Canine COL1A2 mutation resulting in C-terminal truncation of pro-alpha2(I) and severe osteogenesis imperfecta

J Bone Miner Res. 2001 Jun;16(6):1147-53. doi: 10.1359/jbmr.2001.16.6.1147.

Abstract

RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 ("CTAG") were replaced with "TGTCATTGG." The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-alpha2(I) suggested comigration with the similarly sized pro-alpha2(I) derived from the mutant allele. Furthermore, a-chains were overhydroxylated and the ratio of alpha1(I):alpha2(I) was 3.2:1, consistent with the presence of alpha1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-alpha2(I) C-propeptide and confirmed a diagnosis of OI.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cells, Cultured
  • Collagen / genetics*
  • Collagen Type I
  • DNA, Complementary / genetics
  • Dogs
  • Female
  • Fibroblasts
  • Frameshift Mutation*
  • Hydroxylation
  • Molecular Diagnostic Techniques
  • Molecular Sequence Data
  • Osteogenesis Imperfecta / genetics*
  • Procollagen / metabolism
  • Protein Subunits

Substances

  • Collagen Type I
  • DNA, Complementary
  • Procollagen
  • Protein Subunits
  • Collagen