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

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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.