Genome‐wide single nucleotide polymorphism‐based autozygosity mapping facilitates identification of mutations in consanguineous families with epidermolysis bullosa

Autozygosity mapping (AM) is a technique utilised for mapping homozygous autosomal recessive (AR) traits and facilitation of genetic diagnosis. We investigated the utility of AM for the molecular diagnosis of heterogeneous AR disorders, using epidermolysis bullosa (EB) as a paradigm. We applied this technique to a cohort of 46 distinct EB families using both short tandem repeat (STR) and genome‐wide single nucleotide polymorphism (SNP) array‐based AM to guide targeted Sanger sequencing of EB candidate genes. Initially, 39 of the 46 cases were diagnosed with homozygous mutations using this method. Independently, 26 cases, including the seven initially unresolved cases, were analysed with an EB‐targeted next‐generation sequencing (NGS) panel. NGS identified mutations in five additional cases, initially undiagnosed due to the presence of compound heterozygosity, deep intronic mutations or runs of homozygosity below the set threshold of 2 Mb, for a total yield of 44 of 46 cases (95.7%) diagnosed genetically.     

Air Pollution and the Risk of Cardiac Defects: A Population-Based Case-Control Study

Previous epidemiologic studies have assessed the role of the exposure to ambient air pollution in the development of cardiac birth defects, but they have provided somewhat inconsistent results. To assess the associations between exposure to ambient air pollutants and the risk of cardiac defects, a population-based case-control study was conducted using 1087 cases of cardiac defects and a random sample of 10,870 controls from 1,533,748 Taiwanese newborns in 2001 to 2007.Logistic regression was performed to calculate odds ratios for 10 ppb increases in O₃ and 10 μg/m³ increases in PM₁₀. In addition, we compared the risk of cardiac defects in 4 categories-high exposure (>75th percentile); medium exposure (75th to 50th percentile); low exposure (<50th–25th percentile); reference (<25th percentile) based on the distribution of each pollutant. The risks of ventricular septal defects (VSD), atrial septal defects (ASD), and patent ductus arteriosus (PDA) were associated with 10 ppb increases in O₃ exposure during the first 3 gestational months among term and preterm babies. In comparison between high PM₁₀ exposure and reference category, there were statistically significant elevations in the effect estimates of ASD for all and terms births. In addition, there was a negative or weak association between SO_2, NO₂, CO, and cardiac defects_.The study proved that exposure to outdoor air O₃ and PM₁₀ during the first trimester of gestation may increase the risk of VSD, ASD, and PDA.     

Longitudinal associations of fundamental movement skills with objectively measured physical activity and sedentariness during school transition from primary to lower secondary school

Objectives

This study aimed to investigate cross-lagged associations of leaping skill and throwing–catching skills with objectively measured moderate-to-vigorous physical activity (MVPA) and sedentary time (ST) during school transition from upper primary (Grade 6) to lower secondary school (Grade 7).

The role of nickel accumulation and epithelial cell proliferation in orthodontic treatment-induced gingival overgrowth

The aim of this study was to investigate the role of nickel in orthodontic treatment-induced gingival hyperplasia. The nickel concentration in gingival tissues with and without overgrowth, histopathology of gingival overgrowth, and epithelial cell proliferation response to different nickel concentrations were analysed. Ten patients receiving orthodontic therapy (eight females and two males, mean age 15.4 years) were included in the study. Hyperplastic and healthy gingiva samples were collected from the same patients. The amount of nickel in the gingival tissue samples was analysed using the atomic absorption spectrometry technique. The tissues removed from hyperplastic areas during gingivectomy were also used for histological analysis. To analyse the effect of nickel on epithelial cell proliferation, four different nickel concentrations (0.5, 2, 5, and 10 microg) were incubated with keratinocyte cells for 11 days. Mann-Whitney U-test, analysis of variance, and Tukey's test were used in the statistical analyses. The results did not show any difference in nickel concentration between the study and control gingiva tissue samples, but histological analysis demonstrated an increase in epithelial thickness and a significant increase (P = 0.031, 0.02, 0.02) in epithelial cell proliferation in response to low-dose nickel concentrations, with a toxic response to a higher dose. In the limitations of this study, it is plausible that the effect of a continuing low-dose nickel release to epithelium is the initiating factor of gingival overgrowth induced by orthodontic treatment.     

Modulation of host matrix metalloproteinases by bacterial virulence factors relevant in human periodontal diseases

OBJECTIVE: Bacterial pathogens involved in periodontal diseases exert a part of their destructive effect by triggering and inducing host cells to elevate their secretion of matrix metalloproteinases (MMPs). Pathogen-secreted phospholipase (PLC) is one bacterial product that may trigger this host response. The roles of exogenous PLC leading to the release, secretion and expression of MMPs by peripheral blood neutrophils (PMNs), cultured epithelial cells of human gingiva and porcine periodontal ligament were investigated. Also the activities of PLC in the diseased and healthy gingival sulcular fluid (crevicular fluid, GCF) and molecular forms of gelatin-ases present in dental plaque were investigated. MATERIALS AND METHODS GCF, salivary and dental plaque samples were analyzed for PLC and proteinase activities. The abilities of PLC to induce PMNs and oral epithelial cells to release and express their MMPs were examined by specific functional, immunological and molecular biology means. RESULTS: PMN-derived MMPs were found to predominate in periodontitis GCF and plaque, and PLC activities were higher in GCF of adult periodontitis patients than in healthy controls. Purified bacterial PLC (1 mU ml^-I) efficiently induced PMN degranulation. PLC also induced MMP expression in the cultured epithelial cells. The strongest response was seen in MMP-9 and less in MMP-2. The induction was dose-dependent in the range of 0.I-1.0 U ml^-1 PI-PLC, and quiescent cultures were more responsive than proliferating ones. PLC induction of MMPs was polar, with increased levels of MMP-9 in the apical region and increased MMP-2 levels secreted in the basal direction. Northern analysis showed a strong increase in mRNA levels of MMP-9 and a smaller increase for MMP-2 and MMP-I. In the second part of the study we investigated the molecular forms of the released MMPs during periodontitis. In bacterial plaque of periodontitis patients the MMP-9 were found to be converted into lower molecular weight forms. Isolated proteinase from Porphyromonas gingivalis (ATCC 33277) was able to convert human proMMPs to their active forms. CONCLUSION: Bacterial PLC may induce degranulation of PMN MMPs and increase MMP expression in oral epithelial cells. The released proteases can be converted into active form by the proteases of plaque bacteria. Thereby, the pathogenic oral bacteria may indirectly participate in the destruction of periodontal tissues.     

A trypsin-like protease from Bacteroides gingivalis: partial purification and characterization

Extracts of cell sonicates of Bacteroides gingivalis were shown to contain proteo-lytic enzymes capable of degrading connective tissue proteins. In this study, neutral proteolytic enzymes, i.e. collagenase and a trypsin-like protease, were isolated. The trypsin-like protease was readily separated from collagenase by affinity chromatography on Benzamidine-Sepharose. Proteases were further purified by gel filtration on Sephacryl S-200; apparent molecular weights of 35 kDa and 70 kDa were obtained for a trypsin-like protease and collagenase, respectively. Further characterization of the potent trypsin-like protease showed that the enzyme was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and benzamidine and by metalloprotease inhibitor EDTA, as well as ascorbic acid. Activation of the enzyme was observed with reducing agents and human serum. The trypsin-like protease was found to be capable of degrading native type IV collagen and denatured type I collagen but not native type I collagen. Thus, we conclude that in addition to collagenase a potent trypsin-like protease from Bacteroides gingivalis may be involved in the etiopathogenesis of periodontal disease. Since the trypsin-like protease is able to degrade the basement membrane collagen (type IV) in the presence of human serum, this enzyme may be a potent virulence factor of Bacteroides gingivalis in relation to invasiveness and connective tissue destruction.     

Hypochlorous acid induced activation of human neutrophil and gingival crevicular fluid collagenase can be inhibited by ascorbate.

Interstitial collagenase either obtained from human neutrophils by phorbol myristate acetate (PMA) induced degranulation or isolated from human gingival crevicular fluid was found to be activated by addition of an oxidative agent, hypochlorous acid (HOCl). Collagenase released by PMA stimulated neutrophils was completely in latent form but underwent partial autoactivation during 16 h incubation at 22 degrees C in the presence of soy bean trypsin inhibitor. The partial autoactivation was potentiated to complete activation of released collagenase after addition of exogenous HOCl. Ascorbate prevented this activation of neutrophil collagenase. Isolated human gingival crevicular fluid collagenase represented an apparent Mr of 70 kD in completely latent form, whereas 70/54 kD enzyme species were detected for partially autoactive form of the enzyme. Western blot analysis of gingival crevicular fluid using a polyclonal antibody raised against purified human neutrophil collagenase revealed the same 70/54 kD molecular forms of the enzyme. The latent gingival crevicular fluid collagenase was also activated by HOCl and this activation could be prevented by ascorbate. Activation of the 70 kD latent collagenase by HOCl as well as by other non-proteolytic activators such as an organomercurial compound (phenylmercuric chloride) and a gold(I) compound (gold thioglucose) was not associated with detectable changes in apparent Mr, whereas trypsin activation resulted in fragmentation of 70 kD enzyme to 54 kD species. Our results provide further evidence for the neutrophil origin of gingival crevicular fluid collagenase and suggest that, in addition to proteolytic activation, oxidative and antioxidative agents seem to be able to regulate neutrophil collagenase activity.     

Genetic heterogeneity of heritable ectopic mineralization disorders in a large international cohort

PurposeHeritable ectopic mineralization disorders comprise a group of conditions with a broad range of clinical manifestations in nonskeletal connective tissues. We report the genetic findings from a large international cohort of 478 patients afflicted with ectopic mineralization.MethodsSequence variations were identified using a next-generation sequencing panel consisting of 29 genes reported in association with ectopic mineralization. The pathogenicity of select splicing and missense variants was analyzed in experimental systems in vitro and in vivo.ResultsA total of 872 variants of unknown significance as well as likely pathogenic and pathogenic variants were disclosed in 25 genes. A total of 159 distinct variants were identified in 425 patients in ABCC6, the gene responsible for pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder. The interpretation of variant pathogenicity relying on bioinformatic predictions did not provide a consensus. Our in vitro and in vivo functional assessment of 14 ABCC6 variants highlighted this dilemma and provided unambiguous interpretations to their pathogenicity.ConclusionThe results expand the ABCC6 variant repertoire, shed new light on the genetic heterogeneity of heritable ectopic mineralization disorders, and provide evidence that functional characterization in appropriate experimental systems is necessary to determine the pathogenicity of genetic variants.     

Epitope mapping of type VII collagen. Identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa.

Epidermolysis bullosa acquisita (EBA) is an acquired blistering skin disease characterized by the presence of IgG autoantibodies that recognize type VII (anchoring fibril) collagen. In this study, we have mapped the antigenic epitopes within the type VII collagen alpha chain by Western immunoblotting analysis with sera from 19 patients with EBA, using bacterial collagenase- or pepsin-resistant portions of type VII collagen and a panel of 12 recombinant fusion proteins corresponding to approximately 80% of the primary sequence of the alpha 1 (VII) collagen polypeptide. These studies identified four major immunodominant epitopes localized within the amino-terminal, noncollagenous (NC-1) domain. In addition to EBA, sera from three patients with bullous systemic lupus erythematosus (BSLE) were tested. The pattern of epitopes recognized by these sera were similar to those noted with EBA, suggesting that the same epitopes could serve as autoantigens in both blistering conditions. In contrast, sera from healthy controls or from patients with unrelated blistering skin diseases did not react with type VII collagen epitopes. Collectively, the results indicate that the immunodominant epitopes in EBA and BSLE lie within the noncollagenous regions of type VII collagen. The precise role of the circulating autoantibodies in the pathogenesis of these blistering diseases remains to be elucidated. Conceivably, however, such antibodies could disrupt the assembly of type VII collagen into anchoring fibrils and/or interfere with their interactions with other extracellular matrix molecules within the cutaneous basement membrane zone.