Single base mutation in the type III procollagen gene that converts the codon for glycine 883 to aspartate in a mild variant of Ehlers-Danlos syndrome IV

• Published in: The Journal of biological chemistry 1989-11, Vol.264 (32), p.19313-19317
• Main Authors: Tromp, G, Kuivaniemi, H, Stolle, C, Pope, F M, Prockop, D J
• Format: Article
• Language: English
• Subjects: Adult; Aspartic Acid; Base Sequence; Biological and medical sciences; Codon - genetics; DNA - genetics; DNA-Directed DNA Polymerase; Ehlers-Danlos syndrome; Ehlers-Danlos Syndrome - genetics; Female; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Genes; Genetic Variation; Glycine; Humans; Male; Middle Aged; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis. Repair; Mutation; Polymerase Chain Reaction; procollagen; Procollagen - genetics; Restriction Mapping; RNA, Messenger - genetics; Skin - metabolism; Templates, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)47303-1

Experiments were carried out to test the hypothesis that a 19-year-old proband with a mild variant of Ehlers-Danlos syndrome type IV had a mutation in the gene for type III procollagen. cDNA and genomic DNA were analyzed by using the polymerase chain reaction and cloning of the products into M13 filamentous phage. A mutation was found that converted the codon for glycine 883 of the triple-helical domain in one allele for type III procollagen to a codon for aspartate. The polymerase chain reaction introduced a few artifactual single base substitutions. Also, it was difficult to distinguish copies from the two alleles in many of the M13 clones. Therefore, several different strategies and analyses of about 50,000 nucleotide sequences in a series of clones were used to demonstrate that the mutation in the codon for glycine 883 was the only mutation in coding sequences for the triple-helical domain of type III procollagen that could have contributed to the phenotype. The same mutation in the codon for glycine 883 in one allele for type III procollagen was found in the proband's 52-year-old father who also had a mild variant of Ehlers-Danlos syndrome type IV. The type III procollagen synthesized by the proband's fibroblasts was analyzed by polyacrylamide gel electrophoresis. Less type III procollagen was secreted by the proband's fibroblasts than by control fibroblasts. Also, the thermal stability of the type III procollagen synthesized by the proband's fibroblasts was lower than the thermal stability of normal type III procollagen as assayed by brief protease digestion. The results, therefore, demonstrated that the single base mutation that converted the codon of glycine 883 to a codon for aspartate destabilized the entire triple helix of type III procollagen and probably accounted for the mild phenotype of Ehlers-Danlos syndrome type IV seen in the proband and her father.