Search

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias resulting in short-limbed dwarfism, joint pain, and stiffness. PSACH and the largest proportion of autosomal dominant MED (AD-MED) results from mutations in cartilage oligomeric matrix protein (COMP); however, AD-MED is genetically heterogenous and can also result from mutations in matrilin-3 (MATN3) and type IX collagen (COL9A1, COL9A2, and COL9A3). In contrast, autosomal recessive MED (rMED) appears to result exclusively from mutations in sulphate transporter solute carrier family 26 (SLC26A2). The diagnosis of PSACH and MED can be difficult for the nonexpert due to various complications and similarities with other related diseases and often mutation analysis is requested to either confirm or exclude the diagnosis. Since 2003, the European Skeletal Dysplasia Network (ESDN) has used an on-line review system to efficiently diagnose cases referred to the network prior to mutation analysis. In this study, we present the molecular findings in 130 patients referred to ESDN, which includes the identification of novel and recurrent mutations in over 100 patients. Furthermore, this study provides the first indication of the relative contribution of each gene and confirms that they account for the majority of PSACH and MED., (© 2011 Wiley Periodicals, Inc.)

Multiple epiphyseal dysplasia (MED) is a clinically variable and genetically heterogeneous disease that is characterized by mild short stature and early onset osteoarthritis. Autosomal dominant forms are caused by mutations in the genes that encode type IX collagen, cartilage oligomeric matrix protein, and matrilin-3: COL9A1, COL9A2, COL9A3, COMP, and MATN3, respectively. Splicing mutations have been identified in all three genes encoding type IX collagen and are restricted to specific exons encoding an equivalent region of the COL3 domain in all three alpha(IX) chains. MED has been associated with mild myopathy in some families, in particular one family with a COL9A3 mutation and two families with C-terminal COMP mutations. In this study we have identified COL9A2 mutations in two families with MED that also have osteochondritis dissecans and mild myopathy. This study therefore extends the range of gene-mutations that can cause MED-related myopathy. (c) 2010 Wiley-Liss, Inc.

Multiple epiphyseal dysplasia (MED) is a clinically variable and genetically heterogeneous chondrodysplasia characterized by mild to moderate short stature and early onset osteoarthritis. Some forms of MED result from mutations in the gene encoding the cartilage structural protein matrilin-3 (MATN3). The majority of MATN3 mutations affect conserved residues within the beta-sheet of the single A-domain of matrilin-3. These mutations cause the protein to misfold and prevent its secretion from the rER, both in vitro and in vivo. More recently a single mutation (p.Phe105Ser) has been identified within the alpha1-helix of the A-domain, but its affect on the structure and/or function of matrilin-3 is unknown. In this paper we describe the characterization of two additional alpha-helical mutations (p.Ala173Asp and p.Lys231Asn) and show that both p.Phe105Ser and pAla173Asp prevent the secretion of A-domain in vitro. In contrast, p.Lys231Asn does not prevent the secretion of matrilin-3 A-domain, nor does it disrupt the structure of this domain or inhibit its binding to type II or type IX collagen. Therefore, despite extensive biochemical analysis the disease mechanism of p.Lys231Asn remains unresolved and care should be taken in counseling for these types of mutation in MATN3., ((c) 2007 Wiley-Liss, Inc.)

Pseudoachondroplasia (PSACH) is an autosomal-dominant osteochondrodysplasia due to mutations in the gene encoding cartilage oligomeric matrix protein (COMP). Clinical diagnosis of PSACH is based primarily on family history, physical examination, and radiographic evaluation, and is sometimes extremely difficult, particularly in adult patients. Genetic diagnosis based on DNA sequencing, on the other hand, can be expensive, time-consuming, and intensive because COMP mutations may be scattered throughout the gene. However, there is evidence that decreased plasma COMP concentration may serve as a diagnostic marker in PSACH, particularly in adult patients. Here, we report the serum and/or plasma COMP concentration-based differential diagnosis of a family with affected adult members. The mean serum and/or plasma COMP concentrations of the three affected family members alive (0.69+/-0.15 and/or 0.81+/-0.08 microg/ml, respectively) were significantly lower than those of an age-compatible control group of 21 adults (1.52+/-0.37 and/or 1.37+/-0.36 microg/ml, respectively; P<0.0001). Bidirectional fluorescent DNA sequencing-based genetic diagnosis of these patients revealed a heterozygous mutation for the nucleotide change 1532A>G in exon 14 of the COMP gene, resulting in a substitution of amino acid 511 from aspartic acid to glycine in COMP. Thus, serum and/or plasma COMP concentration may be suggested as an additional diagnostic marker to aid clinical and radiographic findings in suspected cases of PSACH.

Skeletal dysplasias are difficult to diagnose for the nonexpert. In a previous study of patients with multiple epiphyseal dysplasia (MED), we identified cartilage oligomeric matrix protein (COMP) mutations in only 36% of cases and suspected that the low-mutation detection rate was partially due to misdiagnosis. We therefore instituted a clinical-radiographic review system, whereby all cases were evaluated by a panel of skeletal dysplasia experts (European Skeletal Dysplasia Network). Only those patients in whom the diagnosis of MED was confirmed by the panel were screened for mutations. Under this regimen the mutation detection rate increased to 81%. When clinical-radiological diagnostic criteria were relaxed the mutation rate dropped to 67%. We conclude that expert clinical-radiological review can significantly enhance mutation detection rates and should be part of any diagnostic mutation screening protocol for skeletal dysplasias.

Congenital cholesteatoma has a different pathophysiology than acquired cholesteatoma in that these patients rarely have eustachian tube dysfunction. This likely accounts for their reasonable preoperative hearing and their lack of complications or recurrences postoperatively. The most important factor is early detection. Treatment remains surgical removal.

Multiple epiphyseal dysplasia (MED) is a relatively common skeletal dysplasia that can present in childhood with a variable phenotype of short stature and pain and stiffness in the large joints, and often progresses to early-onset osteoarthritis in adulthood. Mutations in the matrilin-3 gene (MATN3) have recently been shown to underlie some forms of autosomal dominant MED. To date all MED mutations in matrilin-3 cluster in the single A-domain, suggesting that they may disrupt the structure and/or function of this important domain. To determine the effects of MATN3 mutations on the structure and function of matrilin-3 we expressed both normal and mutant matrilin-3 in mammalian cells. Wild-type (wt) matrilin-3 was efficiently secreted into conditioned medium, whereas mutant matrilin-3 was retained and accumulated within the cell. Furthermore, when the mutant A-domains were examined individually, they existed primarily in an unfolded conformation. Co-immunoprecipitation experiments demonstrated that the mutant A-domains were specifically associated with ERp72, a chaperone protein known to be involved in mediating disulfide bond formation. Light microscopy of cartilage from an MED patient with a MATN3 mutation showed the presence of intracellular material within the chondrocytes, whilst the overall matrix appeared normal. On electron micrographs, the inclusions noted at the light microscopy level appeared to be dilated cisternae of rough endoplasmic reticulum and immunohistochemical analysis confirmed that the retained protein was matrilin-3. In summary, the data presented in this paper suggest that MED caused by MATN3 mutations is the result of an intracellular retention of the mutant protein., (Copyright 2005 Wiley-Liss, Inc.)

Pseudoachondroplasia (PSACH) and some forms of multiple epiphyseal dysplasia (MED) result from mutations in the gene encoding cartilage oligomeric matrix protein (COMP). COMP is a large pentameric glycoprotein found predominantly in the extracellular matrix of cartilage, tendon, and ligament. As a modular protein, it is composed of a coiled-coil domain, four type II (T2) repeats, eight type III (T3) repeats, and a large globular C-terminal domain (CTD). The majority (>85%) of COMP mutations causing PSACH or MED are found in the exons encoding the T3 repeats, and the disease mechanism has been characterised in detail. Much less is known about disease-causing mutations in the CTD; in 10 years only seven mutations have been identified. In this study, we describe eight novel and two recurrent mutations that we have recently identified in patients with PSACH or MED. Interestingly, these mutations result in a spectrum of disease, ranging from mild MED to severe PSACH. Mapping of all known COMP CTD mutations on a three-dimensional model of the C-terminal domain shows that the CTD mutations cluster in two distinct regions. These regions are probably important in stabilising the T3-CTD structure and mediating intra- or intermolecular interactions.

Multiple epiphyseal dysplasia (MED) is a clinically and genetically heterogeneous chondrodysplasia. Mutations in six genes (COMP, COL9A1, COL9A2, COL9A3, MATN3 and DTDST) have been reported, but the genotype-phenotype correlations and the proportions of cases due to mutations in these genes are still not well characterized. We performed a clinical, radiological and molecular analysis of known MED genes on 29 consecutive MED patients. The mutation analysis resulted in identification of the DTDST mutation in four patients (14%), the COMP mutation in three (10%) and the MATN3 mutation in three (10%). Thus, a disease-causing mutation was identified in 10 patients altogether (34%). The phenotypic features observed in the patients with mutations were in accordance with previously described phenotypes, but two new distinct phenotypic entities were identified in patients in whom no mutation was found. One of them was characterized by severe, early-onset dysplasia of the proximal femurs with almost complete absence of the secondary ossification centres and abnormal development of the femoral necks. The other phenotype was characterized by 'mini-epiphyses', resulting in severe dysplasia of the proximal femoral heads. The findings suggest that mutations in the known genes are not the major cause of MED and are responsible for less than half of the cases. The existence of additional MED loci is supported by the exclusion of known loci by mutation analysis and finding of specific subgroups among these patients.

Multiple epiphyseal dysplasia (MED) is characterized by pain and stiffness in joints and delayed and irregular ossification of epiphyses. Causative mutations have been recognized in six different genes. We have identified disease-causing mutations in the gene encoding matrilin-3, an extracellular matrix protein, in seven families with autosomal dominant MED. Review of the clinical and radiographic features in 12 of the affected family members shows a uniform pattern of skeletal anomalies in all patients with considerable degree of variability in severity, both between and within families. The characteristic clinical findings are onset of symptoms in early childhood with predominance of knee and hip related complaints, normal stature, and early-onset osteoarthritis. Radiographs show small and irregular epiphyses and mild metaphyseal irregularities and striations, especially at the knees and hips and mild spinal changes. Despite overlap, both clinically and radiographically, with other forms of MED, the described features may help to differentiate this particular form from other entities within the MED spectrum., (Copyright 2003 Wiley-Liss, Inc.)

The observation that TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine family, induces apoptosis in a number of different tumor cell types led us to compare the tumoricidal effects of TRAIL to those of other TNF family molecules on human melanoma cells. We found that a high proportion of the melanoma cell lines tested were killed by TRAIL, whereas all the melanoma lines were resistant to the other TNF family cytokines tested. TRAIL-induced death was characterized by caspase activation and cellular protein cleavage within minutes of TRAIL addition, and death could be completely inhibited by the caspase inhibitors Ile-Glu-Thr-Asp (IETD) and Val-Ala-Asp (VAD), indicating the presence of a TRAIL receptor signaling pathway similar to that identified for Fas and TNF receptors. Specific TRAIL receptor expression was determined by RT-PCR, and the presence of mRNA encoding the "protective" TRAIL receptors did not correspond to resistance or sensitivity to TRAIL-induced apoptosis. Addition of protein synthesis inhibitors to TRAIL-resistant melanomas rendered them sensitive to TRAIL, indicating that the presence or the absence of intracellular apoptosis inhibitors may mediate resistance or sensitivity to TRAIL-mediated apoptosis. Expression of one such inhibitor, FLICE-inhibitory protein (FLIP), was highest in the TRAIL-resistant melanomas, while being low or undetectable in the TRAIL-sensitive melanomas. Furthermore, addition of actinomycin D to TRAIL-resistant melanomas resulted in decreased intracellular concentrations of FLIP, which correlated with their acquisition of TRAIL sensitivity. Collectively, our results indicate that TRAIL-induced apoptosis occurs through a caspase signaling cascade and that resistance is controlled by intracellular regulators of apoptosis.

The effects of several different schedules of primary reinforcement were compared in a picture-naming task with retarded children. In Experiment I, number of correct responses and learning rate were higher under fixed-ratio schedules than under continuous reinforcement. In Experiment II, number of correct responses and learning rate tended to be greater under in intermediate than under low or high fixed-ratio schedules. In Experiment III, number of correct responses was higher under interlocking schedules, in which the response requirement increased with time following the previous reinforcement, than under comparable fixed-ratio schedules. Learning rates were generally low and, perhaps because of this, not very different under the two types of schedules in this experiment. Accuracy (i.e., proportion of trials on which correct responses occurred) was typically high and insensitive to variations in schedule and schedule parameter throughout each experiment.

Human pancreas is the source of an alanine aminopeptidase (HPAA) that is unique to pancreas and is readily distinguishable from the liver, kidney, and duodenal alanine aminopeptidases. Each of these three aminopeptidases appears in small quantities in blood and therefore may consitute tissue/organ specific marker enzymes. In this study alanine aminopeptidase from pancreas has been purified. Pancreas alanine aminopeptidase was resolved upon purification with ion exchange chromatography into three isoenzymes. Gel filtration chromatography of these isoenzymes indicates that their molecular weights are near 235 000 daltons. The isoenzymes contain a firmly bound divalent cation that could be removed with EDTA only at temperatures above 40 degrees C (at which the enzymes were stable) and below 52 degrees C (at which thermal denaturation begins to take place). Treatment with EDTA at 4 degrees C yields fully active enzymes, which, however may be stimulated approximately 200% by the addition of Co2+ at 10(-4) mol/l. This cobalt stimulation is easily reversed by dialysis of the stimulated isoenzymes against deionized water. The pancreas alanine aminopeptidases were not inhibited by tosyl-phenylalanine chloromethylketone or by tosylleucine chloromethylketone, whereas phenylalanine chloromethylketone was inhibitory. The Km values for methionyl-, arginyl-, leucyl-, alanyl-, and isoleucyl-beta-naphthylamide for each isoenzyme are statistically identical and the average values are 0.36, 0.60, 0.69, 1.25, and 1.29 X 10(-4) mol/l, respectively. The kcat values are 2.50, 1.58, 0.82, 0.38, and 0.19 X 10(4) sec-1, respectively.