Mutations in type I collagen genes resulting in osteogenesis imperfecta in humans

Osteogenesis imperfecta (OI), commonly known as "brittle bone disease", is a dominant autosomal disorder characterized by bone fragility and abnormalities of connective tissue. Biochemical and molecular genetic studies have shown that the vast majority of affected individuals have mutations in either the COL1A1 or COL1A2 genes that encode the chains of type I procollagen. OI is associated with a wide spectrum of phenotypes varying from mild to severe and lethal conditions. The mild forms are usually caused by mutations which inactivate one allele of COL1A1 gene and result in a reduced amount of normal type I collagen, while the severe and lethal forms result from dominant negative mutations in COL1A1 or COL1A2 which produce structural defects in the collagen molecule. The most common mutations are substitutions of glycine residues, which are crucial to formation and function of the collagen triple helix, by larger amino acids. Although type I collagen is the major structural protein of both bone and skin, the mutations in type I collagen genes cause a bone disease. Some reports showed that the mutant collagen can be expressed differently in bone and in skin. Since most mutations identified in OI are dominant negative, the gene therapy requires a fundamentally different approach from that used for genetic-recessive disorders. The antisense therapy, by reducing the expression of mutant genes, is able to change a structural mutation into a null mutation, and thus convert severe forms of the disease into mild OI type I.     

Homozygous osteogenesis imperfecta unlinked to collagen I genes

Summary In a consanguineous pedigree in which a severe type of osteogenesis imperfecta was segregating as an autosomal recessive trait, analysis of genetic markers for both collagen I structural loci COL1A1 and COL1A2 showed that the phenotype was unlinked to either locus.     

Physical activity in youth with osteogenesis imperfecta type I

Introduction:

Individuals with Osteogenesis Imperfecta (OI) type I often show muscular weakness. However, it is unclear whether muscular weakness is a consequence of physical inactivity or a result of the disease itself. The aim was to assess muscle function in youth with OI type I and evaluate physical activity (PA).

Methods:

Fourteen children with OI type I (mean age [SD]: 12.75 [4.62] years) were compared to 14 age- and gender-matched controls (mean age [SD]: 12.75 [4.59] years). Muscle force and power were determined through mechanography. PA and daily energy expenditure were measured with an accelerometer and a questionnaire.

Results:

Compared to controls, children with OI type I had lower muscle force and power. OI type I children were as active as their healthy counterparts.

Conclusions:

Children and adolescents with OI type I and their healthy counterparts did not reached daily recommendations of PA. Given their muscle function deficit, youth with OI type I would benefit to reach these recommendations to prevent precocious effect of aging on muscles.     

Studies on type I collagen in skin fibroblasts cultured from twins with lethal osteogenesis imperfecta

Studies on type I procollagen produced by skin fibroblasts cultured from twins with lethal type II of osteogenesis imperfecta (OI) showed that biosynthesis of collagen (measured by L-[5-(3)H]proline incorporation into proteins susceptible to the action of bacterial collagenase) was slightly increased as compared to the control healthy infant. SDS/PAGE showed that the fibroblasts synthesized and secreted only normal type I procollagen. Electrophoretic analysis of collagen chains and CNBr peptides showed the same pattern of electrophoretic migration as in the controls. The lack of posttranslational overmodification of the collagen molecule suggested a molecular defect near the amino terminus of the collagen helix. Digestion of OI type I collagen with trypsin at 30 degrees C for 5 min generated a shorter than normal alpha2 chain which melted at 36 degrees C. Direct sequencing of an asymmetric PCR product revealed a heterozygous single nucleotide change C-->G causing a substitution of histidine by aspartic acid in the alpha2 chain at position 92. Pericellular processing of type I procollagen by the twin's fibroblasts yielded a later appearance of the intermediate pC-alpha1(I) form as compared with control cells.     

Hammerhead ribozymes selectively suppress mutant type I collagen mRNA in osteogenesis imperfecta fibroblasts

Ribozymes are a promising agent for the gene therapy of dominant negative genetic disorders by allele-specific mRNA suppression. To test allele-specific mRNA suppression in cells, we used fibroblasts from a patient with osteogenesis imperfecta (OI). These cells contain a mutation in one α1(I) collagen allele which both causes the skeletal disorder and generates a novel ribozyme cleavage site. In a preliminary in vitro assay, ribozymes cleaved mutant RNA substrate whereas normal substrate was left intact. For the studies in cell culture we generated cell lines stably expressing active (AR) and inactive (IR) ribozymes targeted to mutant α1(I) collagen mRNA. Quantitative competitive RT–PCR analyses of type I collagen mRNA, normalized to β-actin expression levels, revealed that the level of mutant α1(I) collagen mRNA was significantly decreased by ~50% in cells expressing AR. Normal α1(I) collagen mRNA showed no significant reduction when AR or IR was expressed from the pHβAPr-1-neo vector and a small (10–20%) but significant reduction when either ribozyme was expressed from the pCI.neo vector. In clonal lines derived from cells expressing AR the level of ribozyme expression correlated with the extent of reduction in the mutant:normal α1(I) mRNA ratio, ranging from 0.33 to 0.96. Stable expression of active ribozyme did not affect cell viability, as assessed by growth rates. Ribozyme cleavage of mutant mRNA results in a reduction in mutant type I collagen protein, as demonstrated by SDS–urea–PAGE. This is the first report of ribozymes causing specific suppression of an endogenous mutant mRNA in cells derived from a patient with a dominant negative genetic disorder.     

Studies of type I collagen (COL1A1) alpha1 chain in patients with osteogenesis imperfecta

Nucleotide sequences of exon 51, adjacent intron areas, and regulatory region of the alpha1 chain of type I collagen (COL1A1) gene were analyzed in 41 patients with osteogenesis imperfecta (OI) from 33 families and their 68 relatives residing at Bashkortostan Republic (BR). Six mutations (four nonsense mutations c.967G > T (p.Gly323X), c.1081C > T (p.Arg361X), c.1243C > T (p.Arg415X), and c.2869C > T (p.Gln957X)) in patients of the Russian origin and two mutations with open reading frame shift c.579delT (p.Gly194ValfsX71), and c.2444delG (p.Gly815AlafsX293)) in patients with OI of Tatar ethnicity as well as 14 single nucleotide polymorphisms in the COL1A1 gene were revealed. Mutations c.967G > T (p.Gly323X) and three alterations in the nucleotide sequence c.544-24C > T, c.643-36delT, and c.957 + 10insA were described for the first time.     

Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta.

Cultured skin fibroblasts from seven consecutive cases of lethal perinatal osteogenesis imperfecta (OI) expressed defects of type I collagen metabolism. The secretion of [14C]proline-labelled collagen by the OI cells was specifically reduced (51-79% of control), and collagen degradation was increased to twice that of control cells in five cases and increased by approx. 30% in the other two cases. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that four of the OI cell lines produced two forms of type I collagen consisting of both normally and slowly migrating forms of the alpha 1(I)- and alpha 2(I)-chains. In the other three OI cell lines only the 'slow' alpha (I)'- and alpha 2(I)'-chains were detected. In both groups inhibition of the post-translational modifications of proline and lysine resulted in the production of a single species of type I collagen with normal electrophoretic migration. Proline hydroxylation was normal, but the hydroxylysine contents of alpha 1(I)'- and alpha 2(I)'-chains purified by h.p.l.c. were greater than in control alpha-chains. The glucosylgalactosylhydroxylysine content was increased approx. 3-fold while the galactosylhydroxylysine content was only slightly increased in the alpha 1(I)'-chains relative to control alpha 1(I)-chains. Peptide mapping of the CNBr-cleavage peptides provided evidence that the increased post-translational modifications were distributed throughout the alpha 1(I)'- and alpha 2(I)'-chains. It is postulated that the greater modification of these chains was due to structural defects of the alpha-chains leading to delayed helix formation. The abnormal charge heterogeneity observed in the alpha 1 CB8 peptide of one patient may reflect such a structural defect in the type I collagen molecule.     

Structural heterogeneity of type I collagen triple helix and its role in osteogenesis imperfecta

We investigated regions of different helical stability within human type I collagen and discussed their role in intermolecular interactions and osteogenesis imperfecta (OI). By differential scanning calorimetry and circular dichroism, we measured and mapped changes in the collagen melting temperature (DeltaTm) for 41 different Gly substitutions from 47 OI patients. In contrast to peptides, we found no correlations of DeltaTm with the identity of the substituting residue. Instead, we observed regular variations in DeltaTm with the substitution location in different triple helix regions. To relate the DeltaTm map to peptide-based stability predictions, we extracted the activation energy of local helix unfolding (DeltaG) from the reported peptide data. We constructed the DeltaG map and tested it by measuring the H-D exchange rate for glycine NH residues involved in interchain hydrogen bonds. Based on the DeltaTm and DeltaG maps, we delineated regional variations in the collagen triple helix stability. Two large, flexible regions deduced from the DeltaTm map aligned with the regions important for collagen fibril assembly and ligand binding. One of these regions also aligned with a lethal region for Gly substitutions in the alpha1(I) chain.     

Detection of mutations in human type I collagen mRNA in osteogenesis imperfecta by indirect RNase protection

A method for detecting a wide variety of mutations within type I collagen has been developed and evaluated on a series of patients with osteogenesis imperfecta. RNA, extracted from the nuclear and cytoplasmic compartment of cultured fibroblasts from affected individuals, is hybridized with antisense single-stranded cDNA to the alpha 1(I) mRNA. The hybrid is digested with RNase A and T1 under varying degrees of stringency. The resistant RNA bands are separated by electrophoresis in agarose, transferred to nitrocellulose, and hybridized with antisense cRNA colinear with the protecting probe. This approach is capable of detecting previously defined mutations such as 252-base pair deletion and a 1-base pair mismatch within the alpha 1(I) mRNA. The method appears to be particularly useful in detecting abnormalities of RNA processing that behave as an insert or deletion within the mature mRNA. The procedure should be generally applicable for the identification and localization of any mutation within an entire gene if the gene is expressed as an RNA and a complete cDNA for the mRNA is available.     

Multiexon deletion in an osteogenesis imperfecta variant with increased type III collagen mRNA

Recently, the dermal fibroblasts (ATCC CRL 1262) of a lethal perinatal variant of osteogenesis imperfecta have been used for the first molecular characterization of a collagen gene defect (Chu, M. L., Williams, C. J., Pepe, G., Hirsch, J. L., Prockop, D. J., and Ramirez, F. (1983) Nature (Lond.) 304, 78-80). These studies revealed that the patient was heterozygous for an internal deletion of approximately 500 base pairs in the pro-alpha 1(I) collagen gene, consistent with previous investigations indicating that CRL 1262 fibroblasts equally synthesized a normal and a shortened pro-alpha 1(I) chain (Barsh, G. S., and Byers, P. H. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 5142-5146). Cloning and analysis of the affected allele of CRL 1262 has now indicated that the deletion is contained between two introns of the pro-alpha 1(I) gene and results in the elimination of three exons of the triple helical domain. Furthermore, the termini of the rearrangement are located within two short inverted repeats suggesting that the self-complementary nature of these DNA elements may have favored the formation of a DNA secondary structure intermediate which, in turn, served as substrate for the deletion. Evidence are also presented for an elevated Type III collagen mRNA content in the patient fibroblasts.     

Analysis of cyanogen bromide peptides of type I collagen from a patient with lethal osteogenesis imperfecta.

The CNBr peptides of type I collagen from bone of a patient with lethal osteogenesis imperfecta and age-matched controls were isolated by molecular-sieve chromatography and their amino acid compositions were determined. No differences were found between the compositions of the peptides from the patient and those from the controls, except for an increase in the degree of hydroxylation of lysine in all peptides from the patient. Type I collagen CNBr peptides from chick-embryo skin [Barnes, Constable Morton & Kodicek (1971) Biochem. J. 125, 925--928] and guinea-pig scar tissue [Shuttleworth, Forrest & Jackson (1975) Biochim. Biophys. Acta 379, 207--216] also have an increased degree of hydroxylation of lysine with an otherwise normal amino acid composition, and it was believed that this could be an embryonic form of collagen. As a similar collagen was present in the bones of the patient studied, it seems possible that the same 'embryonic' collagen is synthesized during development, in repair process and also in genetic disorders of collagen metabolism.     

Dental findings in osteogenesis imperfecta: I. Occurrence and expression of type I dentinogenesis imperfecta

Sixty-eight patients with osteogenesis imperfecta (OI) classified according to Sillence were evaluated for dentinogenesis imperfecta (DI). Orthopantomograms of 51 of the 68 were examined. Type I DI was recognized in 22 patients from 16 families. DI was observed in 4/45 patients with type I OI, in one of two patients with type III, and in 13/16 patients with type IV OI. Four of the five patients with an unidentified type of OI had DI. The expression of type I DI was variable. Discoloration and pulpal obliteration were the major manifestations. Teeth from 14 patients from 12 families were studied histologically. Eight of the 14 patients were from six families who had clinical and/or radiographic evidence of DI. Irregularity of the dentin matrix and tubular pattern in the circumpulpal dentin and normal mantle dentin were observed. Interfamilial variability was greater than intrafamilial variability. The expression of DI was mild in one family with type I OI. There was no further relation between the type of OI and the severity of DI.     

Two novelCOL1A1 mutations in patients with osteogenesis imperfecta (OI) affect the stability of the collagen type I triple-helix

Osteogenesis imperfecta (OI) is a bone dysplasia caused by mutations in theCOL1A1 andCOL1A2 genes. Although the condition has been intensely studied for over 25 years and recently over 800 novel mutations have been published, the relation between the location of mutations and clinical manifestation is poorly understood. Here we report missense mutations inCOL1A1 of several OI patients. Two novel mutations were found in the D1 period. One caused a substitution of glycine 200 by valine at the N-terminus of D1 in OI type I/IV, lowering collagen stability by 50% at 34°C. The other one was a substitution of valine 349 by phenylalanine at the C-terminus of D1 in OI type I, lowering collagen stability at 37.5°C. Two other mutations, reported before, changed amino residues in D4. One was a lethal substitution changing glycine 866 to serine in genetically identical twins with OI type II. That mutated amino acid was near the border of D3 and D4. The second mutation changed glycine 1040 to serine located at the border of D4 and D0.4, in a proband manifesting OI type III, and lowered collagen stability at 39°C (2°C lower than normal). Our results confirm the hypothesis on a critical role of the D1 and D4 regions in stabilization of the collagen triple-helix. The defect in D1 seemed to produce a milder clinical type of OI, whereas the defect in the C-terminal end of collagen type caused the more severe or lethal types of OI.     

Perinatal lethal osteogenesis imperfecta (OI type II): a biochemically heterogeneous disorder usually due to new mutations in the genes for type I collagen.

To resolve uncertainty concerning the inheritance of the perinatal lethal form of osteogenesis imperfecta (OI type II), we collected family data and radiographs for 71 probands and analyzed the collagens synthesized by dermal fibroblastic cells cultured from 43 of the probands, 19 parental pairs, and single parents of each of four additional probands. In 65 families for which there were complete data on sibship size, there was recurrence of the OI type II phenotype in five families such that six (8.6%) of 70 sibs were affected. In two families with recurrence, the radiographic phenotype was milder than that for the remainder; and one of those families was consanguinous, suggesting autosomal recessive inheritance. In the remaining three families there was no evidence of consanguinity, but in one of them the structure was compatible with gonadal mosaicism in the mother. In studies of collagens synthesized by cells from 43 infants, we identified two probands with separate rearrangements in an allele of one of the genes of type I collagen; but in the rest there were subtle mutations that disrupted the normal triple-helix structure of type I collagen molecules. In two probands we identified de novo mutations; in 16 additional families cells from the parents made only normal collagens, compatible with new mutations in their offsprings. These findings indicate that the OI type II phenotype is biochemically heterogeneous, that the majority result from new dominant mutations in the genes encoding type I collagen, and that some recurrences can be accounted for by gonadal mosaicism in one of the parents.     

Molecular findings in Brazilian patients with osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a genetic disorder of increased bone fragility and low bone mass. Severity varies widely, ranging from intrauterine fractures and perinatal lethality to very mild forms without fractures. Most patients with a clinical diagnosis of OI have a mutation in the COL1A1 or COL1A2 genes that encode the a chains of type I procollagen, the major protein in bones. Hence, the aim of the present study was to identify mutations in the COL1A1 gene in 13 unrelated Brazilian OI patients. This is the first molecular study of OI in Brazil. We found 6 mutations, 4 of them novel (c.1885delG, p.P239A, p.G592S, p.G649D) and 2 previously described (p.R237X and p.G382S). Thus, the findings show that there are no prevalent mutations in our sample, and that their distribution is similar to that reported by other authors, with preponderance of substitutions for glycine in the triple helix domain, causing OI types II, III and IV.     

A frameshift mutation results in a truncated nonfunctional carboxyl-terminal pro alpha 1(I) propeptide of type I collagen in osteogenesis imperfecta

A codon frameshift mutation caused by a single base (U) insertion after base pair 4088 of prepro alpha 1(I) mRNA of type I procollagen was identified in a baby with lethal perinatal osteogenesis imperfecta. The mutation was identified in fibroblast RNA by a new method that allows the direct detection of mismatched bases by chemical modification and cleavage in heteroduplexes formed between mRNA and control cDNA probes. The region of mismatches was specifically amplified by the polymerase chain reaction and sequenced. The heterozygous mutation in the amplified cDNA most likely resulted from a T insertion in exon 49 of COL1A1. The frameshift resulted in a truncated pro alpha 1(I) carboxyl-terminal propeptide in which the amino acid sequence was abnormal from Val1146 to the carboxyl terminus. The propeptide lacked Asn1187, which normally carries an N-linked oligosaccharide unit, and was more basic than the normal propeptide. The distribution of cysteines was altered and the mutant propeptide was unable to form normal interchain disulfide bonds. Some of the mutant pro alpha 1(I)' chains were incorporated into type I procollagen molecules but resulted in abnormal helix formation with over-hydroxylation of lysine residues, increased degradation, and poor secretion. Only normal type I collagen was incorporated into the extracellular matrix in vivo resulting in a tissue type I collagen content approximately 20% of that of control (Bateman, J. F., Chan, D., Mascara, T., Rogers, J. G., and Cole, W. G. (1986) Biochem. J. 240, 699-708).     

Altered processing of alpha (I) collagen in a case of lethal osteogenesis imperfecta

Cultured skin fibroblasts from a newborn with the lethal perinatal form of Osteogenesis imperfecta synthesized an over-hydroxylated form of pro alpha 1 (I) chain. The analysis of the CNBr peptides showed that over-hydroxylation occurred all along the molecule.     

Osteogenesis imperfecta murine: interaction between type I collagen homotrimers

Types I, II, and III collagens are believed to have evolved from the same homotrimer ancestor and they have substantial sequence homology, but type I molecules are alpha1(I)(2)alpha2(I) heterotrimers, unlike homotrimeric types II and III. It is believed that the alpha2(I) chain first appeared in lower vertebrates and that it plays a particularly important role in bone formation. For instance, spontaneous mutations resulting in non- functional alpha2 chains and formation of type I homotrimers cause severe bone pathology (osteogenesis imperfecta) in humans and in animals. However, the exact role of the alpha2 chain is not known. Here, we report measurements of intermolecular forces between collagen helices in native and reconstituted fibers composed of type I homotrimers, heterotrimers and their mix. For comparison, we report forces between type II homotrimers in reconstituted fibers. In agreement with previous studies, we find that the absence of the alpha2 chain reduces temperature-favored attraction between collagen helices, either because of the difference in amino acid sequence of the alpha1 and alpha2 chains or because of more extensive post-translational modification of homotrimers. We find that forces between helices in fibers from type I (as well as type II) homotrimers are not sensitive to pH between pH 6 and 7.5, in contrast to type I heterotrimers. Apparently, the effect of pH is related to extra histidine residues present on alpha2 chains but not on alpha1 chains. Finally, our measurements indicate that the alpha2 chain is responsible for binding some soluble compound(s), possibly glycosaminoglycans, whose displacement results, e.g., in the loss of tendon crystallinity. The ability of the alpha2 chain to bind non-collagen matrix components may be particularly important for bone matrix formation and mineralization.     

Two pregnancies after preimplantation genetic diagnosis for osteogenesis imperfecta type I and type IV

Osteogenesis imperfecta (OI) is an autosomal dominant genetic disorder characterized by the presence of brittle bones and decreased bone mass (osteopenia), as a result of mutations in the genes that encode the chains of type I collagen, the major protein of bone. The clinical features of the disease range from death in the perinatal period to normal life span with minimal increase in fractures. The present report describes two polymerase chain reaction (PCR)-based assays allowing preimplantation genetic diagnosis (PGD) on the one hand for OI type I, the mildest form, and on the other hand for OI type IV, which is intermediate in severity between OI type I and OI type III. In the couple referred for PGD for OI type I, the female partner carried a 1-bp deletion in exon 43 of the COL1A1 gene, resulting in a premature stop codon in exon 46. The synthesis of too little type I procollagen results from such a non-functional or COL1A1 null allele. In the other couple, referred for PGD for OI type IV, the male partner carried a G to A substitution in exon 19 of the COL1A2 gene, which results in an abnormal gene product due to an alphaGly247 (GGT) to Ser (AGT) substitution (G247S). Both mutations result in the loss of a specific restriction enzyme recognition site and can therefore be detected by PCR amplification followed by restriction fragment analysis. PCR amplification of genomic DNA of the parents-to-be with one of the two primers fluorescently labelled, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified and restricted fragments, allowed a distinction between the healthy and affected genotypes. PCR on single Epstein-Barr-virus (EBV)-transformed lymphoblasts resulted in acceptable amplification efficiencies (87% and 85% for OI type I and OI type IV respectively) and the allele drop-out (ADO) rate was assessed at 11.5% and 11.1% for OI type I and OI type IV respectively. With research blastomeres, 100% amplification rates were obtained and no contamination was observed in the blank controls, which validated the tests for clinical application. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal genotype of the non-affected parent. For OI type I, two frozen-thawed ICSI-PGD cycles and two fresh ICSI-PGD cycles were carried out for the same couple. The transfer of two unaffected embryos in the last cycle resulted in a twin pregnancy. A twin pregnancy was also achieved in one clinical ICSI-PGD cycle for OI type IV.