RESUMEN
Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease caused by pathogenic variations in the DMD gene. There is a need for robust DMD biomarkers for diagnostic screening and to aid therapy monitoring. Creatine kinase, to date, is the only routinely used blood biomarker for DMD, although it lacks specificity and does not correlate with disease severity. To fill this critical gap, we present here novel data about dystrophin protein fragments detected in human plasma by a suspension bead immunoassay using two validated anti-dystrophin-specific antibodies. Using both antibodies, a reduction of the dystrophin signal is detected in a small cohort of plasma samples from DMD patients when compared to healthy controls, female carriers, and other neuromuscular diseases. We also demonstrate the detection of dystrophin protein by an antibody-independent method using targeted liquid chromatography mass spectrometry. This last assay detects three different dystrophin peptides in all healthy individuals analysed and supports our finding that dystrophin protein is detectable in plasma. The results of our proof-of-concept study encourage further studies in larger sample cohorts to investigate the value of dystrophin protein as a low invasive blood biomarker for diagnostic screening and clinical monitoring of DMD.
Asunto(s)
Distrofia Muscular de Duchenne , Proteómica , Femenino , Humanos , Anticuerpos , Biomarcadores , Cromatografía Liquida , Distrofia Muscular de Duchenne/genética , Proteómica/métodos , Distrofina/sangreRESUMEN
Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin in muscle, and while all patients share the primary gene and biochemical defect, there is considerable patient-patient variability in clinical symptoms. We sought to develop multivariate models of serum protein biomarkers that explained observed variation, using functional outcome measures as proxies for severity. Serum samples from 39 steroid-naïve DMD boys 4 to <7 years enrolled into a clinical trial of vamorolone were studied (NCT02760264). Four assessments of gross motor function were carried out for each participant over a 6-week interval, and their mean was used as response for biomarker models. Weighted correlation network analysis was used for unsupervised clustering of 1305 proteins quantified using SOMAscan® aptamer profiling to define highly representative and connected proteins. Multivariate models of biomarkers were obtained for time to stand performance (strength phenotype; 17 proteins) and 6 min walk performance (endurance phenotype; 17 proteins) including some shared proteins. Identified proteins were tested with associations of mRNA expression with histological severity of muscle from dystrophinopathy patients (n = 28) and normal controls (n = 6). Strong associations predictive of both clinical and histological severity were found for ERBB4 (reductions in both blood and muscle with increasing severity), SOD1 (reductions in muscle and increases in blood with increasing severity) and CNTF (decreased levels in blood and muscle with increasing severity). We show that performance of DMD boys was effectively modeled with serum proteins, proximal strength associated with growth and remodeling pathways and muscle endurance centered on TGFß and fibrosis pathways in muscle.
Asunto(s)
Biomarcadores/sangre , Distrofina/sangre , Distrofia Muscular de Duchenne/sangre , Niño , Preescolar , Humanos , Masculino , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/patología , Oligonucleótidos , Fenotipo , Pregnadienodioles/administración & dosificación , Índice de Severidad de la Enfermedad , Esteroides/metabolismoRESUMEN
The response of rainbow trout (Oncorhynchus mykiss, Walbaum) towards probiotics present in the feed was investigated by examining the proteome of serum as a measure of the acute phase response (APR). Proteomic analysis by two-dimensional electrophoresis (2D) concurrently with mass spectrometry was used to detect APR related proteins in rainbow trout serum following feeding with probiotics Aeromonas sobria GC2 and Bacillus sp. JB-1. Three candidate proteins increased following use of GC2, and were putatively identified as NADH dehydrogenase, dystrophin and mKIAA0350. Conversely, one of the proteins, which were induced following use of JB-1 was identified as transferrin.
Asunto(s)
Reacción de Fase Aguda/sangre , Enfermedades de los Peces/sangre , Oncorhynchus mykiss/sangre , Probióticos/farmacología , Proteómica/métodos , Reacción de Fase Aguda/inmunología , Aeromonas/inmunología , Animales , Acuicultura/métodos , Bacillus/inmunología , Distrofina/sangre , Electroforesis en Gel Bidimensional/veterinaria , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/prevención & control , NADH Deshidrogenasa/sangre , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/veterinaria , Transferrina/metabolismoRESUMEN
Cardiac involvement is common in skeletal muscles disorders associated with dystrophin defect. It has been suggested however, that X-linked dilated cardiomyopathies with minimal or absent skeletal disease are distinct entities, resulting from mutations in cardiac-specific regions of dystrophin gene. This study presents a unique observation of phenotypic variability in monozygotic triplets with Becker's muscular dystrophy. The expressions of the disease range from severe peripheral myopathy to severe congestive heart failure. No deletion in dystrophin gene was observed and the mechanisms responsible for selective impairment of morphologically and functionally different muscles in three monozygotic siblings remain unclear.
Asunto(s)
Cardiomiopatía Dilatada/diagnóstico , Cardiomiopatía Dilatada/etiología , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/etiología , Trillizos , Adulto , Biomarcadores/sangre , Cardiomiopatía Dilatada/genética , Creatina Quinasa/sangre , Forma MB de la Creatina-Quinasa , Diagnóstico Diferencial , Distrofina/sangre , Ecocardiografía Doppler , Electrocardiografía , Predisposición Genética a la Enfermedad/etiología , Predisposición Genética a la Enfermedad/genética , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/genética , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/fisiopatología , Humanos , Isoenzimas/sangre , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/genética , Fenotipo , Índice de Severidad de la Enfermedad , Volumen Sistólico/fisiología , Disfunción Ventricular Izquierda/diagnóstico , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/genéticaRESUMEN
OBJECTIVE: To study the effects of Si-Wu-Tang on serum protein of blood deficient mice b y proteomicstechnique and study the enriching and regulating blood mechanism of Si-Wu-Tang on mocular level. METHOD: The blood deficient mice was induced by using a single dose of 3.5 Gy radiation from a 60Cogamma source, and high resolution two-dimensional polyacryamide gel electrophoresis (2-DE), computer-assisted image analysis, and mass spectrometry were used to detect regulated protein by Si-Wu-Tang. RESULT: 12 lower and 4 higher protein in sera could be recovered by Si-Wu-Tang, 4 protein might be DNA-dependent protein kinase catalytic subunit, Dystrophin, KIF13A, dystonin. They play a part in DNA double-stranded break repair, recombination and modulation of transcription, transportation of mannose-6-phosphate receptor, etc. CONCLUSION: Si-Wu-Tang can regulate serum protein in blood deficient mice, resulting in improving hematopoiesis and lessening irradiated injury.
Asunto(s)
Medicamentos Herbarios Chinos/farmacología , Traumatismos Experimentales por Radiación/sangre , Protectores contra Radiación/farmacología , Irradiación Corporal Total , Animales , Autoantígenos/sangre , Proteínas Portadoras , Proteínas del Citoesqueleto , Combinación de Medicamentos , Medicamentos Herbarios Chinos/aislamiento & purificación , Distonina , Distrofina/sangre , Femenino , Cinesinas/sangre , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso , Colágenos no Fibrilares/sangre , Plantas Medicinales/química , Colágeno Tipo XVIIRESUMEN
White blood cells are isolated from whole blood in silicon-glass 4.5-microliter microchips containing a series of 3.5-micron feature-sized 'weir-type' filters, formed by an etched silicon dam spanning the flow chamber. Genomic DNA targets, e.g., dystrophin gene, can be directly amplified using the polymerase chain reaction (PCR) from the white cells isolated on the filters. This dual function microchip provides a means to simplify nucleic acid analyses by integrating in a single device two key steps in the analytical procedure, namely, cell isolation and PCR.
Asunto(s)
Leucocitos/citología , Reacción en Cadena de la Polimerasa/instrumentación , Separación Celular/métodos , ADN/análisis , Distrofina/sangre , Distrofina/genética , Eritrocitos/citología , Eritrocitos/metabolismo , Vidrio , Hemoglobinas/metabolismo , Humanos , Leucocitos/química , Filtros Microporos , Reacción en Cadena de la Polimerasa/métodos , SilicioRESUMEN
The objectives of this study were to evaluate a novel semiquantitative application of the bioluminescence test for screening newborns for Duchenne muscular dystrophy (DMD) and to use this technique in a pilot national program. The study was performed on the island of Cyprus, which provides ideal conditions for maximizing the prevention rate due to the small size of the country, the well-defined population, and the high degree of awareness of the public concerning genetic diseases. Guthrie spots were obtained through the national screening center for phenylketonuria and congenital hypothyroidism. The bioluminescence method for measuring creatine kinase (CK) in dried blood spots was adapted for use in a semiquantitative way. During the first 6 years of the program (1992-1997), we screened 30,014 samples and found 43 with initially high CK values. We were able to obtain repeat specimens in 35 cases. Of the repeat samples, 30 were found to have normal activity, giving a false-positive rate of 0.10%. Five boys had persistent CK elevations and were confirmed to be DMD or Becker (BMD) cases by DNA analysis and/or dystrophin analysis. The semiquantitative application of the bioluminescence assay of CK that we have introduced has proved to be a fast and reliable method for screening large numbers of samples for DMD. It has a low rate of false positives, which compares favorably with that of other DMD screening programs. Although it is early to evaluate its impact fully, the program seems to be bringing about the anticipated benefits to affected families.
Asunto(s)
Creatina Quinasa/sangre , Distrofina/sangre , Pruebas Genéticas/métodos , Distrofias Musculares/diagnóstico , Tamizaje Neonatal/métodos , Chipre/epidemiología , Análisis Mutacional de ADN , Reacciones Falso Positivas , Humanos , Recién Nacido , Isoenzimas , Mediciones Luminiscentes , Masculino , Distrofias Musculares/epidemiología , Distrofias Musculares/genética , Proyectos PilotoRESUMEN
We developed a method that allows prenatal diagnosis of Duchenne muscular dystrophy using a single nucleated erythrocyte (NRBC) isolated from maternal blood. Maternal blood was obtained at 8 to 20 weeks of gestation. NRBCs were separated with Percoll using a discontinuous density gradient method and then collected by micromanipulator under microscopic observation. The entire genome of a single cell was amplified by primer extension preamplification (PEP). Sex was determined from a small aliquot of the PEP reaction. After an NRBC was determined to be male and confirmed to be of fetal origin, dystrophin exons 4, 8, 12, 45, 48, 50, and 51 were determined from the same PEP reaction. This diagnostic method using maternal blood is safer than amniocentesis or cordocentesis and can be applied to other X-linked diseases.
Asunto(s)
Distrofina/genética , Eritrocitos/patología , Eliminación de Gen , Distrofias Musculares/diagnóstico , Diagnóstico Prenatal/métodos , Secuencia de Bases , Núcleo Celular/patología , Separación Celular , Centrifugación Zonal , Cartilla de ADN , Distrofina/sangre , Embrión de Mamíferos , Femenino , Feto , Edad Gestacional , Humanos , Masculino , Datos de Secuencia Molecular , Distrofias Musculares/sangre , Distrofias Musculares/embriología , Reacción en Cadena de la Polimerasa/métodos , Embarazo , Análisis para Determinación del Sexo , Cromosoma X , Cromosoma YRESUMEN
Eighty-four patients with Duchenne muscular dystrophy (DMD) were examined clinically for hypertrophy and wasting in different muscles, parts of the muscles or muscle groups. Some muscles were examined under mild contraction to bring out any subclinical pseudohypertrophy. Findings revealed infraspinatus muscle hypertrophy to be significantly frequent (88%) and closely second to well known calf hypertrophy (94%). Infraspinatus hypertrophy was noted in 5 such DMD patients in whom calf hypertrophy was unremarkable. The wasting was consistently observed in the muscles forming anterior and posterior axillary folds. In conclusion, infraspinatus muscle hypertrophy and wasting of axillary folds are the important supportive clinical evidences during the examination of DMD patients.
Asunto(s)
Distrofias Musculares/fisiopatología , Adolescente , Niño , Preescolar , Cartilla de ADN , Distrofina/sangre , Distrofina/genética , Eliminación de Gen , Humanos , Hipotonía Muscular , Músculo Esquelético , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Reacción en Cadena de la PolimerasaRESUMEN
We present the association of a distrophinopathy with a case of facioscupulohumeral dystrophy in two individuals belonging to the same family. The discrepancy in the seric creatinphosphokinase (CPK) of the two patients together with certain clinical data suggests the possibility that it is a question of two different processes. This impression was confirmed later through dystrophine analysis and genetic examination techniques. This case drew attention to the vital need today to insist on a combination of genetic examinations and dystrophine analysis when diagnosing muscular dystrophies, thus avoiding mistakes derived from diagnostic assumptions made on the basis of antecedents in the family involving neuromuscular disorders and the consequences that this might have regarding vital prognosis and advice to the family. This is the first case of coincidental association of these two processes within one family that has so far come to our knowledge.
Asunto(s)
Distrofina/análisis , Cara/fisiopatología , Húmero/fisiopatología , Músculos/química , Músculos/fisiopatología , Distrofias Musculares/genética , Distrofias Musculares/fisiopatología , Escápula/fisiopatología , Anticuerpos Monoclonales , Niño , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Creatina Quinasa/sangre , Distrofina/sangre , Humanos , Masculino , Distrofias Musculares/sangre , Cromosoma XRESUMEN
In order to investigate if the same apparent decrease in dystrophin negative fibers with aging observed in mouse mdx female heterozygotes also occurs in carriers of the DMD and BMD gene, we have studied the muscle of 29 DMD carriers (19 adults and 10 young daughters of obligate carriers, including 3 manifesting carriers) and 5 adult asymptomatic heterozygotes for Becker dystrophy (BMD). All young DMD possible carriers and 11 of 24 adult DMB/BMD heterozygotes had increased serum enzymes activities. A population of dystrophin negative fibers, more evident with the use of the C-terminal antibody, was seen in the three manifesting and in a 9-yr-old possible DMD carrier. In the remaining females, a positive immunohistochemical pattern of dystrophin, which did not differ from normal controls, was observed. Our results suggest that: (1) the increased population of dystrophin negative fibers reported in young mdx female heterozygotes was not seen in young DMD carriers, aged 6-17 yr; and (2) abnormalities in dystrophin immunostaining are not easily observed and are more frequent in manifesting carriers, when the muscle is grossly altered.
Asunto(s)
Distrofina/sangre , Heterocigoto , Distrofias Musculares/genética , Adolescente , Adulto , Anciano , Envejecimiento/metabolismo , Niño , Preescolar , Creatina Quinasa/sangre , Distrofina/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Persona de Mediana Edad , Músculos/patología , Distrofias Musculares/sangre , Distrofias Musculares/inmunologíaRESUMEN
In 6 patients with dystrophin-verified Becker muscular dystrophy (BMD), 3 patients had dilated cardiomyopathy (DCM group). The other 3 patients (non-DCM group) also had ECG abnormalities including incomplete right bundle branch block, left ventricular enlargement and intraventricular conduction defect. Between DCM and non-DCM group, there was no prominent difference in ages at onset, mean duration and severity of muscular weakness. Serum CK levels, and molecular weight and amount of dystrophin also showed no significant difference between two groups. On reviewing 14 BMD patients, including 3 present patients with cardiomyopathy, the cardiac symptoms appeared from 4 to 41 years, averaging 17.1 years of age. The mean duration of muscle symptoms was 9 years, ranging from 0 to 33 years. There was no correlation between severity of muscle weakness and cardiomyopathy. Six patients died of heart failure and 3 received cardiac transplantation. Thus there was no characteristic clinical feature in BMD patients with cardiomyopathy except for very poor prognosis. Since the myocardial involvement is not related with clinical severity and duration of the disease, careful observation for cardiac function should be carried out in all BMD patients even in the early stage of muscle weakness.