The primary goals (a) to define the genetic heterogeneity of Marfan Syndrome; (b) to determine the nature of mutations in a well-defined phenotyic-genetic group of osteogenesis imperfecta and (c) to study the expression of proAlpha2(I) chain mutation in cultured bone cells from a patient with osteogenesis imperfecta. We propose to use Restriction Fragment Length Polymorphisms (RFLPs) associated with genes encoding for various collagen chains and elastin, as markers for genetic linkage studies in families with Marfan Syndrome. With these studies, it will be determined if any Marfan Syndrome phenotypes are caused by mutations in the collagen or elastin genes. Affected individuals and their unaffected relatives will be genotyped for various RFLPs and a correlation will be made between the inheritance of the phenotype and the segregation of various RFLP alleles. Furthermore, this approach will allow us to identify asymptomatic carriers of the gene for Marfan Syndrome and offer prenatal diagnosis in informative families. Total genomic DNA will be extracted from leucocytes, restricted with a variety of endonucleases, electrophoresed, transferred on a filter and hybridized with p32 nick translated probes coding for the COL1A1, COL1A2, COL3A1, COL5A2 and elastin genes. We will initiate studies to determine whether a proAlpha2(I) chain deletion from a patient with osteogenesis imperfecta results from a structural gene deletion or from a splicing error. RNA will be extracted from cultured skin fibroblasts for S1 nuclease mapping. The previous experiment will help define the area of the mutant COL1A2 allele to be sequenced. We will also study the protein defect in two other families with osteogenesis imperfecta genetically linked to the COL1A2 gene. Cultured skin fibroblasts will be labelled with H3 proline and the procollagen chains will be studied with the standard protein separation and mapping techniques. We will also establish a bone cell culture to study the expression of a proAlpha2(I) chain mutation from a patient with osteogenesis imperfecta. The long-term goal is to establish the genetic and biochemical heterogeneity of the various connective tissue disorders and also understand the pathogenetic mechanisms involved.
NIH ExPorter
NIH ExPorter