RESUMO
Factor (F) X deficiency is a rare inherited autosomal recessive trait. We report on a patient affected by a severe bleeding diathesis. Mutations were sought by F10 sequence analysis. The consequences of the mutation were characterized by measuring thrombin and FXa formation after triggering the clotting cascade with activated partial thromboplastin time (aPTT) reagent or with phospholipid vesicles plus either tissue factor (TF) or FIXabeta, or with the FX activator from Russell's viper venom (RVV-X). The patient was found to be homozygous for a novel FX p.G51V mutation (G11V of the mature protein) within the omega-loop of the gamma-carboxyglutamic-rich domain. FX activity was markedly reduced (FX:C <1%) in prothrombin time and aPTT assays, and was 15% of normal in the RVV-X assay. The antigen level (FX:Ag) was 75%. TF, alone or in combination with recombinant FVIIa, failed to trigger detectable FXa or thrombin activity in the patient's plasma. FIXabeta also failed to trigger measurable FXa or thrombin production, but activation with RVV-X was only 4-fold less effective in the patient's plasma than in normal plasma. Supplementation with normal FX suggested that FX(G11V) and/or FXa(G11V) might slow the clotting cascade by competition. Overall, the patient's phenotype appears to be due to a very low rate of FX(G11V) activation by TF/FVIIa and FVIIIa/FIXa complexes rather than to FXa(G11V) activity within prothrombinase.
Assuntos
Substituição de Aminoácidos , Deficiência do Fator X/genética , Fator X/química , Mutação de Sentido Incorreto , Valina/metabolismo , Sequência de Aminoácidos , Criança , Consanguinidade , Fator X/genética , Fator X/metabolismo , Fator Xa/biossíntese , Feminino , Teste de Complementação Genética , Homozigoto , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Ligação Proteica , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Trombina/biossínteseRESUMO
The presence of gene lesions in blood coagulation factor X (FX) was investigated in eight FX-deficient patients with severe bleeding symptoms, originating from five unrelated Algerian families (FX coagulant activity <1%, FX antigen ranging from 2% to 16%). A missense mutation (p.Phe31Ser) in the Gla domain was found in homozygous form for all patients but one, who is a compound heterozygote for the Phe31Ser mutation and for a non-sense mutation, Tyr130Term in EGF-2 domain. The haplotypes of FX alleles were determined by the following allelic variants located in the promoter: g.1323_1330delTTGTGA (A1/A2), g.1449T>C, g.1451C>A, upstream to exon 3: g.17257C>T and downstream to exon 3: g.17396A>C. The A1-C-A-T-C haplotype was found on each allele bearing the Phe31Ser mutation in the eight FX deficient patients contrasting with its low frequency (8%) in a control Algerian population (in which the Phe31Ser substitution was absent). The patients came from the same geographical area of Algeria (5/8 are certainly from Kabyle origin) and the haplotype analysis suggests a founder effect. Transient expression study reveals that, for the mutant FX-Phe31Ser, FX antigen level was 60% in conditioned media and 140% in cell lysates compared with the wild type FX. The partial retention and intracellular accumulation of the mutant FX might be due to impaired folding and/or conformational changes, and the discrepancies observed between the FX antigen level in COS-7 cell supernatant (60%) and in the patients plasma (2-16%) to an in vivo increased clearance of the secreted unstable FX mutant.
Assuntos
Fator X/genética , Efeito Fundador , Mutação , Fenilalanina/genética , Serina/genética , Argélia , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Primers do DNA , Haplótipos , HumanosRESUMO
We describe here five F7 mutations found in four patients without bleeding history, despite constitutional coagulation Factor VII (FVII) deficiency. All five mutations are missense and affect the catalytic domain of FVII (A191T, A191V, T239P, R224Q and M298I). The A191V and T239P mutations are novel and were found in homozygous patients with no clinical bleeding tendency. The patient diagnosed with the A191V mutation had a phenotype corresponding to a moderate type 1 FVII deficiency (FVII:C 4%, FVII:Ag 5%). The T239P mutation was found in a patient with mild type 2 FVII deficiency (FVII:C 25%, FVII:Ag 95%). Novel mutations are both in close vicinity to the charge-stabilizing system of FVII. Modeling studies allow understanding in part the molecular basis for the loss of function.
Assuntos
Deficiência do Fator VII/genética , Mutação de Sentido Incorreto , Adulto , Argélia , Domínio Catalítico , Análise Mutacional de DNA , Fator VII/química , Fator VII/genética , Feminino , Hemorragia/genética , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
The molecular basis of severe type I factor (F)VII deficiency was investigated in two Algerian patients. One patient, a 13-year-old-girl who has suffered from severe bleeding since birth, was homozygous for a 7-bp deletion (nt 7774-7780) and a 251-bp insertion (nt 7773-7781) of mitochondrial origin, in IVS 4 acceptor splice site. The other patient, an infant who died from massive intracranial haemorrhage, was homozygous for a transversion in the IVS 7 donor splice site (T9726+2-->G) and a missense mutation in exon 8 (G10588-->A; Arg224-->Gln). In both cases, the deleterious mutations are probably the splice site junction abnormalities impairing mRNA processing. These three lesions have not yet been reported.
