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Type 1 diabetes mellitus (T1DM) and poor glycemic control are risk factors for severe coronavirus disease 2019 (COVID-19). Sotrovimab can treat mild-to-moderate COVID-19 in patients at a high risk of progression to severe COVID-19. However, its safety and efficacy in T1DM patients remain to be elucidated. We report the case of a 12-yr-old patient who was treated with sotrovimab for COVID-19 immediately after treatment for diabetic ketoacidosis (DKA) due to new-onset T1DM. He presented with nausea and sore throat and was diagnosed with severe DKA and COVID-19. A productive cough and sputum developed after admission. On the 3rd day of admission, the DKA resolved, and sotrovimab was administered to prevent exacerbation of COVID-19. Although the blood glucose levels increased after the administration of sotrobimab, there was no recurrence of DKA. Hyperglycemia may be a sotrovimab-related adverse event in T1DM patients. Nevertheless, the benefits of sotrovimab treatment may far outweigh the potential risks. Thus, sotrovimab was considered safe for patients with T1DM immediately after treatment of severe DKA.
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BACKGROUND: Malnutrition measured by the geriatric nutritional risk index (GNRI) was reported to be associated with poor prognosis for patients with peripheral artery disease (PAD). However, the optimal cut-off value of preprocedural GNRI for critical limb ischemia (CLI) and intermittent claudication (IC) is unknown. We aimed to determine its optimal cut-off value for CLI or IC patients requiring endovascular revascularization. METHODS: We explored data of 2246 patients (CLI: n = 1061, IC: n = 1185) registered in the Tokyo-taMA peripheral vascular intervention research COmraDE (TOMA-CODE) registry, which prospectively enrolled consecutive PAD patients who underwent endovascular revascularization in 34 hospitals in Japan from August 2014 to August 2016. The optimal cut-off values of GNRI were assessed by the survival classification and regression tree (CART) analyses, and the survival curve analyses for major adverse cardiovascular and limb events (MACLEs) were performed for these cut-off values. RESULTS: In addition to the first cut-off value of 96.2 in CLI and 85.6 in IC, the survival CART provided an additional cut-off value of 78.2 in CLI and 106.0 in IC for further risk stratification. The survival curve was significantly stratified by the GNRI-based malnutrition status in both CLI [high risk: 47.7% (51/107), moderate: 30.1% (118/392), and low: 10.2% (53/520), log-rank p < 0.001] and IC [high risk: 14.3% (7/49), moderate: 4.5% (29/646), and low: 0.5% (2/407), log-rank p < 0.001]. The multivariate Cox-proportional hazard analysis showed that a higher GNRI was significantly associated with a better outcome in both CLI [hazard ratio (HR) per 1-point increase: 0.97, 95% CI: 0.96-0.98, p < 0.001] and IC (HR: 0.94, 95% CI: 0.91-0.97, p < 0.001). CONCLUSIONS: Preprocedural nutritional status significantly stratified future events in patients with PAD. Given that the optimal cut-off value of GNRI in CLI was almost 10-points lower than that of IC, using a disease-specific cut-off value is important for risk stratification.
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Procedimientos Endovasculares/mortalidad , Evaluación Geriátrica/estadística & datos numéricos , Desnutrición/diagnóstico , Evaluación Nutricional , Enfermedad Arterial Periférica/fisiopatología , Anciano , Anciano de 80 o más Años , Femenino , Evaluación Geriátrica/métodos , Humanos , Japón , Masculino , Desnutrición/complicaciones , Persona de Mediana Edad , Estado Nutricional , Enfermedad Arterial Periférica/complicaciones , Enfermedad Arterial Periférica/cirugía , Valor Predictivo de las Pruebas , Periodo Preoperatorio , Modelos de Riesgos Proporcionales , Valores de Referencia , Sistema de Registros , Medición de Riesgo , Factores de Riesgo , Análisis de SupervivenciaRESUMEN
Feeding behavior in cnidarians has been studied as a model experimental system in physiology and neurobiology. Although the feeding response in cnidarians, such as Hydra, is triggered by chemical signals, the underlying molecular mechanisms that ensure their precise execution are not well understood. It could be largely due to the lack of genetic analysis in cnidarian experimental systems. Cladonema pacificum is a hydrozoan jellyfish that is easy to maintain and cross for genetic analysis in the laboratory. To establish C. pacificum as a model experimental animal in cnidarians, we have been inbreeding strains of jellyfish. Here, we document our progress in developing C. pacificum inbred lines and feeding-defective strains that we isolated in the course of inbreeding. In the inbred lines, an increasing number of feeding-defective strains appeared as descending generations and finally all the F5 progeny showed a feeding-deficient phenotype presumably owing to inbreeding depression. Feeding behaviors of these strains were analyzed by video microscopy and we found that the feeding-defective strains captured prey, but could not kill them. After trapping prey, wild-type medusae contracted their tentacles tightly and then bent the tentacles to bring the prey to the mouth; however, feeding-defective medusae rarely contracted their tentacles and did not bend. These feeding-defective phenotypes are caused by lack of stinging nematocytes in their tentacle batteries. These findings furnish a clue to the regulatory aspects of feeding behavior, but also reveal the mechanisms of stinging nematocyte transport in tentacles.
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Hidrozoos/fisiología , Endogamia , Fenotipo , Animales , Cruzamiento , Conducta AlimentariaRESUMEN
Incidence and neonatal risk factors for short stature in preterm children born small for gestational age (SGA) have not been fully investigated in Japan. In this prospective study, infants born ≤32 weeks' gestational age (GA) from 2004-2015 were enrolled and followed for 3 years. Incidence of short children born SGA and short stature treated with growth hormone (GH) were investigated. Neonatal risk factors were analysed using univariate and multivariate analyses. GA cut-off value was determined using receiver operating characteristic (ROC) curve analyses. Of 604 infants born ≤32 weeks' GA, 76 (13%) were SGA at birth. Twenty-seven infants (36%) developed short stature at age 2 and 14 infants (19%) received GH treatment at age 3. GA, birthweight, birth length, birth head circumference, and chronic lung disease at 36 weeks' corrected GA were determined as risk factors by univariate analyses (p < 0.01). Multivariate analyses only revealed low GA as an independent risk factor. ROC curve analysis determined a cut-off value of 24 weeks' GA. Nineteen percent of preterm SGA infants ≤32 weeks' GA developed short stature treated with GH. A low GA at birth could be an early detection marker for short stature that requires GH treatment in preterm infants born SGA.
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Trastornos del Crecimiento/tratamiento farmacológico , Hormona de Crecimiento Humana/administración & dosificación , Enfermedades del Recién Nacido/tratamiento farmacológico , Recien Nacido Prematuro/crecimiento & desarrollo , Tamaño Corporal , Preescolar , Femenino , Edad Gestacional , Trastornos del Crecimiento/fisiopatología , Humanos , Lactante , Recién Nacido , Enfermedades del Recién Nacido/fisiopatología , Japón , Masculino , Embarazo , Estudios ProspectivosRESUMEN
PURPOSE: Hypoglycemia is a common and life-threatening complication in type 1 diabetes mellitus (T1DM) patients. Current guidelines recommend glucagon for treating hypoglycemia in out-of-hospital settings; however, glucagon is reportedly underused in such patients. We conducted a doctor-oriented, questionnaire-based survey of pediatricians and physicians to determine the glucagon prescription rate and identify the reason(s) for its underuse in T1DM patients. METHODS: A questionnaire was mailed to 415 pediatricians and 200 physicians employed at 66 facilities with >100 general wards throughout Hyogo, Japan. The following variables were surveyed: doctor's specialty, glucagon prescription rate, familiarity with glucagon use guidelines, barriers to prescribing glucagon, and attitude changes after education. RESULTS: After 16 doctors were found to have retired, 599 doctors were enrolled; 305 (187 pediatricians and 118 physicians) returned a completed questionnaire. In all, 45 pediatricians and 104 physicians were treating T1DM patients, of whom 24% and 28% reported prescribing glucagon, respectively. The guideline familiarity rate among pediatricians was lower than that among physicians. The major barrier to prescribing glucagon was the complex preparation procedure required by patients/caregivers. More than half of the doctors who did not prescribe glucagon began doing so after being educated about the guidelines. CONCLUSION: The glucagon prescription rate was low among both pediatricians and physicians in Japan.
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Diabetes Mellitus Tipo 1/sangre , Prescripciones de Medicamentos/estadística & datos numéricos , Glucagón/uso terapéutico , Hipoglucemia/tratamiento farmacológico , Pautas de la Práctica en Medicina/estadística & datos numéricos , Adulto , Niño , Estudios Transversales , Diabetes Mellitus Tipo 1/complicaciones , Femenino , Encuestas de Atención de la Salud , Humanos , Hipoglucemia/sangre , Hipoglucemia/etiología , Japón , MasculinoRESUMEN
Mutations of the hepatocyte nuclear factor 4α (HNF4α) gene give rise to maturity-onset diabetes of the young type 1. Although many such mutations have been identified in affected individuals, part of these mutations has been characterized with regard to their pathological relevance. We here identified a missense mutation (c.773G>A, p.R258H) of HNF4A in a mother and daughter with early-onset diabetes and impaired insulin secretion. In silico simulation and in vitro luciferase reporter analyses showed that the mutation impairs the stability of self-dimerization and the transactivation activity of HNF4α. Although arginine-258 does not appear to participate directly in dimerization, its mutation alters the electrostatic surface potential of the dimer interface. Our results thus suggest that this mutation impairs the function of HNF4α and thereby contributes to the pathogenesis of maturity-onset diabetes of the young type 1.
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Simulación por Computador , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Factor Nuclear 4 del Hepatocito/genética , Mutación Missense , Femenino , Humanos , Técnicas In Vitro , Recién Nacido , PronósticoRESUMEN
BACKGROUND: Duchenne muscular dystrophy (DMD), the most common inherited muscular disease in childhood, is caused by dystrophin deficiency because of mutations in the DMD gene. Although DMD is characterized by fatal progressive muscle wasting, cardiomyopathy is the most important nonmuscle symptom threatening the life of patients with DMD. The relationship between cardiac involvement and dystrophin isoforms has not been analyzed. METHODS AND RESULTS: The results of 1109 echocardiograms obtained from 181 Japanese DMD patients with confirmed mutations in the DMD gene were retrospectively analyzed. Patients showed an age-related decline in left ventricular ejection fraction. Patients were divided by patterns of dystrophin isoform deficiency into 5 groups. The cardiac dysfunction-free survival was significantly higher in the group with mutations in the Dp116 coding region than the others, whereas no significant differences in the other 3 groups. At age 25 years, the cardiac dysfunction-free rate was 0.6 in the Dp116 group, but only 0.1 in others. PCR amplification of Dp116 transcript in human cardiac muscle indicated promoter activation. CONCLUSIONS: Left ventricular ejection fraction in DMD declined stepwise with age. Cardiac dysfunction was less frequent in Dp116-deficient than other patients with DMD. Dp116 transcript was identified in human cardiac muscle for the first time. These results indicate that Dp116 is associated with cardiac involvement in DMD.
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Distrofina/genética , Distrofia Muscular de Duchenne/genética , Función Ventricular Izquierda/fisiología , Adolescente , Adulto , Niño , Preescolar , Supervivencia sin Enfermedad , Distrofina/metabolismo , Humanos , Estimación de Kaplan-Meier , Distrofia Muscular de Duchenne/mortalidad , Distrofia Muscular de Duchenne/patología , Sistemas de Lectura Abierta/genética , Polimorfismo Genético , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estudios Retrospectivos , Adulto JovenRESUMEN
BACKGROUND: Dystrophin Dp71 is one of the isoforms produced by the DMD gene which is mutated in patients with Duchenne muscular dystrophy (DMD). Although Dp71 is expressed ubiquitously, it has not been detected in normal skeletal muscle. This study was performed to assess the expression of Dp71 in human skeletal muscle. METHODS: Human skeletal muscle RNA and tissues were obtained commercially. Mouse skeletal muscle was obtained from normal and DMDmdx mice. Dp71 mRNA and protein were determined by reverse-transcription PCR and an automated capillary Western assay system, the Simple Western, respectively. Dp71 was over-expressed or suppressed using a plasmid expressing Dp71 or antisense oligonucleotide, respectively. RESULTS: Full-length Dp71 cDNA was PCR amplified as a single product from human skeletal muscle RNA. A ca. 70 kDa protein peak detected by the Simple Western was determined as Dp71 by over-expressing Dp71 in HEK293 cells, or suppressing Dp71 expression with antisense oligonucleotide in rhabdomyosarcoma cells. The Simple Western assay detected Dp71 in the skeletal muscles of both normal and DMD mice. In human skeletal muscle, Dp71 was also detected. The ratio of Dp71 to vinculin of human skeletal muscle samples varied widely, indicating various levels of Dp71 expression. CONCLUSIONS: Dp71 protein was detected in human skeletal muscle using a highly sensitive capillary Western blotting system.
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Distrofina/metabolismo , Músculo Esquelético/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Distrofina/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Vinculina/genética , Vinculina/metabolismoRESUMEN
INTRODUCTION: Few long-term cohort studies have addressed changes in the ambulatory capacity of patients with Duchenne muscular dystrophy (DMD), and no reports have evaluated the factors associated with ambulatory capacity in Japanese. METHODS: The longitudinal changes in 10-meter run/walk ability and associated factors were retrospectively investigated using general practice data. The factors associated with loss of this ability before the age of 10â¯years were explored by logistic regression analysis using parameters of genetic mutations, corticosteroid use, the manual muscle test (MMT), and the joint range of motion (ROM). Explanatory variables of MMT grade included hip flexors, knee flexors, and knee extensors; ROM included hip extension, knee extension, and ankle dorsiflexion. RESULTS: Among 418 patients diagnosed with DMD, 145 patients underwent the 10-meter run/walk test between March 1999 and July 2015. The median age at loss of 10-meter walking ability was 10.4 (interquartile range: 9.2-11.3) years. The 10-meter run/walk speed began to decline 3â¯years before the loss of 10-meter walking ability, and the median was <1â¯m/s 1â¯year before the loss of 10-meter walking ability. MMT grade for knee flexors and ROM for hip and knee extension were identified as independent predictors. Based on the change over time of these three items, limitation of the hip extension ROM preceded knee flexor weakness and limitation of the knee extension ROM. CONCLUSIONS: This knowledge can be used in optimizing rehabilitation programs and evaluating effect of treatment for DMD patients.
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Distrofia Muscular de Duchenne/fisiopatología , Caminata , Corticoesteroides/uso terapéutico , Niño , Progresión de la Enfermedad , Prueba de Esfuerzo , Humanos , Japón , Modelos Logísticos , Estudios Longitudinales , Extremidad Inferior/fisiopatología , Masculino , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/epidemiología , Distrofia Muscular de Duchenne/genética , Rango del Movimiento Articular , Estudios RetrospectivosRESUMEN
A Japanese woman aged in her late 30s with severe insulin resistance and bodily features including a triangular face, prominent forehead, small chin, large and low-set ears, and ocular depression was investigated. A similar phenotype was not observed in other family members with the exception of her son, suggesting that the condition was caused by a de novo mutation that was transmitted from mother to son. Exome analysis showed the presence in the proband and her son of a c.1945C>T mutation in PIK3R1, a common mutation associated with SHORT (short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay) syndrome. Administration of a sodium-glucose cotransporter 2 inhibitor lowered the proband's hemoglobin A1c level and allowed a reduction in her insulin dose without treatment-related adverse events including ketoacidosis, exaggerated loss of body mass or hypoglycemia. Sodium-glucose cotransporter 2 inhibitors might thus offer an additional option for the treatment of genetic syndromes of severe insulin resistance.
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Resistencia a la Insulina/genética , Mutación/genética , Fosfatidilinositol 3-Quinasas/genética , Índice de Severidad de la Enfermedad , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Adulto , Fosfatidilinositol 3-Quinasa Clase Ia , Femenino , Humanos , Linaje , Síndrome , Resultado del TratamientoRESUMEN
Duchenne muscular dystrophy (DMD) is a fatal progressive muscle wasting disease of childhood. Titin in sarcomere is digested by calcium dependent protease. To explore muscle damage in DMD, the urinary concentrations of the N-terminal fragment of titin were determined using a newly developed enzyme linked immune sorbent assay kit. The urinary titin concentrations were normalized to creatinine (Cr). A total of 145 urine samples were obtained at a single Japanese hospital from 113 DMD patients aged 3-29years. Normalized urinary titin concentration was 965.8±1011.9 (Mean±SD) pmol/mg Cr in patients with DMD. This was nearly 700-fold higher than healthy children (1.4±0.8pmol/mg Cr). The concentration was significantly higher in DMD than in BMD patients who had significantly higher urinary titin than normal. Urinary titin in DMD patients tended to decrease with age. The median concentration of urinary titin in the youngest (aged 3-7years) and oldest (aged ≥16years) groups was 1468.3 and 411.3pmol/mg Cr, respectively, with significant difference. Urinary concentration of titin correlated significantly with serum creatine kinase concentration, the best-known biomarker of DMD. The N-terminal fragment of titin in urine has potential as a diagnostic and clinical biomarker for DMD.
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Conectina/orina , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/orina , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Conectina/sangre , Creatina Quinasa/sangre , Creatina Quinasa/metabolismo , Humanos , Distrofia Muscular de Duchenne/sangre , Distrofia Muscular de Duchenne/genética , Adulto JovenRESUMEN
BACKGROUND: Glutaryl carnitine (C5DC) in dried blood spots is used as a biomarker for glutaric aciduria type 1 (GA-1) screening. C5DC, however, is the only screening marker for this condition, and various pathological conditions may interfere with C5DC metabolism. Recently, C5DC elevation has been reported in cases of renal insufficiency. METHOD: Five patients who were positive for GA-1 on newborn screening with tandem mass spectrometry between September 2012 and March 2015 at Kobe University Hospital were enrolled in this study. RESULTS: GA-1 was not confirmed on urinary organic acids analysis in any of the patients. C5DC decreased immediately in four patients, but one patient, who had high C5DC for at least 4 months, was diagnosed with bilateral renal hypoplasia. CONCLUSION: In the case of persistently elevated C5DC, renal insufficiency should be considered as a differential diagnosis.
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Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Encefalopatías Metabólicas/diagnóstico , Glutaril-CoA Deshidrogenasa/deficiencia , Tamizaje Neonatal/métodos , Insuficiencia Renal/diagnóstico , Diagnóstico Diferencial , Humanos , Recién Nacido , Espectrometría de Masas en TándemRESUMEN
The Duchenne muscular dystrophy (DMD) gene is one of the largest genes in the human genome. The gene exhibits a complex arrangement of seven alternative promoters, which drive the expression of three full length and four shorter isoforms. Dp116, the second smallest product of the DMD gene, is a Schwann cell-specific isoform encoded by a transcript corresponding to DMD exons 56-79, starting from a promoter/exon S1 within intron 55. The physiological roles of Dp116 are poorly understood, because of its extensive homology with other isoforms and its expression in specific tissues. This review summarizes studies on Dp116, focusing on clinical findings and alternative activation of the upstream translation initiation codon that is predicted to produce Dp118.
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Duchenne and Becker muscular dystrophy (DMD/BMD) are caused by mutations in the dystrophin gene and are characterized by severe and mild progressive muscle wasting, respectively. Short stature has been reported as a feature of DMD in the Western hemisphere, but not yet confirmed in Orientals. Height of young BMD has not been fully characterized. Here, height of ambulant and steroid naive Japanese 179 DMD and 42 BMD patients between 4 and 10 years of age was retrospectively examined using height standard deviation score (SDS). The mean height SDS of DMD was -1.08 SD that was significantly smaller than normal (p < 0.001), indicating short stature of Japanese DMD. Furthermore, the mean height SDS of BMD was -0.27 SD, suggesting shorter stature than normal. Remarkably, the mean height SDS of DMD was significantly smaller than that of BMD (p < 0.0001). In DMD higher incidence of short stature (height SDS < -2.5 SD) was observed in Dp71 subgroup having mutations in dystrophin exons 63-79 than others having mutations in exons 1-62 (27.8% vs. 7.5%, p = 0.017). These suggested that height is influenced by dystrophin in not only DMD but also BMD and that dystrophin Dp71 has a role in height regulation.
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Estatura/genética , Distrofina/genética , Distrofia Muscular de Duchenne/epidemiología , Distrofia Muscular de Duchenne/genética , Mutación , Factores de Edad , Índice de Masa Corporal , Niño , Preescolar , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Estudios RetrospectivosRESUMEN
BACKGROUND: One of the main complications in patients with muscular dystrophies is cardiac dysfunction. The literature on cardiac involvement in patients with Fukuyama congenital muscular dystrophy (FCMD) is limited. AIM: To compare cardiac involvement between patients with FCMD and Duchenne muscular dystrophy (DMD). METHODS: We compared cardiac involvement between 30 patients with FCMD and 181 patients with DMD using echocardiography and serum biomarkers. All patients were receiving regular checkups at Kobe University Hospital. We used single regression analysis to compare echocardiographic parameters, age, and serum biomarkers. RESULTS: Almost all clinical and echocardiographic parameters were lower in patients with FCMD than DMD. The brain natriuretic peptide concentration in patients with FCMD showed no correlation with age or left ventricular ejection fraction (r=0.231, p=0.22 and r=0.058, p=0.76, respectively). A log-rank test revealed that the risk of left ventricular systolic dysfunction was lower in patients with FCMD than DMD (p=0.046, hazard ratio=0.348). CONCLUSION: The clinical progression of cardiac dysfunction is significantly milder in patients with FCMD than DMD, while skeletal muscle involvement is significantly worse in patients with FCMD. These data suggest that the pathophysiological findings of FCMD can be explained by less severe cardiac dysfunction in FCMD than DMD.
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Distrofia Muscular de Duchenne/complicaciones , Distrofia Muscular de Duchenne/fisiopatología , Síndrome de Walker-Warburg/complicaciones , Síndrome de Walker-Warburg/fisiopatología , Adolescente , Biomarcadores/sangre , Niño , Ecocardiografía/métodos , Femenino , Corazón/fisiopatología , Insuficiencia Cardíaca , Humanos , Masculino , Músculo Esquelético/fisiología , Péptidos Natriuréticos/análisis , Péptidos Natriuréticos/sangre , Función Ventricular Izquierda/fisiologíaRESUMEN
BACKGROUND: The clinical features of patients with very early-onset acquired demyelinating syndrome (ADS) with the anti-myelin oligodendrocyte glycoprotein (MOG) antibody are unknown. We investigated the clinical characteristics and described detailed treatment of weekly intramuscular interferon ß-1a (IFNß-1a) in children aged <4years with ADS and the anti-MOG antibody. METHODS: We conducted a retrospective chart review of patients with anti-MOG positivity who were diagnosed as having multiple sclerosis (MS) at <4years of age. RESULTS: Subjects comprised 2 boys and 2 girls. Initial symptoms included ataxia, facial paresis, status epilepticus, and encephalopathy. Abnormal lesions on magnetic resonance imaging scans were often detected in the brainstem and cerebellum as well as the cerebrum. All patients started receiving IFNß-1a at age 3.1-3.5years. The initial doses ranged from 3 to 6µg, which were 1/10-1/5 doses, respectively, for adults. During 0.6-4.3years of IFNß-1a administration, all patients had flu-like symptoms, and 1 patient had an increased liver enzyme level. Although 1 patient discontinued IFNß-1a therapy because of frequent relapses, no patient discontinued therapy due to severe adverse events. CONCLUSIONS: This case series adds novel information regarding the clinical features of children <4years old with ADS and the anti-MOG antibody.
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Autoanticuerpos/sangre , Enfermedades Desmielinizantes/sangre , Enfermedades Desmielinizantes/inmunología , Glicoproteína Mielina-Oligodendrócito/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Edad de Inicio , Preescolar , Enfermedades Desmielinizantes/diagnóstico por imagen , Enfermedades Desmielinizantes/tratamiento farmacológico , Femenino , Humanos , Inyecciones Intramusculares , Interferón beta-1a/uso terapéutico , Imagen por Resonancia Magnética , Masculino , Estudios RetrospectivosRESUMEN
BACKGROUND: The DMD gene encoding dystrophin is mutated in Duchenne muscular dystrophy, a fatal progressive muscle wasting disease. DMD has also been shown to act as a tumor suppressor gene. Rhabdomyosarcoma (RMS) is a mesodermal sarcoma that shares characteristics of skeletal muscle precursors. Products of the DMD gene in RMS have not yet been fully clarified. Here, DMD products were analyzed in CRL-2061 cells established from alveolar RMS. METHODS: The 14-kb long DMD cDNA was PCR amplified as 20 separated fragments, as were nine short intron regions. Dystrophin was analyzed by Western blotting using an antibody against the C-terminal region of dystrophin. RESULTS: Sixteen of the 20 DMD cDNA fragments could be amplified from CRL-2061 cells as muscle cDNA. Three fragments included aberrant gene products, including one in which exon 71 was omitted and one each with retention of introns 40 and 58. In one fragment, extending from exon 70 to 79, no normally spliced product was obtained. Rather, six alternatively spliced products were identified, including a new product deleting exon 73, with the most abundant product showing deletion of exon 78. Although dystrophin expression was expected in CRL-2061 cells, western blotting of cell lysates showed no evidence of dystrophin, suggesting that translation of full-length DMD mRNA was inhibited by intron retention that generated a premature stop codon. Intron specific PCR amplification of nine short introns, showed retention of introns 40, 58, and 70, which constituted about 60, 25 and 9%, respectively, of the total PCR amplified products. The most abundant DMD transcript contained two abnormalities, intron 40 retention and exon 78 skipping. CONCLUSIONS: Intron-specific PCR amplification showed that DMD transcripts contained high levels of introns 40, 58 and 70. Retention of these introns may have been responsible for the lack of dystrophin expression by CRL-2061 cells, thereby abolishing the tumor suppressor activity of dystrophin.
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Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease characterized by dystrophin deficiency from mutations in the dystrophin gene. Antisense oligonucleotide (AO)-mediated exon skipping targets restoration of the dystrophin reading frame to allow production of an internally deleted dystrophin protein with functional benefit for DMD patients who have out-of-frame deletions. After accelerated US approval of eteplirsen (Exondys 51), which targets dystrophin exon 51 for skipping, efforts are now focused on targeting other exons. For improved clinical benefits, this strategy requires more studies of the delivery method and modification of nucleic acids. We studied a nucleotide with a 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), which shows high nuclease resistance and high affinity for complementary RNA strands. Here, we describe the process of developing a 2'-O-methyl RNA(2'-OMeRNA)/ENA chimera AO to induce dystrophin exon 45 skipping. One 18-mer 2'-OMeRNA/ENA chimera (AO85) had the most potent activity for inducing exon 45 skipping in cultured myotubes. AO85 was administered to mdx mice without significant side effects. AO85 transfection into cultured myotubes from 13 DMD patients induced exon 45 skipping in all samples at different levels and dystrophin expression in 11 patients. These results suggest the possible efficacy of AO-mediated exon skipping changes in individual patients and highlight the 2'-OMeRNA/ENA chimera AO as a potential fundamental treatment for DMD.
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Next-generation sequencing (NGS) discloses nucleotide changes in the genome. Mutations at splicing regulatory elements are expected to cause splicing errors, such as exon skipping, cryptic splice site activation, partial exon loss or intron retention. In dystrophinopathy patients, prediction of splicing outcomes is essential to determine the phenotype: either severe Duchenne or mild Becker muscular dystrophy, based on the reading frame rule. In a Vietnamese patient, NGS identified a c.9361+1G>A mutation in the dystrophin gene and an additional DNA variation of A>G at +117 bases in intron 64. To ascertain the consequences of these DNA changes on dystrophin splicing, minigene constructs were prepared inserting dystrophin exon 64 plus various lengths of intron 64. Exon 64 skipping was observed in the minigene construct with 160 nucleotide (nt) of intron 64 sequence with both c.9361+1A and +117G. In contrast, minigene constructs with larger flanking intronic domains resulted in cryptic splice site activation rather than exon skipping. Meanwhile, the cryptic splice site activation was induced even in +117G when intron 64 was elongated to 272 nt and longer. It was expected that cryptic splice site activation is an in vivo splicing outcome.
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Distrofina/genética , Exones/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación/genética , Sitios de Empalme de ARN/genética , Secuencia de Bases , Niño , Preescolar , Humanos , Intrones/genética , Masculino , Empalme del ARN/genéticaRESUMEN
Enterovirus D68 (EV-D68) infection is associated with upper and lower respiratory tract symptoms such as fever, cough, and wheezing. Pediatric patients with EV-D68 infection easily develop more severe respiratory complications compared to patients infected with other species of enterovirus, and consequently, have a higher rate of hospitalization and admission to intensive care units. Therefore, the clinical picture of respiratory complications associated with EV-D68 infection needs to be elucidated. Here, we report a 4-year-old girl of EV-D68 infection that required artificial respiration management within 24 h from the onset of cold symptoms. The patient was diagnosed with interstitial pneumonia on the basis of chest imaging findings with patchy, funicular and frosted glassy shadows, increased blood markers of surfactant protein-A, surfactant protein-D and sialylated carbohydrate antigen KL-6, and increased neutrophils and lymphocytes in the bronchoalveolar lavage. Steroids showed a remarkable effect in her treatment. Further investigations are needed to confirm the efficacy of steroids for interstitial pneumonia due to EV-D68 infection. As rapid deterioration of respiratory status is observed in EV-D68 infection, the possibility of interstitial pneumonia may be considered.