Assuntos
Conversão Gênica , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Fator de von Willebrand/imunologia , Criança , Códon sem Sentido , Fator VIII/administração & dosagem , Fator VIII/efeitos adversos , Saúde da Família , Homozigoto , Humanos , Lactente , Isoanticorpos/sangue , Masculino , Doenças de von Willebrand/complicações , Doenças de von Willebrand/etiologiaRESUMO
The 3' end of the VWF gene was screened in the affected members of 3 different families with type 2A (phenotype IID) von Willebrand disease (vWD). Exons 49 to 52 of the VWF gene were amplified and screened for mutations by chemical cleavage mismatch detection. Mismatched bands were detected in exon 52 of 2 patients and in exon 51 of a third patient. Using direct DNA sequencing, a heterozygous G8562A transition leading to a Cys2008Tyr substitution was found in all the patients in family 1, and a T8561A transversion leading to a Cys2008Ser substitution was found in both patients from family 2. In a patient from a third family, an 8-base deletion from nucleotide 8437 to 8444 was identified in exon 51. The 2 mutations in exon 52 were reproduced by in vitro site-directed mutagenesis of full-length von Willebrand factor (vWF) cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWFs for these 2 mutations exhibited the typical aberrant vWF:Ag multimer pattern seen in the plasma of the patients. These 3 mutations demonstrate the importance of other carboxy-terminal cysteines in addition to the reported Cys2010 residue, in the normal dimerization of vWF, and their essential role in the assembly of normal multimeric vWF. (Blood. 2001;98:674-680)
Assuntos
Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo , Animais , Células COS , Códon sem Sentido , Análise Mutacional de DNA , Dimerização , Saúde da Família , Feminino , Mutação da Fase de Leitura , Genes Dominantes , Humanos , Masculino , Mutação , Fenótipo , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Deleção de Sequência , TransfecçãoRESUMO
Type 2A von Willebrand disease (VWD) is mostly an autosomal dominantly inherited bleeding disorder characterised by a qualitative defect of von Willebrand factor (VWF). Mutation screening was used to screen the whole of VWF gene followed by direct sequencing to detect the mutation in a father and son diagnosed with type 2A (phenotype IIA) von Willebrand disease. A C5219 to A transversion was detected predicting Leucine to Isoleucine substitution in codon 1657. This novel missense mutation which was also identified by MboI restriction enzyme analysis, was found in both patient and his father but not in any other unaffected family member or 50 unrelated normal individuals. This substitution was reproduced by in vitro site directed mutagenesis of full-length VWF cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWF protein exhibited the full spectrum of VWF multimers, suggesting that the abnormal multimer seen in the patient results from increased proteolysis.
Assuntos
Doenças de von Willebrand/genética , Substituição de Aminoácidos , Animais , Células COS , Análise Mutacional de DNA , DNA Complementar/genética , Dimerização , Saúde da Família , Feminino , Hemofilia A/genética , Humanos , Masculino , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Doenças de von Willebrand/classificação , Doenças de von Willebrand/etiologia , Fator de von Willebrand/genéticaRESUMO
Common alleles or polymorphisms form the basis of human diversity, and some of these polymorphisms closely linked on the same chromosome with a defective gene have been used for gene tracking in many genetic disorders. The success of any linkage analysis also relies on the nature of the polymorphisms used, and the most useful are those that can have a large numbers of alleles.
