; コラーゲン遺伝子; DNA多型

1. Analysis of Clooagen Protein Molecules Newly synthesized [3H]proline-labeled proteins produced by skin fibroblasts from eighteen cases of Marfan syndrome, nine cases of Ehlers-Danlos syndrome and seven cases of osteogenesis imperfecta were investigated. We found fibroblasts derived from a patient with osteogenesis imperfecta type II ( lethal form ) produced shortened pro alpha1(I)chain. Further precise analysis revealed that there was a mutaion in CB8 peptide of alpha1(I)chain (a middle portion of triple-herical lesion). In other all cases, we could not detect any abnormalities of type I and III procollagen. However, unusual 185 KDa collagenous protein was synthesized by skin fibroblasts from four patients with marfan syndrome. We identified the 185 KDa band as type IV collagen by immunoprecipitaion and CNBr peptide mapping.2. Gene Analysis Using Collagen Gene Probes DNA purified from fibroblasts were digested with various kinds of restriction enzymes and were electrophoresed on agarose gels followed by transfered to nitrocelluroce membranes. We analysed the DNA with gene probes of human type I procollagen; Hf677, NJ3 and Nj1/4.1 by southern blotting. So far we could not detect any abnormalities in any cases.3. Linkage Study of Marfan Syndrome Families with Type I Procollagen Gene We did linkage analysis using restriction fragment length polymorphism ( RFLP ) in the pro alpha2(I)chain by Southern blotting. Before doing the linkage analysis, we checked allelic frequencies of both probes. Allelic frequencies of MspI RFLP and EcoRI RFLP positive were 83% and 69%, respectively. Analysis showed no linkage in two Marfan families, and the probes were not useful in one family.

1. Clooagen蛋白分子的分析研究了18例马凡综合征、9例Ehlers-Danlos综合征和7例成骨肉瘤患者皮肤成纤维细胞产生的新合成[3 H]脯氨酸标记蛋白。我们发现成纤维细胞来源于成骨细胞II型（致死型）患者产生缩短的pro α 1（I）链。进一步的精确分析表明，CB 8 α 1（I）链肽段（三重螺旋损伤的中间部分）发生了突变。在其他所有情况下，我们不能检测到任何异常的I型和III型前胶原。然而，不寻常的185 kDa的胶原蛋白合成的皮肤成纤维细胞从4例马凡综合征。通过免疫沉淀和CNBr肽图分析，确定185 KDa条带为IV型胶原.使用胶原基因探针的基因分析将从成纤维细胞纯化的DNA用各种限制性内切酶消化，并在琼脂糖凝胶上电泳，然后转移到硝酸纤维素膜上。我们用人Ⅰ型前胶原基因探针Hf 677、NJ 3和Nj 1/4.1进行DNA的Southern印迹分析。到目前为止，我们在任何情况下都没有发现任何异常。马凡氏综合征家系与Ⅰ型前胶原基因的连锁研究我们用限制性片段长度多态性（RFLP）技术对pro α 2（Ⅰ）链进行了连锁分析。在进行连锁分析之前，我们检查了两个探针的等位基因频率。MspI和EcoR Ⅰ RFLP的等位基因频率分别为83%和69%。分析表明，在两个马凡家族中没有连锁，探针在一个家庭中无效。

项目成果

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Yasunori.Okada 等人：FEBS Lett。

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Hajime Sawada: Experimental Cell Research. 171. 94-109 (1987)

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