RESUMO
Non-isotopic single-strand conformation polymorphism (SSCP) and direct sequencing was used for carrier diagnosis in four families of DMD/BMD patients with previously characterized point mutations, leading to the identification of eight carriers and four non-carriers. When the mutation caused a distinctly altered migration pattern of the single strands, in principle, the SSCP-technique allowed determination of carrier status in the extended family of the probands without direct sequencing. However, because SSCP measures a function of not only the mutation, but of the entire sequence of the PCR product, it can lead to false negative and/or false positive diagnoses due to intronic and exonic sequence heterogeneity in the family. As we discovered this pitfall in one of the reported families, we concluded that for carrier testing the SSCP approach must be performed in essential conjunction with an independent assessment of the mutation site by direct sequencing.
Assuntos
Heterozigoto , Distrofias Musculares/genética , Mutação Puntual , Adulto , Sequência de Aminoácidos , Sequência de Bases , DNA/análise , Éxons , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Distrofias Musculares/diagnóstico , Conformação de Ácido Nucleico , Linhagem , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
The article describes responsibility as a term with six relations and four different levels. It analyses the conflicts between different addresses, levels and values and emphasizes the need to integrate the responsibilities of the physician. The term "responsibility" provides a more differentiating analytical tool for conflicts in medical acting than the so-called "principles of biomedical ethics".
Assuntos
Ética Médica , Responsabilidade Legal , Imperícia/legislação & jurisprudência , Equipe de Assistência ao Paciente/legislação & jurisprudência , Confidencialidade/legislação & jurisprudência , Responsabilidade pela Informação/legislação & jurisprudência , Feminino , Alemanha , Humanos , MasculinoRESUMO
In this study we demonstrate that the Deg1 degradation signal of the transcriptional repressor Matalpha2 confers compartment-specific turnover to a reporter protein. Rapid degradation of a Deg1-containing fusion protein is observed only when the reporter is efficiently imported into the nucleus. In contrast, a reporter that is constantly exported from the nucleus exhibits an extended half-life. Furthermore, nuclear import functions are crucial for both Deg1-induced degradation as well as for the turnover of the endogenous Matalpha2 protein. The conjugation of ubiquitin to a Deg1-containing reporter protein is abrogated in mutants affected in nuclear import. Obviously, the Deg1 signal initiates rapid proteolysis within the nucleoplasm, whereas in the cytosol it mediates turnover via a slower pathway. In both pathways the ubiquitin-conjugating enzymes Ubc6p/Ubc7p play a pivotal role. These observations imply that both the cellular targeting of a substrate and the compartment-specific activity of components of the ubiquitin-proteasome system define the half-life of naturally short-lived proteins.
Assuntos
Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Enzimas de Conjugação de Ubiquitina , Ubiquitinas/metabolismo , Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Escherichia coli/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde , Transferases Intramoleculares , Cinética , Ligases/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação , Plasmídeos/metabolismo , Testes de Precipitina , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
Patients scheduled for operation of an abdominal aortic aneurysm are a challenge to the anesthesiologist due to multiple coexisting diseases and serious intraoperative hemodynamic changes caused by cross-clamping. The aim of this study was to investigate the incidence of intra- and postoperative complications and to analyze the coexisting diseases in order to estimate complications and risks. PATIENTS AND METHOD: The charts of 72 patients scheduled for resection of an abdominal aortic aneurysm in 1984 and 1985 were retrospectively analysed. The patients are divided into 6 groups: E: elective operation; K: without pulmonary catheterization; N: emergency operation; R: ruptured aneurysm; D: acute dissection. The statistical analysis was performed by chi-square test. RESULTS: Patients monitored by Swan-Ganz catheter suffered more frequently from chronic obstructive or restrictive pulmonary diseases and coronary heart disease or cardiac failure. INTRAOPERATIVE COMPLICATIONS: Emergency patients showed more than twice as many intraoperative cardiovascular complications than scheduled patients; 3 fatal cases were observed in this group. Renal complications (anuria) occurred in 2% during elective operations and in 30% during emergency operations. POSTOPERATIVE COMPLICATIONS: Most of the postoperative complications - 75% - were associated with the cardiovascular system, followed by disturbances of gas exchange and hypoxemia. Two patients in group E had a short-lasting renal insufficiency; 1 patient died of myocardial infarction 3 weeks postoperatively. Emergency procedures were much more risky, with a 90% incidence of cardiovascular complications; 4 patients died within 5 days, 1 other after 1 week. Patients monitored by Swan-Ganz catheter showed more arrhythmias and hypotension than the others. Atelectasis was seen on X-rays in 46% of emergency patients, 35% of group P, and 2.6% of group K. CONCLUSIONS: Retrospective studies of special and high-risk patients are very useful in assessing the individual clinical standard, despite problems with data acquisition. This study permitted the assessment of perioperative complications and risks in these patients.
Assuntos
Aneurisma Aórtico/cirurgia , Complicações Intraoperatórias , Complicações Pós-Operatórias , Idoso , Idoso de 80 Anos ou mais , Aorta Abdominal , Cateterismo de Swan-Ganz/efeitos adversos , Humanos , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Duchenne muscular dystrophy and its allelic disorder Becker muscular dystrophy are among the most common hereditary human pathologies (1:3500). Two thirds of the genomic alterations responsible for these diseases involve gross gene rearrangements such as deletions, and less frequently duplications. The remaining one third includes point mutations such as deletions, insertions, and substitutions. This study describes four nonpreviously reported polymorphisms in the dystrophin gene by using the polymerase chain reaction/single-strand conformation polymorphism technique and subsequent nonisotopic silver staining.
Assuntos
Distrofina/genética , Polimorfismo Genético , Adolescente , Argentina , Sequência de Bases , Criança , Pré-Escolar , DNA/genética , Humanos , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
Duchenne and Becker muscular dystrophies (DMD/BMD) are caused by mutations in the human dystrophin gene. About two-thirds of DMD/BMD patients exhibit gross rearrangements in the gene whereas the mutations in the remaining one third are thought to be point mutations or minor structural lesions. By means of various progressive PCR-based techniques hitherto a number of point mutations has been described that in most cases should cause premature translational termination. These data indicate a particular functional importance for the C-terminal region of dystrophin and consequently for its gene products Dp 71 and Dp 116. To screen for microheterogeneities in this gene region we applied PCR-SSCP analysis to exons 60-79 of twenty-six DMD/BMD patients without detectable deletions. The study identified seven point mutations and one intron polymorphism. Six point mutations, found in DMD patients, should cause premature translational termination. One point mutation, identified in a BMD patient, results in an amino acid exchange. Five of the DMD patients bearing a point mutation are mentally retarded suggesting that a disruption of the translational reading frame in the C-terminal region is associated with this clinical finding in DMD cases. Therefore our data raise the possibility, that Dp 71 and/or Dp 116, the C-terminal translational products of dystrophin, may be causally involved in cases of mental retardation that are associated with DMD.
Assuntos
Distrofina/genética , Deficiência Intelectual/genética , Distrofias Musculares/genética , Mutação Puntual , Sequência de Aminoácidos , Sequência de Bases , Éxons , Regulação da Expressão Gênica , Rearranjo Gênico , Humanos , Deficiência Intelectual/complicações , Íntrons , Dados de Sequência Molecular , Distrofias Musculares/complicações , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Deleção de SequênciaRESUMO
Approximately one-third of the mutations responsible for Duchenne muscular dytrophy (DMD) do not involve gross rearrangements of the dystrophin gene. Methods for intensive mutation screening have recently been applied to this immense gene, which resulted in the identification of a number of point mutations in DMD patients, mostly translation-terminating mutations. A number of data raised the possibility that the C-terminal region of dystrophin might be involved in some cases of mental retardation associated with DMD. Using single-strand conformation analysis of products amplified by polymerase chain reaction (PCR-SSCA) to screen the terminal domains of the dystrophin gene (exons 60-79) of 20 unrelated patients with DMD or BMD, we detected two novel point mutations in two mentally retarded DMD patients: a 1-bp deletion in exon 70 (10334delC) and a 5' splice donor site alteration in intron 69 (10294 + 1G-->T). Both mutations should result in a premature translation termination of dystrophin. The possible effects on the reading frame were analyzed by the study of reverse transcripts amplified from peripheral blood lymphocytes mRNA and by the protein truncation test.
Assuntos
Distrofina/genética , Deficiência Intelectual/genética , Distrofias Musculares/genética , Mutação Puntual , Sequência de Bases , Análise Mutacional de DNA , Humanos , Dados de Sequência Molecular , Terminação Traducional da Cadeia Peptídica , Reação em Cadeia da Polimerase/métodosRESUMO
More than 30% of Duchenne and Becker muscular dystrophy (DMD/BMD) patients have no gross DNA rearrangements like deletions or duplications. The large size of the coding sequence of the dystrophin gene (11 kilobases) complicates systematic identification of point mutations. Recently reported approaches based on genomic DNA or mRNA show that chemical cleavage of mismatches is an effective but time consuming and technically demanding method for the identification of point mutations in the human dystrophin gene. We have used a fast and convenient system consisting of PCR amplification of genomic DNA, non-isotopic SSCP analysis, and direct sequencing of PCR products for the detection of mutations in exon 13 and adjacent intron sequences. Sixty-eight DMD patients without detectable deletions or duplications were analysed, resulting in the identification of a point mutation in the coding sequence and two polymorphisms in the 5' flanking intron. The C to T change of the first nucleotide in the third triplet leads to a stop codon and seems to be the cause of the functional deficiency of the gene product in this patient.
Assuntos
Distrofina/genética , Éxons , Genes , Mutação Puntual , Polimorfismo Genético , Sequência de Bases , Análise Mutacional de DNA , DNA de Cadeia Simples/genética , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
Studies of the coding region of the neuronal glutamate transporter of 6 amyotrophic lateral sclerosis (ALS) patients and 10 controls show an identical pattern of four reported amino acid variations. No mutations and polymorphisms were detected in 5 sporadic ALS patients and a single patient with the familial form of the disease.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Esclerose Lateral Amiotrófica/genética , Sistema X-AG de Transporte de Aminoácidos , Sequência de Bases , Genes , Código Genético , Humanos , Dados de Sequência Molecular , Neurônios/fisiologiaRESUMO
Carrier determination is important for genetic counselling in DMD/BMD families. The detection of altered PCR amplified dystrophin mRNA fragments owing to deletions, insertions, or point mutations has increased the possibilities of carrier determination. However, problems may occur because of alternative splicing events. Here we present a family with a DMD patient characterised by a deletion of exons 45 to 54. At the mRNA level we detected a corresponding altered fragment which served for carrier determination. The mother and the sister of the patient showed the same altered dystrophin mRNA fragment as the patient and are therefore carriers. In the mother two additional altered dystrophin mRNA fragments were detectable, obviously resulting from alternative splicing in the normal allele. The grandmother and two other related females of the patient possess only the normal mRNA fragment. In a further female we detected an altered fragment owing to an mRNA deletion of exon 44. This fragment is created either by alternative splicing or a new mutation. Therefore, the carrier status of this female is still ambiguous indicating problems in carrier determination by the method of dystrophin mRNA analysis.
Assuntos
Processamento Alternativo , Distrofina/genética , Triagem de Portadores Genéticos/métodos , Distrofias Musculares/genética , RNA Mensageiro/análise , Sequência de Bases , Southern Blotting , Deleção Cromossômica , Feminino , Aconselhamento Genético , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Transcrição GênicaRESUMO
We report the first C-terminal missense mutation in a Duchenne muscular dystrophy patient. A G10227A transition of the dystrophin gene was found which resulted in the substitution of a highly conserved cysteine at position 3340 within the second half of the dystroglycan-binding domain. Residual amounts of 427 kDa dystrophin were detected in western blot analysis of the patient's muscle tissue, and immunohistological examination revealed weak traces of dystrophin on all fibers. Sarcolemmal staining intensity of 43 kDa beta-dystroglycan was also reduced. Mental retardation in our patient and absence of the b-wave in his electroretinogram indicate that central nervous functions of dystrophin isoforms also depend on the presence of cysteine 3340.