Mutation in the carboxy-terminal propeptide of the proα1 (I) chain of type I collagen in a child with severe osteogenesis imperfecta (OI type III): Possible implications for protein folding

A young girl presented with severe type III osteogenesis imperfecta; her otherwise healthy mother also had a mild connective tissue disorder with blue sclerae and recurrent joint dislocations. Skin fibroblast cultures from the child produced both normal and post-translationally overmodified type I collagen. The mutant collagen was poorly secreted but had normal thermal stability. Cyanogen bromide peptide maps of the abnormal protein indicated a C-terminal mutation. The mother's cells produced only normal-appearing collagens. Mismatch analysis and extensive sequencing of cDNAs covering the suspect region did not reveal any potentially causal changes in the triple helical domains of either the α1(I) or α2(I) chains. However, examination of the C-propeptide sequences revealed two heterozygous single base changes in the child. One, an A→C changing threonine to proline at residue 29 of the α2(I) C-propeptide was also present in the mother and maternal grandfather but not in 50 unrelated control individuals. The second, a T→C altered the last amino acid residue of the α1(I) C-propeptide from leucine to proline and had occurred de novo in the affected child. This mutation highlights the importance of the C-propeptides in molecular assembly but it is not clear how such an extreme mutation causes the delay in triple helix formation indicated by the extensive overmodification and reduced secretion of the mutant type I collagen. It may inhibit intrachain disulfide bonding or possibly affect the association of the procollagen chain with an intracellular “chaperone” protein that normally assists the assembly of trimeric procollagen molecules. © 1996 Wiley-Liss, Inc.     

Molecular heterogeneity in osteogenesis imperfecta type I

Osteogenesis imperfecta (OI) type I is characterized by bone fragility without significant deformity, osteopenia, normal stature, blue sclerae, and autosomal dominant inheritance. Dermal fibroblasts from most affected individuals produce about half the expected amount of type I collagen, suggesting that the OI type I phenotype results from a variety of mutations which alter the apparent expression of either COL1A1 or COL1A2, the genes encoding the chains of type I collagen. Short-pulse labeling of dermal fibroblasts with [³H]proline from affected individuals in 19 families indicates that most have alterations in the expected 2:1 synthetic ratio of proα1(I): proα2(I), with most having decreased production of proα1(I). Ratios of COL1A1:COL1A2 mRNA from these individuals, using slot-blot hybridization, indicate that they fall into different groups, but that most have decreased COL1A1 mRNA levels, compared with controls. These data suggest that most of our OI I families have COL1A1 mutations. Copy number and size of the COL1A1 gene by restriction endonuclease analysis of genomic DNA from affected individuals are normal in the families examined. We have identified one 3 generation family in which all affected members have one normal COL1A1 allele and another with a 5 base-pair deletion near the 3′ end of the gene. The deletion creates a shift in the translational reading-frame and predicts the synthesis of an elongated proα1(I) chain. In a second family, a father and a son have a single exon deletion that results from a splicing mutation. Chemical cleavage analysis of amplified cDNA from affected individuals in different regions of the COL1A1 gene, including the promoter, suggests that several individuals have point mutations within the coding region of the gene, while one individual may have a small deletion within the α1(I) carboxyl-terminal propeptide region. Our data provide evidence for significant molecular heterogeneity within the OI type I phenotype and indicate that a variety of mutations can result in decreased synthesis of type I collagen.     

Variable clinical expression in a family with OI type IV due to deletion of three base pairs in COL1A1

We have studied a family with autosomal dominant osteogenesis imperfecta (OI) type IV. Electrophoresis of collagen produced by cultured fibroblasts revealed a slower migrating population of collagen I. Cyanogen bromide peptide mapping localised the structural defect to the area of the αl(l)CB3 peptide. Subsequent sequencing revealed a deletion of nucleotides 1964–1966 in exon 27 of COL1A1. By means of restriction enzyme analysis, the deletion could be detected in all affected family members. This in-frame deletion resulted in the removal of alanine-438 and a Glu437Asp substitution in the proαl (I) collagen chain. Clinical variation was considerable among affected family members. The most consistent clinical features were reduced height and extraosseous manifestations of OI.     

Genetic and biochemical analyses of Israeli osteogenesis imperfecta patients

Osteogenesis imperfecta (OI) is clinically characterized by abnormal bone fragility, with most patients harboring heterozygote germline mutations in the COL1A1 or COL1A2 genes that encode the chains of type I procollagen, the major protein in bone. More than 250 mutations in both genes in OI patients have been reported, mostly missense mutations affecting glycine residues in the triple helical domains of the two chains. These mutations disrupt protein folding and structure, and their effects often can be detected by the analysis of proteins synthesized but cultured fibroblasts or, less often, osteoblasts. In this study, mutational analysis of all the COL1A1 and part of the COL1A2 was performed using exon-specific PCR amplification followed by denaturing gradient gel electrophoresis (DGGE) analysis and complemented by DNA sequencing in 57 Israeli OI patients from 55 unrelated families. Protein analysis was also performed using cultured fibroblasts obtained from a subset of these OI patients. Of 57 OI patients analyzed, 35 had OI type 1, 12 has OI type III, 8 had OI type IV, and 2 had OI type II. Fourteen different pathogenic mutations (10 novel) were identified in the COL1A1 gene: 3 missense, 5 nonsense, 3 insertion/deletion frameshift, 2 splice junction mutations, and 1 in frame deletion. We conclude that COL1A1 mutations underlie a subset of Israeli OI patients, that most commonly in OI type I, the mutations are contained within the COL1A1 gene, and that there are no predominant mutations in Jewish OI patients. Lastly, the use of protein analyses complements genetic analyses.     

A de novo G+1 → a mutation at the α2(I) exon 16 splice donor site causes skipping of exon 16 in the cDNA of one allele of an OI Type IV proband

We have investigated the procollagen, collagen, α2(I) mRNA, and DNA of a proband with type IV OI. The proband synthesized two α2(I) chains, one with normal electrophoretic migration and one more rapidly migrating. The fast α2(I) chain was relatively retained within the cell and was present in collagens synthesized in the presence of α,α′-dipyridyl. The α2(I) cyanogen bromide peptide CB 4-2 contained both normal and rapidly migrating components. Thermal stability of helices containing the rapidly migrating α2(I) chain was reduced 6°C. Parental fibroblast collagens were normal. RNA/RNA hybrids between proband total RNA and antisense riboprobe complementary to α2(I) nt 236–1390 were digested with RNase A and T1. Digestion products seen exclusively in the proband suggested a structural change in the region coding for exons 16-19. The region which hybridized to the riboprobe was amplified using RNA-PCR and subcloned. Multiple restriction enzyme digestions of the two subcloned alleles suggested a structural change localized to the region coding for exons 16-17. Sequencing revealed a deletion of the 54 bp comprising exon 16 in the cDNA of one allele. The region of the proband's genomic DNA spanning exons 15-17 was amplified by PCR. The subcloned genomic fragments of each allele were distinguished by RNA/DNA hybrid analysis using a riboprobe complementary to normal genomic DNA from this region. Sequencing revealed a G⁺¹ → A mutation at the exon 16 donor site in one allele. The mutation eliminates a Styl site. Digestion of PCR fragments amplified from the proband and parental WBC DNA revealed that only the proband had the undigested mutant fragment. © 1993 Wiley-Liss, Inc.     

Osteogenesis imperfecta: Recent findings shed new light on this once well-understood condition

Osteogenesis imperfecta is a systemic heritable disorder of connective tissue whose cardinal manifestation is bone fragility. In approximately 90% of individuals with osteogenesis imperfecta, mutations in either of the genes encoding the pro-α1 or pro-α2 chains of type I collagen (COL1A1 or COL1A2) can be identified. Of those without collagen mutations, a number of them will have mutations involving the enzyme complex responsible for posttranslational hydroxylation of the position 3 proline residue of COL1A1. Two of the genes encoding proteins involved in that enzyme complex, LEPRE1 and cartilage-associated protein, when mutated have been shown to cause autosomal recessive osteogenesis imperfecta, which has a moderate to severe clinical phenotype, often indistinguishable from osteogenesis imperfecta types II or III. Mutations in COL1A1 or COL1A2 which result in an abnormal protein still capable of forming a triple helix cause a more severe phenotype than mutations that lead to decreased collagen production as a result of the dominant negative effect mediated by continuous protein turnover. The current standard of care includes a multidisciplinary approach with surgical intervention when necessary, proactive physiotherapy, and consideration for the use of bisphosphonates all in attempts to improve quality of life.     

Complete COL1A1 allele deletions in osteogenesis imperfecta

PurposeTo identify a molecular genetic cause in patients with a clinical diagnosis of osteogenesis imperfecta (OI) type I/IV.MethodsThe authors performed multiplex ligation-dependent probe amplification analysis of the COL1A1 gene in a group of 106 index patients.ResultsIn four families with mild osteogenesis imperfecta and no other phenotypic abnormalities, a deletion of the complete COL1A1 gene on one allele was detected, a molecular finding that to our knowledge has not been described before, apart from a larger chromosomal deletion detected by fluorescent in situ hybridization encompassing the COL1A1 gene in a patient with mild osteogenesis imperfecta and other phenotypic abnormalities. Microarray analysis in three of the four families showed that it did not concern a founder mutation.ConclusionThe clinical picture of complete COL1A1 allele deletions is a comparatively mild type of osteogenesis imperfecta. As such, multiplex ligation-dependent probe amplification analysis of the COL1A1 gene is a useful additional approach to defining the mutation in cases of suspected osteogenesis imperfecta type I with no detectable mutation.     

A single amino acid substitution (D1441Y) in the carboxyl-terminal propeptide of the proalpha1(I) chain of type I collagen results in a lethal variant of osteogenesis imperfecta with features of dense bone diseases

Osteogenesis imperfecta (OI) is characterised by brittle bones and caused by mutations in the type I collagen genes, COL1A1 and COL1A2. We identified a mutation in the carboxyl-terminal propeptide coding region of one COL1A1 allele in an infant who died with an OI phenotype that differed from the usual lethal form and had regions of increased bone density. The newborn female had dysmorphic facial features, including loss of mandibular angle. Bilateral upper and lower limb contractures were present with multiple fractures in the long bones and ribs. The long bones were not compressed and their ends were radiographically dense. She died after a few hours and histopathological studies identified extramedullary haematopoiesis in the liver, little lamellar bone formation, decreased osteoclasts, abnormally thickened bony trabeculae with retained cartilage in long bones, and diminished marrow spaces similar to those seen in dense bone diseases such as osteopetrosis and pycnodysostosis. The child was heterozygous for a COL1A1 4321G→T transversion in exon 52 that changed a conserved aspartic acid to tyrosine (D1441Y). Abnormal proα1(I) chains were slow to assemble into dimers and trimers, and abnormal molecules were retained intracellularly for an extended period. The secreted type I procollagen molecules synthesised by cultured dermal fibroblasts were overmodified along the full length but had normal thermal stability. These findings suggest that the unusual phenotype reflected both a diminished amount of secreted type I procollagen and the presence of a population of stable and overmodified molecules that might support increased mineralisation or interfere with degradation of bone.     

An exon skipping mutation of a type V collagen gene (COL5A1) in Ehlers-Danlos syndrome.

The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of inherited connective tissue disorders characterised by skin hyperextensibility, joint hypermobility, easy bruising, and cutaneous fragility. Nine discrete clinical subtypes have been classified. We have investigated the molecular defect in a patient with clinical features of Ehlers-Danlos syndromes types I/II and VII. Electron microscopy of skin tissue indicated abnormal collagen fibrillogenesis with longitudinal sections showing a marked disruption of fibril packing giving very irregular outlines to transverse sections. Analysis of the collagens produced by cultured fibroblasts showed that the type V collagen had a population of alpha 1 (V) chains shorter than normal. Peptide mapping suggested a deletion within the triple helical domain. RTPCR amplification of mRNA covering the whole of this domain of COL5A1 showed a deletion of 54 bp. Although six Gly-X-Y triplets were lost, the essential triplet amino acid sequence and C-propeptide structure were maintained allowing mutant protein chains to be incorporated into triple helices. Genomic DNA analysis identified a de novo G+3-->T transversion in a 5' splice site of one COL5A1 allele. This mutation is analogous to mutations causing exon skipping in the major collagen genes, COL1A1, COL1A2, and COL3A1, identified in several cases of osteogenesis imperfecta and EDS type IV. These observations support the hypothesis that type V, although quantitatively a minor collagen, has a critical role in the formation of the fibrillar collagen matrix.     

Three novel type I collagen mutations in osteogenesis imperfecta type IV probands are associated with discrepancies between electrophoretic migration of osteoblast and fibroblast collagen

In three cases of type IV osteogenesis imperfecta (OI), we identified unique point mutations in type I collagen α1(I) Cdna. In two cases, the appearance of dimers indicated the presence of cysteine substitutions in the α1(I) protein chain. Cyanogen bromide digestion localized these cross-links to CB8 and 3, respectively. In the third case, the overmodification pattern of the CNBr peptides was compatible with a substitution in the aa 123–402 region of either type I collagen chain. We identified a unique point mutation in each proband, which resulted in substitutions for glycine residues in a 300-aa region of the α1(I) helix, specifically, Gly to Ala at codon 220 (GGT→GCT), Gly to Cys at codon 349 (GGT→TGT) and Gly to Cys at codon 523 (GGT→TGT). We compared each proband's fibroblast and osteoblast collagen directly, as well as with fibroblast and osteoblast controls. For all cases, the OI osteoblast collagen was more electrophoretically delayed than OI fibroblast collagen. In the patient with G349C, OI fibroblast and osteoblast collagen synthesized in the presence of α,α′-dipyridyl co-migrated on gels, demonstrating that the electrophoretic discrepancy resulted from differences in post-translational modification. Melting temperature curves for stability of the collagen helix yielded an identical T_m for control fibroblast and osteoblast collagen (41.2°C). By contrast, for collagen with the gly349→cys substitution, the T_m of the fibroblast collagen was 1°C lower than the T_m of the osteoblast collagen. These data indicate that the metabolism of mutant collagen might be cell-specific and has significant implications for understanding the phenotype/genotype correlations and the pathophysiology of OI. Hum Mutat 11:395–403, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Two new recurrent nucleotide mutations in the COL1A1 gene in four patients with osteogenesis imperfecta: About one-fifth are recurrent

Previous observations on mutations causing osteogenesis imperfecta (OI) suggested that unrelated patients had private mutations. Here preliminary studies on two patients with type I OI indicated that some mutations in the COL1A1 gene for type I procollagen cannot be detected by analyses of cDNAs. Therefore, we developed a protocol whereby 43 exon and exon flanking sequences of the COL1A1 gene can be amplified by PCR and scanned for mutations by denaturing gradient gel electrophoresis. Two new recurrent nucleotide mutations in the gene were found in four apparently unrelated patients with OI. Analysis of previous publications indicated that up to one-fifth of the mutations causing OI are recurrent in the sense that they were identical in apparently unrelated probands. About 80% of these identical mutations were in CpG dinucleotide sequences. Hum Mutat 9:148–156, 1997. © 1997 Wiley-Liss, Inc.     

OIM and related animal models of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is characterized by fragile bones, skeletal deformity, and growth retardation. This heritable disorder of connective tissue is the result of mutations affecting the COL1A1 and COL1A2 genes of type I collagen. Progress in OI research has been limited because of dependence on human fibroblast and osteoblast specimens and the absence of a naturally occurring animal model for this genetic disorder. Recent technology in molecular biology has led to the development of transgenic models of OI based on site directed mutagenesis of type I collagen genes. OIM is a naturally occurring model which incorporates both the phenotypic and biochemical defects of moderate to severe osteogenesis imperfecta. This powerful tool permits the development of models based on different type I collagen mutations. The collagen type I mutation in OIM is a C propeptide deletion which impairs the production of normal pro-alpha2(I). Tissues in OIM contain only [pro-alpha1(I)]3 homotrimer. Thus, although several animal models are now available for research in osteogenesis imperfecta few are viable or fully mimic human disease disorders. OIM duplicates the phenotype and biochemistry of human disease and has a normal life span.     

Genotypic and phenotypic characterization of Chinese patients with osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a rare hereditary skeletal dysplasia, characterized by recurrent fractures and bone deformity. This study presents a clinical characterization and mutation analysis of 668 patients, aiming to establish the mutation spectrum and to elucidate genotype–phenotype correlations in Chinese OI patients. We identified 274 sequence variants (230 in type I collagen encoding genes and 44 in noncollagen genes), including 102 novel variants, in 340 probands with a detection rate of 90%. Compared with 47 loss‐of‐function variants detected in COL1A1, neither nonsense nor frameshift variants were found in COL1A2 (p < 0.0001). The major cause of autosomal recessive OI was biallelic variants in WNT1 (56%, 20/36). It is noteworthy that three genomic rearrangements, including one gross deletion and one gross duplication in COL1A1 as well as one gross deletion in FKBP10, were detected in this study. Of ten individuals with glycine substitutions that lie towards the N‐terminal end of the triple‐helical region of the α1(I) chain, none exhibited hearing loss, suggesting a potential genotype–phenotype correlation. The findings in this study expanded the mutation spectrum and identified novel correlations between genotype and phenotype in Chinese OI patients.     

Mutation analysis of COL1A1 and COL1A2 in patients diagnosed with osteogenesis imperfecta type I-IV

Osteogenesis Imperfecta (OI) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. To date, seven types of OI have been described, based on clinical phenotype and histological findings. Most patients with a clinical diagnosis of OI type I-IV have a mutation in the COL1A1 or COL1A2 genes which encode the two alpha chains of type I collagen, the major component of the bone matrix. Analysis of COL1A1 and COL1A2 in a cohort of 83 unrelated patients with OI type I-IV identified a total of 62 mutations. Thirty-eight appear novel, 26 in COL1A1, and 12 in COL1A2, and these are described here. The largest group consists of point mutations affecting glycine residues in the triple helical domain of the two alpha chains, predicted to disrupt protein folding and structure. This is in accordance with previously published data. A doublet GC deletion, an unusual 398 base deletion predicted to completely remove exon 20 of COL1A2, and a point mutation resulting in substitution of a conserved cysteine in the C-terminal propeptide are described. In addition rare mutations at the cleavage sites of the C-propeptide and the N-terminal signal peptide are described.     

Perinatal lethal osteogenesis imperfecta.

Perinatal lethal osteogenesis imperfecta is the result of heterozygous mutations of the COL1A1 and COL1A2 genes that encode the alpha 1(I) and alpha 2(I) chains of type I collagen, respectively. Point mutations resulting in the substitution of Gly residues in Gly-X-Y amino acid triplets of the triple helical domain of the alpha 1(I) or alpha 2(I) chains are the most frequent mutations. They interrupt the repetitive Gly-X-Y structure that is mandatory for the formation of a stable triple helix. Most babies have their own private de novo mutation. However, the recurrence rate is about 7% owing to germline mosaicism in one parent. The mutations act in a dominant negative manner as the mutant pro alpha chains are incorporated into type I procollagen molecules that also contain normal pro alpha chains. The abnormal molecules are poorly secreted, more susceptible to degradation, and impair the formation of the extracellular matrix. The collagen fibres are abnormally organised and mineralisation is impaired. The severity of the clinical phenotype appears to be related to the type of mutation, its location in the alpha chain, the surrounding amino acid sequences, and the level of expression of the mutant allele.     

Mutational spectrum of type I collagen genes in Korean patients with osteogenesis imperfecta

Mutations in the type I collagen genes COL1A1 and COL1A2 are responsible for the dominantly inherited connective tissue disorder osteogenesis imperfecta (OI). The severity of OI is diverse, ranging from perinatal lethality to a very mild phenotype that is characterized by normal stature and the absence of deformities. Although there have been several studies on the mutational spectra of COL1A1 and/or COL1A2 in Western populations, very few cases have been reported from Asia. In this study, we investigated 67 unrelated Korean probands with OI and used nucleotide sequence analysis to detect COL1A1 and COL1A2 mutations. Thirty-five different mutations were identified in the two genes, including 24 novel mutations. Among the 35 kinds of detected mutations, 15 were glycine substitutions (seven in COL1A1 and eight in COL1A2), one was a nonsense mutation, four were frameshift mutations in COL1A1, three were in-frame duplications in COL1A2, and 12 were splice site mutations (seven in COL1A1 and five in COL1A2). Until now, mutations in the COL1A1 and COL1A2 genes known to cause OI were unique and rarely repeated in other families. Interestingly, the c.982G>A (p.Gly328Ser) mutation in COL1A2 was found recurrently and was the causative mutation in five independent OI probands. Haplotype analysis of the COL1A2 gene revealed that four probands from five independent OI probands with c.982G>A (p.Gly328Ser) had a common haplotype. Our clinical data showed the heterogeneity even within a specific genotype, which suggested the complex expression of this disease.     

Recurrence of lethal osteogenesis imperfecta due to parental mosaicism for a mutation in the COL1A2 gene of type I collagen. The mosaic parent exhibits phenotypic features of a mild form of the disease.

We have determined that a man, ascertained because he fathered a child with lethal osteogenesis imperfecta (OI) with each of two partners, is mosaic in both his germline and somatic tissues for a mutation in the COL1A2 gene which encodes the pro alpha 2(I) chain of type I procollagen. His dermal fibroblasts were previously shown to synthesize a population of cysteine-containing alpha 2(I) chains that were posttranslationally overmodified. DNA sequence analysis of COL1A2 cDNAs demonstrated that the cysteine-containing chain resulted from a point mutation (G to T) in the first position of the codon for the glycine at residue 472 of the triple helical domain. Genomic DNA from the one available affected infant contained the mutant and normal COL1A2 alleles in equal proportion. Examination of DNA from several tissues of the father showed that the mutant allele was present in approximately 40% of his sperm, 80% of his lymphocytes, and nearly 100% of his dermal fibroblasts. Despite the high level of mosaicism detected in somatic tissues, the only phenotypic manifestation of OI in the proband was that he was shorter than his unaffected male relatives and had mild dentinogenesis imperfecta. Thermal stability of type I collagen molecules containing the substitution was decreased, but to a lesser extent than for a nonlethal cysteine for glycine substitution at residue 259 of alpha 2(I), indicating that this measure of molecular stability may be of limited use in explaining the pathogenesis of osteogenesis imperfecta.