Ehlers-Danlos syndrome (EDS) types I and II, which comprise the classical variety, are well characterized from the clinical perspective, but it has been difficult to identify the molecular basis of the disorder in the majority of affected individuals. Several explanations for this failure to detect mutations have been proposed, including genetic heterogeneity, failure of allele expression, and technical difficulties. Genetic heterogeneity has been confirmed as an explanation for such failure, since causative mutations have been identified in the COL5A1, COL5A2, and tenascin X genes and since they have been inferred in the COL1A2 gene. Nonetheless, in the majority of families with autosomal dominant inheritance of EDS, there appears to be linkage to loci that contain the COL5A1 or COL5A2 genes. To determine whether allele-product instability could explain failure to identify some mutations, we analyzed polymorphic variants in the COL5A1 gene in 16 individuals, and we examined mRNA for the expression of both alleles and for alterations in splicing. We found a splice-site mutation in a single individual, and we determined that, in six individuals, the mRNA from one COL5A1 allele either was not expressed or was very unstable. We identified small insertions or deletions in five of these cell strains, but we could not identify the mutation in the sixth individual. Thus, although as many as one-half of the mutations that give rise to EDS types I and II are likely to lie in the COL5A1 gene, a significant portion of them result in very low levels of mRNA from the mutant allele, as a consequence of nonsense-mediated mRNA decay.

Ehlers-Danlos syndrome (EDS) type VII results from defects in the conversion of type I procollagen to collagen as a consequence of mutations in the substrate that alter the protease cleavage site (EDS type VIIA and VIIB) or in the protease itself (EDS type VIIC). We identified seven additional families in which EDS type VII is either dominantly inherited (one family with EDS type VIIB) or due to new dominant mutations (one family with EDS type VIIA and five families with EDS type VIIB). In six families, the mutations alter the consensus splice junctions, and, in the seventh family, the exon is deleted entirely. The COL1A1 mutation produced the most severe phenotypic effects, whereas those in the COL1A2 gene, regardless of the location or effect, produced congenital hip dislocation and other joint instability that was sometimes very marked. Fractures are seen in some people with EDS type VII, consistent with alterations in mineral deposition on collagen fibrils in bony tissues. These new findings expand the array of mutations known to cause EDS type VII and provide insight into genotype/phenotype relationships in these genes.

Osteogenesis imperfecta (OI) is characterised by brittle bones and caused by mutations in the type I collagen genes, COL1A1 and COL1A2. We identified a mutation in the carboxyl-terminal propeptide coding region of one COL1A1 allele in an infant who died with an OI phenotype that differed from the usual lethal form and had regions of increased bone density. The newborn female had dysmorphic facial features, including loss of mandibular angle. Bilateral upper and lower limb contractures were present with multiple fractures in the long bones and ribs. The long bones were not compressed and their ends were radiographically dense. She died after a few hours and histopathological studies identified extramedullary haematopoiesis in the liver, little lamellar bone formation, decreased osteoclasts, abnormally thickened bony trabeculae with retained cartilage in long bones, and diminished marrow spaces similar to those seen in dense bone diseases such as osteopetrosis and pycnodysostosis. The child was heterozygous for a COL1A1 4321G→T transversion in exon 52 that changed a conserved aspartic acid to tyrosine (D1441Y). Abnormal proα1(I) chains were slow to assemble into dimers and trimers, and abnormal molecules were retained intracellularly for an extended period. The secreted type I procollagen molecules synthesised by cultured dermal fibroblasts were overmodified along the full length but had normal thermal stability. These findings suggest that the unusual phenotype reflected both a diminished amount of secreted type I procollagen and the presence of a population of stable and overmodified molecules that might support increased mineralisation or interfere with degradation of bone.T ype I collagen is the principle protein of bone, skin, ligaments, tendon, and most other connective tissues. It is synthesised as a soluble precursor, procollagen, which consists of two proα1(I) chains and one proα2(I) chain. Each proα chain contains a central obligatory Gly-Xaa-Yaa repeat sequence (the triple helical domain) of more than 1000 residues (in which Xaa and Yaa are any residue other than cysteine or tryptophan), and propeptides at the amino-and carboxyl-termini. The carboxyl-terminal propeptide (Cpropeptide) domains direct chain-chain recognition and alignment of proα chains into correct registration. Assembled procollagen trimers are translocated from the RER to the Golgi apparatus, further modified, packaged, and secreted into the extracellular spaces where they are converted to collagen by proteolytic cleavage of the N-and C-propeptides. Outside the cell, collagen trimers assemble into fibrils, which serve as the main source of mechanical strength in connective tissue and the template for matrix deposition and mineralisation in the bone.Mutations in the COL1A1 and COL1A2 type I collagen genes perturb normal collagen assembly in the cell, secretion from the cell, and fibril assembly in the extracellular spaces, which, collectively, result in the osteogenesis imperfecta (OI) phenotypes.1 2 The spectrum of severity of O...

Triple helix formation is a prerequisite for the passage of type I procollagen from the endoplasmic reticulum and secretion from the cell to form extracellular fibrils that will support mineral deposition in bone. Analysis of cDNA from 11 unrelated individuals with osteogenesis imperfecta (OI) revealed the presence of 11 novel, short in-frame deletions or duplications of three, nine, or 18 nucleotides in the helical coding regions of the COL1A1 and COL1A2 collagen genes. Triple helix formation was impaired, type I collagen alpha chains were post-translationally overmodified, and extracellular secretion was markedly reduced. With one exception, the obligate Gly-Xaa-Yaa repeat pattern of amino acids in the helical domains was not altered, but the Xaa- and Yaa position residues were out of register relative to the amino acid sequences of adjacent chains in the triple helix. Thus, the identity of these amino acids, in addition to third position glycines, is important for normal helix formation. These findings expand the known repertoire of uncommon in-frame deletions and duplications in OI, and provide insight into normal collagen biosynthesis and collagen triple helix formation.

We completed prenatal diagnostic studies from 129 pregnancies at risk for osteogenesis imperfecta (OI). Studies in 107 pregnancies were completed by analysis of collagen synthesized by cells cultured from chorionic villus biopsies and the remaining 22 used direct mutation identification or analysis of polymorphic restriction sites in the COL1A1 gene of type I collagen. The vast majority of studies (n=113) were obtained to identify fetuses with OI type II (the perinatal lethal form) and some fetuses affected with OI type III or IV (the deforming varieties). Of the 50 couples who had had one previous affected pregnancy with the lethal form of OI, one had a second affected pregnancy, a rate of 2 per cent. Two of the seven unaffected couples (28 per cent) who had had two previous affected pregnancies with OI type II had a third affected pregnancy; none of the three with two previous pregnancies with OI type III had a third. Pregnancies at risk for OI type I could not be ascertained reliably by biochemical analysis of cultured CVS cells but were identified by direct analysis of the causative mutation or the use of linked markers in families. All prenatal diagnostic studies were undertaken only after earlier diagnostic studies (biochemical or molecular) had been completed on the proband, a necessary strategy for accurate results. In all pregnancies at risk for OI type II, OI type III, and OI type IV studied with biochemical strategies and in pregnancies at risk for OI type I studied with molecular techniques, there were neither false-negative nor false-positive results. Diagnostic information can be obtained within 20-30 days of biopsy using biochemical techniques and within 10-14 days when molecular strategies are used. 1997 by John Wiley & Sons, Ltd.