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Zeitschriftenartikel zum Thema "Hemophilia – Genetics"
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Knox, David, Christopher Samuel, Janneth Pazmino-Canizares, Chris Barnes, Georgina Floros, Ann Marie Stain und Manuel Carcao. „The Genetics of Hemophilia: Analysis of Patients at the Hospital for Sick Children, Toronto, Canada.“ Blood 108, Nr. 11 (16.11.2006): 1040. http://dx.doi.org/10.1182/blood.v108.11.1040.1040.
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Abstract There is great interest in identifying genetic mutations responsible for hemophilia and in determining if/how mutations correlate with disease phenotype. A hemophilia genetic database was created at SickKids in 2004. At the time <40% of hemophiliacs followed by the clinic had been genotyped. Currently mutations have been identified on 194/236 patients (82%) followed in the clinic. From this we are performing genotype/phenotype correlations. Preliminary analysis has revealed the following novel findings: Most mothers of hemophiliacs are carriers; even when there is a no family history of hemophilia. Of the 199 mothers of the 236 children only 6 have been shown not to be carriers; 205 have been shown to be carriers; 15 have not been tested and 10 are unavailable for testing. We believe that most de novo FIX and FVIII mutations occur in females. Mutations responsible for hemophilia B show poor concordance with disease severity i.e. for any mutation the disease severity is not always the same. One example found in 10 hemophiliacs (5 families) who despite having the same missense mutation in exon H have shown FIX levels anywhere between 0 and 11%. Given that disease severity is assigned according to factor levels these patients have been labeled as either severe (n = 3), moderate (n = 5) or mild (n = 2). Clinically these patients all appear to behave as moderate. This points to the fallibility of using FIX levels (which varies according to patient age and health state) in labeling patients. Other aspects of the hemophilia genetic data base are being analyzed. We believe that a detailed study of the genetics of hemophilia will point to novel findings that will eventually translate into patient care.
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Lawn, Richard M., und Gordon A. Vehar. „The Molecular Genetics of Hemophilia“. Scientific American 254, Nr. 3 (März 1986): 48–54. http://dx.doi.org/10.1038/scientificamerican0386-48.
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Geddes, Valerie A., und Ross T. A. MacGillivray. „The Molecular Genetics of Hemophilia B“. Transfusion Medicine Reviews 1, Nr. 3 (Dezember 1987): 161–70. http://dx.doi.org/10.1016/s0887-7963(87)70018-2.
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Chudley, Albert E., und James C. Haworth. „Genetic landmarks through philately - hemophilia“. Clinical Genetics 56, Nr. 4 (Oktober 1999): 279–81. http://dx.doi.org/10.1034/j.1399-0004.1999.560404.x.
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Gouw, Samantha C., und Karin Fijnvandraat. „Unraveling the genetics of inhibitors in hemophilia“. Blood 121, Nr. 8 (21.02.2013): 1250–51. http://dx.doi.org/10.1182/blood-2012-12-472647.
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Tantawy, Azza A. G. „Molecular genetics of hemophilia A: Clinical perspectives“. Egyptian Journal of Medical Human Genetics 11, Nr. 2 (November 2010): 105–14. http://dx.doi.org/10.1016/j.ejmhg.2010.10.005.
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Astermark, Jan, John Schwarz, Sharyne M. Donfield, Donna M. DiMichele, Bruce M. Ewenstein, Edward D. Gomperts, George W. Nelson et al. „Genetic Factors Associated with Inhibitor Development in Hemophilia A: Initial Results From the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort.“ Blood 114, Nr. 22 (20.11.2009): 217. http://dx.doi.org/10.1182/blood.v114.22.217.217.
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Abstract Abstract 217 Introduction: Both genetic and environmental factors have been implicated as potential risk factors for the development of inhibitory factor VIII (FVIII) antibodies. Previous studies suggest that genetic factors are of major importance. The causative FVIII mutation likely sets the stage for inhibitor risk, with other genetic markers important in determining the final outcome. Data suggest that the process of inhibitor development is complex, involving a variety of immune regulatory genes, several of which have the potential to modify risk. Through a collaboration among three multi-center studies: the Hemophilia Inhibitor Genetics Study (HIGS), the Malmö International Brother Study (MIBS), and the Hemophilia Growth and Development Study (HGDS), a combined cohort was formed to conduct an association study to test the hypothesis that antibody development to FVIII is mediated by immune response genes. Methods: The study includes clinical and laboratory data for 680 people with hemophilia A. Participants from Europe and North America account for 43% and 57% of the population, respectively. Eighty-five percent have severe (<0.01 IU/mL), 10% moderate (0.01 – 0.05% IU/mL), and 4.4% mild (>0.05 – 0.4 IU/mL) hemophilia. The cohort is predominately Caucasian, 81.0%, with 6.2% of African heritage, 8.8% Hispanic, and the remaining 4% of other races and ethnicities. Forty-nine percent have a current, or history of, an inhibitor ≥1 BU. Using the Illumina iSelect platform, 14,626 single nucleotide polymorphisms (SNPs) from 1,081 candidate genes were genotyped. These included immune response and immune modifier genes: cytokines and their receptors, chemokines and their receptors, and pathway genes involved in inflammatory and immune responses. Analyses were completed among the total group and the subgroup with severe hemophilia to identify SNPs associated with inhibitor status. The models were adjusted for population admixture, severity of hemophilia, type of mutation (high vs. low risk), year of birth, and geographic region. Meta analyses were used to obtain single odds ratios (OR) and p-values for the three cohorts. Results: 13,952 of the 14,626 (95.4%) SNPs were successfully genotyped. One hundred fourteen were associated with inhibitor status at the p<0.01 level. Strong SNP associations for the total group were observed in the DOCK2 (OR 0.28, p= 0.00004, and OR 3.9, p=0.0002), MAPK9 (OR 2.0, p=0.0003), F13A1 (OR 0.32, p=0.00007), CD36 (OR 0.56, p=0.0002), and PTPRR (OR 0.51, p=0.0003) genes. For four markers located within the MAPK9, DOCK2, and CD36 genes, the associations were similar, or stronger, for the subgroup with severe hemophilia. Analysis of polymorphisms in the FVIII gene, completion of HLA Class II typing, and haplotype analysis are underway. Conclusions: Our findings suggest that functional pathways involved in a variety of cellular processes will be important in inhibitor development, but these results warrant further study and replication in similarly powered case-control or cohort studies. Disclosures: Ewenstein: Baxter Healthcare: Employment. Spotts:Baxter Healthcare: Employment.
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Fernández, Raquel M., Ana Peciña, Beatriz Sánchez, Maria Dolores Lozano-Arana, Juan Carlos García-Lozano, Rosario Pérez-Garrido, Ramiro Núñez, Salud Borrego und Guillermo Antiñolo. „Experience of Preimplantation Genetic Diagnosis for Hemophilia at the University Hospital Virgen Del Rocío in Spain: Technical and Clinical Overview“. BioMed Research International 2015 (2015): 1–8. http://dx.doi.org/10.1155/2015/406096.
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Hemophilia A and B are the most common hereditary hemorrhagic disorders, with an X-linked mode of inheritance. Reproductive options for the families affected with hemophilia, aiming at the prevention of the birth of children with severe coagulation disorders, include preimplantation genetic diagnosis (PGD). Here we present the results of our PGD Program applied to hemophilia, at the Department of Genetics, Reproduction and Fetal Medicine of the University Hospital Virgen del Rocío in Seville. A total of 34 couples have been included in our program since 2005 (30 for hemophilia A and 4 for hemophilia B). Overall, 60 cycles were performed, providing a total of 508 embryos. The overall percentage of transfers per cycle was 81.7% and the live birth rate per cycle ranged from 10.3 to 24.1% depending on the methodological approach applied. Although PGD for hemophilia can be focused on gender selection of female embryos, our results demonstrate that methodological approaches that allow the diagnosis of the hemophilia status of every embryo have notorious advantages. Our PGD Program resulted in the birth of 12 healthy babies for 10 out of the 34 couples (29.4%), constituting a relevant achievement for the Spanish Public Health System within the field of haematological disorders.
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Chuansumrit, Ampaiwan, Werasak Sasanakul, Ian Williams, Anne Goodeve, Praguywan Kadegasem und Ian Peake. „Comparison of Phenotypic Assessment and Mutation Detection in the Diagnosis of Carrier State in Hemophilia: Identification of 10 Novel Mutations.“ Blood 104, Nr. 11 (16.11.2004): 4020. http://dx.doi.org/10.1182/blood.v104.11.4020.4020.
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Abstract The carrier state in 54 females (A38, B16) at risk from 35 moderate and severe hemophilia families (A25, B10) in Thailand, was determined. They were classified as obligate (A17, B8) and possible (A21, B8) carriers by history taking. The phenotypic assessment was performed in two subsequent blood samples taken one week apart when they were not pregnant or on birth control pills. Then, molecular genetics among hemophiliac patients were performed. Inversion of intron 22 among 25 hemophilia A patients was initially performed. Then, the mutations were intensively detected by using conformation sensitive gel electrophoresis (CSGE). Coding region, intron/exon boun-daries, and 5′ and 3′ regions of the factor VIII and IX genes were amplified in 33 separate reactions in hemophilia A patients without inversion of intron 22 (n=10) and 9 separate reactions in hemophilia B patients (n=10). Samples displaying abnormal CSGE profiles were numbered according to Wood et al and Yoshitake et al. The results revealed that mutation were found at 88% (22/25, 15 inversion of intron 22 and 7 mutations) in hemophilia A and 90% (9/10) in hemophilia B patients. Ten were novel mutations as shown in Table 1. Also, the carrier state assessed by the phenotypic study and mutation detections are shown in Table 2. As a result, the phenotypic assessment alone showed a limitation in the diagnosis of carrier state especially hemophilia B carrier while the mutation detections provide an absolute diagnosis over phenotypic assessment. Table 1. The identified ten novel mutations among Thai hemophilia A and B patients. Table 2. Carrier state assessed by phenotypic study and mutation detections among females at risk. Exon Nucleotide Amino acid FVIII:C/FIX:C (%) Promotor to exon 22 large deletion - FVIII:C < 1 6 680 G>A W208X FVIII: C = 2 8 1264 G>C D403H FVIII:C < 1 12 1820_1821 del CA 588 frame shift FVIII:C < 1 13 2066 T>G L670R FVIII:C = 1 14 4794 A>T E1579D FVIII:C = 1 21 6266 G>A W2070X FVIII:C < 1 A 57_58 del GA −37 frame shift FIX:C < 1 H 31127 T>G C336G FIX:C < 1 H 31171 T>G C350W FIX:C = 3.6 Type of carrier N FVIII:C or FIX:C < 50% FVIII:C/vWF:Ag < 0.6 Inversion intron 22 CSGE Total mutation Obligate hemophilia A 17 35.3% 70.6% 64.7% 17.6% 82.3% Obligate hemophilia B 8 12.5% - - 87.5% 87.5% Possible hemophilia A 21 19.0% 42.8% 33.3% 14.3% 47.6% Possible hemophilia B 8 25.0% - - - 62.5%
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Kehl, Alexandra, Anita Haug Haaland, Ines Langbein-Detsch und Elisabeth Mueller. „A SINE Insertion in F8 Gene Leads to Severe Form of Hemophilia A in a Family of Rhodesian Ridgebacks“. Genes 12, Nr. 2 (21.01.2021): 134. http://dx.doi.org/10.3390/genes12020134.
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Hemophilia A is the most common coagulation factor disorder in humans and dogs. The disease is characterized by the lack or diminished activity of Factor VIII (FVIII), caused by variants in the F8 gene and inherited as an X chromosomal trait. Two related male Rhodesian Ridgebacks were diagnosed with Hemophilia A due to reduced FVIII activity. The purpose of the study was to determine the genetic cause and give breeding advice for the remaining family members in order to eradicate the variant. By Sanger sequencing a short interspersed nuclear element (SINE) insertion in exon 14 of the F8 gene was found. Perfect correlation of this genetic variant with clinical signs of hemophilia A in the family tree, and the lack of this genetic variant in more than 500 unrelated dogs of the same and other breeds, confirms the hypothesis of this SINE being the underlying genetic cause of Hemophilia A in this family. The identification of clinically unaffected female carriers allows subsequent exclusion of these animals from breeding, to avoid future production of clinically affected male offspring and more subclinical female carriers.
Dissertationen zum Thema "Hemophilia – Genetics"
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Firrman, Jenni Ann. „ENHANCEMENT OF hFVIII ACTIVITY THROUGH LC MODIFICATIONS FOR GENE THERAPY OF HEMOPHILIA A“. Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/335863.
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Microbiology and ImmunologyPh.D.
Gene therapy for Hemophilia A (HA) using the recombinant Adeno-associated virus (rAAV) offers an alternative to classic treatment, which consists of FVIII protein infusions. However, due to limitations associated with rAAV and the FVIII protein itself, the end result is a transgene expression below therapeutic limits. One approach to improving the therapeutic value of rAAV gene therapy for HA is to engineer a more active FVIII protein through genetic modifications. Preliminary testing revealed that canine FVIII Light Chain (kLC) enhances coagulation activity, and that it would be possible to improve FVIII activity through modifications of the light chain. Through the process of engineering, evaluation, and negative selection of kLC, a final construct was engineered. The hLC-K12 is a human Light Chain (hLC) construct containing 12 amino acid changes that work together to enhance coagulation activity. A comparison of the FVIII clotting activity to the amount of protein produced determined that hLC-K12 produced a 3.28 fold increase in specific activity over hLC in vitro. Similar in vitro results were observed when hLC-k12 was tested with the X5 heavy chain (X5HC), a heavy chain that has been genetically modified to enhance production. CD4KO/HA mice were injected with a rAAV vector carrying the hLC-K12 gene in conjugation with a rAAV vector carrying the X5HC gene. Replacing the hLC vector with the hLC-K12 vector produced an average 7.43 fold increase in FVIII clotting activity. An ELISA assay revealed no significant difference between productions of the heavy or light chains at any time point. By comparing the clotting activity to the amount of protein produced, it was determined that the increase in coagulation activity was due to an increase in specific activity. In fact, replacing the hLC vector with the hLC-K12 vector resulted in an average 5.8 fold increase in FVIII specific activity. The K12 modifications were evaluated using a single chain FVIII conformation. In vitro, the addition of the K12 mutations to the human heavy chain, hHCK12BDD, resulted in a 4.3 fold increase in clotting activity, but no increase in protein production. There was however, a 3.3 fold increase in specific activity of the protein. Adding the K12 mutations to the X5 heavy chain, X5K12BDD, in vitro, resulted in a 2.7 fold increase in clotting activity and a 1.42 fold increase in specific activity of the protein. Single chain rAAV vectors were packaged and delivered to CD4KO/HA mice. Compared to mice injected with hFVIIIBDD, the hHCK12BDD produced an average 4.6 fold increase in clotting activity. An ELISA revealed no significant difference in production between these two groups. However, mice injected with hHCK12BDD produced FVIII with an average of 4.13 fold increase in specific activity. Similarly, when compared to mice injected with X5FVIIIBDD, the X5K12BDD produced an average 2.14 fold increase in clotting activity. An ELISA assay demonstrated no significant increase in protein production between these two groups. However, when compared to X5BDD, mice injected with the X5K12BDD vector produced FVIII with an average 1.98 fold increase in specific activity. Results demonstrate that the K12 light chain modifications are able to enhance clotting activity of hFVIII both in vitro and in vivo, using either a dual chain or single chain delivery method. In order to determine the mechanism of enhancement, hFVIIIBDD and hHCK12BDD protein was partially purified and tested for activity. Results demonstrated that the hHCK12BDD protein produced a specific activity of 39,153.69 Units/mg, which is a 6.28 fold increase over hFVIIIBDD specific activity, which was 6,237.92 Units/mg. Measurement of conversion from FX to FXa revealed that the hHCK12BDD protein generated a higher amount of FXa at a quicker rate. In conclusion, these results provide evidence that the K12 modifications enhance specific activity through an increase in FXa generation.
Temple University--Theses
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Holt, Erika Tyne. „Perceptions of Severity of Children's Bleeding Disorders: Impact on Parental Quality of Life and Reproductive Decisions“. Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1383060340.
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Landin, Linnéa. „Är genterapi medierad av adenoassocierat virus en effektiv och säker behandling mot hemofili A och B ur ett långsiktigt perspektiv? : En systematisk litteraturstudie“. Thesis, Linnéuniversitetet, Institutionen för kemi och biomedicin (KOB), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-98996.
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Bakgrund - Hemofili A och B är X-kromosombundna blödarsjukdomar, som beror på genetiska avvikelser i de gener som kodar för koagulationsfaktor VIII respektive IX. I dagsläget förlitar sig hemofilipatienter på kontinuerliga intravenösa injektioner med faktorkoncentrat, för att förhindra att potentiellt livshotande blödningar uppstår. Genterapi med rekombinanta adeno-associerade virus (AAV) skulle kunna erbjuda ett kurativt behandlingsalternativ, genom införandet av friska arvsanlag i hepatocyter. Syfte - Syftet med den här litteraturstudien var att undersöka huruvida genterapi medierad av AAV-vektorer är en effektiv och säker behandling mot hemofili A och B ur ett långsiktigt perspektiv. Metod - Studien är genomförd som en systematisk litteraturstudie och är baserad på sex originalartiklar framsökta via databasen PubMed, med sökorden "hemophilia AND gene therapy". Specificerade sökkriterier användes för att underlätta relevansbedömning och valet av artiklar. Resultat - En ökad endogen koagulationsfaktorproduktion kunde påvisas hos majoriteten av studiedeltagarna efter genterapibehandlingarna. Sammantaget observerades också en väsentlig blödningsreducering och en minskad faktorkoncentratanvändning. Störst förbättring noterades i de kohorter som erhållit högre genterapidoser eller den muterade faktor IX Padua-genen. Ingen immunrespons mot transgenprodukten detekterades i någon studie. Däremot sågs ett humoralt immunsvar mot AAV-kapsiden hos samtliga studiedeltagare. En mycket stor variation i T-cellssvar mot AAV-kapsiden kunde noteras. Förhöjda nivåer av alaninaminotransferas (ALAT) var den vanligast förekommande incidenten, men samtliga fall kunde framgångsrikt behandlas med glukokortikoidpreparat. Slutsats - Genterapibehandling med rekombinanta AAV-vektorer mot hemofili A och B förefaller effektiv och säker. Förhöjda ALAT-nivåer återstår dock som en behandlingsproblematik. Längre uppföljningar av fler genterapibehandlade hemofilipatienter krävs, för att kunna dra några definitiva slutsatser, väga risker mot nytta, samt optimera och individanpassa doser.Background - Hemophilia A and B are X-linked bleeding disorders, resulting from defects in the genes encoding coagulation factors VIII and IX respectively. The current treatment for hemophiliacs entails frequent intravenous injections of coagulation factor concentrates, to prevent potentially life-threatening hemorrhages. Gene therapy utilizing recombinant adeno-associated viruses (AAV) could offer a potentially curative treatment option through the introduction of healthy genes into hepatocytes. Aim - The aim of this literature study was to investigate the long-term efficacy and safety of AAV vector-mediated gene therapy for the treatment of hemophilia A and B. Methods - The study is conducted as a systematic literature study and is based on six original articles retrieved from the search engine PubMed, using the key words "hemophilia AND gene therapy". Specific search criteria were used to facilitate the relevance assessment and selection of articles. Results - An increased endogenous coagulation factor synthesis was noted in the majority of the study participants after the gene therapy. Overall, a significant reduction in bleeding episodes and the use of factor concentrates were observed. The greatest improvements were noted in the cohorts that received the higher gene therapy doses or the mutated factor IX Padua gene. None of the study participants had an immunologic response to the transgene product. A humoral immune response against the AAV capsid was seen in all participants though. Large differences in AAV capsid-specific T-cell activation were observed. The most common adverse event was an elevation in the alanine aminotransferase (ALT) level. However, these events could be controlled with glucocorticoids. Conclusions - AAV vector-mediated gene therapy for the treatment of hemophilia A and B had a positive efficacy and safety profile. Although increased ALT levels remain a concern. Monitoring of larger numbers of study participants for longer follow-up periods is necessary for any definite conclusions to be drawn, to weigh risks against benefits and to optimize individual dosing.
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Tedgård, Ulf. „Prenatal diagnosis of haemophilia psychological, social and ethical aspects /“. Malmö : Dept. of Pediatrics, University Hospital of Malmö, University of Lund, 1999. http://catalog.hathitrust.org/api/volumes/oclc/57455671.html.
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Santos, Andrey dos. „Caracterização de aspectos geneticos e imunologicos envolvidos no desenvolvimento de inibidores em hemofilia A e B“. [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/310746.
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Orientador: Margareth Castro OzeloTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Uma complicação decorrente do tratamento da hemofilia é a formação de anticorpos neutralizadores da atividade coagulante do fator VIII ou IX (inibidores). Diversos fatores estão relacionados com o desenvolvimento desses inibidores em indivíduos com hemofilia, incluindo fatores genéticos e ambientais. Entre os fatores genéticos, a mutação associada ao diagnóstico da hemofilia é um fator de risco bem documentado. Recentemente foi observada a maior ocorrência de inibidores em indivíduos da etnia negra. O objetivo deste trabalho foi analisar os aspectos genéticos e não genéticos envolvidos no desenvolvimento de inibidores. Foram incluídos nesse estudo 411 pacientes hemofílicos, sendo 321 com hemofilia A (HA) (238 famílias) e 99 com hemofilia B (HB) (59 famílias). A presença de inibidores foi constatada apenas entre os pacientes HA graves. Do total de 220 HA graves desse estudo, 46 (20,9%) apresentaram inibidor detectado em pelo menos uma ocasião após sua inclusão no estudo. Mutações consideradas de alto-risco para o desenvolvimento de inibidores foram identificadas em 125/220 pacientes HA graves (58,8%), e 33 deles desenvolveram inibidores (26,4%). Considerando o grupo étnico de acordo com traços físicos e ancestralidade, 38% dos pacientes HA graves foram classificados como negros. A incidência de inibidores foi maior nesse grupo de pacientes (31% do total de pacientes HA graves classificados como negros) quando comparada aos pacientes caucasóides (20% do total de pacientes HA graves classificados como caucasóides). Recentemente, foi observado que a maior incidência de inibidores em uma população norte-americana de pacientes com HA, estava relacionada com a presença de determinados haplótipos no gene do fator VIII. Esta observação poderia ser explicada pelo fato dos as proteínas expressas pelos haplótipos que aparecem exclusivamente entre a população negra (denominados H3 e H4), estarem ausentes nos concentrados de fator VIII recombinantes utilizados rotineiramente no tratamento desses pacientes. Em nossa análise a presença desses haplótipos não está relacionada com a maior freqüência de inibidor na população negra desse estudo. Além disso, a distribuição dos diferentes haplótipos do gene do fator VIII, classificados de H1 a H6, foi distinta entre todos os grupos étnicos brasileiros e norte-americanos. Essa observação pode ser explicada pela origem distinta entre os negros que imigraram da África para o Brasil e para a América do Norte, assim como o alto índice de miscigenação de nossa população. Em outra fase desse estudo, foi realizada a análise comparativa da expressão gênica a partir de amostras de RNA mensageiro (RNAm) extraídas em pool leucocitário de pacientes com HA grave, com ou sem a presença de inibidores. Na avaliação que incluiu numa primeira análise pacientes de uma mesma família discordante para a presença de inibidor e, em uma segunda fase, indivíduos não relacionados, foram observados 50 genes mais expressos e 16 genes menos expressos em pacientes com inibidor em comparação aos sem inibidor. Dentre esses genes foram selecionados dez, levando-se em conta sua participação na resposta imune ou sua correlação prévia com o desenvolvimento de inibidores em outros estudos. Pela técnica de PCR em tempo real, observou-se que os genes da interleucina 8 (IL-8) e da cistatina F (CST7) demonstraram ser mais expressos em pacientes com inibidor, enquanto que o gene da interleucina 10 (IL-10) foi menos expresso nesse grupo de pacientes. Dessa forma, nossos resultados fortalecem a idéia de que o mecanismo de desenvolvimento de inibidores em hemofilia é complexo e ainda não totalmente esclarecido e que existe um grande envolvimento de diversos genes relacionados com sistema imune na formação desses inibidores. O estudo em diferentes populações é uma importante etapa para o entendimento dos fatores de risco para o desenvolvimento de inibidores. Esse foi o primeiro trabalho realizado no Brasil incluindo pacientes de diversas regiões e analisando simultaneamente diferentes fatores e seu envolvimento com o desenvolvimento de inibidores. A determinação desses fatores de risco ajudará no futuro a determinar um tratamento diferenciado para o controle e sobretudo prevenção do desenvolvimento de inibidores
Abstract: The most serious complication of the treatment of hemophilia is the development of neutralizing antibodies to coagulation activity of factor VIII or IX (inhibitors). Several risk factors are related to the development of these inhibitors in patients with hemophilia, including genetic and environmental factors. Among genetic factors, the mutation associated with the diagnosis of hemophilia is a risk factor well documented. Recently, it was observed a higher incidence of inhibitors in African ancestry patients. The aim of this study was to analyze the genetic and non-genetic factors involved in the development of inhibitors. The study included 411 hemophilia patients, of which 321 with hemophilia A (HA) and 99 with hemophilia B (HB), belonging to a total of 238 and 59 families, respectively. The inhibitors were observed only in severe HA patients. From the 220 severe HA, 46 (20.9%) had inhibitor. The high risk mutation for the development of inhibitors were identified in 125 / 220 (58.8 %) severe HA patients, and 33 (26.4 %) of them developed inhibitors. Considering the ethnic group according to physical traits and ancestry, 38 % of severe HA patients were classified as black. The incidence of inhibitors is higher in this group of patients (31%) when compared to Caucasian patients (20%). The higher risk of inhibitor among African-Brazilians, could not be explained by the presence of the distinct factor VIII haplotypes, such as H3 and H4, as suggested in previous study. In fact, the prevalence rates of these haplotypes were distinct between Brazilians and North Americans, probably due to the fact that migrations of blacks to Brazil and to North America were originated from different geographic areas of Africa. In another phase of this study, we performed a comparative analysis of gene expression in samples of messenger RNA (mRNA) extracted from leukocytes of inhibitor and non-inhibitor patients with severe HA was performed. The evaluation consisted of an initial analysis of severe HA patients siblings, or from the same family, discordant for inhibitor development and in a second phase a group of unrelated individuals. Using the bioarrays technology 50 genes were upregulated and 16 were downregulated in inhibitor patients compared with non-inhibitor patients. Ten genes were selected among them, which are involved in immune response and were related to inhibitors development in other studies. It was observed by real time PCR that the genes for interleukin 8 (IL-8) and cystatin F (CST7) were upregulated and for interleukin 10 (IL-10) was downregulated in inhibitor patients. In conclusion, our results strengthen the idea that the mechanism of inhibitor development in hemophilia is complex, not clear and there is a large involvement of several genes related to the immune system in the development of these inhibitors. The study in different populations is important to understand the risk factors for the development of inhibitors. This is the first work in Brazil, to study patients from various regions and to performe analysis of different factors and their involvement in the development of inhibitors. The determination of these risk factors will help in the future to determine differential treatment for the control and in particular, for preventing the development of inhibitors
Doutorado
Clinica Medica
Doutor em Clínica Médica
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Gardiner, Courtney Elizabeth Campbell. „Colours of confetti: the role of ABP genes and environmental variables in flower colour polymorphisms of Rhodohypoxis baurii var. confecta“. Thesis, 2019. https://hdl.handle.net/10539/29470.
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A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, South Africa in fulfilment of the requirements for the degree of Master of Science, 2019The study of flower colour is a particularly valuable approach to investigating fundamental evolutionary questions such as the maintenance of variation within species, the role of natural selection and genetic drift in maintaining polymorphisms, and how such polymorphisms contribute to biodiversity. Flower colour is a phenotype that is easily measured and it provides a strong indicator of the outcomes of selection pressures. It is both driven and maintained by either non- pollinator mediated selection agents, pollinator selection agents, or a combination of both. The overall aim of this study was to investigate the maintenance of flower colour using Rhodohypoxis baurii (Baker) Nel. var. confecta Hilliard and Burtt as a study system. This involved understanding the genetic mechanisms that regulate flower colour and examining the environmental variables that drive this trait variation across populations. Molecular and ecological methods were used in combination to understand flower colour by investigating the genes responsible, as well as the environmental selection pressures acting on this phenotype. Rhodoypoxis baurii var. confecta is a Drakensberg near-endemic species that has naturally occurring populations of magenta, pink, and white flower colour morphs. These flower colour morphs are discrete variants that co-exist in single populations and are thus considered to be true polymorphisms. Three populations of R. baurii var. confecta that occur in Sentinel Peak in the Royal Natal National Park, South Africa were studied. Non-pollinator mediated selection agents were investigated as potential drivers of flower variation in R. baurii var. confecta. The frequency of unpigmented (white) and pigmented (pink/magenta) flower colour morphs was measured over the flowering season. No pollinator visitation was observed at populations of R. baurii var. confecta and therefore pollinator-mediated selection was excluded. The extent to which non pollinator selection agents drive this intra-population variation was investigated by measuring the change in soil moisture, temperature, and precipitation over the flowering season. One population shifted from a greater proportion of unpigmented morphs at the beginning of the flowering season to a relatively greater proportion of pigmented morphs at the end of the season. In this population, a positive correlation was found between the proportion of pigmented morphs and soil moisture. This suggests that the accumulation of water in the soil promotes the production of pigmented flowers. The anthocyanin biosynthetic pathway (ABP) is responsible for the production of pigment in R. baurii var. confecta. The ABP is pleiotropic and in addition to pigment production is responsible for traits related to plant physiology and environmental stress response. Consequently, the ABP is cited as being well conserved among angiosperm species. The presence of expression in four ABP genes (CHI, F3H, F3’H, and F3’5’H) was tested for and compared between unpigmented and pigmented R. baurii var. confecta flowers. As there is no sequence data for Rhodohypoxis or any Hypoxidaceae species, primers for this molecular work were designed using closely related monocotyledon ABP gene sequences. Sequencing results showed that the amplified PCR products were not the targeted ABP genes. These results suggested that the designed primers were non-specific. The similarity of the four ABP genes within angiosperm species was investigated. All available complete sequences of the four regions of interest were aligned and sequence similarity was quantified. The results from this alignment indicate that the four investigated ABP gene sequences are polymorphic and are likely not well conserved within angiosperm species as a whole and, to some extent, within monocotyledon and dicotyledon species respectively. This suggests that the basic structure of the ABP is well conserved amongst angiosperm species however, the gene sequences that make up the pathway are polymorphic and less well conserved. The study highlights the importance of non-pollinator mediated selection on the presence and maintenance of flower colour polymorphisms. In addition, it provides some insight on the ABP and its conservation amongst angiosperm species. It also contributes to understanding how flower colour polymorphisms are maintained in natural populations both on a molecular and ecological level and is the foundational work in understanding the polymorphisms of R. baurii var. confecta.
TL (2020)
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Lochan, Anneline. „Genetic factors influencing inhibitor development in a cohort of South African Haemophilia A patients“. Thesis, 2014.
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Haemophilia A (HA) is a an X-linked bleeding disorder that manifests due to a mutation in the F8 gene encoding the coagulation factor VIII (FVIII) protein. Therapeutic management of HA involves intravenous FVIII infusions which are either plasma derived or recombinant concentrates that are administered to prevent or manage bleeding episodes promptly. A critical complication of repeated FVIII replacement therapy is the production of FVIII neutralising inhibitors which affect the coagulation potential of the replacement therapy, thus compromising the ability to manage bleeding episodes. The genetic and environmental factors predisposing to inhibitor development remain uncertain, and require improved understanding to provide optimal patient care and surveillance.
The study firstly aimed to characterise a cohort of South African HA patients in terms of clinical severity, ethnicity, int22 mutation status and inhibitor development; secondly, to explore whether the genetic factors (clinical severity, ethnicity, int22 mutation status, F8 gene haplotype) influence inhibitor development.
A total of 229 probands who had diagnostic HA testing at the Molecular Genetics Laboratory, Division of Human Genetics, of the National Health Laboratory Services (NHLS) and School of Pathology, University of the Witwatersrand, Johannesburg, were included in the study. The majority of patients (91%) in the cohort had severe HA. There were a similar proportion of black and white patients in the cohort. There was a 13% incidence of inhibitor development in the cohort of which 72% were black and 28% were white patients. To investigate the influence of genetic factors on inhibitor development only the probands with known inhibitor status
were included (n=216). It was established that 36% (77/216) of patients were int22 positive of which 20% (15/77) were reported to be inhibitor positive while 10% (14/139) of the int22 negative patients (n=139) were shown to be inhibitor positive. Therefore, the int22 positive patients had a two-fold higher incidence of inhibitor development than int22 negative patients. F8 gene haplotype analysis revealed that the H1 and H2 haplotypes were the most common in the cohort while the H3 and H5 haplotypes were only reported in black patients. Black patients were shown to have a higher prevalence of inhibitor development within each haplotype, thus suggesting that factors other than F8 gene haplotype are important in inhibitor development.
Overall, black int22 positive probands had a significantly higher prevalence (p=0.04) of inhibitor development than white int22 positive and negative patients in the cohort which is suggestive that ethnicity and F8 gene mutation may play a more major role in inhibitor development compared to F8 gene haplotype. Hence, there is a need to identify other genetic factors that may predispose HA patients to inhibitor development.
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Tseng, Su-Cheng, und 曾淑真. „Genetic analysis of hemophilia in Taiwanese by using Long-Distance PCR and DHPLC“. Thesis, 2005. http://ndltd.ncl.edu.tw/handle/86348523368667918572.
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碩士國立臺灣大學
分子醫學研究所
93
Hemophilia A (HA) and hemophilia B (HB), with the deficiency of coagulation factor VIII (FVIII) and IX (FIX) respectively, represent the most common sex-linked inherited bleeding disorders in human. A wide range of different mutations have been identified including the intrachromosomal inversions involving regions in intron 1 and 22 of the FVIII gene as well as many mutation types found in the remaining part of the factor gene, sunch as large and small deletions, insertions, and point mutations. Patients suffering from those disorders and their families bear great financial and social burden, it is very important to prevent recurrence of the diseases. To achieve this goal genetic analysis for carrier screening and prenatal diagnosis is mandatory. We have established a diagnostic strategy consisting of screening for most common mutations in the Factors VIII and Factor IX genes by using long-distance polymerase chain reaction (LD-PCR)and denaturing high performance liquid chromatography (DHPLC). Forty-four affected Taiwanese families including 29 HA and 15 HB families were analyzed. We found eight novel point mutations in 6 HA families and 2 HB families together with two novel small deletion mutations in HA family.The intron 22 inverions in 13 HA families and the remaining mutations have been described previously in HA and HB mutation database. These small mutations were further confirmed by direct sequencing. Neither false positive nor false negative results were found. Our combinatory approach by subcycling LD-PCR and heteroduplex analysis based on DHPLC proves to be a highly informative, rapid and practical means to detect mutations in affected individuals and carriers of hemophilias.
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Lin, Shu-Rong, und 林淑容. „Molecular characterization of genetic defects of hemophilia a in chinese patients from Taiwan“. Thesis, 1993. http://ndltd.ncl.edu.tw/handle/40164540892982001180.
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Lin, Shu-Rung, und 林淑容. „Molecular Characterization of Genetic Defects of Hemophilia A in Chinese patients from Taiwan“. Thesis, 1993. http://ndltd.ncl.edu.tw/handle/12392203444017452310.
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碩士國立臺灣大學
醫事技術學系
81
A型血友病乃因第八因子基因異常所引起,利用聚合■鏈反應(polymerase chain reaction;PCR)及直接核酸序列定位(direct Sequencing)等方 法,可以直接分析病人的第八因子基因. 然而第八因子基因相當巨大,本論 文闡述利用電腦程式設計45對PCR所需的引子(oligonucleotide primers) 來放大第八因子基因的每個特定的exon.接著用單股結構多形性(single strand conformation polymorphism;SSCP)及雙去氧指印法(dideoxy fingerprint -ing;ddF)兩種簡單篩檢方法,可偵測出突變的去氧核糖核酸 片段,這兩種方法都是應用單股去氧核糖核酸的特定形狀(distinct conformation)而偵測出核■酸(nucleotide)的突變. 本研究初步利用 SSCP及ddF鑑定出不正常的 DNA片段,這些DNA片段相對應的exon區域再被 用PCR放大後,定出核酸序列的改變. 在此階段實驗,共分析了87位病人, 其中40位可由SSCP及ddF找出突變型,這些人中5位具輕微至中型症狀,其 餘35位為嚴重型的A型血友病,對於每個病人,本研究均皆找出一個(且僅有 一個)突變型, 而這些突變型中有21個是單一核■酸突變(single nucleotide mutation),其中8個變成nonsens -e,13個變成missense codons.另外19位中有16個是減少(delete)或增加了 (insert)1-11個核■ 酸,其餘3個卻失掉了一大段基因片段. 利用SSCP,本實驗也檢視8個已被確 定的第八因子多形性(polymorphism),其中3個基因多形性的發生率在中國 人與與西方人不同,這3個分別是codon 1241,codon 1269及 codon 2223,其發生率分別為1.2%,15.7%及97.9%.在本論文進行同時,有實驗報導 指出嚴重型病人中約有50%病人其突變型可能是由於intron22有未明的突 變導致其第八因子mRNA不正常.本研究對於其餘47位病人之中的24位分析 其intron 22. 利用反轉錄-聚合■鏈反應(RT-PCR)的方法,證明17個嚴重 的病人的白血球中具有不正常的第八因子mRNA.綜論,本論文有三個重點: 一.是利用PCR-SSCP,ddF及直接核■酸序列分析找出血友病病人確切的突 變型.二.是證明有38%的嚴重型病人其第八因子基因突變是由於intron 22的未明異常,產生不正常的第八因子mRNA而導致無法製造正常的第八因 子蛋白.三.是利用SSCP分析8種第八因子多形性在中國人的發生率.另外, 本研究至少有二個應用:一.是PCR-SSCP,ddF及直接核■酸序列分析的結果 可應用於病人家屬的帶因者鑑定(carrier detection) 及產前診斷( prenatal diagnosis).二.是第八因子基因表現(gene expression)及蛋白 結構功能關係(structur -e function relationships)的研究. The molecular characterization of hemophilia A of Chinese origin was carried out by the polymerase chain reaction (PCR) and direct sequencing of patients' factor VIII genes. Forty- five sets of oligonucleotide primers were designed to allow specific amplification of individual exons and their flanking introns of the factor VIII genes.The single strand conformation polymorphism (SSCP) and dideoxy fingerprinting (ddF) were used as screening methods to detect mutated DNAs.Both techniques reveal single base substitutions in a single-stranded DNA as distinct conformations. The abnormal exon initially identified by SSCP were subsequently amplified and their nucleotide sequence determined.A total of 87 patients from different families were analyzed by PCR-SSCP.Among them,40 cases revealed mutations in the coding regions of their factor VIII genes.A single mutational event was found to be associated with each of the 40 affected individuals.These include 21 cases of single base mutations,16 cases of deletion or insertion of 1-11 nucleotides,and 3 cases of deletion of large DNA fragments. Using PCR-SSCP,the frequencies of 8 of the identified factor VIII polymorphism or silent mutations were also examined among Chinese.The frequencies for codons 1241,1269 and 2223 were found to be 1.2%,15.7% and 97.9% respectively, different from those reported for other populations.As for the 47 cases whose mutational event was not readily detected by PCR-SSCP ,the reverse transcriptase combined with PCR (RT-PCR) method was applied to examine their factor VIII mRNA.In 24 cases analyzed,17 were found to contain unusual mutations in intron 22 which caused failure in RT-PCR amplification.Our study presented in this thesis reveals the direct determination of genetic defects of patients with hemophilia A.This provides a precise diagnosis for carriers of members in the hemophilic families.Moreover,the study will be benefit for the study of factor VIII gene expression and structure-function
Bücher zum Thema "Hemophilia – Genetics"
1
Pieneman, Wouterina Cynthia. Molecular genetics of hemophilia A. [Leiden: University of Leiden, 1998.
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2
Winterflight: A novel. Colorado Springs, Colo: Victor, 2000.
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3
Potts, D. M. Queen Victoria's gene: Haemophilia and the royal family. Stroud: Sutton Pub., 1999.
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4
Potts, D. M. Queen Victoria's gene. Stroud: Alan Sutton, 1995.
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Genetic Disorders Sourcebook: Basic Consumer Health Information About Hereditary Diseases and Disorders, Including Cystic Fibrosis, Down Syndrome, Hemophilia, ... Disease (Health Reference Series). 2. Aufl. Detroit, Mich.: Omnigraphics, 2000.
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International, Symposium on Blood Transfusion (19th 1994 Groningen Netherlands). Hereditary diseases and blood transfusion: Proceedings of the Nineteenth International Symposium on Blood Transfusion, Groningen, 1994. Dordrecht: Kluwer Academic, 1995.
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7
Hoeben, Robert Cornelis. Towards gene therapy for Haemophilia A: Vectors for the expression of blood-clotting factor VIII in vivo. [Netherlands: s.n., 1991.
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8
(Editor), Inge Scharrer, und Wolfgang Schramm (Editor), Hrsg. 32nd Hemophilia Symposium Hamburg 2001: Epidemiology; Genetic Diagnosis of Clotting Disorders; Hemophilia; Hemotherapy in Sepsis; Pediatric Hemostaseology Free Lectures. Springer, 2003.
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9
Winterflight. RiverOak Publishing, 2006.
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10
Bayly, Joseph. Winterflight. Vision House, 1994.
Den vollen Inhalt der Quelle findenBuchteile zum Thema "Hemophilia – Genetics"
1
Herrmann, F. H., K. Wulff, G. Auerswald, J. Astermark, A. Batorova, W. Kreuz, H. Pollmann, A. Ruiz-Saez, L. Salazar-Sanchez und S. Schulman. „Factor VII Deficiency: Clinical Manifestation and Molecular Genetics of 718 Subjects with FVII Gene Mutations“. In 37th Hemophilia Symposium, 238–46. Berlin, Heidelberg: Springer Berlin Heidelberg, 2008. http://dx.doi.org/10.1007/978-3-540-73535-9_51.
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2
Antonarakis, Stylianos E. „The Molecular Genetics of Hemophilia A and B in Man“. In Advances in Human Genetics 1, 27–59. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4613-0987-1_2.
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3
Antonarakis, Stylianos E. „Erratum to: The Molecular Genetics of Hemophilia A and B in Man“. In Advances in Human Genetics 1, 201–3. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4613-0987-1_6.
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4
Schneppenheim, R., S. Krey, G. Auerswald, R. Ganschow, H. Holzhüter, H. J. Klose, W. Kreuz et al. „Human Immunodeficiency Virus-Negative ››High-Risk Patients‹‹ with Hemophilia or Severe von Willebrand Disease Type 3: Coincidence or Genetics?“ In 30th Hemophilia Symposium Hamburg 1999, 191–93. Berlin, Heidelberg: Springer Berlin Heidelberg, 2001. http://dx.doi.org/10.1007/978-3-642-18240-2_24.
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5
Chen, Harold. „Hemophilia A“. In Atlas of Genetic Diagnosis and Counseling, 1319–29. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-2401-1_114.
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Chen, Harold. „Hemophilia A“. In Atlas of Genetic Diagnosis and Counseling, 1–11. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4614-6430-3_114-2.
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7
Astermark, Jan. „Genetic and Environmental Risk Factors for Inhibitor Development“. In Textbook of Hemophilia, 57–61. Oxford, UK: Wiley-Blackwell, 2010. http://dx.doi.org/10.1002/9781444318555.ch8.
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Astermark, Jan. „Genetic and Environmental Risk Factors for Factor VIII Inhibitor Development“. In Textbook of Hemophilia, 48–52. Oxford, UK: John Wiley & Sons, Ltd, 2014. http://dx.doi.org/10.1002/9781118398258.ch6.
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Phadke, Shubha. „Rheumatic Manifestations of Genetic Disorders and Hemophilia“. In Pediatric Rheumatology, 595–609. Singapore: Springer Singapore, 2016. http://dx.doi.org/10.1007/978-981-10-1750-6_47.
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Marinova, M., Ph Westhofen, M. Watzka, B. Pötzsch und J. Oldenburg. „Functional Promoter Polymorphism in the VKORC1 Gene is no Major Genetic Determinant for Vitamin K Dependent Coagulation Factor Activity“. In 37th Hemophilia Symposium, 261–63. Berlin, Heidelberg: Springer Berlin Heidelberg, 2008. http://dx.doi.org/10.1007/978-3-540-73535-9_56.
Der volle Inhalt der QuelleKonferenzberichte zum Thema "Hemophilia – Genetics"
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Antonarakis, E. „The Molecular Genetics of Hemophilia A Stylianos“. In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643980.
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Hemophilia A is a common X linked hereditary disorder of blood coagulation due to deficiency of factor 8. The gene for factor 8 has been cloned and characterized (Nature 312:326-342, 1984). It is divided into 26 exons and 25 introns and spans 186 kb of DNA. The CGNA is 9 kb and codes for 2351 amino acids. The first 19 amino acids comprise the secretory leader peptide and the mature excreted polypeptide consists of 2332 amino acids. The nucleotide sequence of the exons and the exon-intron junctions is known and the complete amino acid sequence has been deducedSeveral laboratories have used cloned factor 8 DNA sequences as probes to characterized mutations that are responsible for hemophilia A in certain pedigrees. These mutations have been characterized by restriction analysis, oligonucleotide hybridization, cloning and sequencing of DNA from appropriate patientsIn about 500 patients with hemophilia A examined, the molecular defect has been recognized in 39. Both gross alterations (mainly deletions) and point mutations of the factor 8 gene have been found.A total of 19 different deletions have been observed. No two unrelated pedigrees share the same exact deletion.The size of the deleted DNA varies from 1.5 kb to more than 210 kb. All but one of these deletions are associated with severe hemophilia A. A deletion of 6 kb that contains exon 22 only is associated with moderate hemophilia. Some deletions are present in patients with inhibitors to factor 8. No correlation of the size or the position of the deletions can be found with the presence of inhibitors to factor 8.A total of 20 point mutations have been characterized. All are recognized by restriction analysis and involve Taq I sites. All are mutations of CpG dinucleotides and generate nonsense or missence codons. Unrelated pedigrees have the same single nucleotide change because of independent origin of the same mutation. In many instances de novo occurrence of a point mutation has been observed. CpG dinucleotides are hot spots for mutation to TG or CA presumably because of spontaneous deamination of methylcytosine. Some point mutations are present in patients with inhibitors but no correlation of the site of mutation and inhibitor formation has been found. The nonsense mutations are present in patients with severe hemophilia A. A missense mutation (Arg Gin) in exon 26 was found in a patient with mild hemophilia while another Arg Gin mutation in exon 24 has been observed in a patient with severe disease. The creation of a donor splice site in IVS 4 of factor 8 gene has been observed in a patient with mild hemophilia.Few DNA polymorphisms within the factor 8 gene and two other closely linked polymorphisms have been used for carrier detection and prenatal diagnosis of hemophilia A. These DNA markers are useful in more than 90% of families at risk for hemophilia A.The author thanks Drs. Gitschier, Din, Olek, Pirastou, Lawn for communication of their data prior to publication.The hemophilia project at Johns Hopkins was supported by an Institutional grant and NIH grant to S.S.A. and Haig H. Kazazian, Jr.
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Suzuki, N., A. Iizuka, T. Nagao, Y. Nakahori, M. Yamada und Y. Nakagome. „CARRIER DETECTION OF HEMOPHILIA A BY DNA ANALYSIS IN AFFECTED JAPANESE FAMILIES“. In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644008.
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Several DNA probes have been isolated to detect Factor VIII gene and a DNA segment which locates veryclose to the gene. They have been successfully used to detect carriers and patients of hemophilia A.We analyzed DNA samples of Japanese population to see whether these probesare also useful for carrier detection of hemophilia A in affected Japanese families, since the size and frequency of allelic fragments detected by a DNA probe are sometimes different in various ethnic groups.A probe of St14 (DXS52) is thought to be one of the best probes for such analysis in Caucasian population because it detects very polymorphic DNA fragments containing a minisatellite. When Taq I digests of Japanese DNA samples were hybridized with Stl4, several DNA fragments with a range from 1.7 kb to 5-5 kb were detected, where .at least 6 fragments were polymorphic. A notable difference between Japanese and Caucasian was that a band of 5-5 kb was variable in Japanese while it was constant in Caucasian. We have so far detected 10 alleles, and about 60% of Japanese women were heterozygous. Using these informationsabout Japanese population, we could detect carriers in several families. Other RFLPs data are necessary to increase information content. Similar studies arein progress using different probes i.e. an extragenic probe ; DX13/Bgl II, and two intragenic probes ; exon 14-26/Bcl I and exon 26/Bgl I. We thank Mandel J.L., Strasbourg, Davies K., Oxford and Genetics Institute, Cambridge for probes.
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Lillicrap, D., A. R. Giles, J. J. A. Holden und B. N. White. „THE RELATIVE EFFICACY OF GENETIC ANALYSIS AND COAGULATION TESTING IN THE DIAGNOSIS OF CARRIERS OF HEMOPHILIA A“. In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644010.
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This study has assessed the relative benefits of restriction fragment length polymorphism (RFLP) linkage and coagulation testing in the diagnosis of carriers of hemophilia A. 221 samples from 55 families have been studied for intragenic and flanking RFLPs. All samples were tested for the Factor VIII intragenic Bell RFLP and for the flanking marker St 14. 83% of obligate carrier females were heterozygous at oneor both of these two polymorphicsites. However, only38% of these women were heterozygous at the intragenic site and might safely be offered prenatal diagnosis using this marker for the hemophilia mutation. Carrier diagnosis was obtained in 52% of 81 potential carriers tested. Diagnosis wasbased on intragenic RFLP information in only 48% of these cases. Genetic diagnosis was possible in 27 atrisk women from families with no prior history of hemophilia. Four of these women were diagnosed as carriers on the basis of a gross Factor VIII gene deletion and the remaining 23 women were identified as non-carriers by the Bell (11) and Stl4 (12) RFLP data. 39 women remained undiagnosed after gene analysis studies. 23 of these women were female relatives of sporadic hemophiliacs and thus RFLP segregation analysis was inappropriate. A further 9 potential carriers were undiagnosed because of homozygosity in key individuals in their families. In 31 potential carriers we have quantitated Factor VIII:C (one stage assay) and vWf:Ag (Laurell and ELISA) and derived probabilities for carrier status. In 3 women there was conflicting genetic and coagulation data. Meanwhile, in 12 undiagnosed women from sporadic families, carrier diagnostic probabilities of > 0.9 were obtained. These studies indicate that optimal carrier detection for hemophilia A requires more intragenic and closely linked RFLPs and the continuance of coagulation testing to assist women from sporadic families.
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Reisner, H. M., E. A. Reisner, D. D. Kostyu, B. C. Lubahn, C. McMillan und G. C. White. „POSSIBLE ASSOCIATION OF HLA AND Gm WITH THE ALLOIMMUNE RESPONSE TO FVIII“. In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644021.
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Between 5 and 15% of individuals with severe hemophilia A are at risk of developing inhibitors (alloantibodies) to FVIII. Genetic factors are important in determining risk, but the nature of these factors is poorly defined. The human immune response to a wide variety of antigens has been associated with the HLA and/or Gm loci. Hence, we have investigated polymorphisms at these two loci in hemophilia A patients with and without inhibitors.DATA SET: To date 127 hemophiliacs have been Gm or HLA typed. Forty-eight are inhibitor positive (I+) based on positive FVIII neutralization assays. This represents about 70% of all I+ hemophiliacs seen at UNC. To prevent familial bias, one member of each of 14 pairs of close relatives was randomly removed from the Data Set without regard to inhibitor status (Data Set 1). Twenty non-white individuals were also removed to constitute Data Set 2. GM TYPING: Samples were typed for Gm antigens 1, 3 and 5. Phenotype frequencies in Data Sets 1 and 2 did not deviate from expected values. I+ hemophiliacs showed an excess of Gm 1 in both Data Sets which was of possible significance (p = .13 and .21 respectively by chi square). Reanalysis of Data Set 2 to include only I- individuals without evidence of either circulating VIII:C or VIII:CAg (N=58) yields a p of .12. HLA TYPING: Analysis on 77 individuals in Data Set 2 (21 I+, 56 I-) has been done. In preliminary typing of HLA A, B, C, DR and DQ no significant differences in antigen frequency were found between the I+ and I- groups. INTERACTION BETWEEN HLA AND GM: A significant excess of Gm 1 I+ individuals was found among all HLA-A2 positive hemophiliacs (N=45, p=.034 by Fisher’s exact test. This was not significant after correction for multiple comparisons). This suggestion of an interaction between HLA-A2 and Gm 1 in determining alloreactivity to FVIII will require further prospective evaluation for confirmation.
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Kojima, T., M. Tanimoto, T. Kamiya, Y. Obata, K. Kurachi und H. Saito. „ANALYSIS OF FACTOR IX GENE IN NORMAL SUBJECTS AND HEMOPHILIA B PATIENTS IN JAPAN“. In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644077.
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We have examined DNA samples from 25 hemophilia B patients (21 B- patients, 2 BR patients and 2 B+ patients) and 51 normal subjects with molecular probes (pHFIX and 2 genomic fragments). By structural gene analysis, 4 out of 7 patients who developed anti-factor IX antibodies were detected to have gross factor IX gene deletion. Although these four patients showed normal pattern of HPRT gene detected by pCDHPRT, the gene deletions were found to expand more than 34kb including with entire factor IX exons. Quantitative Southern blot analysis of factor IX gene of the patient's family members indicated that the gene deletion was inherited in one family, establishing the carrier status of 2 aunts, 2 cousins and one sister. The 'de novo' mutation of factor IX gene was also established in 2 families. Three patients with anti-factor IX antibodies and 17 patients without antibody to factor IX had normal pattern of factor IX gene by several restriction enzyme digestions. Analysis of factor IX gene of three patients with anti-factor IX antibodies and two B+ patients are now underway to detect the unique gene defects which may be responsible for the disease Phenotypes. Common RFLPs in factor IX gene were studied in normal Japanese subjects. More than 80 X chromosomes were analysed with BamHI, Ddel, MspI, TaqI or XmnI digestion, followed by hybridization with pHFIX. RFLPs produced by these enzymes were found to be uncommon or possibly absent in normal Japanese subjects. These results imply that racial differences in the frequency of gene polymorphisms should be seriously considered before initiating the gene counseling by the genetic probes.
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Al-Mohannadi, Anjud Khamis, Sara Deola und Ahmed Malki. „Visualization of Factor Viii with Flow-Cytometry as a tool for Novel Gene Therapy Approach in Hemophilia A“. In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0164.
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Haemophilia A is a genetic X-linked disorder, characterized by coagulation Factor VIII (FVIII) deficiency and leading to pathological bleedings. The disease occurs at a rate of 1 in 5000 males’ births. The treatment is the administration of plasma-derived or recombinant Factor VIII, which is expensive and leads to the development of inhibitory antibodies in around 40% of patients affected by the severe form of the disease. The disease becomes for these patients as life threatening. In new approaches to treat Haemophilia include gene therapy (GT), cells corrected through genetic modifications are used to produce in Haemophilia A patients FVIII protein in a sustained manner, as long-term treatment for this disorder. The cells of choice should be persistent and equipped with themachinery for large protein assembly and secretion. So far, target cells for Haemophilia gene correction are mostly liver cells, although they are highly immunogenic and exposed to immune-mediated destruction after GT. Based on literature evidences, bone marrow transplantation can correct Haemophilia A in mice, providing evidence that Hematopoietic stem cells (HSC) or their progeny are able to produce FVIII. We chose the approach of correcting HSC with lentiviral vectors carrying the FVIII gene cassette. Whereas classically FVIII protein is visualized on adherent cells through immunohistochemistry staining, flow-cytometry (FC) literature publications are very scarce. FC analysis is an attractive method for analysing hematopoietic cells, and in general, a versatile method for protein visualization. However, large proteins as FVIII are difficult to be carefully analysed, and the method requires several steps of optimization. This joint project with Dr. Muhammad Elnaggar, aims to optimize a method to characterize large proteins as FVIII with a reliable FC staining protocol. To this aim, we used cell lines to evaluate the expression and secretion pathways of FVIII, the intracellular requirements to fold and secrete large proteins, and the toxicities of protein accumulation, in case of GT mediated protein overexpression. For this purpose, the FC experiments were performed to optimise the FC protocol for FVIII visualization, by improving blocking efficacy, antibody-labelling efficacy and to ensure accuracy and validity through qPCR and FC double staining. This FC protocol proved its validity and usefulness in visualizing and studying functionally FVIII.
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Ørstavik, K. H., L. Kornstad und H. M. Reisner. „LEWIS BLOOD TYPE HAS AN EFFECT ON THE PLASMA CONCENTRATION OF FACTOR VIII“. In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644062.
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The plasma concentration of factor VIII is influenced by the ABO blood group. Individuals with blood group 0 have a lower concentration of both factor VIII coagulant activity, factor VIII coagulant antigen (VIIICAg) and factor VIII related antigen (VIHRAg) than individuals with group A, B and AB. Thirty percent of the genetic variance of VIIIRAg concentration is due to the ABO blood group. The Lewis substances Lea and Leb are closely related to the A, B and H substances. We therefore examined the effect of the presence of these antigens on the plasma concentration of VIIICAg and VIIIRAg. The material was 74 monozygotic and 84 dizygotic twin pairs and 58 blood donors with ABO blood group 0. VIIICAg was determined by a radioimmunoassay and VIIIRAg was determined by an electroimmunoassay. A higher mean concentration °f VIIICAg (147%) and VIIIRAg (81%) was found in individuals with the Le antigen on their red cell surface compared to the mean concentration of VIIICAg (101%) and VIIIRAg (66%) in individuals who lacked this antigen. The difference was found in individuals with ABO blood group 0 only. Individuals with red cell Lea antigen are non-secretors and individuals who lack this antigen but have the Leb antigen are secretors of the A, B and H substances. The lowest factor VIII concentration was found in group 0 secretors. The effect of the Lewis phenotype on factor VIII concentration is therefore most probably due to an effect of the secretor locus. This finding may have practical implications for the diagnosis of carriers of hemophilia A. it has been shown that information on the ABO blood group improves the discrimination between carriers and normals. We found that the effect of ABO blood group on the total variance of VIIIRAg was higher in secretors (21%) than in non-secretors (8%). Since the frequency of secretors varies widely, it is possible that the importance of the ABO locus in carrier detection is different in different populations. Lewis blood typing in materials of carriers and normals are necessary to determine the effect of the Lewis phenotype in carrier detection.