Instability of Polymeric Skin Collagen in Osteogenesis Imperfecta

The structural polymeric collagen of the skin of 19 patients with osteogenesis imperfecta has been examined. In those with severe bone disease, who often have white sclerae, this collagen fraction is less resistant to depolymerization than that of age-matched controls, though the total amount is normal. In patients with less severe bone disease, whose sclerae are usually blue, the polymeric collagen may have normal stability but the total amount is reduced. These results suggest defective cross-linking of collagen in severe osteogenesis imperfecta.     

Dentinogenesis imperfecta

Dentinogenesis imperfecta is a congenital dentin dysplasia that occurs either isolated or associated with a genetic disorder known as osteogenesis imperfecta. Dentinogenesis imperfecta is inherited in an autosomal dominant pattern. Clinically the teeth color of both dentitions varies from brown to a translucent gray with an opalescent sheen. Shields et al. (1973) proposed a classification of Dentinogenesis imperfecta into three types: type I, associated with osteogenesis imperfecta; type II, hereditary opalescent dentin; type III Brandywine-type. The phenotypes of Dentinogenesis imperfecta are described in regard to their genetic defects, pathology, radiology and histopathology as well as their dental treatment.     

Proteomic analysis of plasma profiles in children with recurrent bone fractures

The aim of the study is proteomic analysis of the plasma profile in children with recurrent bone fractures. The study involved 16 children: 6 patients with recurrent low-energy fractures and normal bone mass and 10 with osteogenesis imperfecta. In the analysis of the protein profile, the two-dimensional protein electrophoresis was used (Ettan DALT II, Amersham Bioscience). The images of protein gels were compared with controls. The protein spots with changed expression were cut from the gel and the amino acid sequence was analyzed with the mass spectrometry method (Q-Tof Premier(TM) API MASS SPECTROMETR, Waters) for protein identification. The most prevalent protein with changed expression, with respect to controls, was haptoglobin observed in 6 patients with a severe form of osteogenesis imperfecta. Increased haptoglobin concentration in these patients was confirmed by the ELISA method. Peptides corresponding to alpha-1 acid glycoprotein and serum amyloid P-component, apolipoprotein A-I, and transthyretin were detected in one, two and three children, respectively. Conclusions: 1) The results show increased haptoglobin which may be suggestive of an inflammatory component taking part in the course of osteogenesis imperfecta. 2) Further studies to explain the possible relationship of this protein with increased bone fragility are necessary.     

The co-existence of primary hyperparathyroidism and osteogenesis imperfecta

A 47-year-old patient suffering from osteogenesis imperfecta was found to have mild hypercalcemia. The latter proved to be due to a parathyroid adenoma. The clinical and laboratory features of this association are summarized, and the implications of serum calcium abnormalities in osteogenesis imperfecta are discussed.     

RNA sequence analysis of a perinatal lethal osteogenesis imperfecta mutation

The perinatal lethal form of osteogenesis imperfecta often results from mutations which disrupt stable assembly, delay secretion, and cause excessive posttranslational modification of type I procollagen molecules. One such mutation was efficiently characterized by an indirect method of RNA sequence analysis. The mutation initially was localized in procollagen by mapping the distribution of abnormal posttranslational modification within the triple helical domain of mutant molecules. Total RNA was isolated from osteogenesis imperfecta cells in culture, cDNA was synthesized using alpha 1(I) and alpha 2(I) specific primers, and fragments of cDNA suspected to harbor the mutation were amplified by the polymerase chain reaction technique and then cloned in M13 vectors. Sequence analysis of the amplified cDNA revealed a new, heterozygous Gly----Val substitution at residue 256 of the triple helical domain of alpha 1(I) chains produced by the perinatal lethal osteogenesis imperfecta cells. The nature and location of the mutation were confirmed by sequence analysis of amplified genomic DNA. A Gly----Val substitution has not previously been associated with the lethal form of osteogenesis imperfecta, and this mutation has the most amino-terminal location within the alpha 1(I) chain triple helical domain reported to date.     

Les complications orthopédiques de l’ostéogenèse imparfaite

Osteogenesis imperfecta is a genetic disease characterized by bone frailty. It is generally caused by an abnormal production of collagen, which is the main fibrous protein of the bone. Collagen is also present in the skin, tendons, the sclera of the eye and dentin. The most frequent manifestation of osteogenesis imperfecta is the occurrence of multiple fractures without major trauma. Severity and timing of the attack varies widely: some patients sustain a significant number of fractures during early childhood which may have a serious impact on growth, while others will have some fractures separated by a few years. In all cases, the bone strength improves in adulthood. The bone fractures cause pain and bone deformities sometimes result in a smaller size. Scoliosis is frequent and associated with painful vertebral collapses. We present a case of osteogenesis imperfecta in a 40-year-old adult and we describe the various orthopaedic complications of the disease, stressing the role of bone scintigraphy in the diagnosis and monitoring of these complications.     

The effects of different cysteine for glycine substitutions within alpha 2(I) chains. Evidence of distinct structural domains within the type I collagen triple helix

Affected individuals from two apparently distinct, mild osteogenesis imperfecta families were heterozygous for a G to T transition in the COL1A2 gene that resulted in cysteine for glycine substitutions at position 646 in the alpha 2(I) chain of type I collagen. A child with a moderately severe form of osteogenesis imperfecta was heterozygous for a G to T transition that resulted in a substitution of cysteine for glycine at position 259 in the COL1A2 gene. Type I collagen molecules containing an alpha 2(I) chain with cysteine at position 259 denaturated at a lower temperature than molecules containing an alpha 2(I) chain with cysteine at position 646. In contrast to cysteine for glycine substitutions in the alpha 1(I) chain, the severity of the osteogenesis imperfecta phenotype is not directly proportional to the distance of the mutation from the amino-terminal end of the triple helix. These findings could be explained if the type I collagen triple helix contains discontinuous domains that differ in their contributions to maintaining helix stability.     

Neural networks for modeling gene-gene interactions in association studies

Background

The monogenic disease osteogenesis imperfecta (OI) is due to single mutations in either of the collagen genes ColA1 or ColA2, but within the same family a given mutation is accompanied by a wide range of disease severity. Although this phenotypic variability implies the existence of modifier gene variants, genome wide scanning of DNA from OI patients has not been reported. Promising genome wide marker-independent physical methods for identifying disease-related loci have lacked robustness for widespread applicability. Therefore we sought to improve these methods and demonstrate their performance to identify known and novel loci relevant to OI.

Results

We have improved methods for enriching regions of identity-by-descent (IBD) shared between related, afflicted individuals. The extent of enrichment exceeds 10- to 50-fold for some loci. The efficiency of the new process is shown by confirmation of the identification of the Col1A2 locus in osteogenesis imperfecta patients from Amish families. Moreover the analysis revealed additional candidate linkage loci that may harbour modifier genes for OI; a locus on chromosome 1q includes COX-2, a gene implicated in osteogenesis.

Conclusion

Technology for physical enrichment of IBD loci is now robust and applicable for finding genes for monogenic diseases and genes for complex diseases. The data support the further investigation of genetic loci other than collagen gene loci to identify genes affecting the clinical expression of osteogenesis imperfecta. The discrimination of IBD mapping will be enhanced when the IBD enrichment procedure is coupled with deep resequencing.     

Increased expression of the gene for the pro alpha 1(IV) chain of basement-membrane procollagen in cultured skin fibroblasts from two variants of osteogenesis imperfecta.

Fibroblasts from two lethal variants of osteogenesis imperfecta were shown to synthesize increased amounts of type IV procollagen. Previous studies established that one of these variants had a non-functional allele for the pro alpha 2 chain of type I procollagen, whereas the other pro alpha 2(I) allele contained a mutation leading to synthesis of shortened pro alpha 2(I) chains. In the two variants, the relative level of mRNA for pro alpha 1(IV) was 31 and 42% of the level of mRNA for pro alpha 1(I) chains. A value of less than 2% was found for a third lethal and four non-lethal variants of osteogenesis imperfecta. Immunofluorescent staining of fibroblasts from the two variants synthesizing increased amounts of type IV procollagen indicated that a homogeneous population of cells synthesized both type IV and type I procollagen. The results suggest that mutations in the type I procollagen genes that result in osteogenesis imperfecta can be associated with increased expression of the genes for type IV procollagen.     

Dental findings in osteogenesis imperfecta: II. Dysplastic and other developmental defects

Orthopantomograms of 49 patients with osteogenesis imperfecta (OI) classified according to Sillence were examined for dysplastic dentin defects and other developmental abnormalities of the teeth. Thistle-tube-shaped pulps in the permanent teeth were observed in five patients. Four of them had pulp stones in several teeth. Apically extended pulp chambers were present in 3/49 patients, and the structure of the mandible was cystic in 1/49. These defects occurred in patients with type I or unclassifiable OI and were mainly not associated with type I dentinogenesis imperfecta (DI). The prevalence rates of invaginations (10.2%) and hypodontia of permanent teeth (18.4%) exceeded those in the normal population. The frequent developmental disturbances of the teeth in OI may be secondary to the connective tissue defect. The relation of the dysplastic defects other than type I DI to OI remains to be clarified.     

Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta

In principle, transplantation of mesenchymal progenitor cells would attenuate or possibly correct genetic disorders of bone, cartilage and muscle, but clinical support for this concept is lacking. Here we describe the initial results of allogeneic bone marrow transplantation in three children with osteogenesis imperfecta, a genetic disorder in which osteoblasts produce defective type I collagen, leading to osteopenia, multiple fractures, severe bony deformities and considerably shortened stature. Three months after osteoblast engraftment (1.5-2.0% donor cells), representative specimens of trabecular bone showed histologic changes indicative of new dense bone formation. All patients had increases in total body bone mineral content ranging from 21 to 29 grams (median, 28), compared with predicted values of 0 to 4 grams (median, 0) for healthy children with similar changes in weight. These improvements were associated with increases in growth velocity and reduced frequencies of bone fracture. Thus, allogeneic bone marrow transplantation can lead to engraftment of functional mesenchymal progenitor cells, indicating the feasibility of this strategy in the treatment of osteogenesis imperfecta and perhaps other mesenchymal stem cell disorders as well.     

Altered triple helical structure of type I procollagen in lethal perinatal osteogenesis imperfecta

Cultured dermal fibroblasts from an infant with the lethal perinatal form of osteogenesis imperfecta (type II) synthesize normal and abnormal forms of type I procollagen. The abnormal type I procollagen molecules are excessively modified during their intracellular stay, have a lower than normal melting transition temperature, are secreted at a reduced rate, and form abnormally thin collagen fibrils in the extracellular matrix in vitro. Overmodification of the abnormal type I procollagen molecules was limited to the NH2-terminal three-fourths of the triple helical domain. Two-dimensional mapping of modified and unmodified alpha chains of type I collagen demonstrated neither charge alterations nor large insertions or deletions in the region of alpha 1(I) and alpha 2(I) in which overmodification begins. Both the structure and function of type I procollagen synthesized by cells from the parents of this infant were normal. The simplest interpretation of the results of this study is that the osteogenesis imperfecta phenotype arose from a new dominant mutation in one of the genes encoding the chains of type I procollagen. Given the requirement for glycine in every third position of the triple helical domain, the mutation may represent a single amino acid substitution for a glycine residue. These findings demonstrate further heterogeneity in the biochemical basis of osteogenesis imperfecta type II and suggest that the nature and location of mutations in type I procollagen may determine phenotypic variation.     

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.     

Metacarpal morphometry in adults with osteogenesis imperfecta.

Osteogenesis imperfecta is often regarded as a form of osteoporosis. In many cases, particularly those in whom the first fracture occurs outside the neonatal period, bones that have not been fractured may appear radiologically normal. In a group of 24 adults with osteogenesis imperfecta the thickness of the metacarpal cortex was normal but their bones were often slender. Osteoporosis is probably not an inevitable feature of such cases, and some of the radiological abnormalities reported may be the results of previous fractures and their treatment.     

Osteogenesis imperfecta type IV. Detection of a point mutation in one alpha 1(I) collagen allele (COL1A1) by RNA/RNA hybrid analysis

We have identified a point mutation in one alpha 1(I) collagen allele (COL1A1) of a child with the type IV osteogenesis imperfecta phenotype. When compared to parental and control samples, skin fibroblasts of the proband synthesized two populations of type I collagen molecules. One population was normal; the other was delayed in secretion and electrophoretic migration due to post-translational overmodification. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated a gradient of overmodification beginning near the carboxyl-terminal CB peptides. This predicts that the mutation delaying helix formation is near the carboxyl-terminal end of one of the component chains of type I collagen. The mRNA of the patient was probed with overlapping antisense riboprobes to type I collagen cDNA. Cleavage of a mismatch in RNA/RNA hybrids of RNase A allowed the location of the mutation to a 225-base pair region of alpha 1(I) cDNA. The mismatch was not present in RNA/RNA hybrids from either parent. This region of both alpha 1(I) alleles of the patient was isolated by screening a lambda ZAP cDNA library. Sequence determination of both alleles demonstrated a single nucleotide change, G----A, resulting in the substitution of a serine for a glycine at amino acid residue 832. This point mutation occurs in the coding region for alpha 1(I) CB6 and is concordant with the protein data. The finding of a glycine substitution in an alpha 1(I) chain of a patient with the milder type IV osteogenesis imperfecta phenotype requires modification of current molecular models for types II and IV osteogenesis imperfecta.     

Distinct biochemical phenotypes predict clinical severity in nonlethal variants of osteogenesis imperfecta.

We reviewed clinical and biochemical findings from 132 probands with nonlethal forms of osteogenesis imperfecta (OI) whose fibroblasts were sent to the University of Washington for diagnostic studies in the years 1981-87. In cells from 86% of probands with nonlethal OI we identified biochemical alterations compatible with heterozygosity for a mutation that affected expression or structure of alpha chains of type I procollagen. We observed two major biochemical phenotypes. Cells from 40 probands (group A) secreted about half the normal amount of normal type I procollagen and no identifiable abnormal molecules; these patients were generally of normal stature, rarely had bone deformity or dentinogenesis imperfecta, and had blue sclerae. Cells from 74 probands (group B) produced and secreted normal and abnormal type I procollagen molecules; these patients were usually short and had bone deformity and dentinogenesis imperfecta, and many had grey or blue-grey sclerae. In cells from an additional 18 probands (group C) we were unable to identify altered type I procollagen synthesis or structure. Detection of these abnormalities has value in the determination of mode of inheritance and in the prediction of clinical severity.     

Transgenic mice that express a mini-gene version of the human gene for type I procollagen (COL1A1) develop a phenotype resembling a lethal form of osteogenesis imperfecta.

A mini-gene version of the human gene for a pro-alpha 1(I) chain of type I procollagen (COL1A1) was prepared that contained -2.5 kilobases of the promoter region and the 5'- and 3'-ends of the gene but lacked a large central region containing 41 exons. The construct was modeled after a sporadic in-frame deletion of the human gene that produced a lethal variant of osteogenesis imperfecta, because it caused synthesis of shortened pro-alpha 1(I) chains that associated with normal pro-alpha 1(I) and pro-alpha 2(I) chains and caused degradation of both the shortened and normal pro-alpha chains through a process called procollagen suicide. The mini-gene was used to prepare transgenic mice. Eight of 15 transgenic mice expressed varying levels of the gene. All except one of the Fo founders were phenotypically normal, but several of the founders were apparently mosaic since they produced F1 progeny that died shortly after birth with a distinctive phenotype. The phenotype included extensive fractures of ribs and long bones similar to the fractures seen in lethal variants of osteogenesis imperfecta. Mice with the lethal phenotype expressed much higher levels of the mini-gene than transgenic mice without the lethal phenotype. Experiments with cultured skin fibroblasts from the transgenic mice demonstrated that shortened pro-alpha 1(I) chains synthesized from the mini-gene became disulfide-linked to pro-alpha 1(I) chains synthesized from the endogenous mouse gene. The results demonstrate that a mutated type I procollagen gene based on the model of procollagen suicide can be used to produce a severe phenotype of osteogenesis imperfecta that is genetically transmitted.     

Nuclear magnetic resonance characterization of peptide models of collagen-folding diseases

Misfolding of the triple helix has been shown to play a critical role in collagen diseases. The substitution of a single Gly by another amino acid breaks the characteristic repeating (Gly-X-Y)n sequence pattern and results in connective tissue disease such as osteogenesis imperfecta. Nuclear magnetic resonance (NMR) studies of normal and mutated collagen triple-helical peptides offer an opportunity to characterize folding and conformational alterations at the substitution site, as well as at positions upstream and downstream of a Gly mutation. The NMR studies suggest that the local sequences surrounding the substitution site, and the renucleation sequences N-terminal to and adjacent to the substitution site, may be critical in defining the clinical phenotype of osteogenesis imperfecta. These studies may pave the way to understanding the mechanism by which a single Gly substitution in collagen can lead to pathological conditions.     

Mutation characteristics in type I collagen genes in Chinese patients with osteogenesis imperfecta

Osteogenesis imperfecta is normally caused by an autosomal dominant mutation in the type I collagen genes COL1A1 and COL1A2. The severity of osteogenesis imperfecta varies, ranging from perinatal lethality to a very mild phenotype. Although there have been many reports of COL1A1 and COL1A2 mutations, few cases have been reported in Chinese people. We report on five unrelated families and three sporadic cases. The mutations were detected by PCR and direct sequencing. Four mutations in COL1A1 and one in COL1A2 were found, among which three mutations were previously unreported. The mutation rates of G>C at base 128 in intron 31 of the COL1A1 gene and G>A at base 162 in intron 30 of the COL1A2 gene were higher than normal. The patients' clinical characteristics with the same mutation were variable even in the same family. We conclude that mutations in COL1A1 and COL1A2 also have an important role in osteogenesis imperfecta in the Chinese population. As the Han Chinese people account for a quarter of the world's population, these new data contribute to the type I collagen mutation map.