RESUMO
INTRODUCTION: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive neuromuscular disease that affects one in 3500 live-born males. The total absence of dystrophin observed in DMD patients is generally caused by mutations that disrupt the reading frame of the DMD gene, and about 80% of cases harbour deletions or duplications of one or more exons. METHODS: We reviewed 284 cases of males with a genetic diagnosis of DMD between 2007 and 2014. These patients were selected from 8 Spanish reference hospitals representing most areas of Spain. Multiplex PCR, MLPA, and sequencing were performed to identify mutations. RESULTS: Most of these DMD patients present large deletions (46.1%) or large duplications (19.7%) in the dystrophin gene. The remaining 34.2% correspond to point mutations, and half of these correspond to nonsense mutations. In this study we identified 23 new mutations in DMD: 7 large deletions and 16 point mutations. CONCLUSIONS: The algorithm for genetic diagnosis applied by the participating centres is the most appropriate for genotyping patients with DMD. The genetic specificity of different therapies currently being developed emphasises the importance of identifying the mutation appearing in each patient; 38.7% of the cases in this series are eligible to participate in current clinical trials.
Assuntos
Distrofia Muscular de Duchenne/genética , Adulto , Análise Mutacional de DNA , Distrofina/genética , Deleção de Genes , Genótipo , Humanos , Masculino , Distrofia Muscular de Duchenne/epidemiologia , Espanha/epidemiologiaRESUMO
X-linked dilated cardiomyopathy is a pure cardiac dystrophinopathy phenotype mainly caused by DMD mutations that present a specific transcription effect in cardiac tissue. We report a 26-year-old male who presented with severe dilated cardiomyopathy and high creatine kinase. The patient did not complain of skeletal muscle weakness. A muscle biopsy showed mild dystrophic changes and a low proportion of dystrophin-negative fibres. A molecular study identified a nonsense DMD mutation (p.Arg2098X) in somatic mosaicism. The ratio of mutant versus normal allele in blood and skeletal muscle suggests selective pressure against mutant muscle cells, a process known as genetic normalization. We hypothesize that this process may have mitigated skeletal muscle symptoms in this patient. This is the second report of a DMD somatic mosaic with evidence of genetic normalization in muscle. Somatic DMD mutations should be considered in patients presenting with idiopathic dilated cardiomyopathy.
Assuntos
Cardiomiopatia Dilatada/genética , Códon sem Sentido/genética , Distrofina/genética , Seleção Genética , Adulto , Western Blotting , Creatina Quinase/sangue , Análise Mutacional de DNA , Primers do DNA/genética , Humanos , Imuno-Histoquímica , Masculino , Repetições de Microssatélites/genética , Mosaicismo , Músculo EsqueléticoRESUMO
BACKGROUND: In some cases, a definitive confirmation of dysferlinopathy cannot be achieved by DNA test, because the mutation is detected in one allele only. PATIENTS AND METHODS: DYSFERLIN expression in skeletal muscle and peripheral blood monocytes (PBM) was studied by Western blot in two unrelated adult patients. The comparative C(T) method (ΔΔC(T) ) was used to calculate relative changes in dysferlin mRNA determined from real-time quantitative PCR experiments. The dysferlin gene was studied by direct sequencing of cDNA and genomic DNA and by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. RESULTS: A comparable severe reduction in dysferlin was demonstrated in both skeletal muscle and PBM. The expression of dysferlin mRNA was significantly reduced. A novel mutation in exon 47 (c.5289G>C) of the dysferlin gene in the heterozygous state, causing an amino acid change (p.Glu1763Asp), was detected in both patients. The MLPA analysis did not reveal any deletion or duplication. CONCLUSIONS: Dysferlin and/or dysferlin mRNA abnormalities are diagnostic for dysferlinopathy when mutational analysis detects a mutation in one allele only. Analysis of dysferlin mRNA can be helpful for distinguishing symptomatic heterozygotes from such patients.
Assuntos
Proteínas de Membrana/genética , Monócitos/patologia , Proteínas Musculares/genética , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Distrofia Muscular do Cíngulo dos Membros/genética , Adulto , Alelos , Western Blotting , Análise Mutacional de DNA , Disferlina , Feminino , Heterozigoto , Humanos , Masculino , Monócitos/metabolismo , Músculo Esquelético/metabolismo , Linhagem , Mutação Puntual , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We report two patients with a new phenotype of dysferlinopathy presenting as congenital muscular disease. Both patients showed weakness in proximal lower limbs and neck flexor muscles at birth. The presence of normal CK levels during the first years should be noted. Initial MRI showed no abnormalities but short-time-inversion-recovery (STIR) sequences revealed a striking myoedema in gastrocnemius and hamstring muscles at the age of 5. Muscle biopsy showed mild dystrophic features and the absence of dysferlin. Dysferlin gene (DYSF) analysis revealed a p.Ala927LeufsX21 mutation in a homozygous state in both siblings. This new phenotype widens the clinical spectrum of dysferlin myopathies.
Assuntos
Predisposição Genética para Doença/genética , Proteínas de Membrana/genética , Proteínas Musculares/genética , Músculo Esquelético/patologia , Doenças Musculares/congênito , Doenças Musculares/genética , Mutação/genética , Substituição de Aminoácidos/genética , Biópsia , Pré-Escolar , Creatina Quinase/análise , Creatina Quinase/metabolismo , Análise Mutacional de DNA , Disferlina , Edema/genética , Edema/patologia , Edema/fisiopatologia , Feminino , Marcadores Genéticos/genética , Testes Genéticos , Genótipo , Humanos , Imageamento por Ressonância Magnética , Masculino , Debilidade Muscular/genética , Debilidade Muscular/patologia , Debilidade Muscular/fisiopatologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Doenças Musculares/fisiopatologia , FenótipoRESUMO
Dysferlin protein is expressed in peripheral blood monocytes. The genomic analysis of the DYSF gene has proved to be time consuming because it has 55 exons. We designed a mutational screening strategy based on cDNA from monocytes to find out whether the mutational analysis could be performed in mRNA from a source less invasive than the muscle biopsy. We studied 34 patients from 23 families diagnosed with dysferlinopathy. The diagnosis was based on clinical findings and on the absence of protein expression using either immunohistochemistry or Western blot of skeletal muscle and/or monocytes. We identified 28 different mutations, 13 of which were novel. The DYSF mutations in both alleles were found in 30 patients and only in one allele in four. The results were confirmed using genomic DNA in 26/34 patients. This is the first report to furnish evidence of reliable mutational analysis using monocytes cDNA and constitutes a good alternative to genomic DNA analysis.
Assuntos
Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Proteínas Musculares/metabolismo , Distrofias Musculares/genética , Mutação , Análise Mutacional de DNA/métodos , Disferlina , Saúde da Família , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/patologia , RNA Mensageiro/genéticaRESUMO
Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetic disorders, all of which impair neuromuscular transmission. Epidemiological data and frequencies of gene mutations are scarce in the literature. Here we describe the molecular genetic and clinical findings of sixty-four genetically confirmed CMS patients from Spain. Thirty-six mutations in the CHRNE, RAPSN, COLQ, GFPT1, DOK7, CHRNG, GMPPB, CHAT, CHRNA1, and CHRNB1 genes were identified in our patients, with five of them not reported so far. These data provide an overview on the relative frequencies of the different CMS subtypes in a large Spanish population. CHRNE mutations are the most common cause of CMS in Spain, accounting for 27% of the total. The second most common are RAPSN mutations. We found a higher rate of GFPT1 mutations in comparison with other populations. Remarkably, several founder mutations made a large contribution to CMS in Spain: RAPSN c.264C > A (p.Asn88Lys), CHRNE c.130insG (Glu44Glyfs*3), CHRNE c.1353insG (p.Asn542Gluf*4), DOK7 c.1124_1127dup (p.Ala378Serfs*30), and particularly frequent in Spain in comparison with other populations, COLQ c.1289A > C (p.Tyr430Ser). Furthermore, we describe phenotypes and distinguishing clinical signs associated with the various CMS genes which might help to identify specific CMS subtypes to guide diagnosis and management.
Assuntos
Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/fisiopatologia , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Miastênicas Congênitas/classificação , Síndromes Miastênicas Congênitas/epidemiologia , Espanha/epidemiologia , Adulto JovemRESUMO
Hutchinson-Gilford progeria syndrome is a very rare but well-characterized genetic disorder that causes premature ageing. Clinical features affect growth, skeleton, body fat, skin, hair and the cardiovascular system. It is caused by mutations in LMNA gene, the most frequent being p.Gly608Gly (c.1824C > T) in exon 11. Here we present a four-year-old HGPS patient who presented several severe strokes and carried a heterozygous LMNA missense mutation in exon 2: p.Glu138Lys. This mutation is located far from the C-terminal region implicated in the posttranslational processing of prelamin A, but it lies within the rod domain of lamin A/C that represents a highly conserved domain specific to nuclear lamins. We hypothesize that this region could be involved in early and severe strokes in HGPS, such as those presented by our patient.