Factor VIII, a component of the intrinsic pathway of blood coagulation, has yet to be purified to homogeneity. It appears that, in vivo, the factor VIII coagulant protein is closely associated with one or more other proteins (factor VHI-related antigen and platelet aggregating factor). The material normally isolated from bovine plasma as 'factor VIII' possesses all three activities and is therefore either a mixture or a complex of the various proteins. In the present study, bovine factor VIII:C was purified approximately fivethousand- fold by a combination of ion-exchange chromatography and fractional precipitation. The factor VIII coagulant activity can be separated from the other activities of the 'factor VIII complex' but the procedures involved are not suitable for preparative use as the factor VIII:C which is obtained is unstable. During coagulation, factor VIII:C is required during the activation of factor X. Studies with purified bovine clotting factors indicate that factor IX_a is the enzyme responsible for the cleavage of factor X, in a calcium-dependent reaction which is stimulated by phospholipid. Factor VIII:C further accelerates the rate at which factor X_a is generated. Preliminary investigations of the kinetic parameters of the reaction indicate that the stimulation by factor VIII:C occurs through a marked increase in the V_max of the reaction; factor VIII:C does not affect the K_m for factor X. The coagulant activity of factor VIII is enhanced by exposure to thrombin, but the 'activated' factor VIII:C which is produced is not itself capable of activating factor X in the absence of factor IX_a. Thus, the 'activation' of factor VIII:C, in contrast to the activation of, for example, factors IX and X, does not appear to result in the formation of an enzyme. That is, factor VIII:C is a non-enzymic, high molecular weight cofactor for factor IX_a.

The role of the nonionic surfactant Tween 80 on the behavior of the therapeutic recombinant protein Factor VIII (rFVIII) was investigated at solid/liquid and air/water interfaces. In order to provide a model system to compare results obtained for the complicated rFVIII-Tween system, a well-characterized globular protein lysozyme was used. The experimental scheme involved the introduction of the protein and Tween to the adsorption substrate in different manners, either lysozyme Tween together or in sequence as lysozyme followed by Tween or vice versa. It was observed that the addition of Tween together with lysozyme reduced the amounts adsorbed at hydrophobic surfaces, while no such reduction was observed on hydrophilic surfaces. A high Tween concentration was required to effect the removal of the lysozyme molecules from the hydrophobic surface and Tween was not effective in removing lysozyme from the hydrophilic surface at any concentration. These results suggest that the Tween-surface interaction is important in determining lysozyme adsorption. Similar observations were made for the rFVIII-Tween system at hydrophobic and hydrophilic silica interfaces. In this case, the presence of interfacial and solution Tween together resulted in complete prevention of rFVIII adsorption. Electrostatic forces were observed to be play an important role in rFVIII adsorption. The rFVIII-Tween interactions at solid interfaces were also evaluated using intrinsic fluorescence and biological activity measurements. Results obtained with respect rFVIII adsorbed mass, and structure or biological activity change upon adsorption, were evaluated in parallel. This parallel evaluation suggested that rFVIII adsorption on hydrophilic, negatively charged surfaces is likely to be highly ordered and oriented in a manner that retains the solvent accessibility of the active sites in rFVIII. On the other hand, rFVIII may adsorb to hydrophobic surfaces in different orientations, with a likelihood of surface induced unfolding. rFVIII-Tween interaction at the air/water interface was investigated separately. Surface tension data recorded for rFVIII-Tween mixtures suggested that Tween dominated the air/water interface as the Tween concentration was increased. Reduced interface-induced unfolding was observed at high Tween concentrations. These results were also thought to contribute to the reduction in rFVIII aggregation typically observed as a result of exposure to the air/water interface. / Graduation date: 2006

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 42-44). Also available on the World Wide Web.

Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair mechanisms cause insertions and deletions, or if a template oligonucleotide can be provided point mutations could be introduced or corrected. However, to accomplish this goal for editing factor VIII mutations, a way to deliver the components of CRISPR/Cas9 into somatic cells is needed. In this study, I confirmed that the CRISPR/Cas9 system was able to create a mutation in the factor VIII gene in zebrafish. I also showed that the components of CRISPR/Cas9 could be piggybacked by vivo morpholino into a variety of blood cells. This study also confirmed that the vivo morpholino did not interfere with the gRNA binding to the DNA, or Cas9 protein inducing the double stranded break.

A Hemofilia A é uma doença sanguínea condicionada por gene localizado no cromossomo X. É causada pela deficiência parcial ou total da atividade do Fator VIII (FVIII), uma glicoproteína plasmática cuja função é necessária para a coagulação normal do sangue. Devido às dificuldades encontradas para o reconhecimento direto da mutação no gene F8, o diagnóstico das portadoras é feito de forma indireta, isto é, por análise de ligação com marcadores polimórficos localizados dentro ou próximos ao gene que permite determinar a co-segregação do haplótipo e da mutação na família sob estudo e, desta maneira, detectar o estado de portadora e, eventualmente, auxiliar no diagnóstico pré-natal. O presente trabalho teve por objetivo avaliar o poder de alguns desses marcadores na diferenciação das populações e determinar o grau de sua informatividade para o diagnóstico e aconselhamento genético de famílias afetadas, bem como verificar o eventual uso forense de tais marcadores. Foram então determinadas as frequências alélicas e haplotípicas, diversidade genética, diferenciação populacional, desequilíbrio de ligação e composição ancestral de quatro microssatélites intragênicos (IVS 1, IVS 13, IVS 22, IVS 25.3), localizados em introns do F8, e um extragênico (IKBKG) em amostras de populações brasileiras urbanas (indivíduos normais de São Paulo, Rio Grande do Sul e Pernambuco), de quilombos (Mimbó, Sítio Velho e Gaucinha localizados no Estado do Piauí) e Ameríndios (Tikúna, Baníwa e Kashináwa). As análises, quando cabível, incluíram um grupo de pacientes hemofílicos. O DNA dos sítios polimórficos foi amplificado por PCR, os produtos separados em PAGE e corado por nitrato de prata. Para as análises estatísticas foram empregados programas já considerados de uso rotineiro. Os parâmetros de diversidade mostraram diferenças entre as amostras populacionais analisadas. Tais diferenças regionais nas frequências alélicas devem ser levadas em conta quando o diagnóstico indireto da Hemofilia A estiver sendo realizado. Com exceção do IKBKG, todos os demais microssatélites apresentaram altas taxas de heterozigose. Usando tais marcadores, o diagnóstico foi possível em 10 das 11 famílias analisadas. Os microssatélites IVS 22, IVS 1, IVS 13, IVS 25.3 e IKBKG foram informativos em 63,6% (7/11), 54.5% (6/11), 54.5% (6/11), 45.5% (5/11) e 18.2% (2/11) dos casos, respectivamente, demonstrando a eficácia do uso desses microssatélites no diagnóstico pré-natal e na identificação de portadoras na população brasileira. / Hemophilia A is a bleeding disorder conditioned by a gene located on the X chromosome and caused by partial or total deficiency of the Factor VIII (FVIII) activity, a plasma glycoprotein whose function is necessary for normal blood clotting. Due to difficulties faced on direct recognition of the F8 gene mutation, carrier diagnosis is done indirectly by linkage analysis with polymorphic markers located within or near the gene. These markers may determine the haplotype and the mutation co-segregation within the studied family, and thus detect the carrier status and possibly assist in prenatal diagnosis. This study aimed to evaluate the power of some of these markers in population differentiation and to determine their degree of informativeness for diagnosis and genetic counseling of affected families, as well as to verify the possible forensic use of such markers. We then determined the allele and haplotype frequencies, genetic diversity, population differentiation, linkage disequilibrium and ancestral composition in Brazilian urban (healthy individuals from São Paulo, Rio Grande do Sul and Pernambuco), quilombo remnant (Mimbó, Sítio Velho and Gaucinha in the State of Piauí) and Amerindian (Tikúna, Baníwa and Kashináwa) population samples by the analysis of four intragenic microsatellites (IVS 1, IVS 13, IVS 22, IVS 25.3), located on F8 introns, and one extragenic (IKBKG ). When appropriated, the analysis included a group of hemophilic patients. DNA polymorphisms were detected by PCR, PAGE and silver nitrate staining. Statistical analysis was implemented by programs already considered in routine use. Diversity parameters showed differences among the populational samples analyzed. Such regional differences in allele frequencies must be taken into account when conduct the indirect diagnosis of Hemophilia A. Except for IKBKG, all other microsatellites showed high rates of heterozygosity. Using these markers, the diagnosis was possible in 10 of the 11 families analyzed. The microsatellites IVS 22, IVS 1, IVS 13, IVS 25.3 and IKBKG were informative in 63.6% (7 / 11), 54.5% (6 / 11), 54.5% (6 / 11), 45.5% (5 / 11 ) and 18.2% (2 / 11) of the cases, respectively, demonstrating the effectiveness of the use of these microsatellites in prenatal diagnosis and on carrier identification in the Brazilian population.

L’administration de Facteur VIII thérapeutique (FVIII) chez les patients hémophiles A entraine l’apparition d’anticorps anti-FVIII appelés « inhibiteurs » chez 30% des hémophiles A sévères. Ceci constitue alors une impasse thérapeutique. Si de nombreuses investigations ont permis de caractériser les effecteurs lymphocytaires T et B impliqués dans cette réponse immunitaire, elles n’ont toutefois pas permis de proposer aux patients des stratégies thérapeutiques pour prévenir l’apparition des inhibiteurs du FVIII. La première partie de ma thèse explore la possibilité de prévenir la réponse immunitaire anti-FVIII en inhibant la capture et l’apprêtement antigénique du FVIII par les cellules présentatrices de l’antigène (CPA). Il avait été montré précédemment que les deux structures hautement mannosylées du FVIII, sur les asparagines 239 et 2118, étaient reconnues par le CD206 exprimé par les cellules dendritiques humaines dérivées de monocytes, et que cette voie d’endocytose menait à l’apprêtement antigénique du FVIII. Je me suis donc intéressée à la possibilité de réduire l’immunogénicité du FVIII en éliminant ces deux glycosylations. La seconde partie de ma thèse porte sur la possibilité de prévenir ou d’éradiquer la réponse immunitaire anti-FVIII en inhibant une molécule de la signalisation du récepteur des lymphocytes B (LB) : la tyrosine kinase de Bruton (BTK). La BTK jouant un rôle central dans la signalisation des LB, l’inhibition de celle-ci a montré un intérêt thérapeutique dans le cas de certaines pathologies malignes et auto-immunes. J’ai donc exploré le potentiel thérapeutique d’un inhibiteur de la BTK dans la réponse immunitaire anti-FVIII. / Administration of therapeutic factor VIII (FVIII) to hemophilia A patients leads to the development of anti-FVIII antibodies called “inhibitors” in 30% of severe hemophilia A patients. Despite a well characterization of T and B effectors cells involved in this immune response, there is still no therapeutic strategy proposed to the patients to prevent the occurrence of FVIII inhibitors. The first part of my thesis explores the possibility to prevent the anti-FVIII immune response by blocking FVIII capture and processing by antigen presenting cells (APC). It has been previously demonstrated that the two highly mannosylated structures on FVIII, on asparagines 239 and 2118, were recognized by the CD206 expressed on human monocyte-derived dendritic cells. This endocytic pathway led to FVIII processing and presentation to T cells. Therefore, I have investigated the possibility to reduce FVIII immunogenicity by eliminating those two glycosylations. The second part of my thesis focuses on the possibility to prevent or eradicate the anti-FVIII immune response by inhibiting a molecule involved in B cell receptor signaling: the Bruton’s tyrosine kinase (BTK). BTK plays a key role in B cells signaling and inhibition of BTK has shown a great interest in B cell malignancies, but also in some auto-immune diseases. Therefore, I have investigated the therapeutic potential of a new BTK inhibitor against the development of the anti-FVIII immune response.

Chronic kidney disease (CKD) affects more than 30 million adults in the U.S. and is strongly associated with cardiovascular events and mortality. Venous thromboembolism (VTE) is the third leading vascular disease, affects up to 900,000 Americans each year and contributes to as many as 100,000 deaths annually. The relationship of CKD and VTE has been described in patients receiving dialysis, kidney transplants recipients and in nephrotic syndrome, however, data supporting the association of VTE in mild to moderate CKD is conflicted. The overall goal of this research was to study the association of CKD and VTE and to understand the mechanisms of this association. To accomplish this goal we studied participants of the Reasons for Geographic and Racial Differences in Stroke (REGARDS) Study, a nationally representative cohort of 30,239 blacks and whites in the U.S.. The first chapter provides a review of the state-of-the science on CKD and VTE and potential mechanisms for this association. We focus on factor VIII as a potential mediator of VTE risk in CKD by reviewing the biochemistry and epidemiology linking factor VIII and CKD. In Chapter 2, we use a cohort study design and a competing risk analysis to determine the risk of VTE with albuminuria (ACR) and with various equations for estimated glomerular filtration rate (eGFR). There was no association of ACR and VTE and the risk of VTE was similar among eGFR equations. Compared to a normal eGFR (>90 ml/min/1.73m2), eGFR < 45 ml/min/1.73m2 was associated with a two-fold risk of VTE. The association of eGFR and unprovoked VTE was similar to the association with provoked VTE. The population attributable fraction of CKD (eGFR<60 ml/min/1.73m2) was modest at 5%. In Chapter 3, we utilize a case-cohort study to determine if biomarkers of inflammation (C-reactive protein) and procoagulation (Factor VIII and D-dimer) attenuate the risk of VTE in CKD. These biomarkers were higher in lower kidney function and were also strongly associated with VTE. Adjustment for factor VIII fully attenuated the risk of VTE in CKD, thus factor VIII is a potential mediator of the association of CKD and VTE. We assessed whether lifestyle factors and medications mitigate the risk of VTE in those with and without CKD. Exercise frequency and use of statins were associated with reduced risk of VTE in the presence and absence of CKD, but normal BMI was associated with reduced VTE risk only in those without CKD. We conclude that CKD is a risk factor for VTE, and findings shed light on the mechanisms of this association. Interventions that might lower VTE risk in CKD patients include exercise and statin therapy, but not weight loss. Factor VIII is a potential mediator of VTE in CKD and deserves further study. We suggest several avenues for future research to explore the relationship of Factor VIII and CKD.

Orientador: Joyce Annichino-Bizzacchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T01:42:43Z (GMT). No. of bitstreams: 1 Bittar_LuisFernando_M.pdf: 1309049 bytes, checksum: 8d7bbcbc4a659300d128bc58336cbb88 (MD5) Previous issue date: 2009 / Resumo: Níveis elevados de fator VIII (FVIII) são um fator de risco independente e prevalente para trombose venosa profunda (TVP), e tem influência do FvW. A ADAMTS13 é responsável pela modulação do tamanho molecular do FvW, clivando os multímeros de altíssimo peso molecular. Alterações moleculares no gene da ADAMTS13 têm correlação com sua atividade. Neste estudo avaliamos a prevalência do polimorfismo A4751G no gene do FvW (região de ligação com a ADAMTS13), os polimorfismos C1797T e C1852G e a mutação C4006T no gene da ADAMTS13 em 435 pacientes com TVP (156M/279F; idade mediana=37) e 580 controles (170M/410F, idade mediana=35). Investigamos a relação entre os genótipos e a dosagem de FVIII e FvW no plasma e o risco de TVP. A dosagem de FVIII:C foi realizada por método coagulométrico de um estágio, e as dosagens de FVIII:Ag e FvW:Ag por método imunoenzimático. As alterações moleculares foram determinadas por PCR e digestão com enzimas específicas, ou SSCP e sequenciamento para confirmação. Pacientes com TVP mostraram níveis significantemente aumentados de FVIII:C (203.7 UI/dl vs. 127 UI/dl; p<0.001), FVIII:Ag (109.6 UI/dl vs. 82.4 UI/dl; p<0.001) e FvW:Ag (154.2 UI/dl vs. 108 UI/dl; p<0.001) quando comparados com o grupo controle. Não houve diferença significativa entre os grupos na prevalência das alterações moleculares estudadas. Os indivíduos com genótipo AG (FvW A4751G) apresentavam níveis significativamente reduzidos de FVIII:C (p=0.04). Embora também tenha demonstrado uma discreta associação com níveis diminuídos de FvW:Ag, esta não foi estatisticamente significativa (p= 0.07). Os indivíduos com genótipo CG (ADAMTS13 C1852G) apresentavam níveis significativamente aumentados de FVIII:Ag (p=0.05) e FvW:Ag (p= 0.01). Apesar da relação com diminuição do FVIII o polimorfismo A4751G não mostrou um efeito protetor para TVP. O polimorfismo ADAMTS13 C1852G está associado à diminuição desta metaloprotease, e sua associação com níveis aumentados de FVIII:Ag e FvW:Ag neste estudo favorece essa hipótese. / Abstract: Elevated levels of factor VIII (FVIII) are a prevalent and independent risk factor for deep venous thrombosis (DVT), and are affected by von Willebrand factor (vWF) levels. ADAMTS13 is responsible for the modulation of the molecular size of vWF, cleaving the ultra large multimers. Mutations and polymorphisms in the ADAMTS13 gene are related with its activity. This study evaluated the prevalence of polymorphism A4751G in the vWF gene, polymorphisms C1797T, C1852G and the mutation C4006T in the ADAMTS13 gene in 435 patients with DVT and 580 healthy controls. Subsequently, we investigated the relationship between the genotypes and plasma levels of FVIII and vWF and DVT risk. FVIII:C was measured by a one-stage clotting method, and FVIII:Ag and vWF:Ag were measured by chromogenic method. The molecular changes were determined by restriction endonucleases or single strand conformation polymorphism followed by sequencing. Statistical test:U Mann-Whitney, = 0.05. Patients with DVT had higher plasma levels of FVIII:C (mean 203,7 UI/dl vs. 127 UI/dl; p<0.001), FVIII:Ag (mean 109,6 UI/dl vs. 82,4 UI/dl; p<0.001) and vWF:Ag (154,2 UI/dl vs. 108 UI/dl; p<0.001) when compared to controls. We observed no statistical difference in the prevalence of all molecular changes studied between patients and controls. A4751G heterozygotes had significantly reduced levels of FVIII:C (p=0,04). Althought there was a slight association with reduced levels of vWF: Ag, this association was not statistically significant (p= 0,07). C1852G heterozygotes had significantly elevated levels of FVIII:Ag (p=0.05) and vWF:Ag (p= 0,01). Despite the relative decline of FVIII:C with the A4751G polymorphism there was no protective effect for DVT. The C1852G polymorphism is associated with a reduction of ADAMTS13, and its association with increased levels of FVIII: Ag and vWF: Ag observed in this study supports this hypothesis. / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Orientador: Margareth Castro Ozelo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T13:42:22Z (GMT). No. of bitstreams: 1 Damian_GuilhermeBenedini_M.pdf: 1979799 bytes, checksum: 8b7242b6fe37a1f5f52bc6f26ca9d829 (MD5) Previous issue date: 2010 / Resumo: A doença de von Willebrand (DVW) é a doença hemorrágica hereditária mais freqüente. Dentre os subtipos relacionados à DVW, o tipo 2N apresenta exclusivamente diminuição da afinidade do fator de von Willebrand (FVW) ao fator VIII (FVIII). Como conseqüência, esses pacientes apresentam redução do FVIII plasmático. Por essa razão, comumente esses casos são diagnosticados erroneamente como portadores de hemofilia A. Em um recente estudo brasileiro, foi observado que cerca de 10% dos casos de hemofílicos A leve sem antecedente familiar prévio para a doença, na verdade tratavam-se de DVW tipo 2N (SIMON & ROISENBERG, 2004). Para o diagnóstico de certeza da DVW tipo 2N, a avaliação molecular é o método mais utilizado, uma vez que os testes laboratoriais confirmatórios não apresentam boa reprodutividade. Esse estudo tem como objetivos fazer a investigação molecular de casos previamente diagnosticados ou com suspeita de DVW tipo 2N baseados na história clínica, familiar e dados laboratoriais. Além disso, pacientes diagnosticados com hemofilia A moderada e leve, foram investigados para a presença das quatro mutações mais freqüentes relacionadas à DVW tipo 2N. Esse estudo avaliou oito casos não relacionados de paciente com suspeita ou diagnóstico de DVW tipo 2N, acompanhados no Hemocentro da UNICAMP (Campinas, SP) e no Hemocentro do Espírito Santo (Vitória, ES). Os oito casos foram investigados para mutações presentes na região que corresponde ao local do sítio de ligação do FVIII ao FVW, localizada entre os éxons 18 a 27 do gene do FVW. Apenas um caso foi conclusivo, com a presença da mutação em homozigose R816W no gene do FVW Essa mutação corresponde à 11% dos casos de DVW tipo 2N. Os outros sete casos foram exaustivamente investigados para mutações nessa região, incluindo o seqüenciamento do DNA genômico e codificante da região entre éxon 18 a 27 do gene do FVW e não foram conclusivos. Esses mesmos casos foram investigados através do seqüenciamento do gene do FVIII, para avaliar a presença de mutação associada à hemofilia A. Em um dos casos do sexo feminino, foi possível a identificação de uma mutação em homozigose G265 +1 G>T no íntron 3 do gene do FVIII. Trata-se de uma mutação não descrita no local de um sítio doador de splice. Foram avaliados ainda 67 pacientes não relacionados com diagnóstico de hemofilia A leve ou moderada para as quatro mutações mais freqüentes relacionadas à DVW tipo 2N. Em nenhum desses casos essas mutações estavam presente, nem mesmo em heterozigose. Em conclusão, na avaliação de oito casos não relacionados com suspeita de DVW tipo 2N, em apenas um caso esse diagnóstico foi confirmado. Em outro caso do sexo feminino foi confirmado o diagnóstico de hemofilia A grave/moderada. Ao contrário do que foi observado em outra população brasileira estuda, entre os pacientes diagnosticados com hemofilia A leve e moderada aqui estudos, as quatro mutações mais freqüentes na região do sítio de ligação do FVIII ao FVW estavam ausentes em todos os casos. O diagnóstico de certeza entre a DVW tipo 2N e hemofilia A é de extrema importância para o tratamento correto desses casos e para o aconselhamento genético. A investigação molecular continua sendo a melhor maneira para a diferenciação desses casos / Abstract: The von Willebrand disease (VWD) is the most frequently hemorrhagic disease. Among the different types of VWD, the type 2N VWD is characterized by a markedly decreased affinity of von Willebrand factor (VWF) to factor VIII (FVIII). As consequence, the clearance of FVIII is accelerated and these patients while maintaining the concentration of VWF and its ability to normal platelet aggregation have reduced plasma FVIII. Frequently these cases are misdiagnosed as carriers of hemophilia A (HA). In a recent Brazilian study, it was observed that about 10% of cases of mild hemophilia A with no family history, it was actually type 2N VWD (SIMON & ROISENBERG, 2004). The molecular investigation is the best method to confirm the diagnosis of type 2N VWD once the confirmatory test, VWF:FVIII binding assay (VWF:FVIIIB) it is not reproducible. This study aims investigate the molecular diagnosis of cases previously diagnosed or with suspect of type 2N VWD, based on clinical and family history, and laboratorial data. Furthermore, patients diagnosed with mild or moderate hemophilia A, were investigated for the presence of the four most frequent mutations related to type 2N VWD. The study evaluated eight unrelated cases with suspect or diagnosis of type 2N VWD followed at Hemocentro UNICAMP (Campinas, SP) and Hemocentro do Espírito Santo (Vitória, ES). The eight cases were investigated for mutations in the region that corresponds to the location of the binding site of FVIII to VWF, located between exons 18 to 27 of the VWF gene. Only one case was conclusive, with the presence of the R816W mutation in the VWF gene. This mutation corresponds to 11% of type 2N VWD. The other seven cases were repeatedly investigated for mutations in this region, including the sequencing of genomic DNA and the coding region of exon 18 to 27 of the VWF gene and were not conclusive. These same cases were investigated by sequencing of the FVIII gene to assess the presence of mutations associated with hemophilia A. In one female case, it was possible to determine the presence in homozigose of a mutation G265 +1 G>T, a donor splicing region in the intron 3 of the FVIII gene. This mutation was not previously described. In addition, we evaluated 67 unrelated patients with diagnosis of mild or moderate hemophilia A for the four most frequent mutations related to type 2N VWD. None of these cases showed the presence of these mutations, even in heterozygosis. In conclusion, the evaluation of eight unrelated cases for type 2N VWD showed that just one case this diagnosis was confirmed. Different to what was observed in another Brazilian population studied, among mild and moderate hemophilia A followed at the Hemocentro UNICAMP, the four mutations in the binding site of FVIII to VWF were absent in all the cases. The confirmatory diagnosis between type 2N VWD and hemophilia A is extremely important for the correct treatment and the appropriate genetic counselling. The molecular investigation remains the best way to differentiate these cases / Mestrado / Clinica Medica / Mestre em Clinica Medica

A Hemofilia A é uma doença sanguínea condicionada por gene localizado no cromossomo X. É causada pela deficiência parcial ou total da atividade do Fator VIII (FVIII), uma glicoproteína plasmática cuja função é necessária para a coagulação normal do sangue. Devido às dificuldades encontradas para o reconhecimento direto da mutação no gene F8, o diagnóstico das portadoras é feito de forma indireta, isto é, por análise de ligação com marcadores polimórficos localizados dentro ou próximos ao gene que permite determinar a co-segregação do haplótipo e da mutação na família sob estudo e, desta maneira, detectar o estado de portadora e, eventualmente, auxiliar no diagnóstico pré-natal. O presente trabalho teve por objetivo avaliar o poder de alguns desses marcadores na diferenciação das populações e determinar o grau de sua informatividade para o diagnóstico e aconselhamento genético de famílias afetadas, bem como verificar o eventual uso forense de tais marcadores. Foram então determinadas as frequências alélicas e haplotípicas, diversidade genética, diferenciação populacional, desequilíbrio de ligação e composição ancestral de quatro microssatélites intragênicos (IVS 1, IVS 13, IVS 22, IVS 25.3), localizados em introns do F8, e um extragênico (IKBKG) em amostras de populações brasileiras urbanas (indivíduos normais de São Paulo, Rio Grande do Sul e Pernambuco), de quilombos (Mimbó, Sítio Velho e Gaucinha localizados no Estado do Piauí) e Ameríndios (Tikúna, Baníwa e Kashináwa). As análises, quando cabível, incluíram um grupo de pacientes hemofílicos. O DNA dos sítios polimórficos foi amplificado por PCR, os produtos separados em PAGE e corado por nitrato de prata. Para as análises estatísticas foram empregados programas já considerados de uso rotineiro. Os parâmetros de diversidade mostraram diferenças entre as amostras populacionais analisadas. Tais diferenças regionais nas frequências alélicas devem ser levadas em conta quando o diagnóstico indireto da Hemofilia A estiver sendo realizado. Com exceção do IKBKG, todos os demais microssatélites apresentaram altas taxas de heterozigose. Usando tais marcadores, o diagnóstico foi possível em 10 das 11 famílias analisadas. Os microssatélites IVS 22, IVS 1, IVS 13, IVS 25.3 e IKBKG foram informativos em 63,6% (7/11), 54.5% (6/11), 54.5% (6/11), 45.5% (5/11) e 18.2% (2/11) dos casos, respectivamente, demonstrando a eficácia do uso desses microssatélites no diagnóstico pré-natal e na identificação de portadoras na população brasileira. / Hemophilia A is a bleeding disorder conditioned by a gene located on the X chromosome and caused by partial or total deficiency of the Factor VIII (FVIII) activity, a plasma glycoprotein whose function is necessary for normal blood clotting. Due to difficulties faced on direct recognition of the F8 gene mutation, carrier diagnosis is done indirectly by linkage analysis with polymorphic markers located within or near the gene. These markers may determine the haplotype and the mutation co-segregation within the studied family, and thus detect the carrier status and possibly assist in prenatal diagnosis. This study aimed to evaluate the power of some of these markers in population differentiation and to determine their degree of informativeness for diagnosis and genetic counseling of affected families, as well as to verify the possible forensic use of such markers. We then determined the allele and haplotype frequencies, genetic diversity, population differentiation, linkage disequilibrium and ancestral composition in Brazilian urban (healthy individuals from São Paulo, Rio Grande do Sul and Pernambuco), quilombo remnant (Mimbó, Sítio Velho and Gaucinha in the State of Piauí) and Amerindian (Tikúna, Baníwa and Kashináwa) population samples by the analysis of four intragenic microsatellites (IVS 1, IVS 13, IVS 22, IVS 25.3), located on F8 introns, and one extragenic (IKBKG ). When appropriated, the analysis included a group of hemophilic patients. DNA polymorphisms were detected by PCR, PAGE and silver nitrate staining. Statistical analysis was implemented by programs already considered in routine use. Diversity parameters showed differences among the populational samples analyzed. Such regional differences in allele frequencies must be taken into account when conduct the indirect diagnosis of Hemophilia A. Except for IKBKG, all other microsatellites showed high rates of heterozygosity. Using these markers, the diagnosis was possible in 10 of the 11 families analyzed. The microsatellites IVS 22, IVS 1, IVS 13, IVS 25.3 and IKBKG were informative in 63.6% (7 / 11), 54.5% (6 / 11), 54.5% (6 / 11), 45.5% (5 / 11 ) and 18.2% (2 / 11) of the cases, respectively, demonstrating the effectiveness of the use of these microsatellites in prenatal diagnosis and on carrier identification in the Brazilian population.