RESUMO
AIM: Dystonia is a medically intractable condition causing twisting or myoclonic movements and abnormal postures. There is an important heterogeneity among etiologies of dystonia. The electrical stimulation of the globus pallidus has been used successfully in primary generalized dystonia. The aim of this study was to examine the long-term efficacy and safety of deep brain stimulation (DBS) in the treatment of primary and secondary generalized dystonia in children and adults. METHODS: Fifty-three patients were included. Electrodes were bilaterally implanted under stereotactic guidance and connected to neurostimulators, subcutaneously inserted. Efficacy was evaluated by comparing scores on the clinical and functional Burke-Marsden-Fahn dystonia rating scales (BMFDRS) before and after implantation. Patients were divided into 3 groups: group 1 comprised 15 patients with DYT1 dystonia; group 2, 17 patients with dystonia of unknown etiology and group 3, 21 patients with secondary dystonia. The mean follow-up was 26.6+/-12.3 months for primary dystonia and 23.1+/-11.8 for secondary dystonia. RESULTS: After 1 year, the improvement of the clinical score is 71% in group 1, 74% in group 2 and 31% in group 3. The functional score was improved by 63% in group 1, 49% in group 2 and 7% in group 3. We did not find any significant difference between children and adults. In secondary dystonia, efficacy of the stimulation is more limited. The efficacy of the stimulation improved with time for the 3 groups. COMCLUSION: Electrical stimulation of the internal globus pallidus proved to be an effective treatment for generalized dystonia and should be considered as first-line therapy.
Assuntos
Distonia/fisiopatologia , Distonia/terapia , Terapia por Estimulação Elétrica , Globo Pálido/fisiopatologia , Adulto , Criança , Feminino , Humanos , Estudos Longitudinais , Masculino , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
Application fields of RT-PCR (reverse transcription-polymerase chain reaction) in clinical diagnosis comprises the assessment of viral load for RNA viruses and the analysis of gene transcription products. RT-PCR is also helpful when large genes have to be sequenced. Developments of quantitative approaches using real-time PCR recently led to a major widening of RT-PCR applications in clinical diagnosis. However, RT reaction is delicate due to its lack of reproducibility and to RNA lability and frequent contamination by DNA. In some cases additional difficulties come from the need to obtain a specific amplification in the presence of homologous sequences which might be present in higher amounts than the sequence of interest. These caveats have to be taken into account, when designing the RT protocol, and when choosing PCR primers and internal and/or external references. This review is aimed at helping the experimental setup of a RT-PCR based assay according to the objectives.
Assuntos
Medicina Clínica/métodos , Técnicas e Procedimentos Diagnósticos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , HumanosRESUMO
Dystonia is not uncommon in childhood, and identification of its etiology is an ultimate aim in the clinical evaluation of dystonia. Advances in neuroimaging, recent identification of gene or loci implicated in dystonic syndromes, and characterisation of new pathological entities (creatine deficiency, biotin-responsive basal ganglia disease) enlarge our understanding of childhood dystonia, and expend its diagnosis spectrum. Awareness of the diverse etiologic categories of childhood-onset dystonia is necessary to accurate diagnosis approach. Clinical examination and cerebral magnetic resonance imaging are the keys of this diagnosis approach. Primary dystonia is defined as syndromes in which dystonia is the sole phenotypic manifestation (especially no cognitive deterioration is observed, and brain MRI is normal); DYT1 dystonia, in which the abnormal gene is located on chromosome 9, is the most frequent childhood-onset primary dystonia; progressive generalisation of the abnormal movements occur in 70p.cent of the patients. Dopa - Responsive Dystonia are characterized by marked diurnal fluctuations of the dystonic symptoms and by their marked and sustained response to dopaminergic therapy; associated parkinsonian signs are usually observed later in the course of the disease. Clinical presentation of DRD might be atypical (mimicking cerebral palsy or isolated limb pain without diurnal fluctuation). DRD is rare, but a trial of L-dopa should be performed on all patients with childhood-onset dystonia, lasting at least one month. Secondary dystonias or heredodegenerative diseases are the most frequent etiology of childhood-onset dystonic syndromes. Among a huge range of heredodegenerative disease, those that are amenable to a specific treatment, such as Wilson's disease or creatine deficiency, should be particularly investigated. The main objective of investigation of dystonia is to identify secondary dystonias or heredodegenerative diseases. Further investigations will be performed according to the clinical characteristics of the dystonia, to the presence of associated neurological or extraneurological symptoms, and according to brain imaging; this approach must be discussed for each single patient. The aim of the diagnosis strategy is the rapid identification of the etiology of dystonia which will lead to accurate treatment and pertinent genetic counselling.
Assuntos
Distúrbios Distônicos/etiologia , Ritmo Circadiano , Diagnóstico Diferencial , Distúrbios Distônicos/diagnóstico , Predisposição Genética para DoençaRESUMO
Autosomal recessive Charcot-Marie-Tooth disease (CMT) type 4 (CMT4) is a complex group of demyelinating hereditary motor and sensory neuropathies presenting genetic heterogeneity. Five different subtypes that correspond to six different chromosomal locations have been described. We hereby report a large inbred Lebanese family affected with autosomal recessive CMT4, in whom we have excluded linkage to the already-known loci. The results of a genomewide search demonstrated linkage to a locus on chromosome 19q13.1-13.3, over an 8.5-cM interval between markers D19S220 and D19S412. A maximum pairwise LOD score of 5.37 for marker D19S420, at recombination fraction [theta].00, and a multipoint LOD score of 10.3 for marker D19S881, at straight theta = .00, strongly supported linkage to this locus. Clinical features and the results of histopathologic studies confirm that the disease affecting this family constitutes a previously unknown demyelinating autosomal recessive CMT subtype known as "CMT4F." The myelin-associated glycoprotein (MAG) gene, located on 19q13.1 and specifically expressed in the CNS and the peripheral nervous system, was ruled out as being the gene responsible for this form of CMT.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Cromossomos Humanos Par 19/genética , Consanguinidade , Doenças Desmielinizantes/genética , Genes Recessivos/genética , Glicoproteína Associada a Mielina/genética , Adolescente , Adulto , Idade de Início , Doença de Charcot-Marie-Tooth/epidemiologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Criança , Pré-Escolar , Mapeamento Cromossômico , Doenças Desmielinizantes/epidemiologia , Doenças Desmielinizantes/fisiopatologia , Progressão da Doença , Feminino , Heterogeneidade Genética , Marcadores Genéticos/genética , Haplótipos/genética , Humanos , Lactente , Recém-Nascido , Islamismo , Líbano , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , LinhagemRESUMO
PURPOSE: To evaluate the occurrence and inheritance of various types of pigmentary retinopathy in patients followed at the outpatient clinic in the university hospital, Montpellier, France. To characterize genes and mutations causing these conditions. METHODS: Ophthalmic examination and various visual tests were performed. Mutations were sought from genomic DNA by PCR amplification of exons associated with single-strand conformation analysis and/or direct sequencing. RESULTS: Among 315 patients over an 8-year period, cases of retinitis pigmentosa (63.2%), Usher's syndrome (10.2%), Stargardt's disease (5.4%), choroideremia (3.2%), Leber's congenital amaurosis (3.2%), congenital stationary night blindness (2.9%), cone dystrophy (2.5%), dominant optic atrophy (1.9%), X-linked juvenile retinoschisis (1.6%), Best's disease (1.6%), and others (4.3%) were diagnosed. In retinitis pigmentosa, inheritance could be determined in 54.2% of the cases including dominant autosomic (26.6%), recessive autosomic (22.6%), and X-linked cases (5%) while it could not be confirmed in 45.7% of the cases (simplex cases in the majority). For the 6 examined genes, mutations were found in 22 out of 182 propositus (12.1%). Analysis of phenotype-genotype correlations indicates that in retinitis pigmentosa, RDS is more frequently associated with macular involvement and retinal flecks, RHO with regional disease, and RPE65 with the great severity of the disease with some cases of Leber's congenital amaurosis. CONCLUSIONS: Identification of genes may help in diagnosis and in genetic counseling, especially in simplex cases with retinitis pigmentosa. In this latter condition, molecular diagnosis will be necessary to rationalize future treatments.
Assuntos
Alquil e Aril Transferases , Mapeamento Cromossômico , Proteínas da Matriz Extracelular/genética , Oftalmopatias Hereditárias/genética , Proteínas do Olho/genética , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso/genética , Proteínas/genética , Degeneração Retiniana/genética , Retinose Pigmentar/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Proteínas de Transporte , Criança , França , Humanos , Mutação , Periferinas , Reação em Cadeia da Polimerase , cis-trans-IsomerasesRESUMO
Dystonia musculorum deformans is an inherited severe disease, with a wide clinical polymorphism. The most severe clinical forms with early onset carry a high risk of life-threatening complications. In the absence of any efficient medical treatment, bilateral pallidotomy has previously been reported to be of value in the management of this disease. We report the first clinical case of a severe early-onset generalized dystonia dramatically improved by a bilateral stimulation of the internal globus pallidus. In November 1996, we proposed this neurosurgical procedure for a 8-year-old girl, who had suffered since the age of 3 from severe generalized dystonia, and who progressively became totally dependent and bedridden. She had been under sedation and permanent controlled respiratory assistance for the last two months. The etiology of the disease remained unknown (the DYT1 mutation was absent). Under general anesthesia, we bilaterally implanted a four-contacts electrode in the internal globus pallidus, using the Leksell's stereotactic frame and a 1.5 tesla MRI control. A dramatic improvement was noted 6 weeks later and led us to connect the two electrodes to neurostimulators inserted under the abdominal skin.
Assuntos
Distonia Muscular Deformante/terapia , Terapia por Estimulação Elétrica , Globo Pálido , Idade de Início , Criança , Distonia Muscular Deformante/epidemiologia , Distonia Muscular Deformante/fisiopatologia , Terapia por Estimulação Elétrica/instrumentação , Terapia por Estimulação Elétrica/métodos , Eletrodos Implantados , Feminino , Globo Pálido/fisiopatologia , Humanos , Técnicas EstereotáxicasRESUMO
PURPOSE: To elucidate the status of a previously described missense mutation (1442A>T) reported in the Rab Escort Protein 1 gene of a patient with choroideremia. METHODS: The base substitution previously described by Donnelly et al. (Hum Mol Genet 1994;3:1017) was first confirmed by direct genomic DNA sequencing. The REP-1 cDNA region encompassing exons 10-14 was then specifically amplified from lymphocyte-derived mRNA. The effect on mRNA splicing of the mutation was analyzed by RT-PCR and cDNA sequencing. RESULTS: The 1442A>T change located at the penultimate nucleotide of exon 11 causes complete skipping of this exon during the processing of REP-1 mRNA. Loss of exon 11 leads to the translation of a premature termination codon within exon 12. CONCLUSION: RT-PCR analyses demonstrated that the 1442A>T transversion previously described as a possible causative missense mutation does act as a splice-site error and gives rise to a truncated REP-1 protein. The virtual absence of any missense mutation found to be responsible for choroideremia makes the RT-PCR-based protein truncation test the most relevant genotypic diagnostic procedure for identifying mutations in the CHM gene.
Assuntos
Alquil e Aril Transferases , Coroideremia/genética , Mutação de Sentido Incorreto , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA Complementar/genética , Éxons/genética , Feminino , Amplificação de Genes , Humanos , Immunoblotting , Linfócitos/metabolismo , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In this study, single sperm typing has been used for high-resolution recombination analysis between the retinoblastoma gene and two closely linked extragenic microsatellites (D13S284 and D13S1307). The analysis of 1198 single sperm from three donors allowed the determination of recombination fractions between RB1.20 and D13S284 and RB1.20 and D13S1307 of 0.022 and 0.033, respectively. These results show that RB1 gene and the two microsatellites are closely linked, which validates their potential use in indirect genetic diagnosis of retinoblastoma.
Assuntos
Repetições de Microssatélites , Recombinação Genética , Proteína do Retinoblastoma/genética , Ligação Genética , Humanos , Masculino , EspermatozoidesAssuntos
Genes do Retinoblastoma , Aconselhamento Genético , Retinoblastoma/genética , Neoplasias Ósseas/epidemiologia , Neoplasias Ósseas/genética , Pré-Escolar , Cromossomos Humanos Par 13/genética , Análise Mutacional de DNA , Feminino , Triagem de Portadores Genéticos , Predisposição Genética para Doença , Haplótipos , Humanos , Lactente , Masculino , Neoplasias Primárias Múltiplas/genética , Osteossarcoma/epidemiologia , Osteossarcoma/genética , Linhagem , Retinoblastoma/classificação , Retinoblastoma/epidemiologia , Retinoblastoma/prevenção & controleRESUMO
PURPOSE: The aim of this study was to define the RT-PCR-PTT parameters for CHM gene analysis and to evaluate its interest as a method for CHM mutation screening. METHODS: The entire CHM coding region was reversed-transcribed in three overlapping cDNA segments (RT-PCR) which were amplified and further analyzed by PTT after in vitro transcription/translation. RESULTS: This strategy enabled us to detect a truncated peptide in each of the 6 unrelated patients from southern France who were investigated. The mutation was further characterized by direct sequencing of the RT-PCR product. CONCLUSION: In CHM gene, all conditions are present to make the RT-PCR-PTT strategy the method of choice for mutation screening. As a result of the simplified protocol described in this study, the families of the patients could benefit from accurate carrier-status assessment.
Assuntos
Alquil e Aril Transferases , Proteínas de Transporte/genética , Coroideremia/diagnóstico , Proteínas do Olho/genética , Triagem de Portadores Genéticos/métodos , Testes Genéticos/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Cromossomo X/genética , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/análise , Proteínas de Transporte/química , Coroideremia/genética , Análise Mutacional de DNA , DNA Complementar/genética , Proteínas do Olho/análise , Proteínas do Olho/química , Feminino , França , Genes Recessivos , Humanos , Masculino , Biossíntese de Proteínas , Análise de Sequência de DNARESUMO
A boy with a Becker muscular dystrophy (BMD) phenotype presented unique muscular dystrophin expression. Western blot analysis showed the presence of two dystrophins of different sizes, i.e., a 400-kDa dystrophin and a 500-kDa form. An immunofluorescent study revealed mosaic expression of these dystrophins in the sarcolemma, with matching alpha-sarcoglycan and beta-dystroglycan staining patterns. DNA and RNA analysis did not reveal any mutation in the dystrophin gene, and the karyotype was normal.
Assuntos
Distrofina/metabolismo , Mosaicismo/fisiopatologia , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Anticorpos Monoclonais/imunologia , Western Blotting , Pré-Escolar , Proteínas do Citoesqueleto/metabolismo , Progressão da Doença , Distroglicanas , Distrofina/química , Distrofina/imunologia , Imunofluorescência , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Peso Molecular , Distrofias Musculares/fisiopatologia , Valores de Referência , Sarcoglicanas , Sarcolema/metabolismoRESUMO
PURPOSE: To characterize the effect on mRNA splicing of a yet undescribed mutation located in intron 13 splice-donor sequence (IVS13 + 3A --> C) in the Rab-Escort-protein 1 gene of a patient with choroideremia. METHODS: The base substitution was firstly detected by the Single Strand conformation analysis from genomic DNA. A REP-1 cDNA region encompassing exons 10-14 was then specifically amplified from lymphocytes-derived mRNA. RESULTS: We could demonstrate that this substitution affects REP-1 RNA processing. The patient revealed only one aberrantly spliced mRNA lacking exon 13 and no normal transcript. CONCLUSION: The skipping of exon 13 results in the creation of a stop codon at the misspliced junction. This is the first case of nucleotide substitution at the +3 position of a splice donor site so far described in choroideremia.
Assuntos
Alquil e Aril Transferases , Proteínas de Transporte/metabolismo , Coroideremia/genética , DNA Recombinante , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Sequência de Bases , Coroideremia/patologia , Sequência Consenso , Éxons/genética , Angiofluoresceinografia , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Data from 6 years of experience in molecular diagnosis of Duchenne (DMD) and Becker (BMD) muscular dystrophy in Southern France are reported. DMD and BMD patients have been extensively analyzed for deletions and for point mutations in the dystrophin gene. By scanning the whole coding sequence as reverse-transcribed from lymphocytes or muscular RNA by the protein truncation test, we have reached a minimum of an 86% detection rate for point mutations responsible for DMD; these mutations consist of nonsense, frameshifting, and splicing mutations. Four of 12 small alterations identified in our sample are novel and described in this study. We also present an improved protocol for the automated detection of fluorescently labeled duplex polymerase chain reactions of six known intragenic microsatellites (Dys II, TG 15, STRs 44, 45, 49, and 50). Accurate sizing of the alleles at each locus was performed, and we elucidated the sequence of several repeat units. Allele frequencies at each of the six microsatellite loci and at one restriction fragment length polymorphism site (intron 16/TaqI) were defined in a sample of normal, DMD, and BMD X chromosomes from Southern France. The determination of the grandparental origin of either deletions or point mutations revealed differences depending on the type of the mutation, with most of the deletions occurring in oogenesis and most of the point mutations occurring in spermatogenesis.
Assuntos
Análise Mutacional de DNA/métodos , Distrofina/genética , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Reação em Cadeia da Polimerase/métodos , Southern Blotting , Feminino , França , Frequência do Gene , Ligação Genética , Humanos , Masculino , Repetições de Microssatélites , Mutação Puntual/genética , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Deleção de Sequência/genética , Fatores Sexuais , Cromossomo X/genéticaAssuntos
Proteínas de Ligação a DNA/genética , Disgenesia Gonadal 46 XY/genética , Proteínas Nucleares , Regiões Promotoras Genéticas , Processos de Determinação Sexual , Fatores de Transcrição , Adulto , Sequência de Aminoácidos , Sequência de Bases , DNA/química , DNA/genética , Análise Mutacional de DNA , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Proteína da Região Y Determinante do SexoAssuntos
Fator VIII/genética , Hemofilia A/genética , Mutação , Transcrição Gênica , Processamento Alternativo , Sequência de Aminoácidos , Inversão Cromossômica , Análise Mutacional de DNA , Mutação da Fase de Leitura , Humanos , Íntrons/genética , Masculino , Dados de Sequência Molecular , Mutação Puntual , RNA Mensageiro/metabolismoRESUMO
By using the single strand conformational analysis to search for point mutations in the choroideremia gene, we have identified an intronic polymorphism within the intron 2 of the CHM gene. We have studied the frequency of this polymorphism in the population from South of France.
Assuntos
Coroideremia/genética , Éxons/genética , Poli T/genética , Splicing de RNA/genética , Coroideremia/diagnóstico , Feminino , França , Frequência do Gene/genética , Humanos , Íntrons , Masculino , Mutação Puntual/genética , Polimorfismo GenéticoRESUMO
Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemopoietic stem cell disorder caused by the absence of glycosyl phosphatidylinositol (GPI)-anchored surface proteins due to a deficient biosynthesis of GPI-anchor. The disease occurs predominantly in adults, and very few cases have been described in children and adolescents. Recent analyses have shown that null mutations in the X-linked PIG-A (phosphatidylinositol glycan-class A) gene are responsible for GPI-anchor deficiency in most PNH adult patients analysed. We report a young male from southern France who was diagnosed with PNH at 12 years of age during follow-up of aplastic anaemia. To further elucidate the molecular basis of PNH occurring in childhood, we used the powerful and rapid protein truncation test to scan for truncative mutations in the entire PIG-A mRNA reverse transcribed and amplified from blood mononuclear cells. The somatic defect responsible for PNH in the patient was found to be a splicing mutation. IVS5+1G-->A, which has previously been described in two Asiatic adults with PNH.
Assuntos
Hemoglobinúria Paroxística/genética , Proteínas de Membrana/genética , Mutação , Criança , Eletroforese em Gel de Poliacrilamida , Humanos , Masculino , Reação em Cadeia da Polimerase , Splicing de RNARESUMO
Heterotaxy is the failure of the developing embryo to establish normal left-right asymmetry, which is often associated with multiple malformations. Previous studies have identified different mutations in the cytoplasmic tail of the connexin 43 (cx 43) gene in six patients from a series of six sporadic cases with defects of laterality and severe heart malformations. These cases showed that of the genes involved in lateralization defects with autosomal recessive transmission, cx 43 was the most important. This result was challenged by two different teams, which, on sequencing only the carboxyl terminal end of the cx 43 gene in 30 patients, found no mutations. To assess the responsibility of the cx 43 gene in human autosomal recessive lateralization defects, we tested its involvement in a selected group of 25 patients (19 familial cases) with a wide variety of lateralization defects and cardiovascular malformations. The whole coding sequence and direct flanking sequences were screened for mutations, both by single strand conformation analysis and direct fluorescent sequencing. We could only detect a single base pair insertion in the 3' untranslated region of one patient. To test the possibility of mutations in other parts of the cx 43 gene, the gene was located onto the physical map of chromosome 6, and flanking polymorphic markers were genotyped. Haplotype analysis excluded the cx 43 gene locus in nearly all of the familial cases of lateralization defects. Thus, our results do not support the suggestion that this gene is implicated in human autosomal recessive lateralization defects.