Collagens are a family of tissue specific structural proteins, whose abnormal biosynthesis may affect the development and integrity of most organs including skin, joints, and vessels (Ehlers-Danlos Syndromes); bone (Osteogenesis Imperfects); and aorta and heart (Marfan's Syndrome). There is tremendous clinical heterogeneity among patients in any category, reflecting the molecular heterogeneity which must be present. Genetic disorders of collagen, and other extracellular matrix components such as elastin and fibronectin, provide an opportunity to understand structure and functional relationships of skin and connective tissue. The cloning of the cDNAs and genes for these proteins plus the development of the polymerase chain reaction provide essential tools for a molecular approach for analysis of mutations. This project will identify molecular genetic defects in selected and previously studied patients with disorders of collagen biosynthesis: Ehlers-Danlos Syndrome and Buschke-Ollendorf Syndrome. Protein biosynthesis SDS PAGE, peptide mapping, Southern and Northern blotting have been and will be used to identify patients for more detailed analysis. Restriction fragment length polymorphisms (RFLPS) will be used to haplotype collagen chain alleles in families to identify the affected chromosome(s). DNA from patients suspected point mutations will be studied using RNAase A protection assays to localize mutations. These mutations will be sequenced by performing 1st strand CDNA synthesis from fibroblast MRNA and amplification by the polymerase chain reaction (PCR). By using unequal molar equivalents of oligomers, single stranded DNA molecules can be amplified in both directions for direct DNA sequencing of mutations. Since collagens are resistant to conventional protein sequencing, a molecular genetic approach to the study of abnormal collagen biosynthesis may be extremely useful, provide information on the structure and significance of collagen in skin morphogenesis, and lead to the discovery of genetic markers and identifiable mutations for prenatal diagnosis of these disorders.
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