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

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

Estudo da variabilidade genética na miopatia central core: aperfeiçoando o screening de mutações no gene ryr1

Introduction: Central core disease (CCD) is characterized by the presence of central core-like areas in muscle fibers. Patients with RYR-related CCD usually have mild or moderate axial and proximal weakness, hypotonia and motor developmental delay. CCD is associated with susceptibility to malignant hyperthermia (MH), and both conditions have been linked to mutations in human RYR1 gene, which encodes a calcium release channel known as ryanodine receptor (RyR1). RYR1 mutational spectrum linked with CCD includes more than 200 described mutations mostly heterozygous dominant missense mutations and minor deletions or duplications. Definite diagnosis of CCD is done by clinical history and evaluation, histopathological analyses of muscle biopsy and, recently, genetic testing of gene?s ?hot spots?, because RYR1 is a large gene and direct sequencing of each exon is laborious. Here, we tested Multiplex Ligation-dependent Probe Amplification (MLPA) as an alternative and simple method for screening patients with histological diagnosis or family history of CCD Objective: To test MLPA technique for screening patients with histological diagnosis or family history of CCD and its precision to detect possible mutations in RYR1 gene, comparing to direct sequencing results of the same exons. Methods: We used DNA database specimens of patients with histological diagnosis or family for MLPA and direct sequencing testing. Results: From 35 patients diagnosed with CCD in three different institutions, 24 (68,6%) had mutations found in direct sequencing and twelve (34,3%) in MLPA. Mutations in exon 101 (c.14582 G>A and c.14581 C>T) were the most prevalent in sequencing and MLPA. The missense mutation c.14256 A>C in exon 98 was the most prevalent polymorphism identified. A new mutation in exon 100 (c.14411 A>C) was detected by direct sequencing. Conclusion: MLPA had shown to be a simple but relevant tool to help diagnosing CCD; moreover, it is especially useful in diagnosing family members of patients with known mutations detectable by MLPA considering the difficulty to detect the mutations in RYR1 gene.Introdução: As miopatias com cores (MCC) têm como principal característica histopatológica a presença de cores, regiões de reduzida atividade oxidativa em fibras tipo I visualizadas na biópsia muscular. A MCC apresenta-se com fraqueza de predomínio proximal leve a moderada, hipotonia e atraso no desenvolvimento motor. A MCC está relacionada à ocorrência de Hipertermia Maligna (HM) e ambas são associadas a mutações no gene RYR1, que codifica o canal de liberação de cálcio denominado receptor de rianodina (RyR1). Atualmente, mais de 200 mutações no gene RYR1 já foram descritas. O diagnóstico definitivo de MCC deve ser realizado a partir de dados da história clínica, análise histopatológica, ressonância magnética e, mais recentemente, testes genéticos dos ?hot spots? do gene. No presente estudo avaliamos o teste Multiplex Ligation-dependent Probe Amplification (MLPA) como um método alternativo simples, rápido e preciso para o screening de pacientes com diagnóstico histológico ou história familiar de MCC. Objetivo: Testar a técnica de MLPA para a realização de screening de pacientes com diagnóstico clínico e histológico de MCC e sua precisão para diagnosticar possíveis mutações no gene RYR1, comparando os resultados obtidos com a técnica rotineira de PCR e sequenciamento da região C-terminal do gene RYR1. Método: Foi utilizado banco de amostras de DNA de pacientes com diagnóstico histológico de MCC para realização de testes de MLPA e PCR com sequenciamento direto. Resultados: Dos 35 pacientes diagnosticados com MCC, 24 (68,6%) tiveram mutações detectadas por PCR e doze (34,3%) por MLPA, sendo que destes 8 foram confirmadospor sequenciamento direto. As mutações no exon 101 c.14582 G>A e c.14581 C>T foram as mais frequentemente encontradas pelos dois métodos. A mutação missense c.14256 A>C no exon 98 foi o polimorfismo mais frequentemente encontrado (seis pacientes). A mutação nova c.14411 A>C no exon 100 foi detectada por sequenciamento direto. Conclusão: O método de MLPA vem surgindo como um teste laboratorial alternativo, simples e de alto rendimento para auxiliar no screening de pacientes e familiares com suspeita de MCC, uma vez que a taxa de identificação das mutações responsáveis pela doença ainda é baixa devido à dificuldade de estudo do gene.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Expression of matrix factors in the process of neovascularization of intervertebral disc

OBJECTIVE: To investigate the effects of proteins products of endothelial cells (ECs) on the annulus fibrosus (AF) cell metabolism in an in vitro culture.METHODS:Human AF cells were expanded in monolayer cultures and treated with proteins from the medium of cell line HMEC-1 (Human Microvascular Endothelial Cells) (125µg/ml). After 72h of treatment RNA was isolated from AF cells for analysis of gene expression and the culture medium was collected for protein expression analysis.RESULTS: The qRT-PCR analysis demonstrated increased gene expression of matrix metalloproteinases (MMPs) in AF cells treated with protein products of endothelial cells compared with cells from control group of AF cells: MMP-1 243.10 times (pCONCLUSIONS: In this study, we observed that the proteins produced by ECs induced the MMPs expression and suppressed the TIMPs as well as the aggrecan in primary cells of the human intervertebral disc, targeting the development of potential treatments for intervertebral disc degeneration and associated discogenic pain.</sec

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

Abstract 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

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

Abstract 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

Post Hoc Subgroup Analysis of the BCause Study Assessing the Effect of AbobotulinumtoxinA on Post-Stroke Shoulder Pain in Adults

Botulinum toxin type A is approved for the focal treatment of spasticity; however, the effectiveness of abobotulinumtoxinA (aboBoNT-A) in patients with shoulder pain who have set reduced pain as a treatment goal is understudied. In addition, some patients encounter delays in accessing treatment programs; therefore, the suitability of aboBoNT-A for pain reduction in this population requires investigation. These factors were assessed in aboBoNT-A-naive Brazilian patients in a post hoc analysis of data from BCause, an observational, multicenter, prospective study (NCT02390206). Patients (N = 49, n = 25 female; mean (standard deviation) age of 60.3 (9.1) years; median (range) time since onset of spasticity of 16.1 (0&ndash;193) months) received aboBoNT-A injections to shoulder muscles in one or two treatment cycles (n = 47). Using goal attainment scaling (GAS), most patients achieved their goal of shoulder pain reduction after one treatment cycle (72.1%; 95% confidence interval: 57.2&ndash;83.4%). Improvements in GAS T-score from baseline, clinically meaningful reductions in pain score at movement, and clinically meaningful increases in passive shoulder abduction angle further improved with repeated treatment more than 4 months later, despite treatment starting at a median of 16.1 months after the onset of spasticity. These findings support the further investigation of aboBoNT-A injections in chronic post-stroke shoulder pain