Analysis of the COL1A1 and COL1A2 Genes by PCR Amplification and Scanning by Conformation-Sensitive Gel Electrophoresis Identifies Only COL1A1 Mutations in 15 Patients with Osteogenesis Imperfecta Type I: Identification of Common Sequences of Null-Allele Mutations

Abstract

Although >90% of patients with osteogenesis imperfecta (OI) have been estimated to have mutations in the COL1A1 and COL1A2 genes for type I procollagen, mutations have been difficult to detect in all patients with the mildest forms of the disease (i.e., type I). In this study, we first searched for mutations in type I procollagen by analyses of protein and mRNA in fibroblasts from 10 patients with mild OI; no evidence of a mutation was found in 2 of the patients by the protein analyses, and no evidence of a mutation was found in 5 of the patients by the RNA analyses. We then searched for mutations in the original 10 patients and in 5 additional patients with mild OI, by analysis of genomic DNA. To assay the genomic DNA, we established a consensus sequence for the first 12 kb of the COL1A1 gene and for 30 kb of new sequences of the 38-kb COL1A2 gene. The sequences were then used to develop primers for PCR for the 103 exons and exon boundaries of the two genes. The PCR products were first scanned for heteroduplexes by conformation-sensitive gel electrophoresis, and then products containing heteroduplexes were sequenced. The results detected disease-causing mutations in 13 of the 15 patients and detected two additional probable disease-causing mutations in the remaining 2 patients. Analysis of the data developed in this study and elsewhere revealed common sequences for mutations causing null alleles.

Molecular mechanism for distinct neurological phenotypes conveyed by allelic truncating mutations

“…This finding, in conjunction with previous results [31][32][33][34][35][36] , suggests that one of the physiological roles of NMD may be to protect against severe disease phenotypes by converting dominant-negative effects to haploinsufficiency. The extent of the beneficial effect may vary depending on both the toxicity of truncated proteins encoded by different genes and the nature of traits.…”

“…First, PTCs are common: nonsense and frameshift mutations are present in approximately one-third of mutations that cause human genetic disease 28,29 . Second, nonsense codons in all internal exons are capable of triggering NMD 23 , which means that mRNA levels will be reduced by most nonsense and frameshift mutations [28][29][30] ; therefore, PTCs generally result in milder phenotypes as compared with missense mutations, as has been postulated in several human diseases including osteogenesis imperfecta 31 , Stickler syndrome 32 and Marfan syndrome 33,34 . Third, on the basis that NMD has a potential role in most disease-causing PTCs, it has been proposed that haploinsufficiency is the most predictable pathogenetic mechanism underlying heterozygous nonsense alleles 29 .…”

Genetic evaluation of suspected osteogenesis imperfecta (OI)

“…Collagen screening and DNA-based testing are available at the University of Washington (www.pathology.washington.edu/clinical/collagen) and DNA-based testing is available at the Tulane University Matrix DNA Diagnostic Lab (http://www.som.tulane.edu/gene_therapy/matrix/matrix_dna_diagnostics.shtml). Test sensitivity is high for both types of tests but it is not clear if the sensitivity is additive 12,13. Mutations in COL1A1 and COL1A2 do not cause OI type V, VI or VII, which account for about 8% of all children with OI.…”

Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans

“…The first class of mutations usually produce premature termination codons in the coding sequence of one COL1A1 allele; these initiate nonsense mediated decay of the mRNA derived from that allele [Genovese and Rowe, 1987;Körkkö et al, 1998;Redford-Badwal et al, 1996;Slayton et al, 2000;Willing et al, 1996Willing et al, , 1992. The ensuing matrix insufficiency results in the mild type I OI clinical picture.…”