Biomedical subjects

D R Eyre

Publications and source records attributed to D R Eyre.

At least 19 recordsLinked to original sources

PubMed1997

Incomplete processing of type II procollagen by a rat chondrosarcoma cell line.

The Swarm rat chondrosarcoma cell line, RCS-LTC, deposits an extracellular matrix that contains the typical type II, IX, and XI collagen phenotype of hyaline cartilage, but the fibrils appear abnormally thin. By N-terminal sequence analysis, the type II collagen from the matrix was shown to have retained its N-propeptides with no evidence of normal processing to type II collagen. Amplification and sequencing of cDNA prepared from the pro alpha1(II) mRNA of these cells showed a normal N-propeptide cleavage site. Furthermore, the type II N-procollagen could be processed to type II collagen by incubation with culture medium from normal chondrocytes. The findings indicate that the RCS-LTC cell line fails to express an active type II procollagen N-proteinase and, therefore, offers a useful culture system in which to study the role of N-propeptide removal in fibrillogenesis.

Amino Acid Sequence↗

PubMed1997

Fibronectin lacking the ED-B domain is a major structural component of tracheal cartilage.

Fibronectin is a highly conserved dimeric glycoprotein found in high concentrations in plasma and widely distributed in low concentrations in the extracellular matrix of tissues. The protein is the product of a single gene, but multiple splicing variants are expressed that show tissue specificity. Three exons (IIIA, IIIB, and V) can be alternatively spliced to produce different fibronectin isoforms. We report here that fibronectin is a remarkably abundant component of the extracellular matrix of bovine tracheal cartilage, increasing with age to more than 20% of the tissue, dry weight. This matrix form of fibronectin is inextractable by 4 M guanidine HCl, indicating that it is a covalently cross-linked structural component. By protein sequence analysis, the main molecular form of fibronectin in bovine tracheal cartilage was shown to lack the ED-B domain encoded by exon IIIB.

Amino Acid Sequence↗

PubMed1997

Osteoclasts generate cross-linked collagen N-telopeptides (NTx) but not free pyridinolines when cultured on human bone.

Urinary excretion of the cross-linked alpha 2(I) N-telopeptide (NTx) of type I collagen has proven in clinical studies to provide a highly responsive and specific index of bone resorption. In order to understand better the biological basis of the specificity and responsiveness of this marker, we examined whether osteoclasts cultured on human bone could generate immunoreactive NTx peptide. Mouse bone marrow cultures stimulated with 1,25 diliydroxyvitamin D3 (1,25(OH)2D3) and hydrocortisone to produce osteoclasts, were cocultured on human bone particles or dentin slices. Aliquots of culture medium were assayed for NTx by enzyme-linked immunosorbent assay (ELISA). NTx was detected in the medium 5 days after the addition of bone and continued to be produced linearly over the 14-day culture period. NTx production required attachment to the bone particles or dentin slices of mononuclear and multinuclear cells that stained for tartrate-resistant acid phosphatase. Surface area of resorbed dentin was highly correlated with medium NTx concentration (R2 = 0.84). Production of NTx was suppressed by the osteoclast inhibitors, calcitonin and alendronate, in a dose-dependent manner. Two other markers of bone resorption, hydroxylysyl pyridinoline and lysyl pyridinoline, were found in peptide linkage in the culture medium but not in free form; indicating that the osteoclasts had degraded the bone collagen to peptides but not to the free cross-linking amino acids.

Acid Phosphatase↗

PubMed1997

Role of type X collagen on experimental mineralization of eggshell membranes.

Type X collagen is a transient and developmentally regulated collagen that has been postulated to be involved in controlling the later stages of endochondral bone formation. However, the role of this collagen in these events is not yet known. In order to understand the function of type X collagen, if any, in the process of biomineralization, the properties of type X collagen in eggshell membranes were further investigated. Specifically, calvaria-derived osteogenic cells were tested for their ability to mineralize eggshell membranes in vitro. Immunohistochemistry with specific monoclonal antibodies was used to correlate the presence or absence of type X collagen or its propeptide domains with the ability of shell membranes to be mineralized. The extent of mineralization was assessed by Von Kossa staining, scanning electron microscopy and energy-dispersive spectroscopy. The results indicate that the non-helical domains of type X collagen must be removed to facilitate the cell-mediated mineralization of eggshell membranes. In this tissue, intact type X collagen does not appear to stimulate or support cell-mediated mineralization. We postulate that the non-helical domains of type X collagen function in vivo to inhibit mineralization and thereby establish boundaries which are protected from mineral deposition.

Amino Acid Sequence↗

PubMed1996

Molecular site specificity of pyridinoline and pyrrole cross-links in type I collagen of human bone.

Compared with soft tissue collagens, bone type I collagen displays a distinctive pattern of covalent cross-linking, with evidence of preferred sites of molecular interaction and a prominence of both immature, divalent cross-links and mature, trivalent cross-links in the adult tissue. In this study the site-specificity of the mature cross-links in human bone collagen was examined. Peptides containing fluorescent pyridinoline cross-links and Ehrlich's-reactive pyrrole cross-links were isolated from a bacterial collagenase digest of demineralized bone matrix. The digest was fractionated by molecular sieve chromatography, monitoring for peptide absorbance, pyridinoline fluorescence, pyrroles by Ehrlich's reagent, and immunoassay for cross-linked N-telopeptides. Individual cross-linked peptides were resolved by ion-exchange and reverse-phase HPLC. Structures were established by NH2-terminal microsequencing, cross-link analysis, electrospray mass spectrometry, and immunoassay. Two, about equally occupied, sites of pyridinoline cross-linking were identified, N-telopeptide to helix and C-telopeptide to helix. Pyrroles were alternative cross-linking products at the same sites, but concentrated (85%) at the N-telopeptide end. Only one combination of chains was cross-linked by pyridinolines at the C-telopeptide to helix site, [alpha1(I)C]2alpha1(I)helix. Several peptide combinations arose from the N-telopeptide to helix site, but the main source of pyridinolines was from the locus, alpha1(I)Nalpha2(I)Nalpha1(I)helix. Pyridinolines linking two alpha1(N) telopeptides were a minor component. Pyrroles were concentrated at the locus, alpha1(I)Nalpha2(I)Nalpha2(I)helix. The cross-link ratio of hydroxylysylpyridinoline to lysylpyridinoline differed between N-telopeptide and C-telopeptide sites, and between the individual interchain combinations. Cross-linked N-telopeptides accounted for two-thirds of the total lysylpyridinoline in bone. N-telopeptide pyridinoline fluorescence was quenched on chromatography, so that reliance on peptide fluorescence alone can underestimate the level of N-telopeptide pyridinoline cross-linking.

Adult↗

PubMed1996

Collagen type IX from human cartilage: a structural profile of intermolecular cross-linking sites.

Type IX collagen, a quantitatively minor collagenous component of cartilage, is known to be associated with and covalently cross-linked to type II collagen fibrils in chick and bovine cartilage. Type IX collagen molecules have also been shown to form covalent cross-links with each other in bovine cartilage. In the present study we demonstrate by structural analysis and location of cross-linking sites that, in human cartilage, type IX collagen is covalently cross-linked to type II collagen and to other molecules of type IX collagen. We also present evidence that, if the proteoglycan form of type IX collagen is present in human cartilage, it can only be a minor component of the matrix, similar to findings with bovine cartilage.

Amino Acid Sequence↗

PubMed1996

Long-term study of the biochemistry and biomechanics of anterior cruciate ligament-patellar tendon autografts in goats.

This study examined the change in type-III collagen concentration and hydroxypyridinium crosslink density of anterior cruciate ligament-patellar tendon autografts and their correlations with Young's modulus of the anterior cruciate autografts and anterior cruciate controls for as long as 3 years after surgery. Fifteen adult female goats (two control and 13 experimental) were tested. Each experimental animal received an anterior cruciate ligament-patellar tendon autograft to the right knee. These animals were tested at 0 (n = 2), 6 (n = 2), 12 (n = 2), and 24 (n = 1) weeks and 1 (n = 3) and 3 (n = 3) years after surgery. After mechanical testing, the anterior cruciate autograft and control tissues were analysed for type-III collagen concentration and hydroxypyridinium crosslink density. The results of sodium dodecyl sulfate gel electrophoresis showed a trend of initial increase in the percentage of type-III collagen in the anterior cruciate ligament autografts and a subsequent decrease after 24 weeks following surgery. There was a nonsignificant (p > 0.05) negative correlation between type-III collagen concentration and Young's modulus. The hydroxypyridinium crosslink density was highest at 1 year after surgery. There was a significant (p < 0.05) positive correlation between hydroxypyridinium crosslink density and Young's modulus in the anterior cruciate autografts and controls. This suggests that hydroxypyridinium crosslink density has a good linear relationship with the material strength of the anterior cruciate ligament autograft and hence could be used as an objective guide for rehabilitation with anterior cruciate autografts.

Animals↗

PubMed1996

Molecular basis and clinical application of biological markers of bone turnover.

An ideal battery of tests would include indices of bone resorption and formation. They should be unique to bone, reflect total skeletal activity, and should correlate with traditional measures of bone remodeling activity, such as radiocalcium kinetics, histomorphometry, or changes in bone mass. Factors that confound their measurement, such as circadian rhythms, diet, age, sex, bone mass, liver function, and kidney clearance rates, should be clearly defined (Fig. 9). To date, no bone marker has been established to meet all these criteria, and each marker may have its own specific advantages and limitations. There are still questions that must be answered before there can be complete confidence in the information gained from measurement of any of the bone markers. Furthermore, it should be emphasized that none of the markers are diagnostic for any particular bone disease and cannot be used for this purpose in individual patients. Nevertheless, recent advances in research and development have provided assays with increased specificity, sensitivity, and availability. Because of this, bone markers can be used for a variety of important purposes: as tools for basic bone biology research, for defining general physiological phenomenon in clinical studies or drug trials, and for following individual patients.

Alkaline Phosphatase↗

PubMed1996

Sublingual testosterone replacement improves muscle mass and strength, decreases bone resorption, and increases bone formation markers in hypogonadal men--a clinical research center study.

To study the effects of androgen replacement therapy on muscle mass and strength and bone turnover markers in hypogonadal men, we administered sublingual testosterone (T) cyclodextrin (SLT; 5 mg, three times daily) to 67 hypogonadal men (baseline serum T, < 8.4 nmol/L) recruited from 4 centers in the U.S.: Torrance (n = 34), Durham (n = 12), New York (n = 9), and Salem (n = 12). Subjects who had received prior T therapy were withdrawn from injections for at least 6 weeks and from oral therapy for 4 weeks. Body composition, muscle strength, and serum and urinary bone turnover markers were measured before and after 6 months of SLT. We have shown previously that this regimen for 60 days will maintain adequate serum T levels and restore sexual function. Total body (P = 0.0104) and lean body mass (P = 0.007) increased with SLT treatment in the 34 subjects in whom body composition was assessed. There was no significant change in total body fat or percent fat. The increase in lean body mass was mainly in the legs; the right leg lean mass increased from 8.9 +/- 0.3 kg at 0 months to 9.2 +/- 0.3 kg at 6 months (P = 0.0008). This increase in leg lean mass was associated with increased leg muscle strength, assessed by leg press (0 months, 139.0 +/- 4.0 kg; 6 months, 147.7 +/- 4.2 kg; P = 0.0038). SLT replacement in hypogonadal men led to small, but significant, decreases in serum Ca (P = 0.0029) and the urinary calcium/creatinine ratio (P = 0.0066), which were associated with increases in serum PTH (P = 0.0001). At baseline, the urinary type I collagen-cross linked N-telopeptides/creatinine ratio [75.6 +/- 7.9 nmol bone collagen equivalents (BCE/mmol] was twice the normal adult male mean (41.0 +/- 3.6 nmol BCE/mmol) and was significantly decreased in response to SLT treatment at 6 months (68.2 +/- 7.7 nmol BCE/mmol; P = 0.0304) without significant changes in urinary creatinine. Serum skeletal alkaline phosphatase did not change. In addition, SLT replacement caused significant increases in serum osteocalcin (P = 0.0001) and type I procollagen (P = 0.0012). Bone mineral density did not change during the 6 months of SLT treatment. We conclude that SLT replacement therapy resulted in increases in lean muscle mass and muscle strength. Like estrogen replacement in hypogonadal postmenopausal females, androgen replacement therapy led to decreased bone resorption and urinary calcium excretion. Moreover, androgen replacement therapy may have the additional benefit of increasing bone formation. A longer term study for several years duration would be necessary to demonstrate whether these changes in bone turnover marker levels will result in increased bone mineral density decreased fracture risks, and reduced frailty in hypogonadal men.

Administration, Sublingual↗

PubMed1995

Structural analysis of cross-linking domains in cartilage type XI collagen. Insights on polymeric assembly.

The collagen framework of hyaline cartilage is based on copolymers of types II, IX, and XI collagens. Previous studies have established specific covalent interactions between types II and IX collagens. The present study examined cross-linking sites in type XI collagen to define better the full heteropolymeric assembly. Pepsinsolubilized type XI collagen was purified from fetal bovine cartilage. The cross-linking amino acids in the preparation were primarily divalent, borohydride-reducible structures; pyridinoline residues were essentially absent. Individual alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains were resolved by high performance liquid chromatography. Telopeptides still attached by cross-links to helical sites were released by periodate oxidation and identified by microsequencing. Analysis of cross-linked peptides isolated from trypsin digest of each alpha-chain identified the attachment helical sites for the telopeptides. A high degree of interchain specificity was evident in the cross-linking between N-telopeptides and the COOH terminus of the triple-helix, consistent with a head-to-tail interaction of molecules staggered by 4D (D = 67 nm) periods. In addition, alpha 1(II) C-telopeptide was linked to the amino-terminal site of the alpha 1(XI) triple helix. In summary, the results show that type XI collagen molecules are primarily cross-linked to each other in cartilage, implying that a homopolymer is initially formed. Links to type II collagen are also indicated, consistent with an eventual cofibrillar assembly. Analysis of cartilage extracts showed that all three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI), had at least in part retained their N-propeptides in cartilage matrix and that the alpha 3 (XI) chain was the IIB splicing variant product of the COL2A1 gene. Of particular note was the finding that the N-telopeptide cross-linking site in both alpha 1(XI) and alpha 2(XI) is located amino-terminal to the putative N-propeptidase cleavage site. This structural feature provides a potential mechanism for the proteolytic depolymerization of type XI collagen by proteases that can cleave between the cross-link and the triple helix (e.g. stromelysin).

Amino Acid Sequence↗

PubMed1995

Long-term treatment of osteopetrosis with recombinant human interferon gamma.

BACKGROUND: Congenital osteopetrosis is a rare osteosclerotic bone disease characterized by both a defect in osteoclastic function and reduced generation of superoxide by leukocytes. The disease is frequently fatal during the first decade of life. A six-month trial of therapy with recombinant human interferon gamma-1b in eight patients with osteopetrosis provided evidence of benefit, prompting this study of more prolonged therapy. METHODS: We studied 14 patients with severe osteopetrosis treated with subcutaneous injections of recombinant human interferon gamma-1b (1.5 micrograms per kilogram of body weight per dose) three times per week for at least 6 months; 11 patients were treated for 18 months. We assessed the effect of therapy by evaluating the patients' clinical status, measuring blood counts and biochemical markers of bone turnover, and performing bone marrow imaging and bone biopsies. RESULTS: After 6 months of therapy, all 14 patients had decreases in trabecular-bone area (determined by histomorphometric analysis of bone-biopsy specimens) and increases in bone marrow space (determined by marrow imaging), and the improvement was sustained in the 11 patients treated for 18 months. The mean (+SD) hemoglobin concentration increased from 7.5 +/- 2.9 to 10.5 +/- 0.3 g per deciliter (P = 0.05), and superoxide generation by granulocyte-macrophage colonies increased (P < 0.001) after 18 months of therapy. In six patients for whom pretreatment data were available, there was a 96 percent decrease in the frequency of infections requiring antibiotic therapy during interferon treatment. There were no side effects necessitating the discontinuation of therapy. CONCLUSIONS: Long-term therapy with interferon gamma in patients with osteopetrosis increases bone resorption and hematopoiesis and improves leukocyte function.

Adult↗

PubMed1995

Changes in cartilage composition and physical properties due to stromelysin degradation.

OBJECTIVE: To determine the effects of stromelysin treatment on biochemical, histologic, and swelling characteristics of intact cartilage explants and to correlate these effects with changes in the functional physical properties of the tissue. METHODS: Bovine articular cartilage explants were cultured for up to 3 days in the presence or absence of recombinant human stromelysin (SLN). Damage to matrix proteoglycans and collagens was assessed and characterized by N-terminal sequencing and Western blot analysis, respectively. Explants were mechanically tested to assess the ability of the tissue to withstand cyclic and static compressive loads. RESULTS: Treatment with SLN resulted in a time- and dose-dependent loss of proteoglycans from cartilage explants, with significant loss seen after 3 days of exposure to 20 nM SLN: Histology indicated that initial loss of proteoglycans occurred in regions near the tissue surface and proceeded inward with increasing time of SLN exposure. SLN treatment resulted in degradation of matrix collagen types IX and II, and a concomitant increase in tissue swelling. This matrix degradation resulted in severe alterations in functional physical properties of the tissue, including compressive stiffness. The initial, focal loss of proteoglycans that resulted from SLN treatment was most accurately detected with high-frequency streaming potential measurements. CONCLUSION: Exposure of intact cartilage to SLN caused specific, molecular-level degradation of matrix molecules, which resulted in changes in the swelling behavior and marked deterioration of functional physical properties of the tissue.

Amino Acid Sequence↗

PubMed1995

Circadian variation in urinary excretion of bone collagen cross-links.

Bone resorption can be evaluated by measuring the urinary excretion of collagen type I cross-linked N telopeptides (NTx). Since it is difficult to obtain (and verify) 24 h urine collections from patients, untimed spot urines are more practical. Such measurements, however, need correction for urine dilution and potentially may vary with collection time since a circadian rhythm in bone metabolism has been reported. This study examined cross-link excretion in urine voids serially collected during a 24 h period from subjects living their normal daily routine (as opposed to a controlled hospital setting). This mimics the situation for walk-in patients visiting a clinician and providing a spot urine. A total of 35 dentists (20 males, 15 females) collected all urine voids separately over a 24 h period. Urines were analyzed for creatinine and NTx. The effects of time of day on the excretion rates of these metabolites (in nmol/h) and on the cross-link:creatinine ratio were assessed. A circadian rhythm was evident in the excretion rate of creatinine with a peak in the late afternoon (18% higher than the 24 h mean, p = 0.0004). The NTx excretion rate peaked in the morning (9% higher than the 24 h mean) but this latter rhythm was not statistically significant (p = 0.31). The NTx:creatinine ratio fell during the day from a high (122% of the 24 h mean) in the early morning to a low in the early evening. This rhythm in the NTx:creatinine ratio in untimed spot urines was statistically significant (p < 0.0001). In conclusion, the NTx:creatinine ratio in spot urines from adult outpatient subjects showed a significant circadian rhythm. Variations in creatinine excretion were the primary cause. Time of day should, therefore, be taken into account when comparing test results of spot urines with normal ranges or with other samples from the same subject.

Adult↗

PubMed1995

Dominant mutations in the type II collagen gene, COL2A1, produce spondyloepimetaphyseal dysplasia, Strudwick type.

The chondrodysplasias are a heterogeneous group of disorders characterized by abnormal growth or development of cartilage. Current classification is based on mode of inheritance as well as clinical, histologic, and/or radiographic features. A clinical spectrum of chondrodysplasia phenotypes, ranging from mild to perinatal lethal, is due to defects in the gene for type II collagen, COL2A1. This spectrum includes Stickler syndrome, Kniest dysplasia, spondyloepiphyseal dysplasia congenita (SEDC), achondrogenesis type II, and hypochondrogenesis. Individuals affected with these disorders exhibit abnormalities of the growth plate, nucleus pulposus, and vitreous humor, which are tissues that contain type II collagen. The Strudwick type of spondyloepimetaphyseal dysplasia (SEMD) is characterized by disproportionate short stature, pectus carinatum, and scoliosis, as well as dappled metaphyses (which are not seen in SEDC). The phenotype was first described by Murdoch and Walker in 1969, and a series of 14 patients was later reported by Anderson et al. The observation of two affected sibs born to unaffected parents led to the classification of SEMD Strudwick as an autosomal recessive disorder. We now describe the biochemical characterization of defects in alpha 1(II) collagen in three unrelated individuals with SEMD Strudwick, each of which is due to heterozygosity for a unique mutation in COL2A1. Our data support the hypothesis that some cases, if not all cases, of this distinctive chondrodysplasia result from dominant mutations in COL2A1, thus expanding the clinical spectrum of phenotypes associated with this gene.

Adult↗

PubMed1995

A radiographic, morphologic, biochemical and molecular analysis of a case of achondrogenesis type II resulting from substitution for a glycine residue (Gly691-->Arg) in the type II collagen trimer.

The type II collagenopathies form a continuous spectrum of clinical severity, ranging from lethal achondrogenesis type II and hypochondrogenesis, through spondyloepiphyseal dysplasia, spondyloepimetaphyseal dysplasia and Kniest dysplasia to the Stickler syndrome and familial precocious osteoarthropathy at the mildest end of the spectrum. We have carried out a radiographic, morphologic, biochemical and molecular study in a case of achondrogenesis type II. Electron micrographs showed inclusion bodies of dilated rough endoplasmic reticulum in the chondrocytes and the presence of sparse collagen fibers in the cartilage matrix. Protein analysis of collagen from cartilage indicated posttranslational overmodification of the major cyanogen bromide peptides, and suggested a mutation near the carboxyl terminus of the type II collagen molecule. Analysis at the DNA level demonstrated that the phenotype was produced by a single base change (G-->C) that resulted in the substitution of glycine691 by arginine in the type II collagen triple helical domain. We confirm previous observations in three cases of hypochondrogenesis that glycine substitutions in the alpha 1(II) chain can result in a phenotype at the most severe end of the type II collagenopathy spectrum.

Abortion, Induced↗

PubMed1995

A multicenter trial of low dose gallium nitrate in patients with advanced Paget's disease of bone.

Gallium nitrate is a potent antiresorptive drug that has been extensively tested in patients with accelerated bone turnover. We have evaluated the effects of this new agent in a pilot multicenter trial of 49 patients with advanced Paget's disease of bone. Patients were randomized to receive 0.05, 0.25, or 0.5 mg/kg.day gallium nitrate administered by sc injection in two 14-day cycles. Serum alkaline phosphatase, fasting 2-h urinary hydroxyproline and N- telopeptide collagen cross-links excretion, and quality of life were assessed every 2 weeks for 12 weeks. The group mean alkaline phosphatase activity at baseline was 854 +/- 100 (+/- SEM) IU/L. The mean changes from baseline to week 12 in serum alkaline phosphatase were +0.5%, -24%, and -31%, respectively, for the three doses tested. The differences for each of the higher dose levels (0.25 and 0.5 mg/kg.day) was statistically significant (P < or = 0.05), and nearly half of the patients treated with the 0.5 mg/kg.day dose achieved a 50% or more reduction in enzyme activity. The nadir value in hydroxyproline excretion occurred at 10 weeks, with mean changes of +9%, -10%, and -17% for the 0.05, 0.25, and 0.5 mg/kg.day doses, respectively; the difference was significant only at the 0.5 mg/kg.day level (P < 0.01). Urinary collagen cross-link excretion showed a significant decrease at the 0.25 and 0.5 mg/kg.day doses. We also observed a definite, but nonsignificant, trend for improved quality of life in patients treated at the highest drug dose. Minor discomfort at the injection site was frequently reported, but did not lead to interruption of therapy. Our results in these patients who had received moderate to extensive prior therapies with other drugs show that cyclical, low dose, sc administration of gallium nitrate is safe and effective for treating patients with advanced Paget's disease of bone.

Adult↗

PubMed1995

Collagen structure and cartilage matrix integrity.

We continue to explore the molecular interactions that characterize the underlying heteropolymeric assembly of types II, IX, and XI collagen in articular cartilage. Structural studies on bovine articular cartilage reveal a high degree of specificity in the interactions of the individual alpha-chains of types IX and XI collagens. The results predict that type IX collagen molecules are not only heavily crosslinked to type II collagen, in an antiparallel orientation, but also to other type IX collagen molecules through specific sites. Type XI collagen molecules are primarily crosslinked to each other in the tissue, but the results are also consistent with type XI-II covalent interactions. We propose that selective proteolysis of types IX and XI collagens is a potential modulator of collagen network architecture.

Animals↗

PubMed1995

An RNA-splicing mutation (G+5IVS20) in the type II collagen gene (COL2A1) in a family with spondyloepiphyseal dysplasia congenita.

Defects in type II collagen have been demonstrated in a phenotypic continuum of chondrodysplasias that includes achondrogenesis II, hypochondrogenesis, spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia, and Stickler syndrome. We have determined that cartilage from a terminated fetus with an inherited form of SEDC contained both normal alpha 1(II) collagen chains and chains that lacked amino acids 256-273 of the triple-helical domain. PCR amplification of this region of COL2A1, from genomic DNA, yielded products of normal size, while amplification of cDNA yielded a normal sized species and a shorter fragment missing exon 20. Sequence analysis of genomic DNA from the fetus revealed a G-->T transversion at position +5 of intron 20; the affected father was also heterozygous for the mutation. Allele-specific PCR and heteroduplex analysis of a VNTR in COL2A1 independently confirmed the unaffected status of a fetus in a subsequent pregnancy. Thermodynamic calculations suggest that the mutation prevents normal splicing of exon 20 by interfering with binding of U1 small-nuclear RNA to pre-mRNA, thus leading to skipping of exon 20 in transcripts from the mutant allele. Electron micrographs of diseased cartilage showed intracellular inclusion bodies, which were stained by an antibody to alpha 1(II) procollagen. Our findings support the hypothesis that alpha-chain length alterations that preserve the Gly-X-Y repeat motif of the triple helix result in partial intracellular retention of alpha 1(II) procollagen and produce mild to moderate chondrodysplasia phenotypes.

Adult↗