Dominant or recessive mutations in the RYR1 gene causing central core myopathy in Brazilian patients.

Central Core Disease (CCD) is an inherited neuromuscular disorder characterized by the presence of cores in muscle biopsy. CCD is caused by mutations in the RYR1 gene. This gene encodes the ryanodine receptor 1, which is an intracellular calcium release channel from the sarcoplasmic reticulum to the cytosol in response to depolarization of the plasma membrane. Mutations in this gene are also associated with susceptibility to Malignant Hyperthermia (MHS). In this study, we evaluated 20 families with clinical and histological characteristics of CCD to identify primary mutations in patients, for diagnosis and genetic counseling of the families. We identified variants in the RYR1 gene in 19/20 families. The molecular pathogenicity was confirmed in 16 of them. Most of these variants (22/23) are missense and unique in the families. Two variants were recurrent in two different families. We identified six families with biallelic mutations, five compound heterozygotes with no consanguinity, and one homozygous, with consanguineous parents, resulting in 30% of cases with possible autosomal recessive inheritance. We identified seven novel variants, four of them classified as pathogenic. In one family, we identified two mutations in exon 102, segregating in cis, suggesting an additive effect of two mutations in the same allele. This work highlights the importance of using Next-Generation Sequencing technology for the molecular diagnosis of genetic diseases when a very large gene is involved, associated to a broad distribution of the mutations along it. These data also influence the prevention through adequate genetic counseling for the families and cautions against malignant hyperthermia susceptibility.

Catabolic effects of endothelial cell-derived microparticles on disc cells: Implications in intervertebral disc neovascularization and degeneration.

Neovascularization of intervertebral discs, a phenomenon considered pathological since normal discs are primarily avascular structures, occurs most frequently in annulus fibrosus (AF) of degenerated discs. Endothelial cells (ECs) are involved in this process, but the mechanism of the interaction between AF and endothelial cells is unclear. In this study, we evaluated the effects on matrix catabolic activity of AF cells by the extracellular endothelial microparticles (EMPs) and soluble protein factors (SUP fraction) produced from ECs. Passage 1 human AF cells grown in monolayer cultures were treated for 72 h with 250 µg of EMPs or SUP fraction isolated from culture of the microvascular endothelial cell line, HEMC-I. Live-cell imaging revealed uptake of EMPs by AF cells. RT-PCR analysis demonstrated increased mRNA expression of MMP-1 (50.3-fold), MMP-3 (4.5-fold) and MMP-13 (5.5-fold) in AF cell cultures treated with EMPs compared to untreated control. Western analysis also demonstrated increased MMP protein expression in EMP-treated AF cells. AF cells treated with the SUP fraction also exhibited a dramatic increase in MMP mRNA and protein expression. Increased MMP expression is primarily due to EMP or SUP stimulation of AF cells since EMPs or SUP fraction alone contained negligible amount of MMPs. Interestingly, MMP activity was elevated in AF cell cultures treated with EMPs but not with SUP. This study revealed enhanced matrix catabolism as a molecular consequence of action of ECs on AF cells via EMPs, which might be expected during neo-angiogenesis of degenerating disc. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1466-1474, 2016.

Expression of matrix factors in the process of neovascularization of intervertebral disc / Expressão de fatores da matriz durante o processo de neovascularização do disco intervertebral / Expresión de factores de la matriz en el proceso de neovascularización del disco intervertebral

To investigate the effects of proteins products of endothelial cells (ECs) on the annulus fibrosus (AF) cell metabolism in an in vitro culture.

Analisar o efeito de produtos proteicos de células endoteliais (CEs) sobre o metabolismo de células de ânulo fibroso (AF) em ambiente controlado de cultura celular in vitro.

Analizar el efecto de los productos de proteína de las células endoteliales (CEs) en el metabolismo celular del anillo fibroso (AF) en sistema in vitro de cultivo controlado.

Silent polymorphisms in the RYR1 gene do not modify the phenotype of the p.4898 I>T pathogenic mutation in central core disease: a case report.

BACKGROUND: Central core disease is a congenital myopathy, characterized by presence of central core-like areas in muscle fibers. Patients have mild or moderate weakness, hypotonia and motor developmental delay. The disease is caused by mutations in the human ryanodine receptor gene (RYR1), which encodes a calcium-release channel. Since the RYR1 gene is huge, containing 106 exons, mutation screening has been limited to three 'hot spots', with particular attention to the C-terminal region. Recent next-generation sequencing methods are now identifying multiple numbers of variants in patients, in which interpretation and phenotype prevision is difficult. CASE PRESENTATION: In a Brazilian Caucasian family, clinical, histopathological and molecular analysis identified a new case of central core disease in a 48-year female. Sanger sequencing of the C-terminal region of the RYR1 gene identified two different missense mutations: c.14256 A > C polymorphism in exon 98 and c.14693 T > C in exon 102, which have already been described as pathogenic. Trans-position of the 2 mutations was confirmed because patient's daughter, mother and sister carried only the exon 98's mutation, a synonymous variant that was subsequently found in the frequency of 013-0,05 of alleles. Further next generation sequencing study of the whole RYR1 gene in the patient revealed the presence of additional 5 common silent polymorphisms in homozygosis and 8 polymorphisms in heterozygosis. CONCLUSIONS: Considering that patient's relatives showed no pathologic phenotype, and the phenotype presented by the patient is within the range observed in other central core disease patients with the same mutation, it was concluded that the c.14256 A > C polymorphism alone is not responsible for disease, and the associated additional silent polymorphisms are not acting as modifiers of the primary pathogenic mutation in the affected patient. The case described above illustrates the present reality where new methods for wide genome screening are becoming more accessible and able to identify a great variety of mutations and polymorphisms of unknown function in patients and their families.

Hipoglicemia reacional: do diagnóstico ao tratamento / Hypoglycemia reactive: from diagnosis to treatment

A hipoglicemia reacional (HR) caracteriza-se por níveis de glicose menores que 60mgdl (3,3mmol/ml) e sintomas simpaticomiméticos observados poucas horas após a ingestão de alimentos. Sua fisiopatologia ainda não está completamente elucidade, porém muitos de seus mecanismos já são conhecidos. Com a revisão da literatura pôde-se comparar a eficiência dos diversos métodos diagnósticos e estabelecer tratamentos mais adequados. A HR deve ser diagnosticada pela observação dos sintomas juntamente à hipoglicemia química, evitando erros diagnósticos. O melhor teste diagnóstico é a dieta hiperglicídica e o tratamento se faz com dieta adequada. Quando esta não é suficiente, a droga de escolha é a acorbose.

Reactive hypoglycemia is due to a biochemical blood glucose less then 3.3mmol/l (60mg/dl) and sympathetic symptoms occurring a few hours after food intake. Although it's pathophysiology is not completely elucidated, many of it's mechanisms are already known. After a literature review, is was possible to compare different diagnose tests in order to propose the most accurate treatment. In order to avoid misdiagnosis, the disease should be diagnosed through the observation of symptoms and chemical hypoglycemia. The best diagnose test is hyperglucidic test, and the main treatment should be diet. If diet itself is not enough, the drug of choise is acarbose.