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CACNA1S-related myopathy, due to pathogenic variants in the CACNA1S gene, is a recently described congenital muscle disease. Disease associated variants result in loss of gene expression and/or reduction of Cav1.1 protein stability. There is an incomplete understanding of the underlying disease pathomechanisms and no effective therapies are currently available. A barrier to the study of this myopathy is the lack of a suitable animal model that phenocopies key aspects of the disease. To address this barrier, we generated knockouts of the two zebrafish CACNA1S paralogs, cacna1sa and cacna1sb. Double knockout fish exhibit severe weakness and early death, and are characterized by the absence of Cav1.1 α1 subunit expression, abnormal triad structure, and impaired excitation-contraction coupling, thus mirroring the severe form of human CACNA1S-related myopathy. A double mutant (cacna1sa homozygous, cacna1sb heterozygote) exhibits normal development, but displays reduced body size, abnormal facial structure, and cores on muscle pathologic examination, thus phenocopying the mild form of human CACNA1S-related myopathy. In summary, we generated and characterized the first cacna1s zebrafish loss-of-function mutants, and show them to be faithful models of severe and mild forms of human CACNA1S-related myopathy suitable for future mechanistic studies and therapy development.
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Canais de Cálcio Tipo L , Doenças Musculares , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Humanos , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Músculo Esquelético/metabolismo , Doenças Musculares/patologia , Mutação , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Although rapid, the evaluation of bone marrow (BM) cellularity is semi-quantitative and largely dependent upon visual estimates. We aimed to construct an automatic quantification method using image analysis software. We used hematoxylin and eosin (HE)-stained specimens of BM biopsies and clots from patients who underwent BM examination at Tottori University Hospital from 2020 to 2022. We compared image analysis (Methods A, B, and C) with visual estimates in pathology reports of 91 HE specimens in 54 cases (29 males, 25 females), including 38 biopsy and 53 clot specimens. Cellularity was visually scored as hypocellular (n = 17), normocellular (n = 44), or hypercellular (n = 30). Compared with the visual estimates, intraclass correlation coefficients for Methods A, B, and C were 0.80, 0.85, and 0.88, respectively. The most appropriate values were obtained with Method C which detected both non-fatty and cell nuclear areas.
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Exertional heat illness (EHI) and malignant hyperthermia (MH) are life threatening conditions associated with muscle breakdown in the setting of triggering factors including volatile anesthetics, exercise, and high environmental temperature. To identify new genetic variants that predispose to EHI and/or MH, we performed genomic sequencing on a cohort with EHI/MH and/or abnormal caffeine-halothane contracture test. In five individuals, we identified rare, pathogenic heterozygous variants in ASPH, a gene encoding junctin, a regulator of excitation-contraction coupling. We validated the pathogenicity of these variants using orthogonal pre-clinical models, CRISPR-edited C2C12 myotubes and transgenic zebrafish. In total, we demonstrate that ASPH variants represent a new cause of EHI and MH susceptibility.
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Transtornos de Estresse por Calor , Hipertermia Maligna , Animais , Cafeína/farmacologia , Proteínas de Ligação ao Cálcio , Humanos , Hipertermia Maligna/genética , Proteínas de Membrana , Oxigenases de Função Mista , Contração Muscular , Fibras Musculares Esqueléticas , Proteínas Musculares , Peixe-Zebra/genéticaRESUMO
Ryanodine receptor type I-related myopathies (RYR1-RMs) are a common group of childhood muscle diseases associated with severe disabilities and early mortality for which there are no available treatments. The goal of this study is to identify new therapeutic targets for RYR1-RMs. To accomplish this, we developed a discovery pipeline using nematode, zebrafish, and mammalian cell models. We first performed large-scale drug screens in C. elegans which uncovered 74 hits. Targeted testing in zebrafish yielded positive results for two p38 inhibitors. Using mouse myotubes, we found that either pharmacological inhibition or siRNA silencing of p38 impaired caffeine-induced Ca2+ release from wild type cells while promoting intracellular Ca2+ release in Ryr1 knockout cells. Lastly, we demonstrated that p38 inhibition blunts the aberrant temperature-dependent increase in resting Ca2+ in myotubes from an RYR1-RM mouse model. This unique platform for RYR1-RM therapy development is potentially applicable to a broad range of neuromuscular disorders.
Muscle cells have storage compartments stuffed full of calcium, which they release to trigger a contraction. This process depends on a channel-shaped protein called the ryanodine receptor, or RYR1 for short. When RYR1 is activated, it releases calcium from storage, which floods the muscle cell. Mutations in the gene that codes for RYR1 in humans cause a group of rare diseases called RYR1-related myopathies. The mutations change calcium release in muscle cells, which can make movement difficult, and make it hard for people to breathe. At the moment, RYR1 myopathies have no treatment. It is possible that repurposing existing drugs could benefit people with RYR1-related myopathies, but trialing treatments takes time. The fastest and cheapest way to test whether compounds might be effective is to try them on very simple animals, like nematode worms. But even though worms and humans share certain genes, treatments that work for worms do not always work for humans. Luckily, it is sometimes possible to test whether compounds might be effective by trying them out on complex mammals, like mice. Unfortunately, these experiments are slow and expensive. A compromise involves testing on animals such as zebrafish. So far, none of these methods has been successful in discovering treatments for RYR1-related myopathies. To maximize the strengths of each animal model, Volpatti et al. combined them, developing a fast and powerful way to test new drugs. The first step is an automated screening process that trials thousands of chemicals on nematode worms. This takes just two weeks. The second step is to group the best treatments according to their chemical similarities and test them again in zebrafish. This takes a month. The third and final stage is to test promising chemicals from the zebrafish in mouse muscle cells. Of the thousands of compounds tested here, one group of chemicals stood out treatments that block the activity of a protein called p38. Volpatti et al. found that blocking the p38 protein, either with drugs or by inactivating the gene that codes for it, changed muscle calcium release. This suggests p38 blockers may have potential as a treatment for RYR1-related myopathies in mammals. Using three types of animal to test new drugs maximizes the benefits of each model. This type of pipeline could identify new treatments, not just for RYR1-related myopathies, but for other diseases that involve genes or proteins that are similar across species. For RYR1-related myopathies specifically, the next step is to test p38 blocking treatments in mice. This could reveal whether the treatments have the potential to improve symptoms.
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Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Doenças Musculares/tratamento farmacológico , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Caenorhabditis elegans , Descoberta de Drogas , Técnicas de Inativação de Genes , Ensaios de Triagem em Larga Escala , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Doenças Neuromusculares/tratamento farmacológico , Preparações Farmacêuticas , Interferência de RNA , Bibliotecas de Moléculas Pequenas , Peixe-ZebraRESUMO
Diffuse alveolar hemorrhage (DAH) is well known as a serious complication of microscopic polyangiitis (MPA). We examined the effectiveness of plasma exchange (PLEX) therapy to reduce mortality in Japanese DAH patients with MPA. This retrospective, double-center, observational cohort study included 20 DAH patients with MPA who were admitted to Juntendo University Hospital or Juntendo Koto Geriatric Medical Center between April 1998 and March 2018. The primary outcome was non-disease-specific mortality. The 1-year survival rate of patients with PLEX therapy (N = 4) was higher than that of patients with conventional therapy (N = 16, 75% and 13%, respectively, P = 0.037). Higher values of the 1996 Five-Factor Score (FFS) and 2009 FFS were associated with increased mortality, with hazard ratios of 2.29 (P = 0.040) and 2.41 (P = 0.043), respectively, by Cox univariate analysis. We investigated PLEX therapy for reducing mortality in DAH patients with MPA, and the 1996 FFS and 2009 FFS were both independent prognostic factors.
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Hemorragia/terapia , Poliangiite Microscópica/terapia , Troca Plasmática/métodos , Alvéolos Pulmonares/patologia , Idoso , Estudos de Coortes , Feminino , Hemorragia/etiologia , Hemorragia/mortalidade , Humanos , Japão , Masculino , Poliangiite Microscópica/complicações , Poliangiite Microscópica/mortalidade , Pessoa de Meia-Idade , Prognóstico , Estudos RetrospectivosRESUMO
BACKGROUND: Adenoid cystic carcinoma (ACC) is a relatively rare malignant neoplasm that occurs in salivary glands and various other organs. Recent studies have revealed that a significant proportion of ACCs harbor gene alterations involving MYB or MYBL1 (mostly fusions with NFIB) in a mutually-exclusive manner. However, its clinical significance remains to be well-established. METHODS: We investigated clinicopathological and molecular features of 36 ACCs with special emphasis on the significance of MYBL1 alterations. Reverse-transcription polymerase-chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH) were performed to detect MYB/MYBL1-NFIB fusions and MYBL1 alterations, respectively. Immunohistochemistry was performed to evaluate MYB expression in the tumors. The results were correlated with clinicopathological profiles of the patients. RESULTS: RT-PCR revealed MYB-NFIB and MYBL1-NFIB fusions in 10 (27.8%) and 7 (19.4%) ACCs, respectively, in a mutually-exclusive manner. FISH for MYBL1 rearrangements was successfully performed in 11 cases, and the results were concordant with those of RT-PCR. Immunohistochemically, strong MYB expression was observed in 23 (63.9%) tumors, none of which showed MYBL1 alterations. Clinicopathologically, a trend of a better disease-specific survival was noted in patients with MYBL1 alterations than in those with MYB-NFIB fusions and/or strong MYB expression; however, the difference was not significant. Interestingly, we found tumors with MYBL1 alterations significantly frequently occurred in the mandibular regions (P = 0.012). Moreover, literature review revealed a similar tendency in a previous study. CONCLUSION: Our results suggest that there are some biological or etiological differences between ACCs with MYB and MYBL1 alterations. Moreover, the frequent occurrence of MYBL1-associated ACC in the mandibular regions suggests that MYB immunohistochemistry is less useful in diagnosing ACCs arising in these regions. Further studies are warranted to verify our findings.
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We report a 38-year-old woman who presented with mild proximal dominant muscle weakness and fatigability that fluctuated during menstruation and treatment with ephedrine-containing medication. The patient had been diagnosed with "congenital myopathy with tubular aggregates" by muscle biopsy at age 19. Her revised diagnosis was congenital myasthenic syndrome (CMS) caused by a mutation in GFPT1 (2p13.3 [MIM 610542], c.722_723insG homozygote, CMS-GFPT1) based on a screening gene analysis. Muscle CT revealed diffuse atrophy of proximal and axial muscles focused on the vastus lateralis, hamstrings, medial gastrocnemius and soleus muscles. Oral administration of pyridostigmine bromide clearly ameliorated weakness and fatigability. This is the first reported case of CMS-GFPT1 in Japan. Since CMS symptoms are reactive to treatment, it is important for clinicians to make an accurate diagnosis at an early stage to improve patient QOL. Tubular aggregates in muscle biopsy and day-to-day fluctuations are important features of the disorder. Quantitative muscle strength measurement was effective for evaluating treatment efficacy.
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Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Síndromes Miastênicas Congênitas/diagnóstico , Adulto , Feminino , Humanos , Japão , Mutação , Síndromes Miastênicas Congênitas/patologia , FenótipoRESUMO
OBJECTIVE: To determine mortality and predictive factors for lower intestinal perforation (LIP) among patients with autoimmune rheumatic diseases. METHODS: This retrospective, single-center, observational study analyzed mortality rates in 31 autoimmune rheumatic disease patients with LIP who were admitted to our hospital from January 2002 to June 2017. The primary outcome was the mortality rate during hospitalization. RESULTS: The median age at the time of LIP was 61 years, and the survival rate at discharge was 64.5%. Eleven patients died of sepsis during hospitalization. Cox univariable analysis for mortality during hospitalization showed that absence of abdominal pain (hazard ratio (HR) 5.61, 95% confidence interval (CI) 1.38-22.9), higher age (HR 1.06, 95% CI 1.01-1.11), chronic kidney disease (HR 6.89, 95% CI 1.85-25.7), systemic vasculitis (HR 3.95, 95% CI 1.14-13.6), higher blood urea nitrogen (HR 1.02, 95% CI 1.01-1.04), higher serum creatinine (HR 1.41, 95% CI 1.06-1.87), and LIP due to malignancy (HR 14.3, 95% CI 1.95-105.1) significantly increased mortality. CONCLUSION: Abdominal pain was absent in 16% of LIP patients with autoimmune rheumatic diseases, and this absence was a poor prognostic factor in this cohort. Moreover, higher age, chronic kidney disease, systemic vasculitis, and LIP due to malignancy were associated with significantly increased mortality. Physicians should be aware of LIP in autoimmune disease patients with higher age, chronic kidney diseases, or systemic vasculitis even if patients reveal mild abdominal symptoms.
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Dor Abdominal/mortalidade , Perfuração Intestinal/mortalidade , Insuficiência Renal Crônica/mortalidade , Doenças Reumáticas/mortalidade , Vasculite Sistêmica/mortalidade , Dor Abdominal/etiologia , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de SobrevidaRESUMO
Objective: Pneumocystis pneumonia (PCP) is a serious complication in patients with rheumatic diseases who are receiving immunosuppressive therapy. These patients have a higher mortality from PCP than those with human immunodeficiency virus. We examined factors associated with poor prognosis in patients with rheumatic diseases and evaluated PCP treatment in this population. Methods: This retrospective, single-center, observational cohort study included 31 patients with rheumatic diseases who were admitted to Juntendo University Hospital for PCP treatment from June 2006 to December 2017. The primary outcome was non-disease-specific mortality at discharge. Results: The median age at PCP diagnosis was 64 years. The survival rate was 61.3% (19/31). Twelve patients died, in all cases due to respiratory failure due to PCP. Among variables at PCP diagnosis and those related to PCP treatment, the presence of coexisting pulmonary diseases and greater glucocorticoid dose at PCP diagnosis were associated with higher mortality. The mortality related to biological agents for PCP was low. Rapid tapering of glucocorticoids improved survivability. Conclusion: In the treatment of PCP in patients with rheumatic diseases, rapid tapering of glucocorticoids was associated with a higher survival rate than the use of conventional therapy.
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Glucocorticoides/uso terapêutico , Pneumonia por Pneumocystis/tratamento farmacológico , Doenças Reumáticas/complicações , Adulto , Idoso , Feminino , Glucocorticoides/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia por Pneumocystis/etiologia , Resultado do TratamentoRESUMO
OBJECTIVE: To determine the molecular factors contributing to progressive cavitating leukoencephalopathy (PCL) to help resolve the underlying genotype-phenotype associations in the mitochondrial iron-sulfur cluster (ISC) assembly system. METHODS: The subjects were 3 patients from 2 families who showed no inconsistencies in either clinical or brain MRI findings as PCL. We used exome sequencing, immunoblotting, and enzyme activity assays to establish a molecular diagnosis and determine the roles of ISC-associated factors in PCL. RESULTS: We performed genetic analyses on these 3 patients and identified compound heterozygosity for the IBA57 gene, which encodes the mitochondrial iron-sulfur protein assembly factor. Protein expression analysis revealed substantial decreases in IBA57 protein expression in myoblasts and fibroblasts. Immunoblotting revealed substantially reduced expression of SDHB, a subunit of complex II, and lipoic acid synthetase (LIAS). Levels of pyruvate dehydrogenase complex-E2 and α-ketoglutarate dehydrogenase-E2, which use lipoic acid as a cofactor, were also reduced. In activity staining, SDH activity was clearly reduced, but it was ameliorated in mitochondrial fractions from rescued myoblasts. In addition, NFU1 protein expression was also decreased, which is required for the assembly of a subset of iron-sulfur proteins to SDH and LIAS in the mitochondrial ISC assembly system. CONCLUSIONS: Defects in IBA57 essentially regulate NFU1 expression, and aberrant NFU1 ultimately affects SDH activity and LIAS expression in the ISC biogenesis pathway. This study provides new insights into the role of the iron-sulfur protein assembly system in disorders related to mitochondrial energy metabolism associated with leukoencephalopathy with cavities.
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SMARCA4-deficient thoracic sarcoma is a recently proposed new entity of soft tissue sarcomas with an undifferentiated round cell morphology that is diagnostically challenging. Here we report a case of this tumor where the diagnosis was established using limited samples and resources. A woman in her early 30s developed two intrathoracic masses. Biopsies for these lesions showed sheets of undifferentiated round/rhabdoid cells that retained SMARCB1 expression. Further analysis revealed a reduced SMARCA4 expression and a complete loss of SMARCA2 expression in tumor cells. Subsequent Sanger sequencing identified a nonsense c.1546A>T (p.516Lys>Ter) mutation in SMARCA4 and confirmed the diagnosis. Our case highlighted clinicopathological correlation and rational use of tissue sections for immunohistochemistry may enable to diagnose this tumor even when only limited samples are available. Recognition of this new entity is important for further understanding of the disease and the future development of specific therapies.
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Biomarcadores Tumorais/genética , DNA Helicases/genética , Análise Mutacional de DNA , Imuno-Histoquímica , Proteínas Nucleares/genética , Sarcoma/diagnóstico , Neoplasias Torácicas/diagnóstico , Fatores de Transcrição/genética , Adulto , Biomarcadores Tumorais/deficiência , Biópsia , Códon sem Sentido , DNA Helicases/deficiência , Feminino , Humanos , Proteínas Nucleares/deficiência , Valor Preditivo dos Testes , Sarcoma/química , Sarcoma/genética , Sarcoma/patologia , Neoplasias Torácicas/química , Neoplasias Torácicas/genética , Neoplasias Torácicas/patologia , Tomografia Computadorizada por Raios X , Fatores de Transcrição/deficiênciaRESUMO
OBJECTIVE: Antibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) have recently been associated with immune-mediated necrotizing myopathy, especially in patients with statin exposure. As the data are very limited concerning phenotypes and treatment in paediatric patients, we aimed to identify the paediatric patients positive for anti-HMGCR antibodies and clarify their features and therapeutic strategies. METHODS: We screened 62 paediatric patients who were clinically and/or pathologically suspected to have inflammatory myopathy for anti-HMGCR antibodies. We further re-assessed the clinical and histological findings and the treatment of the patients positive for anti-HMGCR antibodies. RESULTS: We identified nine paediatric patients with anti-HMGCR antibodies (15%). This was more frequent than anti-signal recognition particle antibodies (four patients, 6%) in our cohort. The onset age ranged from infancy to 13 years. Five patients were initially diagnosed with muscular dystrophy, including congenital muscular dystrophy. Most patients responded to high-dose corticosteroid therapy first but often needed adjuvant immunosuppressants to become stably controlled. CONCLUSION: Paediatric necrotizing myopathy associated with anti-HMGCR antibodies may not be very rare. Phenotypes are similar to those of adult patients, but a chronic slowly progressive course may be more frequent. Some patients share the clinicopathological features of muscular dystrophy indicating that recognizing inflammatory aetiology would be challenging without autoantibody information. On the other hand, most patients responded to treatment, especially those who were diagnosed early. Our results suggest the importance of early autoantibody testing in paediatric patients who have manifestations apparently compatible with muscular dystrophy in addition to those who have typical features of inflammatory myopathy.
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Autoanticorpos/imunologia , Hidroximetilglutaril-CoA Redutases/imunologia , Miosite/imunologia , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miosite/metabolismo , Miosite/patologiaRESUMO
BACKGROUND: Recently, a lot of cases with microcalcifications of the breast are pointed by the images of mammography (MG), because breast screening using MG become common. Although MG is a gold standard modality for detecting microcalcifications, images of ultrasonography (US) are now feasible to detect microcalcifications with recent improvements to ultrasound diagnostic devices. In this report, we analyzed clinical significance of microcalcifications detected with US images in invasive breast carcinoma. METHODS: Eighty-eight patients with invasive breast carcinoma who underwent MG and US before surgery at the Division of Breast and Endocrine Surgery of Tottori University Hospital between January 2012 and August 2013. After reviewing US images, the association between the presence of echogenic spots that indicate microcalcifications and images of MG or pathological findings was assessed. RESULTS: Patients without microcalcifications on US images were significantly more likely to have the Luminal A subtype and a lower nuclear grading. Conversely, patients with microcalcifications on US images were significantly more likely to have higher level of MIB-1 index, lymphovascular invasion, comedonecrosis and lymph node metastasis. The rate of detecting microcalcifications on US images was relatively good, with 81.8% of sensitivity, 94.5% of specificity and 89.8% of diagnostic accuracy. Among the calcifications detected by MG images, detected rate of calcifications with US images was higher in necrotic type (92.6%) than secretory type (33.3%). CONCLUSION: This study suggest that microcalcifications of tumors detected by US images could serve as an useful prediction to evaluate the degree of malignancy for patients with invasive breast carcinoma.
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The aim of this study was to determine the correlation between shear wave velocity (SWV) generated by acoustic radiation force impulse and the pathologic structure of thyroid lesions. A total of 599 thyroid tissue samples were divided into four groups based on pathologic structure: 254 normal thyroid tissue samples as a control, 128 with chronic autoimmune thyroiditis (CAT) that demonstrated diffuse fibrosis, 165 with benign nodules that had high cell density and 52 with papillary thyroid carcinoma (PTC) that showed high cell density and fibrosis. The mean SWVs in each group were as follows: 1.60 ± 0.18 m/s in normal thyroid, 2.55 ± 0.28 m/s in CAT, 1.72 ± 0.31 m/s in benign nodules and 2.66 ± 0.95 m/s in PTC. The SWVs of CAT and PTC were significantly higher than those of normal thyroid, (p < 0.001). SWV was significantly affected by fibrosis.
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Técnicas de Imagem por Elasticidade/métodos , Interpretação de Imagem Assistida por Computador/métodos , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/fisiopatologia , Tireoidite Autoimune/patologia , Tireoidite Autoimune/fisiopatologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Resistência ao Cisalhamento , Estatística como Assunto , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Tireoidite Autoimune/diagnóstico por imagem , Adulto JovemRESUMO
Danon disease is an X-linked disorder clinically characterized by the triad of hypertrophic cardiomyopathy, myopathy, and intellectual disability. Cardiomyopathy is a severe and life-threatening problem, for which cardiac transplantation is the only therapeutic option. The most striking finding in muscle biopsy samples is small basophilic granules scattered in myofibers, which are in fact small autophagic vacuoles surrounded by membranes with sarcolemmal features characterized by the recruitment of sarcolemmal proteins and acetylcholine esterase and by the presence of basal lamina on its luminal side. The mechanism underlying the formation of these autophagic vacuoles with unique sarcolemmal features (AVSF) still remains a mystery and its origin is unknown. In heart, cardiomyocytes show dramatically increased vacuolation and degenerative features, including myofibrillar disruption and lipofuscin accumulation. In brain, pale granular neurons and neurons with lipofuscin-like granules may be seen. Danon disease is caused by loss-of-function mutations in the LAMP2 gene, which encodes lysosome-associated membrane protein 2 (LAMP-2), a single-spanned transmembrane protein localized in the limiting membranes of lysosomes and late endosomes. Most mutations lead to splicing defects or protein truncation, resulting in a loss of transmembrane and/or cytoplasmic domains, leading to LAMP-2 protein deficiency. LAMP-2 is required for the maturation of autophagosomes by fusion with lysosomes; therefore, LAMP-2 deficiency leads to a failure in macroautophagy. There are three LAMP-2 isoforms, LAMP-2A, -2B, and -2C. Clinical features of Danon disease are thought to be mediated by loss of the LAMP-2B isoform which is the major isoform expressed in muscle. It is also known that LAMP-2 plays a role in chaperone-mediated autophagy and RNA- and DNA-targeting autophagy. However, the precise pathophysiological mechanism through which LAMP-2 deficiency causes Danon disease is still not fully understood and its elucidation would promote the development of new therapies.
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Autofagia/genética , Doença de Depósito de Glicogênio Tipo IIb/genética , Doença de Depósito de Glicogênio Tipo IIb/patologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Humanos , FenótipoRESUMO
OBJECTIVE: To determine the genetic variants in patients with dystroglycanopathy (DGP) and assess the pathogenicity of these variants. METHODS: A total of 20 patients with DGP were identified by immunohistochemistry or Western blot analysis. Whole-exome sequencing (WES) was performed using patient samples. The pathogenicity of the variants identified was evaluated on the basis of the phenotypic recovery in a knockout (KO) haploid human cell line by transfection with mutated POMGNT2 cDNA and on the basis of the in vitro enzymatic activity of mutated proteins. RESULTS: WES identified homozygous and compound heterozygous missense variants in POMGNT2 in 3 patients with the milder limb-girdle muscular dystrophy (LGMD) and intellectual disability without brain malformation. The 2 identified variants were located in the putative glycosyltransferase domain of POMGNT2, which affected its enzymatic activity. Mutated POMGNT2 cDNAs failed to rescue the phenotype of POMGNT2-KO cells. CONCLUSIONS: Novel variants in POMGNT2 are associated with milder forms of LGMD. The findings of this study expand the clinical and pathologic spectrum of DGP associated with POMGNT2 variants from the severest Walker-Warburg syndrome to the mildest LGMD phenotypes. The simple method to verify pathogenesis of variants may allow researchers to evaluate any variants present in all of the known causative genes and the variants in novel candidate genes to detect DGPs, particularly without using patients' specimens.
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We sought to elucidate the correlation between shear wave velocity (SWV) and fibrosis in thyroid by precisely assessing pathological structures inside 5 × 5 mm(2) regions of interest (ROIs) of resected specimens, under conditions that excluded physical artifacts. The materials were unselected thyroid and lymph node specimens resected during thyroid surgery. Immediately after surgery, fresh unfixed thyroid and metastatic lymph node specimens were suspended in gel phantoms, and SWV was measured. Upon pathological examination of each specimen, the extent of fibrosis was graded as none, moderate, or severe. A total of 109 specimens were evaluated: 15 normal thyroid, 16 autoimmune thyroiditis, 40 malignant nodules, 19 benign thyroid nodules, and 19 metastatic lymph nodes. When all specimens were classified according to the degree of fibrosis determined by pathological imaging, the mean SWV was 1.49 ± 0.39 m/s for no fibrosis, 2.13 ± 0.66 m/s for moderate fibrosis, and 2.68 ± 0.82 m/s for severe fibrosis. The SWVs of samples with moderate and severe fibrosis were significantly higher than those of samples without fibrosis. The results of this study demonstrate that fibrosis plays an important role in determining stiffness, as measured by SWV in thyroid.
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Fibrose/diagnóstico por imagem , Doenças da Glândula Tireoide/diagnóstico por imagem , Glândula Tireoide/diagnóstico por imagem , Técnicas de Imagem por Elasticidade/métodos , Técnicas de Imagem por Elasticidade/normas , Fibrose/fisiopatologia , Humanos , Doenças da Glândula Tireoide/fisiopatologia , Glândula Tireoide/fisiopatologiaRESUMO
OBJECTIVES: To identify gene mutations in patients with dystroglycanopathy and prove pathogenicity of those mutations using an in vitro cell assay. METHODS: We performed whole-exome sequencing on 20 patients, who were previously diagnosed with dystroglycanopathy by immunohistochemistry and/or Western blot analysis. We also evaluated pathogenicity of identified mutations for phenotypic recovery in a DAG1-knockout haploid human cell line transfected with mutated DAG1 complementary DNA. RESULTS: Using exome sequencing, we identified compound heterozygous missense mutations in DAG1 in a patient with asymptomatic hyperCKemia and pathologically mild muscular dystrophy. Both mutations were in the N-terminal region of α-dystroglycan and affected its glycosylation. Mutated DAG1 complementary DNAs failed to rescue the phenotype in DAG1-knockout cells, suggesting that these are pathogenic mutations. CONCLUSION: Novel mutations in DAG1 are associated with asymptomatic hyperCKemia with hypoglycosylation of α-dystroglycan. The combination of exome sequencing and a phenotype-rescue experiment on a gene-knockout haploid cell line represents a powerful tool for evaluation of these pathogenic mutations.
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Creatina Quinase/metabolismo , Distroglicanas/genética , Distroglicanas/metabolismo , Doenças Metabólicas/genética , Distrofias Musculares/genética , Mutação/genética , Adulto , Linhagem Celular Transformada , Estudos de Coortes , Distrofina/metabolismo , Feminino , Estudos de Associação Genética , Glicosilação , Humanos , Laminina/metabolismo , Masculino , Doenças Metabólicas/complicações , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Distrofias Musculares/complicações , Distrofias Musculares/diagnóstico , Fenótipo , Transporte Proteico/genética , TransfecçãoRESUMO
The store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel is activated by diminished luminal Ca(2+) levels in the endoplasmic reticulum and sarcoplasmic reticulum (SR), and constitutes one of the major Ca(2+) entry pathways in various tissues. Tubular aggregates (TAs) are abnormal structures in the skeletal muscle, and although their mechanism of formation has not been clarified, altered Ca(2+) homeostasis related to a disordered SR is suggested to be one of the main contributing factors. TA myopathy is a hereditary muscle disorder that is pathologically characterized by the presence of TAs. Recently, dominant mutations in the STIM1 gene, encoding a Ca(2+) sensor that controls CRAC channels, have been identified to cause tubular aggregate myopathy (TAM). Here, we identified heterozygous missense mutations in the ORAI1 gene, encoding the CRAC channel itself, in three families affected by dominantly inherited TAM with hypocalcemia. Skeletal myotubes from an affected individual and HEK293 cells expressing mutated ORAI1 proteins displayed spontaneous extracellular Ca(2+) entry into cells without diminishment of luminal Ca(2+) or the association with STIM1. Our results indicate that STIM1-independent activation of CRAC channels induced by dominant mutations in ORAI1 cause altered Ca(2+) homeostasis, resulting in TAM with hypocalcemia.
Assuntos
Canais de Cálcio/genética , Hipocalcemia/genética , Fibras Musculares Esqueléticas/patologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Adulto , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Criança , Pré-Escolar , Células HEK293 , Heterozigoto , Humanos , Masculino , Fibras Musculares Esqueléticas/metabolismo , Mutação de Sentido Incorreto , Miopatias Congênitas Estruturais/complicações , Proteína ORAI1 , Linhagem , Molécula 1 de Interação EstromalRESUMO
BACKGROUND: Renal carcinomas associated with Xp11.2 translocations/TFE3 gene fusions are rare subtypes of renal neoplasm that predominantly occur in younger individuals. There are very few reports describing the cytologic features of these tumors. CASE: A 27-year-old man presented with hematuria and was found to have a mass in the lower part of the right kidney. Cytology of catheterized urine obtained from the right renal pelvis showed clusters of cells with abundant clear or eosinophilic granular cytoplasm, large round nuclei and prominent nucleoli. Papillary clusters containing thin fibrous stroma were occasionally seen. Voided urine cytology showed similar cell clusters but degeneration made the features obscure. Nephroureterectomy revealed a renal tumor showing a mixed papillary and nested architecture. The diagnosis was confirmed by immunohistochemistry and fluorescence in situ hybridization. CONCLUSION: The present case indicates that the characteristic features of these tumor subtypes can be retained in urine cytology. Cytology may be enough to suspect these tumors as part of the differential diagnosis when the patient's age and imaging findings are taken into account and may facilitate further studies for a definitive diagnosis.