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.     

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.     

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.     

In vitro expression of osteoblastic markers in cells isolated from normal fetal and postnatal human bone and from bone of patients with osteogenesis imperfecta

We studied the expression of osteoblastic markers in cultured cells isolated from the bone of 15 patients with different clinical forms of osteogenesis imperfecta (OI) and of seven fetal and postnatal controls. Cultured bone cells of ten OI patients produced abnormal collagen type I. Similar to controls, OI bone cells produced predominantly collagen type I with traces of collagen types III and V. The 1,25(OH)₂ vitamin D₃-stimulated synthesis of osteocalcin, a specific osteoblastic marker protein, was similar in OI bone cells and age-matched controls. Bone cells from fetal controls and from patients with the perinatal lethal OI type II produced less osteocalcin than bone cells from postnatal controls and surviving OI patients. OI bone cells responded to parath.yroid hormone (PTH) by increased production of cAMP similar to controls. Bone cells from fetal controls and from OI type II donors showed a decreased response to PTH. Activity of the bone-liver-kidney isoenzyme alkaline phosphatase (AP) was detected in all control and OI bone cells. The expression of all osteoblastic markers was similar in bone cells producing abnormal collagen type I. These observations show that OI bone cells in vitro express a pattern of osteoblastic markers similar to age-matched control bone cells indicating that osteoblastic differentiation is not altered by the underlying defects of collagen type I metabolism in OI bone cells. © 1993 Wiley-Liss, Inc.     

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.     

Identification of a novel COL1A1 frameshift mutation, c.700delG,in a Chinese osteogenesis imperfecta family

Osteogenesis imperfecta (OI) is a family of genetic disorders associated with bone loss and fragility. Mutations associated with OI have been found in genes encoding the type I collagen chains. People with OI type I often produce insufficient α1-chain type I collagen because of frameshift, nonsense, or splice site mutations in COL1A1 or COL1A2. This report is of a Chinese daughter and mother who had both experienced two bone fractures. Because skeletal fragility is predominantly inherited, we focused on identifying mutations in COL1A1 and COL1A2 genes. A novel mutation in COL1A1, c.700delG, was detected by genomic DNA sequencing in the mother and daughter, but not in their relatives. The identification of this mutation led to the conclusion that they were affected by mild OI type I. Open reading frame analysis indicated that this frameshift mutation would truncate α1-chain type I collagen at residue p263 (p.E234KfsX264), while the wild-type protein would contain 1,464 residues. The clinical data were consistent with the patients’ diagnosis of mild OI type I caused by haploinsufficiency of α1-chain type I collagen. Combined with previous reports, identification of the novel mutation COL1A1-c.700delG in these patients suggests that additional genetic and environmental factors may influence the severity of OI.     

Incorporation of type I collagen molecules that contain a mutant alpha 2(I) chain (Gly580-->Asp) into bone matrix in a lethal case of osteogenesis imperfecta.

To understand more directly the tissue defect in osteogenesis imperfecta (OI), bone matrix was analyzed from an infant with lethal OI (type II) of defined mutation (collagen alpha 2(I)Gly580-->Asp). Pepsin-solubilized alpha 1(I) and alpha 2(I) chains and derived CNBr-peptides migrated more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared with normal human controls. The peptide alpha 2(I)CB3,5, predicted to contain the mutation site, ran as a retarded doublet band and was purified by high performance liquid chromatography and digested with V8 protease. Two peptides with amino-terminal sequences beginning at residue 576 of the alpha 2(I) chain were isolated. One had the normal sequence. The other differed in that aspartic acid replaced glycine at residue 580 as predicted from cDNA analysis, and in having an unhydroxylated proline at residue 579. From yields on microsequencing and the relative intensities of the two forms of alpha 2(I)CB3,5 on SDS-polyacrylamide gel electrophoresis, the ratio of mutant to normal alpha 2(I) chains in the infant's bone matrix was 0.7/1. Although the effects of an efficient incorporation of mutant chains on the properties of the bone matrix are unknown, it may be that in this OI case the tissue abnormalities result more from the presence of mutant protein than from an underexpression of matrix.     

Variable expression of osteogenesis imperfecta in a nuclear family is explained by somatic mosaicism for a lethal point mutation in the alpha 1(I) gene (COL1A1) of type I collagen in a parent.

Fibroblasts from a man with a mild form of osteogenesis imperfecta (OI) and from his son with perinatal lethal OI (OI type II) produced normal and abnormal type I procollagen molecules. The abnormal molecules synthesized by both cell strains contained one or two pro alpha 1(I) chains in which the glycine at position 550 of the triple-helical domain was substituted by arginine as the result of a G-to-A transition in the first base of the glycine codon. Cells from the mother produced only normal type I procollagen molecules. By allele-specific oligonucleotide hybridization to amplified genomic sequences from paternal tissues we determined that the mutant allele accounted for approximately 50% of the COL1A1 alleles in fibroblasts, 27% of those in blood, and 37% of those in sperm. These findings demonstrate that the father is mosaic for the potentially lethal mutation and suggest that the OI phenotype is determined by the nature of the mutation and the relative abundance of the normal and mutant alleles in different tissues. Furthermore, the findings make it clear that some individuals with mild to moderate forms of OI are mosaic for mutations that will be lethal in their offspring.     

Perinatal lethal osteogenesis imperfecta (OI type II): a biochemically heterogeneous disorder usually due to new mutations in the genes for type I collagen.

To resolve uncertainty concerning the inheritance of the perinatal lethal form of osteogenesis imperfecta (OI type II), we collected family data and radiographs for 71 probands and analyzed the collagens synthesized by dermal fibroblastic cells cultured from 43 of the probands, 19 parental pairs, and single parents of each of four additional probands. In 65 families for which there were complete data on sibship size, there was recurrence of the OI type II phenotype in five families such that six (8.6%) of 70 sibs were affected. In two families with recurrence, the radiographic phenotype was milder than that for the remainder; and one of those families was consanguinous, suggesting autosomal recessive inheritance. In the remaining three families there was no evidence of consanguinity, but in one of them the structure was compatible with gonadal mosaicism in the mother. In studies of collagens synthesized by cells from 43 infants, we identified two probands with separate rearrangements in an allele of one of the genes of type I collagen; but in the rest there were subtle mutations that disrupted the normal triple-helix structure of type I collagen molecules. In two probands we identified de novo mutations; in 16 additional families cells from the parents made only normal collagens, compatible with new mutations in their offsprings. These findings indicate that the OI type II phenotype is biochemically heterogeneous, that the majority result from new dominant mutations in the genes encoding type I collagen, and that some recurrences can be accounted for by gonadal mosaicism in one of the parents.     

High proportion of mutant osteoblasts is compatible with normal skeletal function in mosaic carriers of osteogenesis imperfecta

Individuals with mosaicism for the autosomal dominant bone dysplasia osteogenesis imperfecta (OI) are generally identified by having more than one affected child. The mosaic carriers have both normal and mutant cell populations in somatic and germline tissues but are unaffected or minimally affected by the type I collagen mutation that manifests clinically in their heterozygous offspring. We determined the proportion of mutant osteoblasts in skeletal tissue of two mosaic carriers who each have a COL1A1 mutation in a high proportion of dermal fibroblasts. Both carriers had normal height and bone histology; the first carrier had normal lumbar spine measurements (L1-L4), as determined by dual-energy x-ray absorptiometry (Z = +1.17). In cultured cells from the first carrier, studied by labeled PCR and single-cell PCR over successive passages, the collagen mutation was present in 85% of fibroblasts and 50% and 75% of osteoblasts from her right iliac crest and left patella, respectively, with minimal selection. The second carrier was studied by PCR amplification of DNA from autopsy paraffin blocks. The proportion of heterozygous cells was 40% in calvarium, 65% in tracheal ring, and 70% in aorta. Thus, in OI, substantially normal skeletal growth, density, and histology are compatible with a 40%-75% burden of osteoblasts heterozygous for a COL1A1 mutation. These data are encouraging for mesenchymal stem-cell transplantation, since mosaic carriers are a naturally occurring model for cell therapy.     

Hammerhead ribozymes selectively suppress mutant type I collagen mRNA in osteogenesis imperfecta fibroblasts

Ribozymes are a promising agent for the gene therapy of dominant negative genetic disorders by allele-specific mRNA suppression. To test allele-specific mRNA suppression in cells, we used fibroblasts from a patient with osteogenesis imperfecta (OI). These cells contain a mutation in one α1(I) collagen allele which both causes the skeletal disorder and generates a novel ribozyme cleavage site. In a preliminary in vitro assay, ribozymes cleaved mutant RNA substrate whereas normal substrate was left intact. For the studies in cell culture we generated cell lines stably expressing active (AR) and inactive (IR) ribozymes targeted to mutant α1(I) collagen mRNA. Quantitative competitive RT–PCR analyses of type I collagen mRNA, normalized to β-actin expression levels, revealed that the level of mutant α1(I) collagen mRNA was significantly decreased by ~50% in cells expressing AR. Normal α1(I) collagen mRNA showed no significant reduction when AR or IR was expressed from the pHβAPr-1-neo vector and a small (10–20%) but significant reduction when either ribozyme was expressed from the pCI.neo vector. In clonal lines derived from cells expressing AR the level of ribozyme expression correlated with the extent of reduction in the mutant:normal α1(I) mRNA ratio, ranging from 0.33 to 0.96. Stable expression of active ribozyme did not affect cell viability, as assessed by growth rates. Ribozyme cleavage of mutant mRNA results in a reduction in mutant type I collagen protein, as demonstrated by SDS–urea–PAGE. This is the first report of ribozymes causing specific suppression of an endogenous mutant mRNA in cells derived from a patient with a dominant negative genetic disorder.     

Defects of type I procollagen metabolism correlated with decrease of prolidase activity in a case of lethal osteogenesis imperfecta.

We have studied the structure and metabolism of type I procollagen in a case of perinatal lethal osteogenesis imperfecta (OI) type II. Cultured skin fibroblasts from the proband synthesized both normal and abnormal forms of type I procollagen. Some abnormal, overmodified molecules were secreted by OI cells, although less efficiently than normal molecules from control cells. The OI fibroblasts accumulated large amounts of abnormal proalpha1(I) and proalpha2(I) chains intracellularly. The extracellular collagenolytic activity was decreased compared to control cells. Furthermore, OI cells produced less type I procollagen and demonstrated lower capacity to synthesize DNA than control cells. We have found that in contrast to prolinase activity, the activity of prolidase (an enzyme essential for collagen synthesis and cell growth) is also significantly reduced in OI cells. No differences were found in the amount of the enzyme protein recovered from both the OI and control cells. However, we found that expressions of beta1 integrin and insulin-like growth factor-I receptor (receptors known to play an important role in up regulation of prolidase activity) were decreased in OI cells compared to control cells. The decrease in prolidase activity may provide an important mechanism of altered cell growth and collagen metabolism involved in producing the perinatal lethal form of the OI phenotype.     

Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta.

Cultured skin fibroblasts from seven consecutive cases of lethal perinatal osteogenesis imperfecta (OI) expressed defects of type I collagen metabolism. The secretion of [14C]proline-labelled collagen by the OI cells was specifically reduced (51-79% of control), and collagen degradation was increased to twice that of control cells in five cases and increased by approx. 30% in the other two cases. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that four of the OI cell lines produced two forms of type I collagen consisting of both normally and slowly migrating forms of the alpha 1(I)- and alpha 2(I)-chains. In the other three OI cell lines only the 'slow' alpha (I)'- and alpha 2(I)'-chains were detected. In both groups inhibition of the post-translational modifications of proline and lysine resulted in the production of a single species of type I collagen with normal electrophoretic migration. Proline hydroxylation was normal, but the hydroxylysine contents of alpha 1(I)'- and alpha 2(I)'-chains purified by h.p.l.c. were greater than in control alpha-chains. The glucosylgalactosylhydroxylysine content was increased approx. 3-fold while the galactosylhydroxylysine content was only slightly increased in the alpha 1(I)'-chains relative to control alpha 1(I)-chains. Peptide mapping of the CNBr-cleavage peptides provided evidence that the increased post-translational modifications were distributed throughout the alpha 1(I)'- and alpha 2(I)'-chains. It is postulated that the greater modification of these chains was due to structural defects of the alpha-chains leading to delayed helix formation. The abnormal charge heterogeneity observed in the alpha 1 CB8 peptide of one patient may reflect such a structural defect in the type I collagen molecule.     

Osteogenesis imperfecta type I: molecular heterogeneity for COL1A1 null alleles of type I collagen.

Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 "null" allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5' donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon, that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype.     

Consistent linkage of dominantly inherited osteogenesis imperfecta to the type I collagen loci: COL1A1 and COL1A2

The segregation of COL1A1 and COL1A2, the two genes which encode the chains of type I collagen, was analyzed in 38 dominant osteogenesis imperfecta (OI) pedigrees by using polymorphic markers within or close to the genes. This was done in order to estimate the consistency of linkage of OI genes to these two loci. None of the 38 pedigrees showed evidence of recombination between the OI gene and both collagen loci, suggesting that the frequency of unlinked loci in the population must be low. From these results, approximate 95% confidence limits for the proportion of families linked to the type I collagen genes can be set between .91 and 1.00. This is high enough to base prenatal diagnosis of dominantly inherited OI on linkage to these genes even in families which are too small for the linkage to be independently confirmed to high levels of significance. When phenotypic features were compared with the concordant collagen locus, all eight pedigrees with Sillence OI type IV segregated with COL1A2. On the other hand, Sillence OI type I segregated with both COL1A1 (17 pedigrees) and COL1A2 (7 pedigrees). The concordant locus was uncertain in the remaining six OI type I pedigrees. Of several other features, the presence or absence of presenile hearing loss was the best predictor of the mutant locus in OI type I families, with 13 of the 17 COL1A1 segregants and none of the 7 COL1A2 segregants showing this feature.     

Mutations near amino end of alpha1(I) collagen cause combined osteogenesis imperfecta/Ehlers-Danlos syndrome by interference with N-propeptide processing

Patients with OI/EDS form a distinct subset of osteogenesis imperfecta (OI) patients. In addition to skeletal fragility, they have characteristics of Ehlers-Danlos syndrome (EDS). We identified 7 children with types III or IV OI, plus severe large and small joint laxity and early progressive scoliosis. In each child with OI/EDS, we identified a mutation in the first 90 residues of the helical region of alpha1(I) collagen. These mutations prevent or delay removal of the procollagen N-propeptide by purified N-proteinase (ADAMTS-2) in vitro and in pericellular assays. The mutant pN-collagen which results is efficiently incorporated into matrix by cultured fibroblasts and osteoblasts and is prominently present in newly incorporated and immaturely cross-linked collagen. Dermal collagen fibrils have significantly reduced cross-sectional diameters, corroborating incorporation of pN-collagen into fibrils in vivo. Differential scanning calorimetry revealed that these mutant collagens are less stable than the corresponding procollagens, which is not seen with other type I collagen helical mutations. These mutations disrupt a distinct folding region of high thermal stability in the first 90 residues at the amino end of type I collagen and alter the secondary structure of the adjacent N-proteinase cleavage site. Thus, these OI/EDS collagen mutations are directly responsible for the bone fragility of OI and indirectly responsible for EDS symptoms, by interference with N-propeptide removal.