RESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (≤8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4-8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1-8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.
Asunto(s)
Haplotipos/genética , Distrofia Muscular Facioescapulohumeral/diagnóstico , Distrofia Muscular Facioescapulohumeral/genética , Secuencias Repetidas en Tándem/genética , Adulto , Anciano , Brasil/epidemiología , Cromosomas Humanos Par 4/genética , Femenino , Pruebas Genéticas , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Polimorfismo GenéticoRESUMEN
Myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (proximal muscular myopaty/DM2) are caused by similar dynamic mutations at two distinct genetic loci. The two diseases also lead to similar phenotypes but different clinical severity. Dysregulation of alternative splicing has been suggested as the common pathogenic mechanism. Here, we investigate the molecular differences between DM1 and DM2 using reverse transcriptase-polymerase chain reaction of troponin T (TnT) and the insulin receptor (IR), as well as immunoblotting of TnT in muscle biopsies from DM1 and DM2 patients. We found that: (a) slow TnT was encoded by two different transcripts in significantly different ratios in DM1 and DM2 muscles; (b) DM2 muscles exhibited a higher degree of alternative splicing dysregulation for fast TnT transcripts when compared to DM1 muscles; (c) the distribution of TnT proteins was significantly skewed towards higher molecular weight species in both diseases; (d) the RNA for the insulin-independent IR-A isoform was significantly increased and appeared related to the fibre-type composition in the majority of the cases examined. On the whole, these data should give a better insight on pathogenesis of DM1 and DM2.
Asunto(s)
Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Trastornos Miotónicos/genética , Receptor de Insulina/genética , Troponina T/genética , Adulto , Empalme Alternativo/genética , Estudios de Casos y Controles , Humanos , Persona de Mediana Edad , Fibras Musculares Esqueléticas/clasificación , Trastornos Miotónicos/clasificación , Trastornos Miotónicos/metabolismo , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN/análisis , Receptor de Insulina/metabolismo , Valores de Referencia , Troponina T/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Dystroglycanopathy (α-DG) is a relatively common, clinically and genetically heterogeneous category of congenital forms of muscular dystrophy (CMD) and limb-girdle muscular dystrophy (LGMD) associated with hypoglycosylated α-dystroglycan. To date, mutations in at least 19 genes have been associated with α-DG. One of them, GMPPB, encoding the guanosine-diphosphate-mannose (GDP-mannose) pyrophosphorylase B protein, has recently been associated with a wide clinical spectrum ranging from severe Walker-Warburg syndrome to pseudo-metabolic myopathy and even congenital myasthenic syndromes. We re-sequenced the full set of known disease genes in 73 Italian patients with evidence of either reduced or nearly absent α-dystroglycan to assess genotype-phenotype correlations in this cohort. We used innovative bioinformatic tools to calculate the effects of all described GMPPB mutations on protein function and attempted to correlate them with phenotypic expressions. RESULTS: We identified 13 additional cases from 12 families and defined seven novel mutations. Patients displayed variable phenotypes including less typical pictures, ranging from asymptomatic hyperCKemia, to arthrogryposis and congenital clubfoot at birth, and also showed neurodevelopmental comorbidities, such as seizures and ataxic gait, as well as autism-spectrum disorder, which is seldom described in clinical reports of dystroglycanopathies. We also demonstrated that few mutations recur in the Italian GMPPB-mutated population and that alterations of protein stability are the main effects of GMPPB missense variants. CONCLUSION: This work adds to the data on genotype-phenotype correlations in α-DG and offers new bionformatic tools to provide the conceptual framework needed to understand the complexity of these disorders.
Asunto(s)
Distrofia Muscular de Cinturas/metabolismo , Distrofias Musculares/metabolismo , Adulto , Anciano , Estudios Transversales , Distroglicanos/metabolismo , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Distrofias Musculares/genética , Distrofia Muscular de Cinturas/genética , Mutación/genética , Mutación Missense/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Adulto JovenRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is characterized by extreme variability in symptoms, with females being less severely affected than males and presenting a higher proportion of asymptomatic carriers. The sex-related factors involved in the disease are not known. Here, we have utilized myoblasts isolated from FSHD patients (FSHD myoblasts) to investigate the effect of estrogens on muscle properties. Our results demonstrated that estrogens counteract the differentiation impairment of FSHD myoblasts without affecting cell proliferation or survival. Estrogen effects are mediated by estrogen receptor ß (ERß), which reduces chromatin occupancy and transcriptional activity of double homeobox 4 (DUX4), a protein whose aberrant expression has been implicated in FSHD pathogenesis. During myoblast differentiation, we observed that the levels and activity of DUX4 increased progressively and were associated with its enhanced recruitment in the nucleus. ERß interfered with this recruitment by relocalizing DUX4 in the cytoplasm. This work identifies estrogens as a potential disease modifier that underlie sex-related differences in FSHD by protecting against myoblast differentiation impairments in this disease.
Asunto(s)
Estradiol/fisiología , Estrógenos/fisiología , Proteínas de Homeodominio/metabolismo , Distrofia Muscular Facioescapulohumeral/metabolismo , Mioblastos/fisiología , Diferenciación Celular , Células Cultivadas , Receptor beta de Estrógeno/metabolismo , Expresión Génica , Humanos , Distrofia Muscular Facioescapulohumeral/patología , Transporte de Proteínas , Activación TranscripcionalRESUMEN
BACKGROUND: Hereditary spastic paraplegia (HSP) is a group of genetically heterogeneous disorders characterized by progressive spasticity of the lower limbs. Mutations in the SPG4 gene, which encodes spastin protein, are responsible for up to 45% of autosomal dominant cases. OBJECTIVE: To search for disease-causing mutations in a large series of Italian patients with HSP. DESIGN: Samples of DNA were analyzed by direct sequencing of all exons in SPG4. Samples from a subset of patients were also analyzed by direct sequencing of all exons in SPG3A, SPG6, SPG10, and SPG13. SETTING: Molecular testing facility in Italy. PATIENTS: Sixty unrelated Italian patients with pure (n = 50) and complicated (n = 10) HSP. MAIN OUTCOME MEASURES: Mutations in SPG4, SPG3A, SPG6, SPG10, and SPG13. RESULTS: We identified 12 different mutations, 8 of which were novel, in 13 patients. No mutations of any of the other HSP genes tested were found in 15 patients with sporadic pure HSP who did not have mutations in the SPG4 gene. CONCLUSIONS: The overall rate of mutation in the SPG4 gene within our sample was 22%, rising to 26% when only patients with pure HSP were considered. The negative result obtained in 15 patients without mutations in SPG4 in whom 4 other genes were analyzed (SPG3A, SPG6, SPG10, and SPG13) indicate that these genes are not frequently mutated in sporadic pure HSP.
Asunto(s)
Adenosina Trifosfatasas/genética , Mutación , Paraplejía Espástica Hereditaria/genética , Adulto , Anciano , Análisis Mutacional de ADN/métodos , Exones , Salud de la Familia , Femenino , Humanos , Italia , Masculino , Persona de Mediana Edad , Paraplejía Espástica Hereditaria/clasificación , EspastinaRESUMEN
AIMS: To report on the diagnostic features, management, and clinical outcome after different treatments of Wilson's disease patients followed over a mean period of 15 years. PATIENTS: Thirty-five patients with Wilson's disease referred to the University of Padova's Department of Gastroenterology for diagnosis or treatment were observed for a mean 15 years. The diagnosis was based on clinical symptoms, laboratory tests (ceruloplasmin, urinary, and hepatic copper concentrations), and uptake of the radiostable isotope Cu into the plasma protein pool. Hepatic Cu content was measured by regular follow-up biopsies. Neurologic outcome after therapy was assessed using a newly developed scoring system. RESULTS: Twenty-three (65.7%) patients presented with liver disease; 12 (34.3%) had mixed neurologic and hepatic involvement. All patients had been initially treated with either penicillamine (23) or zinc sulfate (12). The neurologic symptoms became worse or remained stationary in 75% of those treated with penicillamine, whereas zinc treatment improved these symptoms in 90% of treated cases. Both treatments were effective in improving the hepatic symptoms. No differences in hepatic Cu content emerged between follow-up biopsies in either treatment group. Six patients (26%) had to abandon the penicillamine treatment due to side effects. In all, 4 patients underwent liver transplantation, which was successful in 3, with a mean survival after transplantation of 4.6 years; the fourth, who had a severe neurologic impairment, died of central pontine myelinolysis. CONCLUSIONS: Penicillamine and zinc can effectively treat Wilson's disease, though the side effects of penicillamine may be severe enough to prompt its suspension. Liver transplantation remains the treatment of choice for end-stage liver disease.
Asunto(s)
Degeneración Hepatolenticular/diagnóstico , Degeneración Hepatolenticular/terapia , Adolescente , Adulto , Alanina Transaminasa/sangre , Alanina Transaminasa/efectos de los fármacos , Biomarcadores/sangre , Biomarcadores/orina , Ceruloplasmina/efectos de los fármacos , Ceruloplasmina/metabolismo , Quelantes/administración & dosificación , Quelantes/efectos adversos , Niño , Preescolar , Cobre/orina , Femenino , Estudios de Seguimiento , Degeneración Hepatolenticular/metabolismo , Humanos , Isótopos/orina , Italia/epidemiología , Trasplante de Hígado , Imagen por Resonancia Magnética , Masculino , Penicilamina/administración & dosificación , Penicilamina/efectos adversos , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/mortalidad , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , Tomografía Computarizada por Rayos X , Oligoelementos/uso terapéutico , Resultado del Tratamiento , Zinc/uso terapéuticoRESUMEN
BACKGROUND: Subjects with facioscapulohumeral muscular dystrophy (FSHD) do not generally suffer from significant cardiac symptoms. Although with heterogeneous results, studies reported to date indicate that heart alterations unrelated to cardiomyopathy are possible in FSHD. PATIENTS AND METHODS: We describe the findings of a multicenter investigation aimed at detecting cardiac abnormalities in 83 FSHD patients, 44 males and 39 females with a mean age of 47 years. All patients underwent clinical heart examination, 12-lead electrocardiography and 24-hour Holter monitoring; echocardiography was also performed on most patients. RESULTS: Among the 83 patients, 62 with no cardiovascular risk factors were identified. Ten of them manifested clinical or subclinical cardiac involvement: 5 reported symptoms represented mostly by frequent palpitations secondary to supraventricular arrhythmia and another 5 exhibited electrocardiographic signs of short runs of supraventricular paroxysmal tachycardia. In the absence of cardiovascular risk factors, we found symptoms or signs of heart involvement of mainly arrhythmic origin in 10 of our 83 FSHD patients (12%). CONCLUSIONS: Considering our data and those available in the literature as a whole, arrhythmic alterations seem to be detected more frequently than expected in FSHD patients.
Asunto(s)
Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/epidemiología , Distrofia Muscular Facioescapulohumeral/complicaciones , Distrofia Muscular Facioescapulohumeral/epidemiología , Adolescente , Adulto , Anciano , Arritmias Cardíacas/genética , Ecocardiografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/métodos , Distrofia Muscular Facioescapulohumeral/genética , Examen Neurológico/métodos , Factores de Riesgo , Índice de Severidad de la EnfermedadRESUMEN
The diagnosis of limb girdle muscular dystrophy (LGMD) type 2A (due to mutations in the gene encoding for calpain-3) is currently based on protein analysis, but mutant patients with normal protein expression have also been identified. In this study we investigated 150 LGMD patients with normal calpain-3 protein expression, identified gene mutations by an allele-specific polymerase chain reaction test, and analyzed the mutant calpain-3 catalytic activity. Four different mutations were found in eight patients (5.5%): a frame-shifting deletion (550 A del) and three missense (R490Q, R489Q, R490W). Patients with normal calpain-3 protein expression on Western blot are a considerable proportion (20%) of our total LGMD2A population. While in control muscle the calpain-3 Ca(++)-dependent autocatalytic activity was evident within 5 minutes and was prevented by ethylene diaminetetraacetic acid, in all mutant patient samples the protein was not degraded, indicating that the normal autocatalytic function had been lost. By this new functional test, we show that conventional protein diagnosis fails to detect some mutant proteins, and prove the pathogenetic role of R490Q, R489Q, R490W missense mutations. We suggest that these mutations impair protein activity by affecting interdomain protein interaction, or reduce autocatalytic activity by lowering the Ca(++) sensitivity.