Molecular analysis of mucopolysaccharidosis type VI in Poland, Belarus, Lithuania and Estonia

Mucopolysaccharidosis VI (MPS VI) is a rare autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Over 130 ARSB gene mutations have been identified thus far and most mutations are unique to individual families. We aimed to analyze the spectrum of mutations in the ARSB gene responsible for the disorder in Poland, Belarus and Baltic States. Twenty one families with MPS VI patients, in whom diagnosis was confirmed biochemically and enzymatically, were studied. Direct sequencing of patient genomic DNA was used to identify ARSB mutations. In total, fourteen different disease-causing mutations were found. Three novel mutations included insertion c.375_376insT, a missense mutation c.499G>A (p.G167R) and deletion/insertion c.750_754delinsCCTGAAGTCAAG. We also report 11 previously described mutations (p.A33V, p.W57C, p.Q88X, p.T92K, p.Q97X, p.R152W, p.R160Q, p.R160X, p.Y210C, p.Y266S, p.G302R). The mutation p.R152W was present at a high prevalence of 50% (21/42) the mutated alleles in this group of patients. High prevalence of p.R152W mutation in Poland, Belarus and Baltic States indicates a possible founder effect and suggests that screening for this mutation may be appropriate in MPS VI patients from this region. Our study has also provided evidence to support genotype-phenotype correlation.     

Toll-Like Receptors Expression and NF-κB Activation in Peritoneal Leukocytes in Morphine-Mediated Impairment of Zymosan-Induced Peritonitis in Swiss Mice

Zymosan-induced peritonitis represents a well-described model of acute inflammation. The binding of zymosan with its specific Toll-like receptors (TLR2 and TLR6) on leukocytes initiates activation and phosphorylation of nuclear factor (NF)-κB, which leads to accumulation of NF-κB p65 subunits in the nucleus and subsequently up-regulation of the proinflammatory cytokine genes expression. Intraperitoneal co-administration of zymosan and morphine significantly inhibits peritonitis in several strains of mice by decreasing the influx of exudatory cells; however, mechanisms of this action still remain unclear. We aimed to verify the effects of morphine on NF-κB and TLRs expression at messenger RNA and protein levels during the early stages of zymosan-induced peritonitis. Peritonitis was induced by a single injection of zymosan A or zymosan supplemented with morphine in Swiss mice. At selected time points, after stimulation, peritoneal leukocytes were harvested. The TLRs and NF-κB expression was assessed by real-time PCR and flow cytometry. In comparison with the mice injected with zymosan only, morphine co-injection significantly decreased the expression of phospho-NF-κB and TLR2 in all investigated immunocompetent cells as well as up-regulated the levels of nitric oxide (NO) in peritoneal fluid. Moreover, supplementation of zymosan with morphine altered the TLR, NF-κB and some proinflammatory cytokines (keratinocyte-derived chemokine, tumor necrosis factor-α) gene expression during ongoing inflammation. We may postulate that after morphine stimulation peritoneal leukocytes recognize less effectively zymosan antigens because of impaired TLRs expression. The lower TLR expression attenuates TLR-mediated signal transduction, which prevents NF-κB activation. Additionally, during zymosan-induced peritonitis, morphine may modulate the NF-κB expression, at least partially, by an up-regulated release of NO, as suggested by others.     

Changed phagocytic activity and pattern of Fcγ and complement receptors on blood monocytes in sarcoidosis

We have recently revealed that mycobacterial heat shock proteins (Mtb-hsp), involved in forming of immune complexes (CIs), can induce immune response in sarcoidosis (SA). The complexemia may result from inappropriate phagocytosis and clearance of CIs by monocytes with following persistent antigenemia and granuloma formation. Because an aberrant expression of receptors for Fc fragment of immunoglobulin G (FcγR) and complement receptors (CR) on monocytes can be involved in this process, we have evaluated the expression of FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and CR1 (CD35), CR3 (CD11b), CR4 (CD11c) receptors on blood CD14(+) monocytes and its phagocytic activity in 24 patients with SA and 20 healthy volunteers using flow cytometry. We found significantly increased expression of all examined FcγR and decreased expression of CD35 and CD11c on CD14(+) monocytes in SA patients vs controls. Significantly increased percentage of CD14(+)CD16(+)CD35(-), CD14(+)CD64(+)CD35(+), CD14(+)CD64(+)CD11b(+), CD14(+)CD64(+)CD11c(+) and decreased of CD14(+)CD32(-)CD35(+), CD14(+)CD32(-)CD11b(+), CD14(+)CD32(-)CD11c(+) monocytes' phenotypes was revealed in SA. The total number and percentage of phagocyting monocytes was significantly increased in SA as compared with controls. In conclusion, altered expression of FcγR and CR on CD14(+) monocytes and its increased phagocytic activity may be responsible for high antigen load, persistent antigenemia and immunocomplexemia in SA patients.     

The Localization of the Supraorbital Notch or Foramen is Crucial for Headache and Supraorbital Neuralgia Avoiding and Treatment

The aim of this study was to provide the morphological and morphometric data of the supraorbital foramina or notches related to sex, side, and the climatic conditions where the population lived. It was hypothesized that the distribution of the occurrence and location of these openings depends on climatic conditions in which the population lived. Orbits from 866 dried skulls obtained from three climatic regions: warm, temperate, and cold were examined. The examination concentrated on the configuration (notch/foramen) and on the distances to the reference points: nasion, frontomalare orbitale, infraorbital foramen and the superior orbital rim. In 14.3% of cases a smooth supraorbital rim was observed while different variants of the structures were observed in 85.7% of the cases. In cold climatic conditions, supraorbital foramina were found in the highest frequency (35.4%). In warm and temperate climates, the observed frequencies of supraorbital foramen were the lowest (18.8% and 19.9%, respectively). Frequency of supraorbital notches was the lowest of those skulls from a cold climate (44.0%) and the highest in those from a warm climate (59.0%). These results support the hypothesis that the occurrence of the supraorbital notches is greater in populations from warm compared with cold regions. This would provide a greater exit route for the neurovascular bundle and this may be related to the thermoregulatory processes in the supraorbital region. Furthermore, knowledge of precise locations of supraorbital structures is important when a supraorbital nerve block is given, for example, in the treatment of migraine headaches. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Evidence for HNRNPH1 being another gene for Bain type syndromic mental retardation

The HNRNPH2‐associated disease (mental retardation, X‐linked, syndromic, Bain type [MRXSB, MIM #300986]) is caused by de novo mutations in the X‐linked HNRNPH2 gene. MRXSB has been described in six female patients with dysmorphy, developmental delay, intellectual disability, autism, hypotonia and seizures. The reported HNRNPH2 mutations were clustered in the small domain encoding nuclear localization signal; in particular, the p.Arg206Trp was found in four independent de novo events. HNRNPH1 is a conserved autosomal paralogue of HNRNPH2 with a similar function in regulation of pre‐mRNAs splicing but so far it has not been associated with human disease. We describe a boy with a disease similar to MRXSB in whom a novel de novo mutation c.616C>T (p.Arg206Trp) in HNRNPH1 was found (ie, the exact paralogue of the recurrent HNRNPH2 mutation). We propose that defective function of HNRNPH2 and HNRNPH1 nuclear localization signal has similar clinical consequences. An important difference between the two diseases is that the HNRNPH1‐associated syndrome may occur in boys (as in the case of our proband) which is well explained by the autosomal (chr5q35.3) rather than X‐linked localization of the HNRNPH2 gene.     

A sustainable solution for the activities of the European network for surveillance of congenital anomalies: EUROCAT as part of the EU Platform on Rare Diseases Registration

The European Commission through its Directorates-General Joint Research Centre (DG JRC) and Health and Food Safety (DG SANTE) is developing the European Platform on Rare Diseases Registration (EU RD Platform) with the objective to set European-level standards for data collection and data sharing. In the field of rare diseases the EU RD Platform will be a source of information on available rare disease patient data with large transnational European coverage. One main function of the EU RD Platform is to enable interoperability for the >600 existing RD registries in Europe. The second function is to offer a sustainable solution for two large European surveillance networks: European Surveillance of Congenital Anomalies (EUROCAT) and Surveillance of Cerebral Palsy in Europe (SCPE).EUROCAT is European network of population-based registries for the epidemiological surveillance of congenital anomalies. It covers about one third of the European birth population. The Central Database contains about 800,000 cases with congenital anomalies among livebirths, stillbirths and terminations of pregnancy, reported using the same standardised classification and coding. These high quality data enables epidemiological surveillance of congenital anomalies, which includes estimating prevalence, prenatal diagnosis and perinatal mortality rates and the detection of teratogenic exposures among others. The network also develops recommendations for primary prevention in the Rare Diseases National Plans for medicinal drugs, food/nutrition, lifestyle, health services, and environmental pollution.The network has received the European Commission's support since its inception. In order to offer a sustainable solution for the continuation of EUROCAT activities, it was agreed that EUROCAT would become part of the EU RD Platform. In 2015, the European level-coordination activities and the Central Database were transferred to the DG JRC, where the JRC-EUROCAT Central Registry is now located. This paper describes the functioning of EUROCAT in the new setting, and gives an overview of the activities and the organisation of the JRC-EUROCAT Central Registry.     

GM1 gangliosidosis and Morquio B disease: expression analysis of missense mutations affecting the catalytic site of acid β‐galactosidase

Alterations in GLB1, the gene coding for acid β‐D‐galactosidase (β‐Gal), can result in GM1 gangliosidosis (GM1), a neurodegenerative disorder, or in Morquio B disease (MBD), a phenotype with dysostosis multiplex and normal central nervous system (CNS) function. While most MBD patients carry a common allele, c.817TG>CT (p.W273L), only few of the >100 mutations known in GM1 can be related to a certain phenotype. In 25 multiethnic patients with GM1 or MBD, 11 missense mutations were found as well as one novel insertion and a transversion causing aberrant gene products. Except c.602G>A (p.R201H) and two novel alleles, c.592G>T (p.D198Y) and c.1189C>G (p.P397A), all mutants resulted in significantly reduced β‐Gal activities (<10% of normal) upon expression in COS‐1 cells. Although c.997T>C (p.Y333H) expressed 3% of normal activity, the mutant protein was localized in the lysosomal‐endosomal compartment. A homozygous case presented with late infantile GM1, while a heterozygous, juvenile case carried p.Y333H together with p.R201H. This allele, recently found in homozygous MBD, gives rise to rough endoplasmic reticulum (RER)‐located β‐Gal precursors. Thus, unlike classical MBD, the phenotype of heterozygotes carrying p.R201H may rather be determined by poorly active, properly transported products of the counter allele than by the mislocalized p.R201H precursors. Hum Mutat 30, 1–8, 2009. © 2009 Wiley‐Liss, Inc.     

Altered apoptosis of inflammatory neutrophils in MMP-9-deficient mice is due to lower expression and activity of caspase-3

Matrix metalloproteinase 9 (MMP-9) is a Zn²⁺-dependent endopeptidase that degrades some of the components of basement membranes and extracellular matrix and thus participates in leukocyte infiltration during inflammation. In a model of zymosan peritonitis, neutrophil infiltration in MMP-deficient (MMP-9^−/−) mice was significantly weaker at the time of their maximal influx in wild-type mice (6 h). However, during the late stages of peritonitis (24 h) an extended accumulation of neutrophils was observed in MMP-9^−/− versus the wild-type mice. Recently, we reported that the ratio of apoptosis of inflammatory leukocytes is impaired in MMP-9^−/− mice during late peritonitis and the process depends on COX-1-driven PGE₂. Here we scrutinized the alterations in apoptotic mechanisms by comparisons between MMP-9^−/− and the wild-type mice. Altered apoptosis occurred only during late (24 h) peritonitis and concerned only neutrophils, and not macrophages, mast cells or lymphocytes. Furthermore, expression and activity of caspases was altered in MMP-9^−/− animals, delayed for caspase-8 and -9, and decreased in the case of caspase-3. Also the expression of Bax/Bcl-2 proteins was changed in MMP-9^−/− mice. These changes, and in particular the impaired neutrophil apoptosis and weaker caspase-3 activity, were restored by the selective COX-1 inhibition. We conclude that in mice lacking MMP-9 the enhanced COX-1-PGE₂ decreases caspase-3 expression and activity leading to impaired apoptosis of inflammatory neutrophils resulting in abnormal accumulation of the cells at the inflammatory focus. The data also reinforce the notion that MMP-9 is a key enzyme in neutrophil biology.     

Constitutive membrane expression of proteinase 3 (PR3) and neutrophil activation by anti-PR3 antibodies

Antineutrophil cytoplasm autoantibodies with specificity for proteinase 3 (PR3) are thought to play a major role in the pathogenesis of Wegener's granulomatosis (WG), presumably by their potential to activate neutrophils. In patients with WG, high expression of PR3 on the surface of nonprimed neutrophils is associated with an increased incidence and rate of relapse. In this study, we analyzed the functional significance of constitutive PR3 expression for neutrophil activation as induced by anti-PR3 antibody. Therefore, primed and nonprimed neutrophils were stimulated with the monoclonal anti-PR3 antibody PR3G-3. Activation was measured as actin polymerization by the phalloidin assay as an early, detectable activation event and oxidative burst by the dihydrorhodamine assay, as a late, detectable activation event. In contrast to the oxidative burst, we found that anti-PR3 antibody-induced actin polymerization could be triggered in neutrophils without priming with tumor necrosis factor alpha (TNF-alpha). In addition, a correlation was found between the level of PR3 expression on the surface of these nonprimed neutrophils and the degree of actin polymerization. However, after priming with TNF-alpha, no correlation was found between membrane expression of PR3 and the level of actin polymerization or respiratory burst as induced by anti-PR3 antibody. These data suggest that the presence of PR3 on the surface of nonprimed neutrophils has consequences for their susceptibility to the initial activation step by anti-PR3 antibodies. These data may be relevant in view of the observed relation between membrane expression of PR3 on nonprimed neutrophils of patients with WG and their susceptibility for relapses.