title: A single base mutation in type I procollagen (COL1A1) that converts glycine alpha 1-541 to aspartate in a lethal variant of osteogenesis imperfecta: detection of the mutation with a carbodiimide reaction of DNA heteroduplexes and direct sequencing of products of the PCR.
abstract: Skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta synthesized both apparently normal type I procollagen and a type I procollagen that had slow electrophoretic mobility because of posttranslational overmodifications. The thermal unfolding of the collagen molecules as assayed by protease digestion was about 2 degrees C lower than normal. It is surprising, however, that collagenase A and B fragments showed an essentially normal melting profile. Assay of cDNA heteroduplexes with a new technique involving carbodiimide modification indicated a mutation at about the codon for amino acid 550 of the alpha 1(I) chain. Subsequent amplification of the cDNA by the PCR and nucleotide sequencing revealed a single-base mutation that substituted an aspartate codon for glycine at position alpha 1-541 in the COL1A1 gene. The results here confirm previous indications that the effects of glycine substitutions in type I procollagen are highly position specific. They also demonstrate that a recently described technique for detecting single-base differences by carbodiimide modification of DNA heteroduplexes can be effectively employed to locate mutations in large genes.
authors.affiliation: Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
authors.forename: J P
authors.initials: JP
authors.lastname: Zhuang
authors.forename: C D
authors.initials: CD
authors.lastname: Constantinou
authors.forename: A
authors.initials: A
authors.lastname: Ganguly
authors.forename: D J
authors.initials: DJ
authors.lastname: Prockop
mesh_terms: D000328:Adult; D001224:Aspartic Acid / Q000235:genetics*; D001483:Base Sequence; D002234:Carbodiimides / Q000737:chemistry; D002478:Cells, Cultured; D005260:Female; D005313:Fetal Death; D005804:Genes, Lethal; D005998:Glycine / Q000235:genetics*; D006801:Humans; D007231:Infant, Newborn; D007234:Infant, Premature; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes; D009693:Nucleic Acid Hybridization; D010013:Osteogenesis Imperfecta / Q000175:diagnosis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011347:Procollagen / Q000235:genetics*
chemical_list: D002234:Carbodiimides; D009692:Nucleic Acid Heteroduplexes; D011347:Procollagen; D001224:Aspartic Acid; D005998:Glycine