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.     

Osteogenesis imperfecta due to recurrent point mutations at CpG dinucleotides in the COL1A1 gene of type I collagen

Summary Most individuals with osteogenesis imperfecta (OI) are heterozygous for dominant mutations in one of the genes that encode the chains of type I collagen. Each of the more than 30 mutations characterized to date has been unique to the affected member (s) of the family. We have determined that two individuals with a progressive deforming variety of OI, OI type III, have the same new dominant mutation [1(I)gly154 to arg] and that two unrelated infants with perinatal lethal OI, OI type II, share a second new dominant muation [1(I)gly1003 to ser]. These mutations occurred at CpG dinucleotides, in a manner consistent with deamination of a methylated cytosine residue, and raise the possibility that CpG dinucleotides are common sites of recurrent mutations in collagen genes. Further, these findings confirm that the OI type-III phenotype, previously thought to be inherited in an autosomal recessive manner, can result from new dominant mutations in the COL1A1 gene of type-I collagen.     

Osteogenesis imperfecta type IV. Detection of a point mutation in one alpha 1(I) collagen allele (COL1A1) by RNA/RNA hybrid analysis

We have identified a point mutation in one alpha 1(I) collagen allele (COL1A1) of a child with the type IV osteogenesis imperfecta phenotype. When compared to parental and control samples, skin fibroblasts of the proband synthesized two populations of type I collagen molecules. One population was normal; the other was delayed in secretion and electrophoretic migration due to post-translational overmodification. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated a gradient of overmodification beginning near the carboxyl-terminal CB peptides. This predicts that the mutation delaying helix formation is near the carboxyl-terminal end of one of the component chains of type I collagen. The mRNA of the patient was probed with overlapping antisense riboprobes to type I collagen cDNA. Cleavage of a mismatch in RNA/RNA hybrids of RNase A allowed the location of the mutation to a 225-base pair region of alpha 1(I) cDNA. The mismatch was not present in RNA/RNA hybrids from either parent. This region of both alpha 1(I) alleles of the patient was isolated by screening a lambda ZAP cDNA library. Sequence determination of both alleles demonstrated a single nucleotide change, G----A, resulting in the substitution of a serine for a glycine at amino acid residue 832. This point mutation occurs in the coding region for alpha 1(I) CB6 and is concordant with the protein data. The finding of a glycine substitution in an alpha 1(I) chain of a patient with the milder type IV osteogenesis imperfecta phenotype requires modification of current molecular models for types II and IV osteogenesis imperfecta.     

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.     

Osteogenesis imperfecta murine: interaction between type I collagen homotrimers

Types I, II, and III collagens are believed to have evolved from the same homotrimer ancestor and they have substantial sequence homology, but type I molecules are alpha1(I)(2)alpha2(I) heterotrimers, unlike homotrimeric types II and III. It is believed that the alpha2(I) chain first appeared in lower vertebrates and that it plays a particularly important role in bone formation. For instance, spontaneous mutations resulting in non- functional alpha2 chains and formation of type I homotrimers cause severe bone pathology (osteogenesis imperfecta) in humans and in animals. However, the exact role of the alpha2 chain is not known. Here, we report measurements of intermolecular forces between collagen helices in native and reconstituted fibers composed of type I homotrimers, heterotrimers and their mix. For comparison, we report forces between type II homotrimers in reconstituted fibers. In agreement with previous studies, we find that the absence of the alpha2 chain reduces temperature-favored attraction between collagen helices, either because of the difference in amino acid sequence of the alpha1 and alpha2 chains or because of more extensive post-translational modification of homotrimers. We find that forces between helices in fibers from type I (as well as type II) homotrimers are not sensitive to pH between pH 6 and 7.5, in contrast to type I heterotrimers. Apparently, the effect of pH is related to extra histidine residues present on alpha2 chains but not on alpha1 chains. Finally, our measurements indicate that the alpha2 chain is responsible for binding some soluble compound(s), possibly glycosaminoglycans, whose displacement results, e.g., in the loss of tendon crystallinity. The ability of the alpha2 chain to bind non-collagen matrix components may be particularly important for bone matrix formation and mineralization.     

Studies of type I collagen (COL1A1) alpha1 chain in patients with osteogenesis imperfecta

Nucleotide sequences of exon 51, adjacent intron areas, and regulatory region of the alpha1 chain of type I collagen (COL1A1) gene were analyzed in 41 patients with osteogenesis imperfecta (OI) from 33 families and their 68 relatives residing at Bashkortostan Republic (BR). Six mutations (four nonsense mutations c.967G > T (p.Gly323X), c.1081C > T (p.Arg361X), c.1243C > T (p.Arg415X), and c.2869C > T (p.Gln957X)) in patients of the Russian origin and two mutations with open reading frame shift c.579delT (p.Gly194ValfsX71), and c.2444delG (p.Gly815AlafsX293)) in patients with OI of Tatar ethnicity as well as 14 single nucleotide polymorphisms in the COL1A1 gene were revealed. Mutations c.967G > T (p.Gly323X) and three alterations in the nucleotide sequence c.544-24C > T, c.643-36delT, and c.957 + 10insA were described for the first time.     

Osteogenesis imperfecta type IV: evidence of abnormal triple helical structure of type I collagen

Summary Skin fibroblasts from a patient with mild osteogenesis imperfecta (OI) type IV synthesize two populations of type I procollagen molecules. One population contains pro1(I) and pro2(I) chains that migrate normally in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a second population contains only slower migrating pro1(I) and pro2(I) chains. The total amount of type I procollagen made by OI cells and the ratio of pro1(I): pro2(I) is normal. When labeled under conditions that inhibit post-translational modification of pro chains, the OI cells produce only single populations of pro1(I) and pro2(I) chains indicating that the apparent increased molecular weight of some OI pro chains is due to excessive post-translational modification rather than peptidyl insertions. Peptide maps indicate that excessive post-translational modification occurs along the entire triple helical segment of some 1(I) and 2(I) chains produced by OI cells. The effect of the mutation is to lower the melting temperature of the molecules containing slow migrating 1(I) and 2(I) chains to 39.5°C (compared to 41.5°C for control), and to delay secretion of the overmodified type I procollagen from OI cells. These data are consistent with a mutation near the carboxyl-terminal end of the triple helical domain which delays triple helical formation and renders all chains available for further post-translational modification amino-terminal to the mutation. Such alterations in triple helical structure, thermal stability, and secretion previously associated only with the lethal OI type II phenotype are thus also seen in the mild OI type IV phenotype.     

A novel mutation causes a perinatal lethal form of osteogenesis imperfecta. An insertion in one alpha 1(I) collagen allele (COL1A1)

We have identified an infant with the perinatal lethal form of osteogenesis imperfecta (type II) whose cells synthesize in equal amounts two different pro alpha 1(I) chains of type I procollagen: one chain is normal in length, the other contains an insertion of approximately 50-70 amino acid residues within the triple helical domain defined by amino acids 123-220. The structure of the insertion is consistent with duplication of an approximately 600-base pair segment in one allele of the alpha 1(I) gene (COL1A1). These cells synthesize normal type I procollagen molecules as well as molecules that contain one or two mutant chains. Unlike type I procollagen molecules synthesized by cells from most other infants with osteogenesis imperfecta type II which contain increased lysyl hydroxylation and hydroxylysyl glycosylation along the triple helical domain, the abnormal molecules synthesized by these cells are not overmodified. The lethal effect of this mutation may result from secretion of about one-quarter the normal amount of normal type I procollagen and secretion of a large amount of a molecule which has a lowered melting temperature, is extended asymmetrically, and which has altered structure in domains important for cross-link formation and bone mineralization.     

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.     

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.     

Ehlers-Danlos syndrome type IV: cosegregation of the phenotype to a COL3A1 allele of type III procollagen

Summary Ehlers-Danlos syndrome (EDS) type IV is a rare and catastrophic genetic disorder of the connective tissue. Individuals from two families with this disorder were studied for a restriction fragment length polymorphism (RFLP) associated with the COL3A1 gene. Our results suggested cosegregation of the EDS type IV phenotype with a COL3A1 RFLP allele. Biochemical studies in cultured skin fibroblasts indicated the presence of different mutations affecting the stability and secretion of the pro1(III) chains of type III procollagen in the two families, thus suggesting that EDS type IV is biochemically heterogeneous. Our data demonstrated the feasibility of molecular diagnosis in this condition using COL3A1 gene related RFLPs.     

Osteogenesis imperfecta mutations may probe vital functional domains (e.g. proteoglycan binding sites) of type 1 collagen fibrils

Osteogenesis imperfecta (OI) is a disease characterized by bone malformations caused by mutations in type 1 collagen. Since many of the 338 possible glycine mutations have not been observed in clinical practice, is this due to chance alone? Because only 83 mutations have been reported in 126 patients, we conclude that many mutations are absent from clinical data for non-random causes. Mutations affecting vital intermolecular interactions in the extracellular matrix (e.g. potential collagen binding sites for proteoglycans) may result in non-viable fetuses that do not progress to clinical status. Some mutations may be silent because they do not significantly affect normal function. The total number of clinically active mutations that will be observed may be far fewer than the potential 338 maximum. © 1997 John Wiley & Sons, Ltd.     

Mutational analysis of COL1A1 and COL1A2 genes among Estonian osteogenesis imperfecta patients

Background

Osteogenesis imperfecta (OI) is a rare bone disorder. In 90% of cases, OI is caused by mutations in the COL1A1/2 genes, which code procollagen α1 and α2 chains. The main aim of the current research was to identify the mutational spectrum of COL1A1/2 genes in Estonian patients. The small population size of Estonia provides a unique chance to explore the collagen I mutational profile of 100% of OI families in the country.

Methods

We performed mutational analysis of peripheral blood gDNA of 30 unrelated Estonian OI patients using Sanger sequencing of COL1A1 and COL1A2 genes, including all intron-exon junctions and 5′UTR and 3′UTR regions, to identify causative OI mutations.

Results

We identified COL1A1/2 mutations in 86.67% of patients (26/30). 76.92% of discovered mutations were located in the COL1A1 (n = 20) and 23.08% in the COL1A2 (n = 6) gene. Half of the COL1A1/2 mutations appeared to be novel. The percentage of quantitative COL1A1/2 mutations was 69.23%. Glycine substitution with serine was the most prevalent among missense mutations. All qualitative mutations were situated in the chain domain of pro-α1/2 chains.

Conclusion

Our study shows that among the Estonian OI population, the range of collagen I mutations is quite high, which agrees with other described OI cohorts of Northern Europe. The Estonian OI cohort differs due to the high number of quantitative variants and simple missense variants, which are mostly Gly to Ser substitutions and do not extend the chain domain of COL1A1/2 products.