Collagenase in mineralized tissues of human teeth

A collagenase cleaving native type I [14C]collagen but inactive against the synthetic substrate Pz-Pro-Leu-Gly-Pro-D-Arg was extracted from mineralized human dental tissue. The enzyme specifically degrades native collagen into characteristic products (3/4) and (1/4). Its apparent molecular mass of 68 kDa is relatively high in comparison with collagenases from other oral tissues. The enzyme is a metalloproteinase inhibited by low concentrations of the chelating agents EDTA, 1, 10-phenanthroline, alpha alpha'-dipyridyl, and not affected by diisopropylfluorophosphate, soybean trypsin inhibitor, and p-chloromercuribenzoate. It is stable to lyophilization and can be stored at-20 degrees C for at least 6 months.     

Lethal osteogenesis imperfecta. Definition and heterogeneity

The lethal form of osteogenesis imperfecta must be more clearly defined than it is. Early death of the child is not a sufficient criteria since it is observed in other forms compatible with survival. The authors therefore insist on the innumerable fractures of the ribs giving the particular aspect described as "bamboo ribs". These are observed in practically all cases. Heterogeneity however is undoubtful and an X-ray film is reproduced showing thin ribs without fractures in another exceptional lethal form. Genetic and very recent biochemical investigations suggest a new heterogeneity of the lethal form, even well defined clinically and radiologically. Contrary to earlier and frequent statements, it results most often from a dominant mutation, while recessive inheritance is much rarer, therefore the overall risk of recurrence much below 25%.     

Orthopaedic and dental abnormalities in osteogenesis imperfecta: a review of the literature

Osteogenesis imperfecta is one of the most commonly recognized inheritable disorders of the connective tissue leading to bone fragility. Usually it is associated to a genetic mutation inducing a reduction in collagen quality and entity production. It involves either modification in dentin formation or multiple bone fractures. The authors reviewed the clinical aspects of these disorders, focusing on oral and orthopaedic concerns, especially related to the histological features of the fracture callus, with respect to new trends in pharmacological and surgical treatments of bone fractures. Surgical treatment varies, according to the age of the patient. In children, surgical orthopaedic procedures include multiple osteotomies and the use of telescopic rods. Medical therapy has always to be associated to surgery and is designed to reduce the incidence of fractures, to increase growth velocity and to ally pain in order to improve mobility and independence. Bisphosphonates (BP) are considered potent inhibitors of bone resorption decreasing the osteoclast population and its activity and bone turn over.     

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.     

Collagen defects in lethal perinatal osteogenesis imperfecta.

Quantitative and qualitative abnormalities of collagen were observed in tissues and fibroblast cultures from 17 consecutive cases of lethal perinatal osteogenesis imperfecta (OI). The content of type I collagen was reduced in OI dermis and bone and the content of type III collagen was also reduced in the dermis. Normal bone contained 99.3% type I and 0.7% type V collagen whereas OI bone contained a lower proportion of type I, a greater proportion of type V and a significant amount of type III collagen. The type III and V collagens appeared to be structurally normal. In contrast, abnormal type I collagen chains, which migrated slowly on electrophoresis, were observed in all babies with OI. Cultured fibroblasts from five babies produced a mixture of normal and abnormal type I collagens; the abnormal collagen was not secreted in two cases and was slowly secreted in the others. Fibroblasts from 12 babies produced only abnormal type I collagens and they were also secreted slowly. The slower electrophoretic migration of the abnormal chains was due to enzymic overmodification of the lysine residues. The distribution of the cyanogen bromide peptides containing the overmodified residues was used to localize the underlying structural abnormalities to three regions of the type I procollagen chains. These regions included the carboxy-propeptide of the pro alpha 1(I)-chain, the helical alpha 1(I) CB7 peptide and the helical alpha 1(I) CB8 and CB3 peptides. In one baby a basic charge mutation was observed in the alpha 1(I) CB7 peptide and in another baby a basic charge mutation was observed in the alpha 1(I) CB8 peptide. The primary defects in lethal perinatal OI appear to reside in the type I collagen chains. Type III and V collagens did not appear to compensate for the deficiency of type I collagen in the tissues.     

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.     

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.     

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.     

Strangulated diaphragmatic hernia in a patient with osteogenesis imperfecta.

Hernias are common in patients with osteogenesis imperfecta, but to our knowledge there have been no reports of diaphragmatic hernias in such patients. We describe a patient with osteogenesis imperfecta in whom a diaphragmatic hernia contained a strangulated segment of the splenic flexure of the colon. Resection of the necrotic tissue and transverse colostomy resulted in an uneventful recovery.     

Dominant mutations in familial lethal and severe osteogenesis imperfecta

Summary Four families presenting with familial osteogenesis imperfecta (OI) have been studied: 2 with the lethal type II and 2 with the severe type III form. Fibroblasts of the patients, all issue from non-consanguineous parents, produced normal and abnormal (I) chains. These heterozygous mutations differentiate the recurrent forms from homozygous mutations characteristic of autosomal recessive forms. Although the identity of the mutations could not be determined, such recurrence of autosomal dominant OI is probably the result of germinal mosaicism in one of the parents. Biochemical results were consistent with a somatic mosaicism in the father's fibroblasts in one family. Moreover, our studies show that not only OI type II but also severe OI type III can arise from gonadal mosaicism. We discuss the importance of such a phenomenon for genetic counseling.     

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.