Lethal Osteogenesis Imperfecta Research Articles

Rosemary Extract-Induced Autophagy and Decrease in Accumulation of Collagen Type I in Osteogenesis Imperfecta Skin Fibroblasts.

Osteogenesis imperfecta (OI) is a heterogeneous connective tissue disease mainly caused by structural mutations in type I collagen. Mutant collagen accumulates intracellularly, causing cellular stress that has recently been shown to be phenotype-related. Therefore, the aim of the study was to search for potential drugs reducing collagen accumulation and improving OI fibroblast homeostasis. We found that rosemary extract (RE), which is of great interest to researchers due to its high therapeutic potential, at concentrations of 50 and 100 µg/mL significantly reduced the level of accumulated collagen in the fibroblasts of four patients with severe and lethal OI. The decrease in collagen accumulation was associated with RE-induced autophagy as was evidenced by an increase in the LC3-II/LC3-I ratio, a decrease in p62, and co-localization of type I collagen with LC3-II and LAMP2A by confocal microscopy. The unfolded protein response, activated in three of the four tested cells, and the level of pro-apoptotic markers (Bax, CHOP and cleaved caspase 3) were attenuated by RE. In addition, the role of RE-modulated proteasome in the degradation of unfolded procollagen chains was investigated. This study provides new insight into the beneficial effects of RE that may have some implications in OI therapy targeting cellular stress.

Current Overview of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI), or brittle bone disease, is a heterogeneous disorder characterised by bone fragility, multiple fractures, bone deformity, and short stature. OI is a heterogeneous disorder primarily caused by mutations in the genes involved in the production of type 1 collagen. Severe OI is perinatally lethal, while mild OI can sometimes not be recognised until adulthood. Severe or lethal OI can usually be diagnosed using antenatal ultrasound and confirmed by various imaging modalities and genetic testing. The combination of imaging parameters obtained by ultrasound, computed tomography (CT), and magnetic resource imaging (MRI) can not only detect OI accurately but also predict lethality before birth. Moreover, genetic testing, either noninvasive or invasive, can further confirm the diagnosis prenatally. Early and precise diagnoses provide parents with more time to decide on reproductive options. The currently available postnatal treatments for OI are not curative, and individuals with severe OI suffer multiple fractures and bone deformities throughout their lives. In utero mesenchymal stem cell transplantation has been drawing attention as a promising therapy for severe OI, and a clinical trial to assess the safety and efficacy of cell therapy is currently ongoing. In the future, early diagnosis followed by in utero stem cell transplantation should be adopted as a new therapeutic option for severe OI.

Lethal osteogenesis imperfecta type XX caused by compound heterozygous frameshift mutations in MESD

Lethal osteogenesis imperfecta type XX caused by compound heterozygous frameshift mutations in MESD

Explanation for mild and severe osteogenesis imperfecta phenotypes due to splice variants at c.2029-1 in COL1A1

We report a fetus aborted at 18 weeks of gestation with lethal osteogenesis imperfecta (OI) carrying a de novo a canonical splice site variant c.2029G > T in COL1A1 (NM_000088.3) identified by exome sequencing. The cDNA change at 2029th position (c.2029G > A and c.2029G > C) of COL1A1 was already reported to cause milder forms of OI. We demonstrate the functional consequences of two single nucleotide transitions, i.e. Guanine to Thymine (G > T) and Guanine to Adenine (G > A) of COL1A1ng. We show the milder OI phenotype caused by the variant c.2029-1G > A is due to haploinsufficiency and severe form of OI caused by the variant, c.2029-1G > T is due to the dominant negative effect on COL1A1 protein.

Perspectives on the evolution of genetic counselling: Experience over three decades in a family with recurrent lethal osteogenesis imperfecta

Perspectives on the evolution of genetic counselling: Experience over three decades in a family with recurrent lethal osteogenesis imperfecta

Fetal Skeletal Dysplasias: Radiologic-Pathologic Classification of 72 Cases

Objective The aim of this study was to classify the fetal skeletal dysplasias (FSD) in a series of affected fetuses based on radio-pathologic criteria. Materials and methods: We gathered clinicopathologic data of 72 cases which were diagnosed among 5995 autopsies performed over a 8-year period. Results: The prevalence of FSD was 1.2:100 autopsies. The overall sex ratio (M:F) was 1.25. Gestational age was between 17 and 24 weeks in 60% of cases. The FSD were classified into 13 distinct pathologic groups. Four major groups were identified: (1) Osteogenesis imperfecta (21 cases, 29%); (2) FGFR3 chondrodysplasia (18 cases, 25%); (3) Ciliopathies (9 cases, 12%); and (4) Sulfation disorders (7 cases, 10%). Thanatophoric dysplasia type 1 and lethal osteogenesis imperfecta were the most common skeletal dysplasias. Conclusion: Our study demonstrates the usefulness of the radio-pathologic examination in the diagnosis and accurate classification of the FSD, thus enabling better targeting of genetic counseling.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2β1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2β1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2β1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

A homozygous pathogenic missense variant broadens the phenotypic and mutational spectrum of CREB3L1-related osteogenesis imperfecta.

The cyclic adenosine monophosphate responsive element binding protein 3-like 1 (CREB3L1) gene codes for the endoplasmic reticulum stress transducer old astrocyte specifically induced substance (OASIS), which has an important role in osteoblast differentiation during bone development. Deficiency of OASIS is linked to a severe form of autosomal recessive osteogenesis imperfecta (OI), but only few patients have been reported. We identified the first homozygous pathogenic missense variant [p.(Ala304Val)] in a patient with lethal OI, which is located within the highly conserved basic leucine zipper domain, four amino acids upstream of the DNA binding domain. In vitro structural modeling and luciferase assays demonstrate that this missense variant affects a critical residue in this functional domain, thereby decreasing the type I collagen transcriptional binding ability. In addition, overexpression of the mutant OASIS protein leads to decreased transcription of the SEC23A and SEC24D genes, which code for components of the coat protein complex type II (COPII), and aberrant OASIS signaling also results in decreased protein levels of SEC24D. Our findings therefore provide additional proof of the potential involvement of the COPII secretory complex in the context of bone-associated disease.

Recurrence of perinatal lethal osteogenesis imperfecta in sibships: Parsing the risk between parental mosaicism for dominant mutations and autosomal recessive inheritance

PurposeRecurrence of lethal osteogenesis imperfecta in families results from either dominant (parental mosaicism) or recessive inheritance. The proportion of these two mechanisms is not known, and determination of the contribution of each is important to structure genetic counseling for these families. MethodsWe measured the recurrence rate of lethal osteogenesis imperfecta after the birth of an affected infant. We determined the rate of parental mosaicism in a subset of families in which we had identified dominant mutations. In 37 families in which two or more affected infants were born, we identified mutations and determined the proportion that resulted from recessive inheritance. ResultsThe recurrence rate after the first affected pregnancy was 1.3%. The rate of parental mosaicism in families in which a dominant mutation was identified in a first affected child was 16%. In 37 families with two affected infants, 26 had dominant mutations, seven had recessive mutations, and we failed to find mutations in four. The overall recurrence rate for couples after two or more affected infants was 32%; 27% for families with parental mosaicism, 31% for recessive mutations, and 50% for families with no identified mutation. ConclusionsIn most populations, recurrence of lethal osteogenesis imperfecta usually results from parental mosaicism for dominant mutations, but the carrier frequency of recessive forms of osteogenesis imperfecta will alter that proportion. Mutation identification is an important tool to assess risk and facilitate prenatal or Genet Med 2011:13(2):125–130.

Osteogenesis imperfecta: questions and answers

Considerable attention has recently been focused on the pathogenesis, diagnosis and treatment of osteogenesis imperfecta. Two new genes have been defined in patients with recessive severe or lethal osteogenesis imperfecta types. Diagnostic concerns involve testing procedures, either skin biopsies or DNA analysis. Bisphosphonates have been accepted as 'standard of care' for children with osteogenesis imperfecta. However, questions remain as to the selection of patients for treatment, effectiveness in fracture prevention, which bisphosphonates should be used and the duration of treatment. Orthopedic intervention occurs on several levels: including the immediate treatment of fractures, the treatment of scoliosis and the use of intramedullary rods. The discovery of mutations involving CRTAP and LEPRE1 genes in severe/lethal and recessively inherited osteogenesis imperfecta has provided partial answers to questions about 'other' osteogenesis imperfecta genes in patients with an osteogenesis imperfecta phenotype but no COL1A1 and COL1A2 mutations. Current experience suggests that DNA analysis is a better test for diagnosis as compared with dermal biopsy. There are no standardized guidelines for initiating bisphosphonate treatment in children. Recent data suggest either intravenous or oral bisphosphonates are effective, but differences exist between different bisphosphonates. Two recent reports document the paucity of evidence-based data regarding the effectiveness of bisphosphonate treatment in fracture prevention. This report will update the medical and orthopedic approaches to care for children with osteogenesis imperfecta.

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

AbstractAutosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [α1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.

Deficiency of Cartilage-Associated Protein in Recessive Lethal Osteogenesis Imperfecta

Classic osteogenesis imperfecta, an autosomal dominant disorder associated with osteoporosis and bone fragility, is caused by mutations in the genes for type I collagen. A recessive form of the disorder has long been suspected. Since the loss of cartilage-associated protein (CRTAP), which is required for post-translational prolyl 3-hydroxylation of collagen, causes severe osteoporosis in mice, we investigated whether CRTAP deficiency is associated with recessive osteogenesis imperfecta. Three of 10 children with lethal or severe osteogenesis imperfecta, who did not have a primary collagen defect yet had excess post-translational modification of collagen, were found to have a recessive condition resulting in CRTAP deficiency, suggesting that prolyl 3-hydroxylation of type I collagen is important for bone formation.

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--&gt;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.

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.

Type I Collagen Triplet Duplication Mutation in Lethal Osteogenesis Imperfecta Shifts Register of α Chains throughout the Helix and Disrupts Incorporation of Mutant Helices into Fibrils and Extracellular Matrix

The majority of collagen mutations causing osteogenesis imperfecta (OI) are glycine substitutions that disrupt formation of the triple helix. A rare type of collagen mutation consists of a duplication or deletion of one or two Gly-X-Y triplets. These mutations shift the register of collagen chains with respect to each other in the helix but do not interrupt the triplet sequence, yet they have severe clinical consequences. We investigated the effect of shifting the register of the collagen helix by a single Gly-X-Y triplet on collagen assembly, stability, and incorporation into fibrils and matrix. These studies utilized a triplet duplication in COL1A1 exon 44 that occurred in the cDNA and gDNA of two siblings with lethal OI. The normal allele encodes three identical Gly-Ala-Hyp triplets at aa 868-876, whereas the mutant allele encodes four. The register shift delays helix formation, causing overmodification. Differential scanning calorimetry yielded a decrease in T(m) of 2 degrees C for helices with one mutant chain and a 6 degrees C decrease in helices with two mutant chains. An in vitro binary co-processing assay of N-proteinase cleavage demonstrated that procollagen with the triplet duplication has slower N-propeptide cleavage than in normal controls or procollagen with proalpha1(I) G832S, G898S, or G997S substitutions, showing that the register shift persists through the entire helix. The register shift disrupts incorporation of mutant collagen into fibrils and matrix. Proband fibrils formed inefficiently in vitro and contained only normal helices and helices with a single mutant chain. Helices with two mutant chains and a significant portion of helices with one mutant chain did not form fibrils. In matrix deposited by proband fibroblasts, mutant chains were abundant in the immaturely cross-linked fraction but constituted a minor fraction of maturely cross-linked chains. The profound effects of shifting the collagen triplet register on chain interactions in the helix and on fibril formation correlate with the severe clinical consequences.

Transparent Bone and Concave Ribs

Osteogenesis imperfecta (OI), a rare connective tissue deformity, can be classified into four categories, one of which (type II) is lethal. Lethal OI can be detected with the use of ultrasound prenatally. The authors studied nine OI cases, seven type II OI cases, and two nonlethal cases. All seven type II OI cases had very short limbs. In six of the seven cases, the back wall of the limb bone was visible (transparent bone sign). In four of the seven cases, concave ribs were visualized in the transverse plane. An incidental finding, beaking of the anterior portion of the skull, was noted in five cases. The transparent bone sign and concave ribs were not observed in the two nonlethal cases of OI.

Four new cases of lethal osteogenesis imperfecta due to glycine substitutions in COL1A1 and genes

Four new cases of lethal osteogenesis imperfecta due to glycine substitutions in COL1A1 and genes

A Gly421-&gt;Asp substitution causes unusual electrophoretic mobility ofα2(I) chain in a lethal case of Osteogenesis Imperfecta. † 602

Biochemical analysis of type I collagen synthesized by cultured skin fibroblasts was performed to characterized the defect in a patient affected by lethal Osteogenesis Imperfecta (OI), a dominant heritable brittle bone disorder caused by mutations in one of the two genes coding for the α1 and α2 chains of type I collagen. The SDS-urea-PAGE of procollagen and collagen revealed a broad α1(I) chain, a normal α2(I) and anα2(I) migrating half way between α1 and α2. Procollagen and collagen of media and cell layers, synthesized in the presence ofαα′-dipyridyl, revealed both normal and slower α2(I) suggesting an insertion in COL1A2 gene. CNBr peptide digestion of collagen yielded overmodified α1(I) CB3 and CB7 peptides and a delayed migration of the α2(I) CB3-5 peptide. A delayed CB3-5 persisted also afterαα′-dipyridyl treatment, supporting an alteration ofα2(I) size. The mutation was localized, by this data, between aa 353-551 in α2(I) (CB 3-5). The sequence of this region revealed a G->A transition at nt 1671 in one allele, changing Gly421->Asp in oneα2(I) chain. Procollagen processing was normal. The Tm of the slowα2(I) collagen was 2iC lower than the control, indicating a decrease in triple helix stability. The mutant protein was incorporated in the extracellular matrix synthesized by long term cultured fibroblasts.

Tracing the pathway between mutation and phenotype in osteogenesis imperfecta: isolation of mineralization-specific genes.

The brittleness of bone in people with lethal (type II) osteogenesis imperfecta, a heritable disorder caused by mutations in the type I collagen genes, arises from the deposition of abnormal collagen in the bone matrix. The inability of the abnormal collagen to participate in mineralization may be caused by its failure to interact with other bone proteins. Here, we have designed a strategy to isolate the genes important for mineralization of collagen during bone formation. Cells isolated from 16-day embryonic chick calvaria and seeded post-confluence in culture deposited a mineralized matrix over a period of 2 weeks. Chick skin fibroblasts seeded and cultured under the same conditions did not mineralize. Using RT-PCR, we prepared short cDNAs (approximately 300 bp) corresponding to the 3' ends of mRNA from fibroblasts and separately from the mineralizing calvarial cells. Subtractive cDNA hybridization generated a pool of cDNAs that were specific to mineralizing calvarial cells but not to fibroblasts. Screening of 100,000 plaques of a chick bone ZAP Express cDNA library with this pool of mineralizing-specific cDNAs identified ten clones which comprised full-length cDNAs for the bone proteins osteopontin (eight of the ten positives), bone sialoprotein II (one of the ten positives), and cystatin (one of the ten positives). cDNAs for type I collagen, fibronectin, alkaline phosphatase, house-keeping genes, and other genes expressed in fibroblasts were not identified in this preliminary screen. The pool of short cDNAs is likely to comprise cDNAs for further bone-specific genes and will be used to screen the entire bone cDNA library of 4.2 million clones.

Substitutions of aspartic acid for glycine-220 and of arginine for glycine-664 in the triple helix of the pro alpha 1(I) chain of type I procollagen produce lethal osteogenesis imperfecta and disrupt the ability of collagen fibrils to incorporate crystalline hydroxyapatite.

We identified two infants with lethal (type II) osteogenesis imperfecta (OI) who were heterozygous for mutations in the COL1A1 gene that resulted in substitutions of aspartic acid for glycine at position 220 and arginine for glycine at position 664 in the product of one COL1A1 allele in each individual. In normal age- and site-matched bone, approximately 70% (by number) of the collagen fibrils were encrusted with plate-like crystallites of hydroxyapatite. In contrast, approximately 5% (by number) of the collagen fibrils in the probands' bone contained crystallites. In contrast with normal bone, the c-axes of hydroxyapatite crystallites were sometimes poorly aligned with the long axis of fibrils obtained from OI bone. Chemical analysis showed that the OI samples contained normal amounts of calcium. The probands' bone samples contained type I collagen, overmodified type I collagen and elevated levels of type III and V collagens. On the basis of biochemical and morphological data, the fibrils in the OI samples were co-polymers of normal and mutant collagen. The results are consistent with a model of fibril mineralization in which the presence of abnormal type I collagen prevents normal collagen in the same fibril from incorporating hydroxyapatite crystallites.