Homozygosity for a Missense Mutation in SERPINH1, which Encodes the Collagen Chaperone Protein HSP47, Results in Severe Recessive Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is characterized by bone fragility and fractures that may be accompanied by bone deformity, dentinogenesis imperfecta, short stature, and shortened life span. About 90% of individuals with OI have dominant mutations in the type I collagen genes COL1A1 and COL1A2. Recessive forms of OI resulting from mutations in collagen-modifying enzymes and chaperones CRTAP, LEPRE1, PPIB, and FKBP10 have recently been identified. We have identified an autosomal-recessive missense mutation (c.233T>C, p.Leu78Pro) in SERPINH1, which encodes the collagen chaperone-like protein HSP47, that leads to a severe OI phenotype. The mutation results in degradation of the endoplasmic reticulum resident HSP47 via the proteasome. Type I procollagen accumulates in the Golgi of fibroblasts from the affected individual and a population of the secreted type I procollagen is protease sensitive. These findings suggest that HSP47 monitors the integrity of the triple helix of type I procollagen at the ER/cis-Golgi boundary and, when absent, the rate of transit from the ER to the Golgi is increased and helical structure is compromised. The normal 3-hydroxylation of the prolyl residue at position 986 of the triple helical domain of proα1(I) chains places the role of HSP47 downstream from the CRTAP/P3H1/CyPB complex that is involved in prolyl 3-hydroxylation. Identification of this mutation in SERPINH1 gives further insight into critical steps of the collagen biosynthetic pathway and the molecular pathogenesis of OI.     

A novel splicing mutation in COL1A1 gene caused type I osteogenesis imperfecta in a Chinese family

Osteogenesis imperfect (OI) is a heritable connective tissue disorder with bone fragility as a cardinal manifestation, accompanied by short stature, dentinogenesis imperfecta, hyperlaxity of ligaments and skin, blue sclerae and hearing loss. Dominant form of OI is caused by mutations in the type I procollagen genes, COL1A1/A2. Here we identified a novel splicing mutation c.3207+1G>A (GenBank ID: JQ236861) in the COL1A1 gene that caused type I OI in a Chinese family. RNA splicing analysis proved that this mutation created a new splicing site at c.3200, and then led to frameshift. This result further enriched the mutation spectrum of type I procollagen genes.     

Novel quantitative trait loci for central corneal thickness identified by candidate gene analysis of osteogenesis imperfecta genes

Osteogenesis imperfecta (OI) is a rare connective tissue disorder caused by mutations in the type I collagen genes, COL1A1 and COL1A2, and is characterised by low bone mass and bone fragility. In this study, we explored the relationship between type 1 collagen genes and the quantitative trait central corneal thickness (CCT). CCT was measured in a cohort of 28 Australian type I OI patients and mean CCT was found to be significantly lower compared to a normal population (P < 0.001). We then investigated CCT and corneal collagen fibril diameter and density in a mouse model of OI with a col1a2 mutation. Mean CCT was significantly lower in mutant mice (P = 0.002), as was corneal collagen fibril diameter (P = 0.034), whilst collagen fibril density was significantly greater in mutants (P = 0.034). Finally, we conducted a genetic study to determine whether common single nucleotide polymorphisms (SNPs) in COL1A1 and COL1A2 are associated with CCT variation in the normal human population. Polymorphism rs2696297 (P = 0.003) in COL1A1 and a three SNP haplotype in COL1A2 (P = 0.007) were all significantly associated with normal CCT variation. These data implicate type 1 collagen in the determination of CCT in both OI patients and normal individuals. This provides the first evidence of quantitative trait loci that influence CCT in a normal population and has potential implications for investigating genes involved in glaucoma pathogenesis, a common eye disease in which the severity and progression is influenced by CCT.     

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.     

A Missense Mutation in the SERPINH1 Gene in Dachshunds with Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a hereditary disease occurring in humans and dogs. It is characterized by extremely fragile bones and teeth. Most human and some canine OI cases are caused by mutations in the COL1A1 and COL1A2 genes encoding the subunits of collagen I. Recently, mutations in the CRTAP and LEPRE1 genes were found to cause some rare forms of human OI. Many OI cases exist where the causative mutation has not yet been found. We investigated Dachshunds with an autosomal recessive form of OI. Genotyping only five affected dogs on the 50 k canine SNP chip allowed us to localize the causative mutation to a 5.82 Mb interval on chromosome 21 by homozygosity mapping. Haplotype analysis of five additional carriers narrowed the interval further down to 4.74 Mb. The SERPINH1 gene is located within this interval and encodes an essential chaperone involved in the correct folding of the collagen triple helix. Therefore, we considered SERPINH1 a positional and functional candidate gene and performed mutation analysis in affected and control Dachshunds. A missense mutation (c.977C>T, p.L326P) located in an evolutionary conserved domain was perfectly associated with the OI phenotype. We thus have identified a candidate causative mutation for OI in Dachshunds and identified a fifth OI gene.     

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.     

COL1A1 mutation analysis in Lithuanian patients with osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a generalised disorder of connective tissue characterised by an increased fragility of bones and also manifested in other tissues containing collagen type I, by blue sclera, hearing loss, dentinogenesis imperfecta, hyperextensible joints, hernias and easy bruising. OI is dominantly inherited and results in >90% OI cases, caused by mutations in one of the two genes COL1A1 or COL1A2 coding for type I procollagen. The Lithuanian OI database comprises 147 case records covering the period of 1980 - 2001. Clinical and genealogical analysis of OI cases/families from Lithuania available for examination revealed 18 familial cases of OI type I and 22 sporadic cases: OI type II (3 cases), OI type III (11 cases) and OI type I (8 cases). As a result of their molecular genetic investigation, 11 mutations were identified in the COL1A1 gene in 13 unrelated patients. Of them, nine mutations (E500X, G481A, c.2046insCTCTCTAG, c.1668delT, c.1667insC, c.4337insC, IVS19+1G > A, IVS20-2A > G, IVS22-1G > T) appeared to be novel, i.e. not yet registered in the Human Type I and Type III Collagen Mutations Database (http://www.le.ac.uk/genetics/collagen).     

A Mutation in the 5'-UTR of IFITM5 Creates an In-Frame Start Codon and Causes Autosomal-Dominant Osteogenesis Imperfecta Type V with Hyperplastic Callus

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disorder associated with bone fragility and susceptibility to fractures after minimal trauma. OI type V has an autosomal-dominant pattern of inheritance and is not caused by mutations in the type I collagen genes COL1A1 and COL1A2. The most remarkable and pathognomonic feature, observed in ∼65% of affected individuals, is a predisposition to develop hyperplastic callus after fractures or surgical interventions. To identify the molecular cause of OI type V, we performed whole-exome sequencing in a female with OI type V and her unaffected parents and searched for de novo mutations. We found a heterozygous de novo mutation in the 5′-untranslated region of IFITM5 (the gene encoding Interferon induced transmembrane protein 5), 14 bp upstream of the annotated translation initiation codon (c.−14C>T). Subsequently, we identified an identical heterozygous de novo mutation in a second individual with OI type V by Sanger sequencing, thereby confirming that this is the causal mutation for the phenotype. IFITM5 is a protein that is highly enriched in osteoblasts and has a putative function in bone formation and osteoblast maturation. The mutation c.−14C>T introduces an upstream start codon that is in frame with the reference open-reading frame of IFITM5 and is embedded into a stronger Kozak consensus sequence for translation initiation than the annotated start codon. In vitro, eukaryotic cells were able to recognize this start codon, and they used it instead of the reference translation initiation signal. This suggests that five amino acids (Met-Ala-Leu-Glu-Pro) are added to the N terminus and alter IFITM5 function in individuals with the mutation.     

Distinct involvement of the Jun-N-terminal kinase and NF-kappaB pathways in the repression of the human COL1A2 gene by TNF-alpha

We used a gene knockout approach to elucidate the specific roles played by the Jun-N-terminal kinase (JNK) and NF-κB pathways downstream of TNF-α in the context of α(2) type I collagen gene (COL1A2) expression. In JNK₁^−/−-JNK₂^−/− (JNK^−/−) fibroblasts, TNF-α inhibited basal COL1A2 expression but had no effect on TGF-β-driven gene transactivation unless jnk1 was introduced ectopically. Conversely, in NF-κB essential modulator^−/− (NEMO^−/−) fibroblasts, lack of NF-κB activation did not influence the antagonism exerted by TNF-α against TGF-β but prevented repression of basal COL1A2 gene expression. Similar regulatory mechanisms take place in dermal fibroblasts, as evidenced using transfected dominant-negative forms of MKK4 and IKK-α, critical kinases upstream of the JNK and NF-κB pathways, respectively. These results represent the first demonstration of an alternate usage of distinct signaling pathways by TNF-α to inhibit the expression of a given gene, COL1A2, depending on its activation state.     

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.     

Novel PAX9 and COL1A2 Missense Mutations Causing Tooth Agenesis and OI/DGI without Skeletal Abnormalities

Inherited dentin defects are classified into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia (DD). The genetic etiology of DD-I is unknown. Defects in dentin sialophosphoprotein (DSPP) cause DD type II and DGI types II and III. DGI type I is the oral manifestation of osteogenesis imperfecta (OI), a systemic disease typically caused by defects in COL1A1 or COL1A2. Mutations in MSX1, PAX9, AXIN2, EDA and WNT10A can cause non-syndromic familial tooth agenesis. In this study a simplex pattern of clinical dentinogenesis imperfecta juxtaposed with a dominant pattern of hypodontia (mild tooth agenesis) was evaluated, and available family members were recruited. Mutational analyses of the candidate genes for DGI and hypodontia were performed and the results validated. A spontaneous novel mutation in COL1A2 (c.1171G>A; p.Gly391Ser) causing only dentin defects and a novel mutation in PAX9 (c.43T>A; p.Phe15Ile) causing hypodontia were identified and correlated with the phenotypic presentations in the family. Bone radiographs of the proband’s dominant leg and foot were within normal limits. We conclude that when no DSPP mutation is identified in clinically determined isolated DGI cases, COL1A1 and COL1A2 should be considered as candidate genes. PAX9 mutation p.Phe15Ile within the N-terminal β-hairpin structure of the PAX9 paired domain causes tooth agenesis.     

PPIB Mutations Cause Severe Osteogenesis Imperfecta

Deficiency of cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1(P3H1) has been reported in autosomal-recessive lethal or severe osteogenesis imperfecta (OI). CRTAP, P3H1, and cyclophilin B (CyPB) form an intracellular collagen-modifying complex that 3-hydroxylates proline at position 986 (P986) in the α1 chains of collagen type I. This 3-prolyl hydroxylation is decreased in patients with CRTAP and P3H1 deficiency. It was suspected that mutations in the PPIB gene encoding CyPB would also cause OI with decreased collagen 3-prolyl hydroxylation. To our knowledge we present the first two families with recessive OI caused by PPIB gene mutations. The clinical phenotype is compatible with OI Sillence type II-B/III as seen with COL1A1/2, CRTAP, and LEPRE1 mutations. The percentage of 3-hydroxylated P986 residues in patients with PPIB mutations is decreased in comparison to normal, but it is higher than in patients with CRTAP and LEPRE1 mutations. This result and the fact that CyPB is demonstrable independent of CRTAP and P3H1, along with reported decreased 3-prolyl hydroxylation due to deficiency of CRTAP lacking the catalytic hydroxylation domain and the known function of CyPB as a cis-trans isomerase, suggest that recessive OI is caused by a dysfunctional P3H1/CRTAP/CyPB complex rather than by the lack of 3-prolyl hydroxylation of a single proline residue in the α1 chains of collagen type I.     

Transforming Growth Factor β1 Induces the Expression of Collagen Type I by DNA Methylation in Cardiac Fibroblasts

Transforming growth factor-beta (TGF-β), a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1) expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β₁. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs) were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β₁ upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β₁ for 48 h. TGF-β₁ treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β₁ can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β₁-induced COL1A1 gene expression.     

ER stress-mediated apoptosis in a new mouse model of osteogenesis imperfecta

Osteogenesis imperfecta is an inherited disorder characterized by increased bone fragility, fractures, and osteoporosis, and most cases are caused by mutations affecting the type I collagen genes. Here, we describe a new mouse model for Osteogenesis imperfecta termed Aga2 (abnormal gait 2) that was isolated from the Munich N-ethyl-N-nitrosourea mutagenesis program and exhibited phenotypic variability, including reduced bone mass, multiple fractures, and early lethality. The causal gene was mapped to Chromosome 11 by linkage analysis, and a C-terminal frameshift mutation was identified in the Col1a1 (procollagen type I, alpha 1) gene as the cause of the disorder. Aga2 heterozygous animals had markedly increased bone turnover and a disrupted native collagen network. Further studies showed that abnormal proα1(I) chains accumulated intracellularly in Aga2/+ dermal fibroblasts and were poorly secreted extracellularly. This was associated with the induction of an endoplasmic reticulum stress-specific unfolded protein response involving upregulation of BiP, Hsp47, and Gadd153 with caspases-12 and −3 activation and apoptosis of osteoblasts both in vitro and in vivo. These studies resulted in the identification of a new model for Osteogenesis imperfecta, and identified a role for intracellular modulation of the endoplasmic reticulum stress-associated unfolded protein response machinery toward osteoblast apoptosis during the pathogenesis of disease.