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A recurrent point mutation in the fibroblast growth factor receptor 3 (FGFR3) gene that converts proline 250 into arginine is commonly associated with coronal craniosynostosis and has allowed definition of a new syndrome on a molecular basis. Sixty-two patients with sporadic or familial forms of coronal craniosynostosis were investigated for the P250R FGFR3 mutation. It was identified in 20 probands originating from 27 unrelated families (74%), while only 6/35 sporadic cases (17%) harboured the mutation. In both familial and sporadic cases, females were significantly more severely affected than males. Hence, while 68% of females carrying the P250R mutation showed brachycephaly, only 35% of males had the same phenotype. In the most severe forms of the disease, the association of bicoronal craniosynostosis with hypertelorism and marked bulging of the temporal fossae were common hallmarks that might be helpful for clinical diagnosis. Taken together, these results indicate that the P250R FGFR3 mutation is mostly familial and is associated with a more severe phenotype in females than in males. The sex related severity of the condition points to the possible implication of modifier genes in this syndrome.

The effects of porcine interleukin-1 (IL-1) alpha on collagen production were studied in cultured human rheumatoid synovial cells. Addition of 0.05-5 ng of IL-1/ml into the cultures resulted in a dose-dependent decreased rate of collagen released into the medium over 24 h. To determine whether this inhibition was due to secondary action of prostaglandin E2 (PGE2) secreted in response to IL-1, cultures were incubated in presence of various inhibitors of arachidonate metabolism. Depending on the cell strains, these inhibitors were able to suppress or diminish the effect of IL-1, suggesting that PGE2 is involved in the mechanism. Depression of collagen production caused by IL-1 mainly affected type I collagen and therefore led to a change in the type I/type III collagen ratio in the extracellular medium. Steady-state levels of mRNA for types I and III procollagens were estimated by dot-blot hybridization and compared with the amounts of respective collagens produced in the same cultures. IL-1 generally increased procollagen type I mRNA, but to a variable extent, as did indomethacin (Indo). Depending on the cell strain, the combination of indo and IL-1 could elevate the mRNA level of type I procollagen compared with Indo alone. These results did not correlate with the production rate of collagen in the medium, which was diminished by exposure to IL-1. The level of mRNA for collagen type III was not greatly changed by incubation with IL-1, and a better correlation was generally observed with the amount of type III collagen found in the medium. These data suggest that an additional control mechanism at translational or post-translational level must exist, counterbalancing the stimulatory effect of IL-1 on collagen mRNA transcription. It is likely that IL-1 could modulate the production of collagen in synovial cells by an interplay of different mechanisms, some of them limiting the effect of primary elevation of the steady-state mRNA level.

Achondrogenesis type 1B is an autosomal recessive, lethal chondrodysplasia caused by mutations in the gene encoding a sulfate/chloride antiporter of the cell membrane (Superti-Furga, A., Hästbacka, J., Wilcox, W. R., Cohn, D. H., van der Harten, J. J., Rossi, A., Blau, N., Rimoin, D. L., Steinmann, B., Lander, E. S., and Gitzelmann, R.(1996) Nat. Genet. 12, 100-102). To ascertain the consequences of the sulfate transport defect on proteoglycan synthesis, we studied the structure and sulfation of proteoglycans in cartilage tissue and in fibroblast and chondrocyte cultures from a fetus with achondrogenesis 1B. Proteoglycans extracted from epiphyseal cartilage and separated on agarose gels migrated more slowly than controls and stained poorly with alcian blue. The patient's cultured cells showed reduced incorporation of [35S]sulfate relative to [3H]glucosamine, impaired uptake of sulfate, and higher resistance to chromate toxicity compared to control cells. Epiphyseal chondrocytes cultured in alginate beads synthesized proteoglycans of normal molecular size as judged by gel filtration chromatography, but undersulfated as judged by ion exchange chromatography and by the amount of nonsulfated disaccharide. High performance liquid chromatography analysis of chondroitinase-digested proteoglycans showed that sulfated disaccharides were present, although in reduced amounts, indicating that at least in vitro, other sources of sulfate can partially compensate for sulfate deficiency. A t1475c transition causing a L483P substitution in the eleventh transmembrane domain of the sulfate/chloride antiporter was present on both alleles in the patient who was the product of a consanguineous marriage. The results indicate that the defect of sulfate transport is expressed in both chondrocytes and fibroblasts and results in the synthesis of proteoglycans bearing glycosaminoglycan chains which are poorly sulfated but of normal length.

Thanatophoric dysplasia (TD) is a lethal skeletal disorder caused by recurrent mutations in the fibroblast growth factor receptor 3 (FGFR 3) gene. The mitogenic response of fetal TD I chondrocytes in primary cultures upon stimulation by either FGF 2 or FGF 9 did not significantly differ from controls. Although the levels of FGFR 3 mRNAs in cultured TD chondrocytes were similar to controls, an abundant immunoreactive material was observed at the perinuclear level using an anti-FGFR 3 antibody in TD cells. Transduction signaling via the mitogen-activated protein kinase pathway was assessed by measuring extracellular signal-regulated kinase activity (ERK 1 and ERK 2). Early ERKs activation following FGF 9 supplementation was observed in TD chondrocytes (2 min) as compared with controls (5 min) but no signal was detected in the absence of ligand. By contrast ligand-independent activation of the STAT signaling pathway was demonstrated in cultured TD cells and confirmed by immunodetection of Stat 1 in the nuclei of hypertrophic TD chondrocytes. Moreover, the presence of an increased number of apoptotic chondrocytes in TD fetuses was associated with a higher expression of Bax and the simultaneous decrease of Bcl-2 levels. Taken together, these results indicate that FGFR 3 mutations in TD I fetuses do not hamper chondrocyte proliferation but rather alter their differentiation by triggering premature apoptosis through activation of the STAT signaling pathway.

Synthetic glycolipids prepared by esterification of various sugars and sorbitol, and containing various numbers of saturated or unsaturated fatty acid residues as well as bacterial lipid A and lipopolysaccharide, were tested for mitogenicity of splenic cells of Fischer rats and Swiss mice and for the augmentation of humoral immune response against sheep red blood cells in these species. Subsequently a few of the humoral immune-response-enhancing glycolipids were compared with non-enhancers in their anti-tumour activity against 13762 rat mammary carcinoma in inbred Fischer 344 rats and Ehrlich tumour in Swiss mice. They were given systemically after tumour inoculation and intratumourally in squalene and Tween emulsion after intradermal MAC tumour development. It was observed that certain structural characteristics in glycolipids with respect to the type of sugar, the type and number of fatty-acid residues were needed for their adjuvant action of the humoral arm of the immune response. Although humoral immune-response enhancers were somewhat superior to non-enhancers in their anti-tumour activity, the correlation coefficient demonstrated a lack of significant concordance. It is concluded that glycolipids selected for their ability to augment humoral immune responses against standard antigens need not be suspect as tumour-enhancers on the grounds that they would elicit blocking antibodies in vivo against tumour-associated antigens.

Two different mutations were found in two unrelated probands with lethal chondrodysplasias, one with achondrogenesis type II and the other with the less severe phenotype of hypochondrogenesis. The mutations in the COL2A1 gene were identified by denaturing gradient gel electrophoresis analysis of genomic DNA followed by dideoxynucleotide sequencing and restriction site analysis. The proband with achondrogenesis type II had a heterozygous single-base mutation that substituted aspartate for glycine at position 310 of the alpha 1(II) chain of type II procollagen. The proband with hypochondrogenesis had a heterozygous single-base mutation that substituted serine for glycine at position 805. Type II collagen extracted from cartilage from the probands demonstrated the presence of type I collagen and a delayed electrophoretic mobility, indicating post-translational overmodifications. Analysis of CNBr peptides showed that, in proband 1, the entire peptides were overmodified. Examination of chondrocytes cultured in agarose or alginate indicated that there was a delayed secretion of type II procollagen. In addition, type II collagen synthesized by cartilage fragments from the probands demonstrated a decreased thermal stability. The melting temperature of the type II collagen containing the aspartate-for-glycine substitution was reduced by 4 degrees C, and that of the collagen containing the serine-for-glycine substitution was reduced by 2 degrees C. Electron microscopy of the extracellular matrix from the chondrocyte cultures showed a decreased density of matrix and the presence of unusually short and thin fibrils. Our results indicate that glycine substitutions in the N-terminal region of the type II collagen molecule can produce more severe phenotypes than mutations in the C-terminal region. The aspartate-for-glycine substitution at position 310, which was associated with defective secretion and a probable increased degradation of collagen, is the most destabilizing mutation yet reported in type II procollagen.

We have used the polymerase chain reaction and single strand conformation polymorphism (SSCP) methods to analyse the COL10A1 gene, which encodes collagen type X, in DNA samples from patients with metaphyseal dysplasia type Schmid (SMCD) and other related forms of metaphyseal dysplasia. Five cases of SMCD were sporadic and three others were familial. Abnormal SSCP profiles were observed in six instances. In two families, the altered pattern segregated with the phenotype. The heterozygous mutations corresponded to a glycine substitution by glutamic acid at position 595 and to an asparagine substitution by lysine at position 617. In one sporadic case, the sequence studies demonstrated that the individual was heterozygous for a single base deletion (del T 1908) that produced a premature stop codon. Three additional mutations were single base substitutions that affected highly conserved residues at positions 597, 644 and 648. In two additional individuals with SMCD, in two patients with unclassifiable forms of metaphyseal dysplasia, and in one family with epiphyso-metaphyseal dysplasia, SSCP analysis detected neutral polymorphisms in the entire coding sequence of the gene but no mutations. Our results demonstrate that mutations in the carboxy-terminal region of collagen X are specific for the SMCD phenotype. Mutations appear to be clustered into three small subdomains: one of them is rich an aromatic residues, the second includes the putative N-linked oligosaccharide attachment site and the third contains mostly hydrophilic residues. The absence of clinical variability between patients carrying heterozygous single base substitutions or small deletions suggests that, in both instances, the mutant collagen chains either fail to be incorporated into stable trimers or disturb type X collagen assembly.

A three generation family with Stickler syndrome is reported. Affected patients exhibited myopia with frequent retinal detachment or glaucoma. Most of them had characteristic facial dysmorphism, the Pierre-Robin sequence being observed in four individuals. Neonatal radiological signs of the Weissenbacher-Zweymüller syndrome were also noticed but early arthopathy was not reported in adults. Restriction fragment length polymorphism studies with the type II collagen gene (COL2A1) showed a recombination event between the disease locus and COL2A1, thus excluding collagen type II as the candidate gene. Although the calculation of the likelihood of genetic heterogeneity versus homogeneity based on 10 families was not statistically significant, we suggest that a second locus is probably involved in this highly variable syndrome.

The characterization of the fibrillar collagens and the cellular control of their spatial deposition were studied in fish scales using immunofluorescence, electron microscopy, electrophoretic and HPLC analyses, immunoprecipitation and hybridization with cDNA probes. This study was carried out on undisturbed and regenerating scales in situ and in organ and cell cultures from regenerating scales. The hyposquamal scleroblasts forming a pseudoepithelium show an apico-basal polarization and synthesize thick collagen fibrils (100 nm) organized in a plywood pattern as long as the integrity of the cell-cell and cell-collagenous matrix contacts are preserved. In culture, scleroblasts become fibroblast-like and produce an unordered meshwork of thin collagen fibrils (30 nm). Comparison of the synthesized collagens in culture with those extracted from the scales indicates that culture conditions modify fibrillogenesis but do not change the expression of fibrillar collagen genes. Type I collagen, the prédominent component, is associated with the minor type V collagen. Type HI collagen was not present. In type I collagen, a third chain, α3 chain, was identified. The ratio between the 3 chains suggests the coexistence of two heterotrimers (αl(I))2 α2(1) and αl(I) α2(1) α3(I). Analysis by HPLC and electrophoresis of the cyanogen bromide-derived peptides obtained from the purified α3 chain support the hypothesis that αl(I) and α3(I) chains are encoded by two different genes. The presence of the two types of heterotrimers in vivo as well as in vitro could correspond to an innate property of the goldfish scleroblasts. Despite the fact that teleost cyanogen bromide-derived peptides differ from those of higher vertebrates, homologies with the mammalian collagen genes (human, for example) are sufficient to allow the detection of mRNA transcripts for al(I), α2(I) and α2(V) from confluent scleroblast cultures with human probes.

Human fibroblasts with mutated type 1 collagen have marked defective adhesive capacities on exogenous type I collagen and exogenous fibronectin in comparison to normal fibroblasts. This defective cell adhesion could be partly explained by the decreased level of cell surface receptors of the β1-integrin family, i.e., the α2 integrin subunit for type I collagen and the α5 integrin subunit for fibronectin, observed in pathological fibroblasts. However, it appeared that the presence of altered collagen interfered both with fibronectin biosynthesis and with its surface expression. Using a binding assay on immobilized fibronectin, we demonstrated that the mutated collagen had a weaker binding to fibronectin. In addition, the pathological fibroblasts plated on a mixture of normal exogenous type I collagen and fibronectin exhibited the same maximal level of adhesion as control fibroblasts. These results indicate that fibroblasts with the mutated collagen exhibit a decreased binding to normal fibronectin, a modification of synthesis and surface expression of fibronectin, and, finally, altered adhesive capacities.

We have used the three-dimensional culture system in alginate beads to redifferentiate human articular chondrocytes which were first expanded on a plastic support. After 15 days in alginate beads, electron microscopy showed that cells had synthesized an extracellular matrix containing collagen fibrils. Electrophoreticanalysis of proline-labeled cells demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (proα₁II, p₀α₁II, and p_nα₁II). After pepsin digestion a small amount of collagen type XI was also detected. These results were confirmed by Northern blot analysis of total RNAs. Hybridization with collagen cDNA probes coding for the α₁(II) and α₁(I) chains of collagen types II and I showed that chondrocytes cultured in alginate expressed mainly α₁(II) mRNA, whereas α₁(I) mRNA transcripts were almost undetectable. Such a result was observed even after several passages on plastic flasks, suggesting that dedifferentiated cells were able to revert to a chondrocytic phenotype in this three-dimensional system. However, SV40-transformed chondrocytes were not able to redifferentiate in alginate as no α₁(II) mRNAs were detected. Total RNA was converted into cDNA by reverse transcription and amplified by polymerase chain reaction. This technique was employed to amplify mRNAs specific for collagen type II and type X and the large aggregating proteoglycan aggrecan. Two transcripts resulting from an alternative splicing of the complement regulatory protein (CRP)-like domain of aggrecan were originally identified in chondrocytes in monolayers. Like intact cartilage, chondrocytes in alginate expressed only the larger transcript with the CRP domain, whereas the two transcripts were equally expressed in SV40-transformed chondrocytes. Thus, the alginate system appears to represent a relevant model for the redifferentiation of human chondrocytes, especially when only a small cartilage biopsy is available, and could prove useful for pulse-chase studies of patients with skeletal chondrodysplasias. However it was unable to restore the chondrocytic phenotype in virally transformed cells. Copyright 1994, 1999 Academic Press

No abstract.

Chondrocytes enzymaticaly extracted from the resting zone of GP cartilage from prepubertal rabbits, were incubated in 10% FCS-Dulbecco's medium for 48 h.They were then grown for 20 days in serum-free defined medium containing 100 ng/ml FGF as unique growth factor. Chondrocytes differentiation was observed at day 10 to 12 when cells were dividing as multilayered colonies and at day 15 to 20 when cells hypertrophied and developped important Golgi apparatus and extracellular matrix as observed by electron microscopy. At both stages of differentiation, Type II collagen was representing 95% of the newly synthesized collagenic proteins as measured after incorporation of [3H]-Pro during 20 h. The specific binding of 125I-IGFI (gift of Dr L. Van den Brande) was studied separately on dividing and hypertrophic Chondrocytes. The 50% displacement was the same at both stages of differentiation at 4.5×10-9concentration of unlabelled IGFI. The presence of several types of receptors was suggested by Scatchard analyses with similar high affinity constant (Ka=3.3×109M-1) in dividing and hypertrophic Chondrocytes. By contrast in dividing cells the maximal specific binding observed at 15°C and pH 8 was 3 times higher than in hypertrophic cells (6% and 2% respectively) and the number of specific binding sites was 1.5 times higher than in hypertrophic cells. These results suggest that GP resting Chondrocytes can divide and hypertrophy in culture while they synthesize Type II collagen and possess a number of IGFI receptors which was decreasing during the differentiation process.