Disorder of collagen metabolism in a patient with osteogenesis imperfecta (lethal type): increased degree of hydroxylation of lysine in collagen types I and III

Abstract. Types I, II and III collagen were isolated from calvarium, skin and cartilage from a patient with recessive lethal osteogenesis imperfecta. The distribution of the various collagen types was normal in all three tissues. The α-chains were purified by molecular sieve and ion-exchange chromatography and were found to differ from the corresponding α-chains of age-matched controls only in that the α1(I), α2 and α1 (III) chains contained higher amounts of hydroxylysine with proportionally less lysine. α1(II) was normal. The excess hydroxylysine residues were all glycosylated in the case of α1(I) chains, but only partly so for the α2 chains. Similar observations were made with collagen from fetuses at various stages of development. In these fetuses, however, the increase in the degree of hydroxylation of lysine in αl(I), α2 and αl(III) varied with age, being highest in the youngest fetus. Seen in the context of embryonic development, the collagen of the patient would correspond to that of a fetus younger than 18 weeks, and one could speculate that the defect seen in this patient is the result of a disturbed process of maturation of connective tissue.     

Osteogenesis imperfecta type IV. Biochemical confirmation of genetic linkage to the pro alpha 2(I) gene of type I collagen.

Fibroblasts from two affected members of a large pedigree in which osteogenesis imperfecta (OI) type IV is genetically linked to the pro alpha 2(I) gene of type I collagen synthesize two populations of pro alpha 2(I) chains. One population is normal; the second population appears to have a deletion of about 10 amino acid residues from the middle of the triple helical domain. The mutation in pro alpha 2(I) causes increased posttranslational modification in the amino-terminal half of some pro alpha 1(I) chains, lowers the melting temperature of type I collagen molecules that incorporate a mutant pro alpha 2(I) chain, and prevents or delays the secretion of those molecules from fibroblasts in cell culture. On the basis of this study and linkage studies in additional families, it appears that the OI type IV phenotype is often the result of heterozygosity for mutations in pro alpha 2(I) that alter the triple helical structure of type I collagen.     

Abnormal alpha 2-chain in type I collagen from a patient with a form of osteogenesis imperfecta.

Dermal fibroblasts in culture from a woman with a mild to moderate form of osteogenesis imperfecta synthesize two species of the pro alpha 2-chain of type I procollagen. One chain is normal. The abnormal chain has a slightly faster mobility than normal during electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Analysis of cyanogen bromide peptides of the pro alpha-chain, the alpha-chain, and of the mammalian collagenase cleavage products of the pro alpha- and alpha-chains indicates that the abnormality is confined to the alpha 2(I)CB4 fragment and is consistent with loss of a short triple-helical segment. Type I collagen production was decreased, perhaps because the molecules that contained the abnormal chain were unstable, with a resultant alteration in the ratio of type III to type I collagen secreted into culture medium. Collagen fibrils in bone and skin had a normal periodicity but their diameters were 50% of control; the bone matrix was undermineralized. The structural abnormality in the alpha 2(I)-chain in this patient may affect molecular stability, intermolecular interactions, and collagen-mineral relationships that act to decrease the collagen content of tissues and affect the mineralization of bone.     

Diminished type I collagen synthesis and reduced alpha 1(I) collagen messenger RNA in cultured fibroblasts from patients with dominantly inherited (type I) osteogenesis imperfecta.

Type I osteogenesis imperfecta (OI) is characterized clinically by a moderate fracture frequency with minimal bone deformity and dominant inheritance. Previous studies of the collagenous proteins synthesized by dermal fibroblasts obtained from unrelated patients with this form of OI suggested that the biochemical basis of the disease was reduced production of type I collagen. This study was designed to determine if this biochemical finding segregated with the disease within an individual family. Dermal fibroblast strains were established from three generations of a family having the typical features of type I OI. Analysis of the collagenous proteins made in culture revealed an elevated alpha 1(III) to alpha 1(I) collagen type ratio and an elevated alpha 1(I) to alpha 2(I) collagen chain ratio. The procollagen that accumulated in the medium reflected these ratios to the same degree. Total collagen synthesis was significantly reduced in affected family members. Therefore, the most striking abnormality in affected members was a 50-75% reduction of type I collagen production. Furthermore, the ratio of the alpha 1(I)/alpha 2(I) collagen messenger RNA (mRNA), measured by dot hybridization, was one-half of the value of uninvolved family members and unrelated controls. Since the reduction in the production of type I collagen and the altered alpha 1(I)/alpha 2(I) mRNA ratio clearly segregated with affected individuals within this family, these biochemical measurements may be a useful genetic marker for type I OI.     

Hydroxylation of collagen type I: evidence that both lysyl and prolyl residues are overhydroxylated in osteogenesis imperfecta

Abstract. The composition of the collagens secreted into the media of fibroblast cultures of 39 patients with osteogenesis imperfecta (OI) was the same in controls and OI cultures. An abnormal migration pattern of collagens upon SDS-PAGE was evident in one third of the cultures investigated. Lysyl and prolyl hydroxylation of HPLC-purified α 1(1) chains was elevated in about 60% of cultures. The degree of hydroxylation was highest in the lethal forms. The extent of lysyl and prolyl hydroxylation showed a strong correlation ( r = 0.74, P < 0.001). While high levels of hydroxylation are frequently observed in OI patients, a direct correlation between lysyl or prolyl hydroxylation and fracture rate or growth retardation could not be established.     

Increased serum concentration of type IV collagen peptide and type III collagen peptide in hyperthyroidism.

Serum concentration of type IV collagen peptide, the 7S domain of type IV collagen (type IV collagen 7S) and the amino terminal propeptide of type III procollagen (type III procollagen peptide) is thought to be a useful marker of progressive liver disease. In the present study, serum levels of these collagens in patients with thyroidal diseases with normal liver function were assayed. Increased levels in the hyperthyroid state and relatively decreased levels in the hypothyroid state were observed. The increased levels in hyperthyroidism was most prominent in type IV collagen peptide. The increased level became normal in the subsidence of hyperthyroidism by treatment with anti-thyroid drug. A positive correlation between serum type IV collagen peptide levels and serum thyroid hormone levels such as T4, T3, free T4 and free T3 was observed. These facts show that serum type IV collagen peptide may be influenced by not only liver disease but also serum thyroid hormone levels. Type IV collagen peptide may provide a useful biochemical marker of hyperthyroid state.     

Treatment of poor linear growth in a patient with osteogenesis imperfecta type III

INTRODUCTION: Osteogenesis imperfecta (OI) is a group of bone and connective tissue disorders arising from a genetic defect. CASE SUMMARY: An 11-year-old boy with OI had >50 fractures before initiating treatment with bisphosphonate (BPT) in 1999; many of these fractures required surgery. Despite improvement in bone mineral density (from 0.260 g/cm(2) in 1999 to 0.547 g/cm(2) in 2002), linear growth did not improve with BPT. Somatropin depot (SD) therapy was started in May 2002 to improve the child's poor linear growth (PLG). The rate of growth was 0.011 cm/d before SD initiation and 0.015 cm/d during SD therapy, a 36% increase in growth rate (standard physiologic rate, 0.014 cm/d). The number of fractures did not increase with the addition of SD. Insulin-like growth factor 1 levels, as well as renal and hematologic parameters indicative of safety, did not change. DISCUSSION: Growth hormone use in mild to moderate OI has produced promising results, as in the present study. However, its long-term effectiveness and its potential for helping patients attain adequate adult stature have been questioned. CONCLUSIONS: Short-term use of SD in the treatment of this case of PLG appeared to be effective and well tolerated. Future work is necessary to determine the long-term safety profile and effectiveness of SD therapy in children with PLG who have OI.     

Antisense oligodeoxynucleotides selectively suppress expression of the mutant alpha 2(I) collagen allele in type IV osteogenesis imperfecta fibroblasts. A molecular approach to therapeutics of dominant negative disorders.

We are investigating the use of antisense oligodeoxynucleotides to selectively suppress expression of the mutant type I collagen allele in osteogenesis imperfecta (OI). In this report, we target a human collagen mutation in its natural cellular context. We used cultured fibroblasts from a case of type IV OI, in which the mutant alpha 2(I) allele produces mRNA with exon 16 deleted due to a point mutation in the splice donor site. Lipid-mediated transfection was used to deliver antisense, sense and missense phosphorothioates targeted to both the abnormal mRNA exon 15/17 junction and the nuclear level point mutation. Significant suppression of the mutant protein chain and mRNA was achieved with antisense oligonucleotide to both mRNA and nuclear levels. Mutant protein was suppressed to 44-47% and mutant alpha 2(I) mRNA to 37-43% of their levels in control cells, indicating decreased mRNA as the basis for suppression. Selectivity of mutant allele suppression was better with an mRNA target: suppression was sequence specific and normal mRNA was expressed at 79% of its level in untreated cells. With a nuclear target, significant suppression of mutant mRNA occurred not only with antisense and sense, but also with missense oligonucleotide, which suppressed mutant mRNA to 60% of its level in untreated cells. We also investigated the time course of suppression of protein and mRNA in response to a 4 h transfection of antisense oligonucleotide. From 24-72 h after transfection, mutant protein was suppressed to approximately 50% of its untreated level and suppression of mutant message was significantly greater than that of normal message. The suppression achieved in these studies is insufficient for clinical intervention, but our results provide support for further development of antisense therapy as an approach to the treatment of dominant negative disorders.     

Frameshift mutation near the 3'' end of the COL1A1 gene of type I collagen predicts an elongated Pro alpha 1(I) chain and results in osteogenesis imperfecta type I.

Osteogenesis imperfecta (OI) is a heterogeneous disorder of type I collagen of which OI type I, an autosomal dominant condition, is the mildest and most common form. Affected individuals have blue sclerae, normal stature, bone fragility without significant deformity and osteopenia. Fibroblasts from most affected individuals produce about half the expected amount of structurally normal type I collagen as a result of decreased synthesis of one of its constituent chains, pro alpha 1(I), but the nature of the mutations which result in OI type I are unknown. We describe a three generation family with OI type I in which all affected members have one normal COL1A1 allele and another from which the intragenic Eco RI restriction site near the 3' end of the gene is missing. Amplification by polymerase chain reaction and sequence determination of the normal allele and of the mutant allele in the domain that normally contains the Eco RI site demonstrated a 5-bp deletion from the mutant allele. The deletion changes the translational reading-frame beginning at the Eco RI site and predicts the synthesis of a pro alpha 1(I) chain that extends 84 amino acids beyond the normal termination. Although the mutant pro alpha 1(I) chain is synthesized in an in vitro translation system, we are unable to detect its presence in intact cells, suggesting that it is unstable and rapidly destroyed in one of the cell's degradative pathways. Our analysis of individuals with OI type I from 20 families indicates that this is a unique mutation and suggests that the phenotype can result from multiple mechanisms that decrease the synthesis of normal type I procollagen molecules, including those that alter protein stability.     

A case of chondrodysplasia punctata with features of osteogenesis imperfecta type II.

The osteogenesis imperfecta syndromes constitute a group of heterogeneous, heritable skeletal dysplasias. Of the 4 types, type II is the most severe, with an incidence of 1 per 55,000. It is characterized by malformed bones secondary to abnormal collagen type I synthesis. Affected fetuses are divided into 3 groups: A, B, and C. All groups have long bones described as "wrinkled" or "crumpled" secondary to repeated fractures. Many bones also show evidence of demineralization, which is especially evident in the bones of the face and calvaria. In groups A and C, the chest is generally small, with thickened and shortened ribs, and each rib has characteristic "beading" patterns secondary to repeated fracturing. Sonography has traditionally been successful in the diagnosis of osteogenesis imperfecta at an early gestational age. Chondrodysplasia punctata describes a heterogeneous group of skeletal disorders characterized by abnormal mineralization of bones during gestation. There are many different causes of it, but some of the specific subtypes include rhizomelic, X-linked dominant (also known as Conradi-Hünermann syndrome), X-linked recessive, and tibia-metacarpal. We report a case of severe X-linked dominant chondrodysplasia punctata, which sonographically had common features with osteogenesis imperfecta type II.     

Measurement of autoimmune response against collagen types I, III, and IV by enzyme-linked immunosorbent assay, and its application in infective endocarditis

In these enzyme-linked immunosorbent assays for determination of autoantibodies (IgG, IgM) against collagen type I (ACA I), III (ACA III), and IV (ACA IV), we use commercially available antigen preparations. Inhibition curves showed limited cross-reactivity between these different collagen preparations, the major interference being observed after addition of collagen type III in the ACA I procedures. Imprecision (CVs) for high- and low-titer samples ranged between 1.5% and 7.9% (within-run) and 5.5% and 12.7% (between-run) for ACA I, between 2.2-8.7% and 4.5-10.7% for ACA III, and between 1.3-5.9% and 7.4-11.6% for ACA IV. Significantly increased humoral immune response against collagen types I and III (P less than 0.001) could be demonstrated during the first month of infective endocarditis. In contrast, only borderline increases, however constant, of autoimmune response against basement-membrane collagen (ACA IV) were noticed during 90 days of follow-up.     

An osteopenic nonfracture syndrome with features of mild osteogenesis imperfecta associated with the substitution of a cysteine for glycine at triple helix position 43 in the pro alpha 1(I) chain of type I collagen.

Mutations affecting the pro alpha 1(I) or pro alpha 2(I) collagen genes have been identified in each of the major clinical types of osteogenesis imperfecta. This study reports the presence of a heritable connective tissue disorder in a family with an osteopenic syndrome which has features of mild osteogenesis imperfecta but was considered idiopathic osteoporosis in the proband. At age 38, while still premenopausal, she was found to have osteopenia, short stature, hypermobile joints, mild hyperelastic skin, mild scoliosis, and blue sclerae. There was no history of vertebral or appendicular fracture. Hip and vertebral bone mineral density measurements were consistent with marked fracture risk. Delayed reduction SDS-PAGE of pepsin-digested collagens from dermal fibroblast cultures demonstrated an anomalous band migrating between alpha 1(I) and alpha 1(III). This band merged with the normal alpha-chains upon prereduction, indicating an unexpected cysteine residue. Cyanogen bromide peptide mapping suggested that the mutation was in the smaller NH2-terminal peptides. cDNA was reverse transcribed from mRNA and amplified by the polymerase chain reaction. A basepair mismatch between proband and control alpha 1(I) cDNA hybrids was detected by chemical cleavage with hydroxylamine:piperidine. The cysteine substitution was thus localized to alpha 1(I) exon 9 within the cyanogen bromide 4 peptide. Nucleotide sequence analysis localized a G----T point mutation in the first position of helical codon 43, replacing the expected glycine (GGT) residue with a cysteine (TGT). The prevalence of similar NH2-terminal mutations in subjects with this phenotype which clinically overlaps idiopathic osteoporosis remains to be determined.