Severe Form Of Osteogenesis Imperfecta Research Articles

Biallelic variants in MESD, which encodes a WNT-signaling-related protein, in four new families with recessively inherited osteogenesis imperfecta.

SummaryThe bone disorder osteogenesis imperfecta (OI) is genetically heterogeneous. Most affected individuals have an autosomal dominant disorder caused by heterozygous variants in either of the type I collagen genes (COL1A1 or COL1A2). To date, two reports have linked Mesoderm Development LRP Chaperone (MESD) to autosomal recessive OI type XX. Four different biallelic pathogenic variants in MESD were shown to cause a progressively deforming phenotype, associated with recurrent fractures and oligodontia in five individuals in five families. Recently, compound heterozygosity for a frameshift predicted to lead to a premature termination codon in exon 2 of the 3-exon gene and a second frameshift in the terminal exon in MESD were detected in three stillbirths in one family with severe OI consistent with the neonatal lethal phenotype. We have identified four additional individuals from four independent families with biallelic variants in MESD: the earlier reported c.632dupA (p.Lys212Glufs∗19) and c.676C>T (p.Arg226∗)—which are associated with a severe form of OI—and one new pathogenic variant, c.603-606delTAAA (p.Asn201Lysfs∗15), which causes a neonatal lethal form of OI. MESD acts in the WNT signaling pathway, where it is thought to play a role in the folding of the WNT co-receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/LRP6) and in chaperoning their transit to the cell surface. Our report broadens the phenotypic and genetic spectrum of MESD-related OI, provides additional insight into the pathogenic pathways, and underscores the necessity of MESD for normal WNT signaling in bone formation.

Male but not female mice with severe osteogenesis imperfecta are partially protected from high-fat diet-induced obesity

Previously we have shown that young mice with a dominant severe form of osteogenesis imperfecta (OI), caused by mutated collagen type I, exhibit an altered glucose/insulin metabolism and energy expenditure along with elevated levels of osteocalcin, a bone-derived hormone involved in the regulation of whole-body metabolism. This study aimed to examine the long-term effects of a western diet in these OI mice. Male and female OI mice and wild type littermates (WT) were fed a high-fat diet (HFD) or a matched low-fat diet (LFD) for 26 weeks. HFD-induced obesity was observed in male and female WT and female OI mice, but not in male OI mice. HFD-fed WT and OI mice of both sexes developed hyperglycemia and glucose intolerance, but the degree of glucose intolerance was significantly lower in male and female OI mice compared to sex- and diet-matched WT mice. Indirect calorimetry revealed increased movement of male OI mice on HFD compared to LFD and, while HFD lowered energy expenditure in WT mice, energy expenditure was not changed in OI mice. Further, HFD-fed male OI mice demonstrated a diet-induced increased expression of the thermogenesis genes, Ucp1 and Pgc1α, in brown adipose tissue. On LFD, total and Gla-13 osteocalcin levels were similar in 30-week-old WT and OI mice, but on HFD, both were significantly higher in OI mice than WT. Thus, male OI mice respond to HFD with increased movement, energy expenditure, brown adipose tissue thermogenesis, and higher levels of osteocalcin, resulting in partial protection against HFD-induced obesity.

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.

Co-occurrence of heterozygous mutations in COL1A1 and SERPINF1 in a high-risk pregnancy complicated by osteogenesis imperfecta

Mutations in several genes, including SERPRINF1 and COL1A1, have been associated with the development of osteogenesis imperfecta (OI). Here, we reported the co-occurrence of a rare heterozygous variant (c.167C>G p.Ala56Gly) in SERPRINF1 and a novel heterozygous mutation (c.1634G>A p.Gly545Asp) in COL1A1 in a foetus with a severe form of OI. Bioinformatics modelling revealed that the effect of the mutation on SPERINF1 is neutral. In contrast, the mutation in COL1A1 is deleterious. It is predicted to cause distortion of the α (1) chain of the type I collagen and results in structural instability of the protein. Therefore, a novel dominant variant of COL1A1 likely underlies the severe foetal pathology observed, although we do not exclude the possibility that the heterozygous mutations in SERPINF and COL1A1 may interact and co-ordinately cause pathogenesis. This novel COL1A1 mutation is recommended to be included in the diagnostic panels for OI.

Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.

Osteogenesis imperfecta in children: Indosesian experiences

Medical management of osteogenesis imperfecta (OI), a genetic disorder of connective tissue characterized by reduced bone mass and frequent fractures, is focused on maximizing patients’ mobility and function. Many methods of pharmacological treatment have been introduced for the severe forms of OI, including calcium, anabolic steroids, growth hormone, and calcitonin, without consistent benefit. Recent studies reported beneficial effect of intravenous and oral bisphosphonates on moderate to severe form of OI in children. This report is to review OI cases in Indonesian children and to know the effect of bisphosphonate treatment for them. Data on age, gender, age at diagnosis, availability of bisphosphonate treatment, and its effects were collected from Indonesian OI children registry, which documented from April to June 2012. Fourteen centers which have members of pediatric endocrinology chapter, Indonesian Pediatric society, were involved in obtaining data for the registry. Forty-six OI cases (17 female), who were diagnosed at the age of 7 days-11 years old and mostly categorized as type 3 of OI, were recorded. Two patients were died before commencing treatment. There were twenty-seven (27/46) patients received bisphosphonates, which were administered to moderate to severe cases. Two patients,who were treated, were died due to hyperthermia after the first day of pamidronate infusion, and pneumonia aspiration. Twenty-five patients received intravenous pamidronate and 3 oral alendronate. Liver and renal function test, and serum electrolyte were evaluated before and after treatment. Most patients reported decreased irritability, increased muscle tone and mobility. In addition, despite hyperthermia in one case, no serious adverse effects documented. Although some patients had government health insurance for the poor, unfortunately, the medicine, which is considered expensive for the patients, is not yet included in the drug list of government insurance. Most of registered OI cases in Indonesian children were type 3. More than half of the cases underwent treatment with bisphosphonates with beneficial effects, although long-term follow up and well-documented data are required.

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. 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.

A novel splicing mutation in FKBP10 causing osteogenesis imperfecta with a possible mineralization defect

Osteogenesis imperfecta (OI) is a group of hereditary disorders characterized by bone fragility and osteopenia, with a broad spectrum of clinical severity. The majority of cases are dominantly inherited and due to mutations in type I collagen genes, whereas recessive forms are less frequent and attributable to mutations in different genes involved in collagen I post translational modifications and folding (prolyl-3-hydroxylase complex, SERPINH1, FKBP10). We report the case of a patient with an initially mild and then progressively severe form of osteogenesis imperfecta due to a novel homozygous splicing mutation in FKBP10 (intron 8 c.1399+1G>A), which results in aberrant mRNA processing and consequent lack of FKBP65 chaperone. Although this mutation does not affect collagen type I post translational modifications in dermal fibroblasts, the histomorphometric pattern of our patient's bone sample showed a mineralization defect possibly due to the mutation in FKBP10.

Severe osteogenesis imperfecta caused by a small in‐frame deletion in CRTAP

Mutations of proteins involved in posttranslational modification of collagen type I can cause osteogenesis imperfecta (OI) inherited in a recessive pattern. The cartilage-associated protein (CRTAP) is part of a heterotrimeric complex (together with prolyl-3-hydroxylase-1 [P3H1] and cyclophilin B) that 3-hydroxylates the alpha 1 chain of collagen type I at proline residue 986 and plays a collagen chaperon role. CRTAP mutations usually cause severe OI. We report on a patient with OI and a homozygous in-frame deletion in CRTAP and a severe form of OI. The girl was born with markedly deformed long bones. Despite intravenous bisphosphonate treatment, she developed multiple vertebral compression fractures and severe scoliosis and at 4 years of age was able to sit only with support. Although CRTAP transcript levels were normal in the patient's fibroblasts, protein levels of both CRTAP and P3H1 were severely reduced. The degree of 3-hydroxylation at proline residue 986 was also decreased. This report characterizes a patient with a CRTAP small in-frame deletion. We are unaware of prior reports of this finding. We suggest that this deletion affects crucial amino acids that are important for the interaction and/or stabilization of CRTAP and P3H1.

Genetic basis for skeletal disease. Osteogenesis imperfecta

Osteogenesis imperfecta (OI) is characterized by its bone fragility. But the spectrum of the severity of the fragility is widely distributed. Recently, amino-bisphosphonate has been introduced into the treatment of the bone fragility of OI. Apparently, the treatment may reduce the frequency of the fractures in severe form of OI. However, its efficacy has not been supported by strong evidences, such as randomized controlled clinical trial. This section describes the pathophysiology and new pathogenetic mechanism of OI, and describes recent evidence for the efficacy of BP treatment in OI.

Results of a prospective pilot trial on mobility after whole body vibration in children and adolescents with osteogenesis imperfecta

Objective: To evaluate the effect of whole body vibration on the mobility of long-term immobilized children and adolescents with a severe form of osteogenesis imperfecta. Osteogenesis imperfecta is a hereditary primary bone disorder with a prevalence from 1 in 10000 to 1 in 20000 births. Most of these children are suffering from long-term immobilization after recurrent fractures. Due to the immobilization they are affected by loss of muscle (sarcopenia) and secondary loss of bone mass. Subjects: Whole body vibration was applied to eight children and adolescents (osteogenesis imperfecta type 3, N=5; osteogenesis imperfecta type 4, N=3) over a period of six months. Interventions and results: Whole body vibration was applied by a vibrating platform (Galileo Systems) constructed on a tilting-table. Success of treatment was assessed by measuring alterations of the tilting-angle and evaluating the mobility (Brief Assessment of Motor Function). All individuals were characterized by improved muscle force documented by an increased tilting-angle (median = 35 degrees) or by an increase in ground reaction force (median at start=30.0 [N/kg] (14.48—134.21); median after six months = 146.0 [N/kg] (42.46—245.25). Conclusions: Whole body vibration may be a promising approach to improve mobility in children and adolescents severely affected with osteogenesis imperfecta.

Tomographic Imaging of Collagen-Mineral Interaction: Implications for Osteogenesis Imperfecta

The novel method of high voltage electron microscopic tomography (3D) has been applied for the first time to examine ultrastructural features and spatial relations between collagen fibrils and mineral crystals in a mouse mutant (oim/oim) which replicates a moderate to severe form of osteogenesis imperfecta. The animal produces collagen consisting of the alpha1(I) homotrimer and has a brittle calcified skeleton. Three-dimensional image reconstructions of the Achilles tendons, which were found to mineralize in the mutant mice, revealed that their composite crystals were different in their structural appearance and spatial association with collagen compared to that determined in normal calcified tissues. These results indicate that the nature of the organic matrix of a mineralizing tissue critically influences the formation, structure, and location of the constituent mineral and, further, the data are interpreted as suggesting that the unusual structural and organizational interaction between mineral and collagen underlies the inherent brittleness and weakness of calcification in this model of osteogenesis imperfecta.

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.

Percutaneous nailing in the management of osteogenesis imperfecta

Marked deformities of the skeleton may develop in the severe form of osteogenesis imperfecta. Internal fixation can be effective in the treatment and prevention of fractures and deformities. Eight years ago, axial correction of deformities of the femora and tibiae by manual osteoclasis was carried out in a 7 year old girl in whom osteogenesis imperfecta had produced severe bowing of the long bones of the legs. Internal metal splints were introduced percutaneously to lie partially in, and partially alongside, the bone, to obtain fixation in the correct position. This treatment resulted in the prevention of refracture of the legs, while correction was maintained.

Substitution of Serine for α1(I)-Glycine 844 in a Severe Variant of Osteogenesis Imperfecta Minimally Destabilizes the Triple Helix of Type I Procollagen: The effects of glycine substitutions on thermal stability are either position of amino acid specific

Abstract Recent reports have demonstrated that a series of probands with severe osteogenesis imperfecta had single base mutations in one of the two structural genes for type I procollagen that substituted amino acids with bulkier side chains for glycine residues and decreased the melting temperature of the triple helix. Here we demonstrate that the type I procollagen synthesized by cultured fibroblasts from a proband with a severe form of osteogenesis imperfecta consisted of normal molecules and molecules over-modified by post-translational reactions. The thermal stability of the intact type I collagen was normal as assayed by protease digestion under conditions in which a decrease in thermal stability was previously observed with eight other substitutions for glycine in the alpha 1(I) chain. In contrast, the thermal stability of the one-quarter length B fragment generated by digestion with vertebrate collagenase was decreased by 2-3 degrees C under the same conditions. Nucleotide sequencing of cDNAs and genomic DNA established that the proband had a substitution of A for G in one allele of the pro alpha 1(I) gene that converted the codon for alpha 1-glycine 844 to a codon for serine. The results also established that the alpha 1-serine 844 was the only mutation that could account for the decrease in thermal stability of the collagenase B fragment. There are at least two possible explanations for the failure of the alpha 1-serine 844 substitution to decrease the thermal stability of the collagen molecule whereas eight similar mutations decreased the melting temperature. One possibility is that the effects of glycine substitutions are position specific because not all glycine residues make equivalent contributions to cooperative blocks of the triple helix that unfold in the predenaturation range of temperatures. A second possible explanation is that substitutions of glycine by serine have much less effect on the stability of protein than the substitutions by arginine, cysteine, and aspartate previously studied.

Early semiclosed intramedullary stabilization in osteogenesis imperfecta.

Fourteen patients with severe osteogenesis imperfecta (OI) were treated operatively by closed or semiclosed intramedullary stabilization, with nonextensible rods and nails. Altogether, 55 bones (both upper and lower limbs) were stabilized primarily. In infants with a severe form of OI (Sillence classification), the operative technique was modified significantly from those presented earlier in the literature. Early intramedullary stabilization, even soon after birth, seems justified in selected patients with severe OI. The possibilities for motor development are improved and later insertion of telescoping nails becomes easier.

Abnormal procollagen synthesis in fibroblasts from three patients of the same family with a severe form of osteogenesis imperfecta (type III)

Dermal fibroblast cultures from three siblings with a severe form of osteogenesis imperfecta were established in order to analyze their procollagen and collagen synthesis. Cell strains from clinically normal consanguineous parents (first cousins), were also obtained for comparison. Total collagen production in culture media was diminished by 55% in the patients fibroblasts and to a lesser extent in the parents. This decrease was specific for collagenous proteins. From polyacrylamide gel electrophoresis, it appeared that the three children had not only the same defective secretion of pro α 1(I) molecules but that their pro α 1(I) migrated slightly faster than the parental and control counterparts. Analysis of secretion confirmed a reduced rate in procollagen synthesis and the absence of intracellular storage. Upon pepsin treatment, extracellular α 1(I) and α 2(I) chains were found in the expected ratio of 2:1 and migrated normally, suggesting that the altered mobility of pro α 1(I) chains was related to COOH or NH 2 terminal propeptides. In agreement with the reduced type I collagen production, an increase in the α 1( III) α 1( I) ratio was also detected. Furthermore, after a 2.5-h labelling followed by alkylation with iodoacetamide, free intracellular pro α 2(I) and α 1(I) chains were detected in the absence of reduction, consistent with an abnormal intracellular ratio of pro α 1( I) pro α 2( I) that was measured after dithiothreitol reduction. Analysis of intracellular collagen chains from parental strains following a 4-h incubation demonstrated that pro α 1(I) appeared as a doublet, one band with normal mobility and a less intense band migrating faster and corresponding to the defective chain found in the patients. Absence of the abnormal molecules in culture media was related to the demonstration of a defective collagen secretion by parental fibroblasts. Correlation between these biochemical findings and clinical data strongly support a recessive inheritance of the disease that could be classified as a type III form of osteogenesis imperfecta. Patients would be homozygous for the same defective allele and the asymptomatic parents would most likely be heterozygous carriers of the mutation. Although the exact location of the alteration is not yet elucidated, a splicing mutation is suggested.

Nuclease S1 mapping of a homozygous mutation in the carboxyl-propeptide-coding region of the pro alpha 2(I) collagen gene in a patient with osteogenesis imperfecta.

The molecular defect in a patient with a moderately severe form of osteogenesis imperfecta was characterized by nuclease S1 mapping. Single-stranded 5' and 3' end-labeled DNA probes coding for 80% of the carboxyl-propeptide of the pro alpha 2(I) collagen gene were hybridized to mRNA isolated from cultured fibroblasts of the patient and his parents. Nuclease S1 digestion revealed a homozygous mutation in the patient and a heterozygous pattern in the consanguineous parents. As a result of the defect in the gene, none of the pro alpha 2(I) chains synthesized by the patient's fibroblasts were incorporated into a type I procollagen heterotrimer consisting of two pro alpha 1(I) chains and one pro alpha 2(I) chain. Cultured skin fibroblasts from the patient have previously been shown to secrete only pro alpha 1(I) trimers. As shown here, fibroblasts from both parents, who do not have osteogenesis imperfecta, secrete both pro alpha 1(I) trimers and normal type I procollagen. A further observation was that synthesis of pro alpha 2(I) chains was decreased in fibroblasts from the patient and his parents. The decrease in the synthesis of pro alpha 2(I) chains is not caused by decreased transcription of the pro alpha 2(I) collagen alleles, since the pro alpha 1(I)/pro alpha 2(I) mRNA ratios were normal in the patient and his parents.

852 BONEMINERAL TURNOVER IN OSTEOGENESIS IMPERFECTA

A young boy with the severe form of osteogenesis imperfecta was treated with NaF for 8 years (1 mg F/kg/d). The frequency of fractures decreased somewhat, but we could detect no gross improvement in bone density. Based on F balance studies and analysis of a bone biopsy, his body F burden was estimated to be about 5 gm at the end of the treatment period (age 14 yr). NaF treatment was abruptly stopped, and during the next 4 1/2 years a number of 24hr urine samples were analyzed for F. After the first few days, during which only 28 mg F were excreted, fluoride excretion declined exponentially: 10% of the body F burden was lost with a half-time of 5 months, 90% with one of 9 years. This latter value is within the range observed by others for the elimination of F and other “bone-seekers” (Sr, Pb, Ra) in normal subjects. Since approximately 99% of body F is located in the mineral phase of bone this element can serve as a tracer for bone mineral. Based on the extended observations on our patient, it would appear that bone mineral turnover is normal in this disease.

Abnormal collagen metabolism in cultured cells in osteogenesis imperfecta.

Cells obtained from normal human skin synthesize predominantly type I collogen in culture. Cells obtained from the skin of an infant with a severe form of osteogenesis imperfecta were found to synthesize as much type III as type I collagen. Decreased synthesis of type I collagen could explain the tissue fragility observed in this case.