RESUMO
DNA from nine hemophilia B patients who produce anti-factor IX inhibitors (antibodies), including two brothers, was analyzed by the Southern blotting method and hybridization with factor IX cDNA, intragenomic, and 3'-flanking probes. Two inhibitor patients were shown to have total deletions of the factor IX gene. Two other inhibitor patients, the brothers, were shown to have a presumably identical complex rearrangement of the factor IX gene involving two separate deletions. The first deletion is of approximately 5.0 kb and removes exon e. The second deletion is between 9 and 29 kb and removes exons g and h but leaves exon f intact. An abnormal Taq I fragment at one end of the deletion junctions acted as a marker for the inheritance of hemophilia B in the patients' family. Five other inhibitor patients have a structurally intact factor IX gene as detected by this method. Our studies indicate that whereas large structural factor IX gene defects predispose hemophilia B patients to developing an anti-factor IX inhibitor, the development of an inhibitor can be associated with other defects of the factor IX gene.
Assuntos
DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II , Fator IX/genética , Hemofilia B/genética , Enzimas de Restrição do DNA , Éxons , Humanos , Masculino , Mutação , Hibridização de Ácido Nucleico , Regiões Promotoras GenéticasRESUMO
von Willebrand disease (VWD) is a bleeding disorder caused by inherited defects in the concentration, structure, or function of von Willebrand factor (VWF). VWD is classified into three primary categories. Type 1 includes partial quantitative deficiency, type 2 includes qualitative defects, and type 3 includes virtually complete deficiency of VWF. VWD type 2 is divided into four secondary categories. Type 2A includes variants with decreased platelet adhesion caused by selective deficiency of high-molecular-weight VWF multimers. Type 2B includes variants with increased affinity for platelet glycoprotein Ib. Type 2M includes variants with markedly defective platelet adhesion despite a relatively normal size distribution of VWF multimers. Type 2N includes variants with markedly decreased affinity for factor VIII. These six categories of VWD correlate with important clinical features and therapeutic requirements. Some VWF gene mutations, alone or in combination, have complex effects and give rise to mixed VWD phenotypes. Certain VWD types, especially type 1 and type 2A, encompass several pathophysiologic mechanisms that sometimes can be distinguished by appropriate laboratory studies. The clinical significance of this heterogeneity is under investigation, which may support further subdivision of VWD type 1 or type 2A in the future.
Assuntos
Doenças de von Willebrand/sangue , Doenças de von Willebrand/fisiopatologia , Proteínas ADAM/fisiologia , Proteína ADAMTS13 , Humanos , Modelos Biológicos , Fenótipo , Estrutura Terciária de Proteína , Doenças de von Willebrand/classificação , Doenças de von Willebrand/diagnóstico , Fator de von Willebrand/metabolismoRESUMO
Incubation of a factor VIII-rich fraction of plasma with a high concentration of salt confirmed the production of both high (HMW) and low (LMW) molecular weight factor VIII clotting activity (FVIIIC) as determined by agarose gel filtration but with considerable overlap. The electrophoretic mobility of factor VIII related protein (FVIIIRP) detected by precipitating rabbit antiserum was not affected by this treatment and LMW FVIIIC devoid of FVIIIRP was apparently produced. At low concentration the reducing agent dithiothreitol (DTT) altered the electrophoretic mobility of FVIIIRP. At higher concentrations it altered both its mobility and antigenicity and an LMW FVIIIRP was produced. Contrary to the findings of other workers no LMW FVIIIC devoid of FVIIIRP was produced. In further studies factor VIII-rich plasma fraction was treated with sepharose beads to which had been coupled a non-coagulation inhibitory precipitating rabbit antibody to FVIIIRP. Both FVIIIRP and FVIIIC were taken up by the beads but after elution with 1.5 M NaCl, FVIIIC of LMW and devoid of FVIIIRP was selectively removed. Antisera raised to LMW FVIIIC produced with 1.5 M NaCl either by the gel filtration or affinity chromatography methods inhibited FVIIIC and precipitated with HMW factor VIII-rich fractions. The results were consistent with the possibility that factor VIII clotting activity and FVIIIRP exist in plasma as a non-covalently bound complex.
Assuntos
Fator VIII , Antígenos , Cromatografia de Afinidade , Ditiotreitol/farmacologia , Humanos , Imunoeletroforese , Peso Molecular , Concentração Osmolar , Precipitinas , Conformação Proteica/efeitos dos fármacosRESUMO
Patients with the haemophilia B Leyden phenotype show a distinct pattern of factor IX expression characterized by a post-pubertal increase in FIX levels and the remission of clinical symptoms in adult life. This phenotype has previously been linked to single base mutations within transcription factor binding sites in a region of approximately 40 bp around the major start point of transcription of the FIX gene. Here we report a novel mutation in this region within the transcription factor C/EBP binding site at +1 to +18. The mutation is a single base pair deletion from a triplet of thymine residues at +6 to +8. We show that the extent to which this mutation disrupts the binding of C/EBP to its binding site is less marked than the disruption caused by the +13 A-->G mutation of FIX Norwich (1). This correlates with age-matched phenotypic data we have available for the patient reported here and that of FIX Norwich.
Assuntos
Composição de Bases/genética , Fator IX/genética , Deleção de Genes , Hemofilia B/genética , Regiões Promotoras Genéticas , Sequência de Bases , Sítios de Ligação , Criança , Pré-Escolar , Humanos , Dados de Sequência Molecular , Fenótipo , Transcrição GênicaRESUMO
A cohort of 79 previously untreated patients (PUPs) with moderate-severe haemophilia A (baseline Factor VIII < or =2%) were enrolled in a study to evaluate the safety, efficacy and immunogenicity of recombinant factor VIII (r-FVIII, Recombinate). Blood samples were obtained retrospectively from a total 55 PUPs who were investigated for the spectrum of FVIII gene mutations responsible for their haemophilia. FVIII gene inversion mutations were found in 27 (49%) patients. Two patients had partial gene deletions. The remaining 26 patients were then screened for mutations in the FVIII gene coding region using conformation sensitive gel electrophoresis. Point mutations were identified in 22 (85%) of the patients and 14 of these mutations were novel. Study subjects were monitored for the development of FVIII inhibitors throughout the study. A total of 23 of the 73 evaluable subjects (including one subject with a low inhibitor titer at baseline) demonstrated an inhibitor on one or more occasions; 11 (15%) were persistent. Inhibitors were detected in patients with partial gene deletions and inversions and in three of eight patients with missense mutations. No inhibitors were found in 11 patients with small insertions or deletions resulting in an alteration of the protein translation reading frame (frameshift mutations). The results corroborate the observation that mutation type is an important determinant of the propensity to develop inhibitory anti-FVIII antibody.
Assuntos
Autoanticorpos/genética , Fator VIII/imunologia , Hemofilia A/genética , Mutação , Formação de Anticorpos , Inversão Cromossômica , Estudos de Coortes , Análise Mutacional de DNA , Avaliação de Medicamentos , Fator VIII/administração & dosagem , Fator VIII/genética , Mutação da Fase de Leitura , Deleção de Genes , Hemofilia A/imunologia , Humanos , Mutação de Sentido Incorreto , Mutação Puntual , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologiaRESUMO
A two site solid phase immunoradiometric assay (IRMA) for VIIIRAg has been developed using a monoclonal antibody as both the solid phase ligand and the radiolabelled antibody. A range of normal plasmas and von Willebrand's disease (vWd) plasmas has been assayed for VIIIRAg by this method and compared with VIIIRAg values measured by an IRMA using polyclonal antibodies and by immunoelectrophoresis and with ristocetin cofactor activity (VIIIRiCoF). It was found that the monoclonal IRMA correlated well with the polyclonal IRMA and the immunoelectrophoretic assay, but the correlation with the VIIIRiCoF assay was relatively poor, in spite of the strong inhibitory effect of the antibody on VIIIRiCoF activity.
Assuntos
Anticorpos Monoclonais , Antígenos/análise , Fator VIII/imunologia , Radioimunoensaio/métodos , Fator VIII/análise , Humanos , Doenças de von Willebrand/sangue , Fator de von Willebrand/análiseRESUMO
The molecular basis of quantitative antithrombin deficiency was investigated in four families predicted to have major antithrombin gene rearrangements. A 1,442 bp deletion and insertion of the sequence 5'T(n = 38-40)GAGACG was characterised in one case. Sequence surrounding the breakpoints contained two perfect, and one imperfect, inverted repeats which may have mediated formation of a stem loop structure on one strand during DNA replication potentiating the deletion. A 9,219 bp deletion spanning introns 2 to 5 was identified in a second family. The identical 6 bp sequence was upstream of each breakpoint and the 5' breakpoint was located in a sequence of the Alu 3 repeat predicted to be susceptible to strand breakage during transcription. This may have promoted misalignment, and deletion, of one of the repeats and the intervening DNA. A novel 1.8 kb antithrombin gene fragment was present in DNA digests from affected members of the third family suggesting a partial antithrombin gene duplication event while in the remaining family, evidence supporting a complete gene deletion was obtained.
Assuntos
Deficiência de Antitrombina III/genética , Antitrombina III/genética , Deleção de Genes , Adolescente , Adulto , Deficiência de Antitrombina III/classificação , Deficiência de Antitrombina III/complicações , Sequência de Bases , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Deleção de Sequência , Trombofilia/complicações , Trombofilia/genética , Trombose Venosa/etiologiaRESUMO
In the course of investigating the molecular basis of protein S deficiency in 31 index cases with thrombophilia, we identified seven kindred where the underlying defect was a novel A to G transition 9 bp upstream of exon 12 in intron K of the PROS1 gene. In all but one case, the mutation caused type I deficiency. One individual had type III deficiency. While ectopic transcript analysis using the BstXI dimorphism in exon 15 failed to detect a transcript from the mutated allele, analysis of transcripts spanning exons 11 and 12 revealed a minor mRNA species. Sequencing confirmed the mutation created a new RNA acceptor site introducing 8 nucleotides of intronic sequence into the mature mRNA. Haplotype analysis of the defective PROS1 alleles in six families revealed the same haplotype in all affected individuals suggesting the presence of a common ancestor. Six of the fourteen relatives with the mutation experienced at least one venous thrombotic event strongly supporting the association of the mutation with venous thrombosis.
Assuntos
Íntrons/genética , Mutação Puntual , Deficiência de Proteína S/genética , Proteína S/genética , Trombofilia/genética , Alelos , Estudos de Coortes , Análise Mutacional de DNA , Fator V/genética , Feminino , Haplótipos , Humanos , Masculino , Linhagem , Reação em Cadeia da Polimerase , Deficiência de Proteína S/epidemiologia , Splicing de RNA , RNA Mensageiro/metabolismo , Trombofilia/epidemiologia , Tromboflebite/epidemiologia , Tromboflebite/genética , Reino Unido/epidemiologiaRESUMO
Using an ELISA-based method to detect type 2N von Willebrand disease (VWD), we found two individuals with absent FVIII binding. Direct sequencing of the FVIII binding region of the von Willebrand factor (VWF) gene showed that one individual had an R854Q substitution whilst the other had a T791M substitution. The very low FVIII binding and the VWF:Ag levels in both individuals suggested a second defect on the other VWF allele. Conformation sensitive gel electrophoresis of polymerase chain reaction amplified DNA was used to detect an additional change in the VWF gene of each patient. Direct sequencing confirmed a previously unreported G to A transition in the donor splice site in intron 25 of both individuals which should result in a null allele. This was confirmed by mRNA analysis. These two individuals therefore have compound heterozygous VWD in which the only expressed allele has a type 2N mutation. In our population, such compound heterozygosity appears to be a significant cause of type 2N VWD.
Assuntos
Mutação Puntual , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Alelos , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Éxons , Fator VIII/metabolismo , Feminino , Expressão Gênica , Heterozigoto , Humanos , Íntrons , Masculino , Fenótipo , Splicing de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doenças de von Willebrand/sangue , Doenças de von Willebrand/classificação , Fator de von Willebrand/metabolismoRESUMO
Examination of the entire von Willebrand factor (VWF) gene for mutations, particularly in types 1 and 3 von Willebrand disease (VWD) is becoming more widely practised. The sequence of the entire VWF gene will soon be compiled as a single sequence. For these reasons, a clearly defined nomenclature to use for numbering the VWF nucleotide and amino acid sequence is required. The following recommendations are made for VWF numbering. VWF cDNA nucleotide sequence should be numbered from the A of the initiator ATG as the +1 position. Genomic DNA should be prefixed with a "g" and also numbered from this position. Amino acid (aa) numbering should be from the initiator methionine as the +1 position with sequential numbering of aa throughout VWF. To avoid confusion with previously used numbering schemes for mature VWF, which started from serine 764 of pre-pro VWF, the use of the single letter amino acid code is recommended.
Assuntos
Terminologia como Assunto , Fator de von Willebrand/genética , Humanos , Mutação , Polimorfismo GenéticoRESUMO
von Willebrand factor (vWF) is a multimeric glycoprotein found in plasma non covalently linked to factor VIII (FVIII). Type 2N von Willebrand disease (vWD) is caused by a mutation in the vWF gene that results in vWF with a normal multimeric pattern, but with reduced binding to FVIII. We have utilised methods for the phenotypic and genotypic detection of type 2N vWD. The binding of FVIII to vWF in 69 patients, 36 with type 1 vWD, 32 with mild haemophilia A and one possible haemophilia A carrier with low FVIII levels was studied. Of these, six were found to have reduced binding (five type 1 vWD, one possible haemophilia A carrier). DNA was extracted from these patients and exons 18-23 of the vWF gene encoding the FVIII binding region of vWF were analysed. After direct sequencing and chemical cleavage mismatch detection, a Thr28Met mutation was detected in two unrelated individuals, one of whom appears to be a compound heterozygote for the mutation and a null allele. No mutations were found in the region of the vWF gene encoding the FVIII binding region of vWF in the other four patients.
Assuntos
Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Alelos , Hemofilia A/sangue , Hemofilia A/genética , Humanos , Técnicas de Sonda Molecular , Mutação , Fenótipo , Doenças de von Willebrand/sangueRESUMO
The presence of the 20210A allele of the prothrombin (PT) gene has recently been shown to be a risk factor for venous thromboembolism. This is probably mediated through increased plasma prothrombin levels. The aim of this study was to compare the prevalence of the prothrombin 20210A allele in control subjects and in subjects with recognised thrombophilia and to establish whether the additional inheritance of the PT 20210A allele is associated with an increased risk of venous thromboembolism. 101 subjects with a history of venous thromboembolism and diagnosed as having either factor V Leiden (R506Q) or heritable deficiencies of protein C, protein S or antithrombin were studied. The prevalence of the PT 20210A allele in this group was compared with the results obtained for 150 control subjects. In addition, the relationships were examined between genetic status and the number of documented thromboembolic episodes, and between plasma prothrombin levels and possession of the PT 20210A allele. 8 (7.9%) of the 101 patients were also heterozygous for the PT 20210A allele. This compares with 0.7% in the control subjects (p = 0.005). After exclusion of patients on warfarin, the mean plasma prothrombin of 113 subjects without 20210A was 1.09 U/ml, as compared with 1.32 U/ml in 8 with the allele (p = 0.0002). Among the 101 patients with either factor V Leiden, protein S deficiency, protein C deficiency or antithrombin deficiency, the age adjusted mean (SD) number of venous thromboembolic episodes at diagnosis was 3.7 (1.5) in those with the PT 20210A allele, as compared with 1.9 (1.1) in those without (p = 0.0001). We have demonstrated that the prevalence of the PT 20210A allele is significantly greater in subjects with venous thrombosis and characterised heritable thrombophilia than in normal control subjects and that the additional inheritance of PT 20210A is associated with an increased risk of venous thromboembolism. We have also confirmed that plasma prothrombin levels are significantly greater in subjects possessing the PT 20210A compared with those who do not.
Assuntos
Genes , Mutação Puntual/genética , Protrombina/genética , Trombofilia/genética , Tromboflebite/genética , Adolescente , Adulto , Idoso , Alelos , Saúde da Família , Feminino , Frequência do Gene , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Mutação Puntual/fisiologia , Protrombina/metabolismo , Embolia Pulmonar/complicações , Embolia Pulmonar/genética , Fatores de Risco , Trombofilia/complicações , Trombofilia/epidemiologia , Tromboflebite/complicações , Tromboflebite/epidemiologia , Reino Unido/epidemiologiaRESUMO
Causative mutations in the factor VIII gene of seven unrelated patients with severe haemophilia A were identified using the mutation screening procedure conformation sensitive gel electrophoresis (1) and characterised by direct sequencing. Female family members of all patients had requested either carrier status determination or prenatal diagnosis. However, lack of the factor VIII gene inversion, a prior family history or informative polymorphisms prevented diagnosis in these families. Identification of a mutation in each family enabled female carrier status to be determined in all cases. Six mutations were previously unreported. One Afro-Caribbean patient had two sequence changes; A670 2G and A6769G. The latter, resulting in Met2238Val and previously reported as a FVIII mutation, was shown to be polymorphic with a 42% heterozygosity rate in an Afro-Caribbean population. Conformation sensitive gel electrophoresis was found to be technically simple and efficient at locating previously unknown FVIII gene mutations.
Assuntos
Análise Mutacional de DNA/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Fator VIII/genética , Triagem de Portadores Genéticos , Testes Genéticos/métodos , Hemofilia A/prevenção & controle , Southern Blotting , Inversão Cromossômica , DNA/genética , Fator VIII/análise , Fator VIII/química , Feminino , Hemofilia A/genética , Humanos , Conformação de Ácido Nucleico , Mutação Puntual , Polimorfismo Genético , Cromossomo X/genéticaRESUMO
The risk of developing factor VIII inhibitor antibodies in haemophilia A may relate both to factor VIII genotype and genes within the HLA complex known to influence immune response. We investigated a cohort of 176 patients with severe haemophilia A and with either high-level inhibitors (> 10BU/ml) or with no history of an inhibitor, stratified according to the presence or absence of the factor VIII gene intron 22 inversion. HLA DRB1, DQA1 and DQB1 polymorphisms were determined by PCR. HLA frequencies form 137 United Kingdom controls were used for comparison. HLA phenotype frequency differences, expressed as odds ratios with 95% confidence intervals were as follows: HLA-DRB*1501, DQB1*0602 and DQA1*0102 were all increased in frequency in patients with inhibitors, only DQA1*0102 reaching statistical significance (OR 2.7, 1.2-5.9). These alleles form part of an established HLA haplotype. The frequencies of HLA-DRB1*1501, DQB1*0602 and DQA1*0102 were particularly raised in patients with inhibitors and a factor VIII gene intron 22 inversion, although again only DQA1*0102 achieved significance (OR 3.1, 1.0-10.1). The frequency of DRB1*01, DQB1*0501, DQA1*0101 were also increased in inhibitor patients lacking the intron 22 inversion although this failed to achieve statistical significance. This data suggests that HLA class II profile constitutes a weak risk factor for developing inhibitor antibodies to factor VIII. This may be more pronounced in patients with an intron 22 inversion.
Assuntos
Fator VIII/imunologia , Genes MHC da Classe II , Antígenos HLA-D/imunologia , Hemofilia A/imunologia , Isoanticorpos/imunologia , Inversão Cromossômica , Estudos de Coortes , Suscetibilidade a Doenças , Fator VIII/uso terapêutico , Frequência do Gene , Genótipo , Antígenos HLA-D/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Cadeias alfa de HLA-DQ , Cadeias beta de HLA-DQ , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Cadeias HLA-DRB1 , Hemofilia A/epidemiologia , Hemofilia A/terapia , Teste de Histocompatibilidade , Humanos , Íntrons/genética , Isoanticorpos/biossíntese , Masculino , Reação em Cadeia da Polimerase , Fatores de Risco , Reino Unido/epidemiologiaRESUMO
Twenty six patients with mild or moderate haemophilia A and inhibitors are described. The inhibitor was detected at a median age of 33 years, after a median of 5.5 bleeding episodes. This usually following intensive replacement therapy. The median presenting inhibitor titre was antihuman 11.6 BU/ml, antiporcine 1.45 BU/ml. Plasma basal factor VIII level declined from a median of 0.08 IU/ml to 0.01 IU/ml following the inhibitor development. This caused spontaneous bleeding in 22 and a bleeding pattern similar to acquired haemophilia in 17. Bleeding was often severe and caused two deaths. The inhibitor disappeared spontaneously, or following immune tolerance induction, in 16 cases after a median of 9 months (range 0.5-46), with a return to the original baseline VIIIC level and bleeding pattern accompanied inhibitor loss. The inhibitor persisted in the remainder of the cases over a median period of 99 months (range 17-433 months) of follow-up. Inhibitors are an uncommon complication of mild haemophilia which frequently persist and may be associated with severe, life-threatening, haemorrhage. Forty-one percent of treated haemophilic family members had a history of factor VIII inhibitors, suggesting a familial predisposition to develop inhibitors in these kindreds. Sixteen patients from 11 families were genotyped. Seven different missense mutations affecting the light chain were detected and two in the A2 domain. Five patients from three families had a mutation causing a substitution of Trp2229 by Cys in the C2 domain which appears to predispose to inhibitor formation since 7 of the 18 affected individuals have a history of inhibitor development.
Assuntos
Autoanticorpos/imunologia , Fator VIII/imunologia , Hemofilia A/imunologia , Adolescente , Adulto , Idoso , Especificidade de Anticorpos , Autoanticorpos/biossíntese , Transfusão de Sangue , Criança , Análise Mutacional de DNA , Desamino Arginina Vasopressina/uso terapêutico , Epitopos/genética , Epitopos/imunologia , Fator VIII/química , Fator VIII/genética , Fator VIII/uso terapêutico , Genótipo , Hemorragia/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Mutação Puntual , Prevalência , Estudos Prospectivos , Conformação Proteica , Estudos RetrospectivosRESUMO
Human inhibitors to procoagulant factor VIII (VIIIC) were tested for possible reactivity with factor VIII related antigen (VIIIRAg) by a modified VIIIRAg immunoradiometric assay (IRMA). Inhibitor IgG's were screened for anti-VIIRAg by competition with I125 labelled VIIIRAg antibodies for common antigenic determinants using either an VIIIRAg concentrate from which factor VIII coagulant antigen (VIIICAg) had been dissociated, or normal plasma (containing VIIIRAg and VIIICAg) as an antigen source. There was no evidence of VIIIRAg antibodies in the five haemophilic VIIIC inhibitor IgG's tested but low titre VIIIRAg antibodies were detected in one spontaneous VIIIC inhibitor IgG.
Assuntos
Anticorpos/imunologia , Antígenos/imunologia , Fator VIII/imunologia , Anticorpos/análise , Ligação Competitiva , Epitopos/análise , Humanos , Imunoglobulina G , Radioimunoensaio , Fator de von WillebrandRESUMO
The frequency of five factor VIII gene intragenic and linked DNA polymorphisms and five factor IX gene intragenic polymorphisms was studied in Thai females. The polymorphisms in the FVIII gene were detected by restriction enzymes BclI, XbaI, BglI and at linked loci DX13 (DXS15) and St14 (DXS52) by BglII and TaqI, respectively, and in the FIX gene by MseI, DdeI, XmnI, TaqI and HhaI. With the exception of the BglI restriction fragment length polymorphism (RFLP), which is absent in Thais, factor VIII polymorphism frequencies were similar in Thais and Caucasians. Combined use of XbaI and TaqI/St14 resulted in a heterozygosity rate of greater than 90% in Thai females. For FIX, the recently described MseI RFLP in the 5' flanking region was the most informative polymorphism in Thais, 43% of females being heterozygous. The other four polymorphisms added little to the overall heterozygosity rate. The appropriate polymorphisms were used to track defective factor VIII and IX genes through 22 Thai pedigrees with haemophilia to enable carrier status to be assigned to female family members. The information obtained during this study will form the basis for carrier detection and prenatal diagnosis of haemophilia A and B by DNA polymorphism analysis in Thailand.
Assuntos
Alelos , Proteínas de Bactérias , Fator IX/genética , Fator VIII/genética , Frequência do Gene , Polimorfismo de Fragmento de Restrição , Desoxirribonucleases de Sítio Específico do Tipo II , Europa (Continente) , Feminino , Triagem de Portadores Genéticos , Hemofilia A/genética , Hemofilia B/genética , Humanos , Masculino , TailândiaRESUMO
A simple, rapid and cheap method for the analysis of variable dinucleotide tandem polymorphisms in introns 13 and 22 of the factor VIII gene has been established. The protocol uses blood spots stored on filter paper (Guthrie spots) and other DNA containing materials as sources of DNA. Following DNA amplification using a thermostable DNA polymerase, products are size fractionated on native polyacrylamide gels and visualized by silver staining. This simplified protocol obviates the use of DNA extraction, reducing time and costs and in addition, reduces the requirement for gel documentation equipment (i.e., photography), as silver staining can be visualized readily without additional equipment and the gels themselves can be stored. These alterations to the analysis enhance the universal applicability of these polymorphisms, as was demonstrated by a comparative study in Caucasian and Thai females.
Assuntos
Repetições de Dinucleotídeos , Fator VIII/genética , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Análise Custo-Benefício , Fator VIII/análise , Fator VIII/economia , Hemofilia A/economia , Hemofilia A/genética , Humanos , LinhagemRESUMO
The assessment of carrier state based on the pedigree and laboratory testing in 55 females from 34 Thai hemophilia families (24 affected by hemophilia A, 10 by hemophilia B) was studied. The laboratory testing included phenotypic analysis (FVIII:C/vWF: Ag ratio, FIX:C) and two types of DNA polymorphisms, restriction fragment length polymorphisms (RFLP) and variable number tandem repeats (VNTR) in/and close to the factor VIII genes (Bcl I, Xba I RFLP, St 14 VNTR) and factor IX genes (Mse I, Dde I RFLP). Fifteen out of seventeen (88%) obligate hemophilia A carriers and one out of five (20%) obligate hemophilia B carriers were diagnosed by phenotypic analysis. All hemophilia A carriers were informative for at least one polymorphism (Bcl I, Xba I or St 14) while 42% of hemophilia B carriers were informative for Mse I RFLP only. DNA polymorphism analysis has advantage over phenotypic analysis since it generally gives an absolute diagnosis when informative. Most DNA polymorphism analyses are performed by PCR technique which is a simple, inexpensive and quick procedure. However, it is limited by non-informativeness and high incidence of new mutations.