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Canine COL1A2 Mutation Resulting in C‐Terminal Truncation of Pro‐α2(I) and Severe Osteogenesis Imperfecta
Author(s) -
Campbell Bonnie G.,
Wootton Joyce A. M.,
Macleod James N.,
Minor Ronald R.
Publication year - 2001
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.2001.16.6.1147
Subject(s) - microbiology and biotechnology , frameshift mutation , osteogenesis imperfecta , chemistry , point mutation , procollagen peptidase , complementary dna , mutant , gel electrophoresis , nonsense mutation , heteroduplex , mutation , type i collagen , biology , genetics , dna , biochemistry , gene , missense mutation , anatomy , endocrinology
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 [ 3 H]proline was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Increased density of pC‐α2(I) suggested comigration with the similarly sized pro‐α2(I) derived from the mutant allele. Furthermore, α‐chains were overhydroxylated and the ratio of α1(I):α2(I) was 3.2:1, consistent with the presence of α1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro‐α2(I) C‐propeptide and confirmed a diagnosis of OI.