No abstract

A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro alpha 1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another lethal osteogenesis imperfecta cell strain (OI26), which has a frameshift mutation altering the sequence of the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande, S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989) J. Biol. Chem. 264, 10960-10964), disturbed procollagen folding and retarded the formation of disulfide-linked trimers. Although assembly was delayed, the presence of slowly migrating, overmodified alpha 1(I) and alpha 2(I) chains indicated that mutant pro alpha 1(I) could associate with normal pro alpha 1(I) and pro alpha 2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted. Further evidence of the aberrant folding of mutant procollagen in OI64 and OI26 was provided by experiments demonstrating that the endoplasmic reticulum resident molecular chaperone BiP, which binds to malfolded proteins, was specifically bound to type I procollagen and was coimmunoprecipitated in the osteogenesis imperfecta cells but not control cells. Experiments with brefeldin A, which inhibits protein export from the endoplasmic reticulum, demonstrated that unassembled mutant pro alpha 1(I) chains were selectively degraded within the endoplasmic reticulum resulting in reduced collagen production by the osteogenesis imperfecta cells. This biosynthetic deficiency was reflected in the inability of OI64 and OI26 cells to produce a substantial in vitro collagenous matrix when grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants showed a marked reduction in collagen accumulation to 20% (or less) of control cultures, comparable to the reduced collagen content of tissues from OI26.

Objective: We aimed to determine the frequency of all known forms of congenital muscular dystrophy (CMD) in a large Australasian cohort. Methods:We screened 101 patients with CMD with a combination of immunofluorescence, Western blotting, and DNA sequencing to identify disease-associated abnormalities in glycosylated ␣-dystroglycan, collagen VI, laminin ␣2, ␣7-integrin, and selenoprotein.Results: A total of 45% of the CMD cohort were assigned to an immunofluorescent subgroup based on their abnormal staining pattern. Abnormal staining for glycosylated ␣-dystroglycan was present in 25% of patients, and approximately half of these had reduced glycosylated ␣-dystroglycan by Western blot. Sequencing of the FKRP, fukutin, POMGnT1, and POMT1 genes in all patients with abnormal ␣-dystroglycan immunofluorescence identified mutations in one patient for each of these genes and two patients had mutations in POMT2. Twelve percent of patients had abnormalities in collagen VI immunofluorescence, and we identified disease-causing COL6 mutations in eight of nine patients in whom the genes were sequenced. Laminin ␣2 deficiency accounted for only 8% of CMD. ␣7-Integrin staining was absent in 12 of 45 patients studied, and ITGA7 gene mutations were excluded in all of these patients. LGMD2I ϭ limb-girdle muscular dystrophy type 2I; MDC1C ϭ congenital muscular dystrophy type 1C; MEB ϭ muscle-eyebrain disease; PVDF ϭ polyvinylidene fluoride; SSCP ϭ single strand conformational polymorphism; UCMD ϭ Ullrich congenital muscular dystrophy; WWS ϭ Walker-Warburg syndrome. Conclusions: