Identification of the oim mutation by dye terminator chemistry combined with automated direct DNA sequencing

Abstract

The homozygous oim/oim mouse, a model of moderate-to-severe human osteogenesis imperfecta, contains a G-nucleotide deletion in the Cola-2 gene (the murine pro alpha(I) collagen gene) that results in accumulation of alpha1(I) homotrimer collagen. Although these mice have a distinctive phenotype that includes multiple fractures and deformities, genotyping is necessary to distinguish them from their wildtype (+/+) and heterozygote (oim/+) littermates. In this study, the dye primer and dye terminator chemistry methods, in combination with automated direct DNA sequencing, were compared for accuracy and ease in genotyping. A total of 82 mice from 14 litters were bred and genotyped; this resulted in 18 +/+, 35 oim/+, and 29 oim/oim mice. The dye primer and dye terminator chemistry methods worked equally well for identification of the deletion mutation and thus the genotype of all of the mice. However, the dye terminator method was found to be superior on the basis of the reduced amount of sample handling and reduced quantity of reagent required.

Novel quantitative trait loci for central corneal thickness identified by candidate gene analysis of osteogenesis imperfecta genes

“…The homozygous mutant mice have a complete deWciency of col1a2 protein and exhibit a phenotype similar to moderate OI in humans, including susceptibility to fractures, low bone density and skeletal deformities (Chipman et al 1993). Heterozygotes exhibit a variable and intermediate phenotype (Camacho et al 1998). Both the wt/oim and oim/ oim mice exhibited a thinner CCT compared to the wt/wt animals, with a notable allele dosage eVect resulting in each oim allele contributing to a reduction in CCT.…”

Abnormal Mineral-Matrix Interactions Are a Significant Contributor to Fragility in oim/oim Bone

“…Upon weaning of the pups at approximately 4 weeks of age, tail snips 1 cm long were obtained for genotype determination [23]. A total of 58 femora from 48 mice were tested either in a three-point bend test or in tension.…”

Canine COL1A2 Mutation Resulting in C-Terminal Truncation of Pro-α2(I) and Severe Osteogenesis Imperfecta

“…Heterozygous oim /+ mice have both heterotrimeric and α1(I)‐homotrimeric type I collagen in the same fibrils (22) . The oim /+ phenotype ranges from clinically normal to severely affected mice that are clinically indistinguishable from oim/oim individuals; the latter is comparable with the severe phenotype seen in this heterozygous Beagle dog (12) . The α1(I)‐homotrimers were the only form of type I collagen in the matrix of a human OI variant in which the last 33 amino acids in pro‐α2(I)‐chains were changed by a homozygous 4‐bp frameshift deletion near the 3′ end of COL1A2 (3) .…”

“…The first of these was a mouse ( oim ) with a frameshift mutation in the region of COL1A2 coding for the C‐propeptide of pro‐α2(I) (10, 11) . Homozygous oim/oim and some oim/+ mice have a severe phenotype analogous to human OI type III (12) . A recent report described abnormalities in type I collagen in three dogs with brittle bone disease, and COL1 mutations have since been identified in two of these animals (13, 14) .…”