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

1. 对18例马凡氏综合征、9例埃勒-丹洛斯综合征和7例成骨不全症皮肤成纤维细胞新合成的[3H]脯氨酸标记蛋白进行了研究。我们发现来自成骨不全II型（致死型）患者的成纤维细胞产生缩短的前α 1(I)链。进一步的精确分析显示，在alpha1(I)链的CB8肽（三轴病变的中间部分）发生了突变。在其他所有病例中，我们未检测到任何I型和III型前胶原的异常。然而，罕见的185 KDa胶原蛋白是由4例马凡氏综合征患者的皮肤成纤维细胞合成的。我们通过免疫沉淀和CNBr肽图谱鉴定185 KDa条带为IV型胶原。从成纤维细胞中纯化的DNA用各种限制性内切酶酶切，在琼脂糖凝胶上电泳，然后转移到硝化纤维素膜上。用基因探针对人I型前胶原进行DNA分析；Hf677、NJ3和Nj1/4.1进行southern印迹。到目前为止，我们没有发现任何异常情况。马凡综合征家族与I型前胶原基因的连锁研究采用Southern blotting法对前α 2(I)链进行限制性内切片段长度多态性（RFLP）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 RFLP和EcoRI RFLP阳性的等位基因频率分别为83%和69%。分析显示两个马凡氏家族无连锁，探针在一个家族中无效。

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.