Assuntos
Códon , Mutação Puntual , Talassemia/genética , Alelos , Análise Mutacional de DNA , Saúde da Família , Feminino , Testes Genéticos , Genótipo , Heterozigoto , Humanos , Masculino , Fenótipo , Talassemia beta/genéticaRESUMO
Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant beta-globin precursor mRNAs (pre-mRNAs). The oligonucleotides restored correct splicing and translation of beta-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2-654/beta(E), IVS2-745/IVS2-745, and IVS2-745/IVS2-1 genotypes. The maximal Hb A level for repaired IVS2-745 mutation was approximately 30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective beta-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach.
Assuntos
Eritrócitos/metabolismo , Terapia Genética , Hemoglobina A/biossíntese , Hemoglobina A/genética , Talassemia beta/sangue , Talassemia beta/terapia , Núcleo Celular/genética , Células Cultivadas , Células Precursoras Eritroides/metabolismo , Imunofluorescência , Globinas/genética , Humanos , Mutação/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Spliceossomos/genética , Fatores de Tempo , Talassemia beta/genéticaRESUMO
We detected Hb D-Los Angeles [beta 121(GH4)Glu-->Gln], the most common hemoglobin variant after Hb S and Hb Lepore-Boston, in six unrelated families in Southern Italy. Ten patients were studied; eight patients were heterozygotes and two were compound heterozygotes for the hemoglobin variant and the beta-thalassemia codon 39 (C-->T) nonsense mutation. The beta-globin gene sequence was characterized by polymerase chain reaction direct sequencing; restriction fragment length polymorphisms were defined by Southern blot analysis. The gene variant, due to the GAA-->CAA substitution at codon 121, was found in association with the 5' subhaplotype [+ - - - -] and the beta-globin gene framework 1; in addition, it was found to be associated with the absence of Ava II/phi beta and Xmn I/5'G gamma, and with the presence of Hpa I/3' beta. This restriction fragment length polymorphism haplotype is common in the Mediterranean area as well as in other populations. The findings are equally compatible with an independent origin in the Mediterranean area or with origin in Asia and subsequent spread to Italy.
Assuntos
Globinas/genética , Hemoglobinas Anormais/genética , Polimorfismo de Fragmento de Restrição , Sequência de Bases , Códon , Heterozigoto , Humanos , Itália , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
We report here a new human alpha-globin gene rearrangement carrying the two normal, alpha 2 and alpha 1, and two hybrid, alpha 1/alpha 2, globin genes in the order 5'-alpha 2-alpha 1/alpha 2-alpha 1/alpha 2-alpha 1-3'. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the beta-thalassaemia IVS 1 nt 1 G----A mutation caused thalassaemia intermedia. We also report a case of an alpha alpha alpha-globin gene rearrangement in the twin of one of the alpha alpha alpha alpha-globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an alpha alpha alpha alpha-anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the alpha-globin gene rearrangements could be of the intrachromosomal rather than the interchromosomal type.
Assuntos
Rearranjo Gênico , Globinas/genética , Família Multigênica/genética , Recombinação Genética/genética , Talassemia/genética , Sequência de Bases , Southern Blotting , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Gêmeos DizigóticosRESUMO
BACKGROUND: Structural hemoglobinopathies usually are inherited as autosomic dominant traits; de novo mutations are uncommon. Analytical and preparative procedures for the characterization of an abnormal hemoglobin are complex and time-consuming. Mass spectrometer analysis allows a rapid identification of the amino acid substitution. METHODS AND RESULTS: A cyanotic 7-year-old girl was found to have 16% methemoglobin. Laboratory data showed the presence of an abnormal hemoglobin, which was isolated by collecting the abnormal peak from DEAE and globin chains from CM52. The amino acid substitution was rapidly identified by FAB mass spectroscopic analysis, leading to the recognition of HbM Hyde Park. These data were confirmed by molecular analysis (Southern blot and DNA sequencing). Neither the parents nor a sister showed any abnormality; non-paternity was excluded by blood group serology and HLA typing. CONCLUSIONS: This is a case of HbM Hyde-Park arising as a de novo mutation. FAB mass spectroscopic analysis is a rapid and useful analytical method for identifying aminoacid substitution.
Assuntos
Cianose/genética , Hemoglobina M/genética , Hemoglobinopatias/genética , Mutação , Southern Blotting , Criança , Análise Mutacional de DNA , Feminino , Humanos , Espectrometria de Massas , Oxiemoglobinas/genéticaRESUMO
In order to evaluate the feasibility of first trimester prenatal diagnosis of beta-thalassaemia by restriction fragment length polymorphism (RFLP) in Campania, one of the most affected regions in Southern Italy, DNA polymorphism analysis was performed on 40 unrelated patients, affected with homozygous beta-thalassaemia, and on their parents. Frequency of the presence of the Hinc II epsilon, Hind III G gamma and A gamma, Hinc II psi beta and 3' psi beta, Ava II psi beta, Ava II beta and Bam HI 3' beta sites have been determined in the beta A and beta thal chromosome samples. In 31 families (over 75%), RFLPs enabled tracing the beta-thalassaemia mutations in both father and mother (100% diagnosis). In the remaining nine families, RFLPs enabled tracing only one of the two mutations (50% diagnosis) because the other parent was found to be homozygous in all the analysed polymorphic sites. Restriction haplotypes, assembled on the basis of linkage analysis, were most heterogeneous, hence a wide heterogeneity of mutations is expected.
Assuntos
Globinas/genética , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Talassemia/genética , Adolescente , Adulto , Criança , Feminino , Frequência do Gene , Haplótipos , Humanos , Itália , Masculino , Mutação , Diagnóstico Pré-Natal , Talassemia/diagnóstico , Talassemia/epidemiologiaAssuntos
Globinas/genética , Hemoglobinopatias/genética , Talassemia/genética , Deleção Cromossômica , Regulação da Expressão Gênica , Genes , Ligação Genética , Haplótipos , Hemoglobinopatias/diagnóstico , Hemoglobinopatias/prevenção & controle , Humanos , Itália , Mar Mediterrâneo , Polimorfismo de Fragmento de Restrição , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNARESUMO
A procedure using fast atom bombardment mass spectrometry was developed for mapping the proteolytic digest of proteins. The procedure was successfully applied to the tryptic peptides of the human beta-globin chain. Almost all the expected peptides were identified by direct analysis of the peptide mixture on the mass spectrometer. Peptide recognition along the beta-globin chain sequence was easily made on the basis of their molecular weight. The general applicability of this mapping procedure in the analysis of haemoglobinopathies was demonstrated by its use for the structural characterization of a variant beta-globin chain.
Assuntos
Globinas/análise , Hemoglobinas Anormais/análise , Sequência de Aminoácidos , Humanos , Espectrometria de Massas , Fragmentos de Peptídeos/análise , TripsinaRESUMO
1. The expected number of patients with Cooley anemia in Naples, as predicted from heterozygote frequencies, is about 300 which is more than that found from hospital statistics. 2. Considering the presence of at least two beta-thalassemic alleles and the coexistence of a significant incidence of the Lepore gene in the population of the area surrounding Naples (the maximum incidence is thought to be in the neighboring province of Caserta), there are at least 6 different genotypes (3 true homozygotes and 3 genetic compounds) that can give rise to clinically important thalassemia syndromes, ranging from thalassemia intermedia to classic Cooley anemia. 3. Since an alpha-thalassemic gene is also present at polymorphic frequency in this population, this gene may coexist by random assortment in subjects who also have a beta-thalassemia syndrome, thus accounting for part of the variability of globin gene expression. 4. It is remarkable that 2 brothers homozygous for Hb Lepore present differences in the non-alpha/alpha ratio. The clinical differences could be explained by the gamma/alpha mRNA ratio close to 1 in one of them.
Assuntos
Hemoglobina M/genética , Hemoglobinas Anormais/genética , Talassemia/genética , Criança , Feminino , Frequência do Gene , Triagem de Portadores Genéticos , Genótipo , Globinas/biossíntese , Hemoglobina M/biossíntese , Humanos , Itália , Masculino , Linhagem , Gravidez , Talassemia/sangue , Talassemia/epidemiologiaRESUMO
A DNA endonuclease has been purified from eggs of Asterias forbesi by a simple four-step-purification procedure. The purified enzyme is at least 96% pure and is free of phosphatase, phosphodiesterase, and RNase. It has a pH optimum of 6.5 and does not require divalent cations. The enzyme produces 3'-phosphoryl and 5'-hydroxyl end groups. The products of exhaustive hydrolysis can be grouped in two fractions. The first fraction, 40%, contains a small amount of mononucleotides and di-, tri-, tetra-, penta-, and hexanucleo-tides. The second fraction, 60%, contains oligonucleotides larger than hexanucleotides.
