Canine COL1A2 mutation resulting in C-terminal truncation of pro-alpha2(I) and severe osteogenesis imperfecta.
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.
Campbell
B G
BG
Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
Wootton
J A
JA
Macleod
J N
JN
Minor
R R
RR
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics*; D024042:Collagen Type I; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D005260:Female; D005347:Fibroblasts; D016368:Frameshift Mutation; D006900:Hydroxylation; D025202:Molecular Diagnostic Techniques; D008969:Molecular Sequence Data; D010013:Osteogenesis Imperfecta / Q000235:genetics*; D011347:Procollagen / Q000378:metabolism; D021122:Protein Subunits
D024042:Collagen Type I; D018076:DNA, Complementary; D011347:Procollagen; D021122:Protein Subunits; D003094:Collagen