Stable Expression of Coagulation Factors by RPS6 Promoter / 中国实验血液学杂志

OBJECTIVE@#To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia.@*METHODS@#Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene.@*RESULTS@#The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups.@*CONCLUSION@#After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.

Analysis of a patient with severe Hemophilia A due to a large duplication of F8 gene / 中华医学遗传学杂志

OBJECTIVE@#To report on a case with severe hemophilia A (HA) due to a large duplication of F8 gene.@*METHODS@#Inversion detection, Sanger sequencing, and multiplex ligation-dependent probe amplification (MLPA) were used to detect the mutation in the proband and his mother.@*RESULTS@#The patient, a 7-year-old boy, was diagnosed with severe HA at 8 months. No inhibitor was developed over 150 exposure days. Intronic inversion detection and Sanger sequencing have failed to identify pathogenic variants, while MLPA revealed a large duplication [Ex 1_22 dup (2 copies)] in the proband, for which his mother was a carrier [Ex 1_22 dup (3 copies)]. Large duplications of the F8 gene have so far been found in 24 HA patients, all of whom had a severe phenotype, only one had a history of inhibitors.@*CONCLUSION@#Large duplications of F8 gene are associated with severe HA. The diagnostic rate for HA may be increased by MLPA.

Analysis of gene inversion in Hemophilia A by Nanopore sequencing / 中华医学遗传学杂志

OBJECTIVE@#To detect gene inversion in two pedigrees affected with Hemophilia A by using Nanopore sequencing technology.@*METHODS@#Peripheral blood samples were taken from members of the two pedigrees. Following extraction of genome DNA, genetic variants of the carriers were detected by Nanopore sequencing and subjected to bioinformatic analysis.@*RESULTS@#Nanopore sequencing has identified the niece of the proband of the pedigree 1 as carrier of Hemophilia A Inv22, and the mother of the proband of the pedigree 2 as carrier of Hemophilia A Inv1, which was consistent with clinical findings. Breakpoint sites in both pedigrees were accurately mapped. Statistical analysis of the sequencing results revealed a large number of variations in the carriers' genomes including deletions, duplications, insertions, inversions and translocations.@*CONCLUSION@#Nanopore sequencing can be used to analyze gene inversions associated with Hemophilia A, which also provided a powerful tool for the diagnosis of diseases caused by gene inversions.

Detection of F8 mutations in carriers and patients with severe hemophilia A: Identification of a novel mutation / Detección de mutaciones en el gen F8 en mujeres portadoras y en pacientes con hemofilia A: Identificación de una mutación nueva

The molecular diagnosis of haemophilia A (HA) patients has many benefits including diagnosis confirmation and inhibitor risk development prediction. In female carries of a mutation, the molecular diagnosis allows for genetic counseling and prenatal diagnosis, which have become part of the comprehensive care for HA in many countries. Therefore, the aim of this study was to determine the F8 mutations in severe HA (sHA) patients and female carriers. In 12 patients with sHA, the presence of the intron 22 and intron 1 inversions was investigated using an inverse and a conventional PCR method, respectively. In patients negative for the inversions, the F8 gene was screened through conformation sensitive gel electrophoresis (CSGE) and further sequencing. The causative mutation was successfully identified in 6/12 patients, including the novel mutation p.G190C. The mothers of these six patients and those of seven other sHA patients molecularly diagnosed in a previous work were investigated for the presence of the genetic alterations found in their sons. All mothers were found to be carriers. This is the first study conducted in Venezuela which directly analyzes the F8 gene in potential carrier mothers to specifically identify the presence of the mutation that was detected in their sons, and complements a previous study on sHA patients. Our findings will facilitate the implementation of regular screening of HA carriers in our country and will allow a better care of bleeding symptoms and genetic counseling.

El diagnóstico molecular de pacientes con hemofilia A (HA) tiene múltiples beneficios, incluyendo la confirmación del diagnóstico y la predicción del riesgo de desarrollar inhibidores. En mujeres portadoras de una mutación, el diagnóstico molecular permite el consejo genético y el diagnóstico prenatal, los cuales son parte de la atención integral de HA en muchos países. Así, el objetivo de este estudio fue determinar mutaciones en el gen F8 en pacientes con HA severa (HAs) y en mujeres portadoras. En 12 pacientes con HAs, la presencia de la inversión del intrón 22 y el intrón 1 fue investigada utilizando una PCR inversa y una convencional, respectivamente. En pacientes negativos para cualquiera de las inversiones, el gen del F8 fue analizado a través de la técnica de electroforesis en gel sensible a conformación (CSGE) y posterior secuenciación. La mutación causante de la enfermedad fue identificada en 6/12 pacientes, incluyendo la mutación nueva p.G190C. Las madres de estos seis pacientes y las de otros siete pacientes de HAs diagnosticados en un estudio previo y fueron investigadas para la presencia de alteraciones genéticas encontradas en sus hijos. Todas las madres resultaron ser portadoras. Éste es el primer estudio realizado en Venezuela donde se analiza directamente el gen F8 en portadoras potenciales para identificar específicamente la presencia de una mutación que fue detectada en sus hijos, y complementa un estudio previo con pacientes HAs. Nuestros hallazgos facilitarán la implementación del análisis regular de portadoras de HA en nuestro país y permitirán un mejor cuidado de los síntomas de sangrado y consejo genético.

Dilataciones endoscópicas del tracto gastrointestinal / Endoscopic dilation of the gastrointestinal tract

Las dilataciones en el tracto gastrointestinal se llevan a cabo para aliviar la obstrucción sintomática, ya sea funcional u orgánica, secundarias a una variedad de patologías tanto benignas como malignas. Con el advenimiento de las nuevas tecnologías, virtualmente toda estenosis digestiva puede ser manejada en forma mínimamente invasiva. Pese a su amplia difusión en la práctica actual, existen pocos estudios controlados que comparen las diferentes modalidades de dilatación. En el presente artículo realizamos una revisión de esta técnica, así como de la evidencia disponible para su aplicación en los diferentes segmentos del tracto gastrointestinal. El futuro de la dilatación incluye el desarrollo de dilatadores que permitan evaluar la dilatación durante su realización. Estos advenimientos, así como la ejecución de estudios controlados prospectivos van a mejorar las indicaciones, beneficios y riesgos para cada uno de los sistemas de dilatación existentes.

The endoscopic dilation of the gastrointestinal tract is carried out to relieve either functional or organic disorders, secondary to a variety of both benign and malignant diseases. With the advent of new technologies, virtually all digestive stenosis can be managed in a minimally invasive way. Despite its wide dissemination in actual practice, there are few controlled studies comparing the different forms of endoscopic dilation. In this article, we review this technique and the evidence available for application in different segments of the gastrointestinal tract. The future of the dilations includes the development of dilators to assess dilation during the procedure. These advents and the implementation of prospective controlled studies will improve the indications, benefits and risks for each of the existing systems of dilations.

Importancia del diagnóstico de portadoras en familias con antecedentes de hemofilia / Importance of the diagnosis of carriers in families with history of hemophilia

La hemofilia es una enfermedad hemorrágica hereditaria ligada al cromosoma X que se presenta debido a mutaciones en los genes del factor VIII (hemofilia A) y el factor IX (hemofilia B), que ocasiona una disminución o deficiencia funcional de estas proteínas en plasma. Sus frecuencias son de 1 en 5 000 y 1 en 30 000 varones recién nacidos vivos, respectivamente. Afecta casi exclusivamente a los varones y las mujeres portadoras presentan un riesgo del 50 por ciento de transmitir la enfermedad a sus hijos, por lo que es importante en las familias con antecedentes de hemofilia identificar las portadoras a través de las consultas de asesoramiento genético, en el cual se brinda información acerca de la enfermedad, la confección del árbol genealógico, el cálculo del riesgo de recurrencia, el diagnóstico molecular y la posibilidad de diagnóstico prenatal en gestantes portadoras. Es imprescindible que el asesoramiento genético constituya un proceso educativo e informativo, nunca impositivo

Hemophilia is a hereditary bleeding disorderX-linked that a rises dueto mutations in the genes offactor VIII(hemophiliaA)andfactor IX(hemophiliaB), which causes a decrease orfunctional deficiency of these proteins inplasma.Their frequencies are 1 in5 000 males and1 in30 000 live births, respectively. It affects males almost exclusively, and female carriers have a50 percent risk of transmitting the disease to their children. So, it is important in families with history of hemophilia that carriers are identified through genetic counseling, which provides information about the disease, making pedigree,calculation of the risk of recurrence, molecular diagnosis and possibility of prenatal diagnosis in pregnant carriers.It is imperative that genetic counselingconstitutes an educational and informative process,never as an imposition

Hemofilia II. Aspectos moleculares y de genética poblacional / Hemophilia II. Molecular and population genetics

La prevalencia de la hemofilia esporádica fue estimada hace más de 40 años y se demostró que aproximadamente un tercio de los casos son de novo. La mayoría de las mutaciones que ocurren en la hemofilia se producen durante la espermatogénesis masculina; en otros casos, los cambios ocurren en los estadios tempranos de desarrollo del embrión o una mutación germinal en la madre. El proceso de inactivación del cromosoma X es al azar. Extensos estudios han evidenciado que son más frecuentes las mutaciones en las meiosis masculinas que en las femeninas, con una proporción global de 3,5/1, especialmente las inversiones de los intrones 22 y 1. Se revisaron aspectos moleculares y bioquímicos de los factores VIII y IX. Destacamos la importancia del dominio B del factor VIII que contribuye a múltiples funciones esenciales, como el control de la calidad de la síntesis, la secreción, la unión con los fosfolípidos plaquetarios, la inactivación y el aclaramiento de la molécula completa

The sporadic hemophilia prevalence was estimated more than 40 years ago and it was shown that approximately a third of the cases are novo. Most of the mutations that occur in hemophilia are produced during the male spermatogenesis; in other cases, they occur in early stages of the embrión development or in the mother a germinal mutation. The X-cromosoma- inactivation process is at random. Extended studies have shown that male meiosis are more frequent than female ones, with a global proportion of 3,5/1, specially introns inversions 22 and 1. There were revised molecular and biochemical aspects of factors VIII and IX. We ruled out the importance of B domain in factor VIII, which contributes to multiple essential functions, as the quality control of synthesis, secretion, union with platelet phospholipids, inactivation and complete clearance of the molecule

Hemofilia: aspectos históricos y genéticos / Hemophilia: historical and genetic aspects

La hemofilia es un trastorno hemorrágico con disminución o ausencia de la actividad procoagulante del factor VIII o del IX. Las primeras descripciones de esta enfermedad son tan antiguas como la propia humanidad. A lo largo de los años, la hemofilia ha sido nombrada enfermedad real debido a que la padecieron diversos miembros de las familias reales europeas. En la actualidad, mediante estudios moleculares, se encontró el defecto genético causante de la enfermedad en los varones hemofílicos de la familia de la Reina Victoria y se encontró que sus descendientes padecieron una hemofilia B severa. El fenotipo de esta enfermedad es hemorrágico; se observan sangramientos en diversos sitios de la economía condicionados fundamentalmente por los niveles del factor deficiente. Existen otros factores que intervienen en las características fenotípicas variables de estos pacientes, entre ellos: las características intrínsecas de los factores VIII/IX, la presencia de genes modificadores y factores ambientales que influyen sobre la severidad de la enfermedad. Se revisa la correlación genotipo-fenotipo en esta enfermedad mediante las mutaciones más frecuentes en cada tipo de hemofilia. En cuanto a la presencia de los inhibidores, se destacan las evidencias actuales en relación con los factores de riesgo relacionados con su aparición y los aspectos moleculares presentes en la variante de hemofilia B Leyden

Hemophilia is a hemorrhagic disorder characterized by decreasing or absence of the procoagulant activity in factor VIII or IX. First descriptions of this disease are as old as mankind itself. During time, hemophilia has been called royal disease since different members of European royal families suffered from it. Currently, by molecular studies, it was found the causing genetic defect of this disease in hemophilic male members of Queen Victoria´s family; and it was found that her descendants suffered a severe hemophilia B. This disease phenotype is hemorrhagic; bleeding in different sites is observed which are mainly conditioned by the levels of the deficient factor. There are other factors participating in the variable phenotype characteristic of these patients, such as: intrinsic characteristics of factors VIII/IX, the presence of modifying genes and environment factors which influence on this disease severity. It is revised here the correlation genotype-phenotype in this disease through the most frequent mutations in each type of hemophilia. Concerning the presence of inhibitors, it is highlighted the current evidences in relation with risk factors related to its emergence and molecular aspects present in hemophilia variant B Leyden

A homozygous female hemophilia A.

BACKGROUND: Hemophilia A (HA), being an X-linked recessive disorder, females are rarely affected, although they can be carriers. AIMS: To study the mutation in F8 gene in an extended family with a homozygous female HA. MATERIALS AND METHODS: All the seven affected members (six males and one female) were initially screened by Conformation Sensitive Gel Electrophoresis (CSGE) and direct DNA sequencing. RESULTS: A homozygous missense mutation c.1315G>A (p.Gly420Ser) was identified in exon 9 of F8 gene in homozygous state in the affected female born of 1° consanguinous marriage and in all the affected male members of the family. Her factor VIII levels was found to be 5.5%, vWF:Ag 120%. CONCLUSION: In India, as consanguineous marriages are very common in certain communities (up to 30%), the likelihood of encountering female hemophilia is higher, although this is the first case of HA out of 1600 hemophilia families registered in our Comprehensive Haemophilia Care Center. Genetic diagnosis in such cases is not necessary as all the male children will be affected and daughters obligatory carriers.

Análisis de las inversiones del intrón 1 y 22 y secuenciación del gen F8 para el diagnóstico genético-molecular de hemofilia A en Chile / Introns 1 and 22 inversions and F8 gene sequencing for molecular diagnosis of hemophilia A in Chile

Background: Hemophilia A is an inherited disorder caused by alterations in factor VIII gene (F8) located on the X-chromosome, the intron 22 inversion being the most common mutation. The rest are predominantly point mutations distributed along this large gene of 26 exons. Aim: To implement a molecular diagnostic test to detect mutations in the F8 gene in Chilean patients with Hemophilia A. Material and Methods: To validate the testing methods, we analyzed samples with intron 22 and intron 1 inversion, and with point mutations previously studied, as well as one subject without Hemophilia. We also studied unrelated Chilean patients with Hemophilia A and their female relatives for carrier testing. Intron 22 and intron 1 inversions were studied by long distance polymerase chain reaction (PCR) and point mutations by sequencing the coding and promoter regions of the F8 gene. Results: The results obtained in all samples used for validation were concordant with those obtained previously. In the Chilean patients, the intron 22 inversion and point mutations previously described were observed. In 6 out of 9 patients with mild Hemophilia A we found the same mutation (Arg2159Cys) in exon 23, which has been described as prevalent in mild Hemophilia A. Conclusions: The analysis of intron 22 and intron 1 inversions, as well as of point mutations in the F8 gene will help us to confirm the diagnosis in patients with severe, moderate and mild Hemophilia A, and also it will allow us to perform carrier testing and to provide better genetic counseling.

Caracterização de aspectos geneticos e imunologicos envolvidos no desenvolvimento de inibidores em hemofilia A e B / Genetic and immunologic aspects related to the development of inhibitors in hemophilia A and B

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...

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.

Polimorfismos intragénicos de los genes de los factores VIII y IX y su utilidad en el diagnóstico indirecto de portadoras de hemofilias A y B / Intragenic polymorphisms of factor VIII and IX genes and their utility in the indirect diagnosis of carriers of haemophilias A and B

Las Hemofilias A y B se consideran enfermedades hereditarias ligadas al sexo debidas a mutaciones en los genes que codifican para los factores VIII y IX respectivamente, ocasionando deficiencia en los niveles de la concentración plasmática de estas proteínas y cuyos roles son los de participar activamente en el mecanismo de la coagulación sanguínea. Se han reportado diversas mutaciones responsables de la alteración de estos genes; razón por la cual resulta poco práctico la aplicación de un método de diagnóstico molecular directo para la identificación de mujeres portadoras, por ello, una estrategia diagnóstica apropiada es el análisis indirecto de polimorfismos ligados al gen. El objetivo de este trabajo fue identificar mujeres portadoras en diversas familias con antecedentes de HA y HB residentes del estado Zulia, en Venezuela, caracterizando polimorfismos intragénicos de los genes del factor VIII y factor IX, los cuales permitieron asignar haplotipos y diagnosticar o descartar el estado portador al 95 por ciento de las mujeres que requerían el estudio para HA y al 100 por ciento para HB.

Haemophilia A and B are considered sex-linked inherited diseases caused by mutations in genes that encode factors VIII and IX, respectively. This results in the deficiency of these proteins plasma levels which are actively involved in the mechanism of blood coagulation. It has been reported that several mutations are responsible for the alteration of these genes, which is why the application of a molecular diagnostic method for the direct identification of female carriers is impractical. An appropriate diagnostic strategy is the indirect analysis of polymorphisms linked to the gene. The aim of this study was to identify female carriers in different families with history of HA and HB that live in Zulia State, Venezuela, characterizing intragenic gene polymorphisms of the clotting factors VIII and IX, which helped to identify and assign haplotypes, to diagnose or to exclude the carrying condition, to 95 percent of women who were needing the study for HA and to 100 percent for HB.

Estudio familiar de las hemofilias A y B: 5 años de experiencia en la detección de portadoras / Family study of hemophilia A and B: 5 years of experience in carrier detection

La hemofilia se caracteriza por ser una enfermedad congénita del trastorno de la coagulación y constituye un desorden recesivo ligado al cromosoma X. El estudio molecular se realiza por estudios indirectos por ser causada por mutaciones heterogéneas en los genes del FVIII y FIX. Se realizó el estudio de 40 familias afectadas con hemofilia A (HA) y 10 hemofilia B (HB). La extracción de ADN se realizó por el método de precipitación salina a 293 muestras de sangre y 19 de líquido amniótico, y se hizo el análisis de los polimorfismos St14, Bcl I y Hind III para la HA y Taq I, Xmn I y Dde I para la HB. Se usó la técnica de PCR. En el caso de la HA se obtuvo el 35 por ciento de informatividad para St14 y Hind III y 32,5 para Bcl 1. El polimorfismo Dde I fue el más informativo para la HB con el 33 por ciento; mientras que Taq I representó el 10 por ciento de informatividad y XmnI el 0 por ciento. Se comprobó que de las 40 familias analizadas con HA, 23 fueron informativas. Por otra parte, fueron informativas 4 familias de las afectadas con HB. Se realizaron 19 diagnósticos prenatales con previa determinación del sexo fetal, incluidos 3 varones enfermos

Hemophilia is a congenital disease of coagulation disorder and it is a recessive disorder linked to X-chromosome. The molecular study is conducted by indirect studies due to it is caused by heterogeneous mutations in gen of FVIII and FIX in 40 families with hemophilia A (HA) and 10 with hemophilia B (HB). DNA extraction was carried out by saline precipitation method in 293 blood samples and 19 samples of amniotic fluid, as well as the analysis of St14, Bcl I and Hind III polymorphism for the AH and Taq I, Xmn I and Dde I for BH. The PCR technique was used. In the caser of AH it was possible to achieve a 35 percent of information for St14 and Hind III and a 32.5 percent for Bcl. Dde polymorphism supplied more information for BH for a 33 percent; whereas the Taq I represented the 10 percent of information and Xmn I the 0 percent. We verified that from the families analyzed with HA, in 23 of them we there was information. Besides, in 4 families affected by HB there was information. A total of 19 prenatal diagnoses were made with a previous determination of fetus sex, including 3 males ill

Haplotipo del gen del factor VIII en el diagnóstico molecular de la hemofilia A: estudio de una familia / Factor VIII gene haplotype in the haemophilia a molecular dagnosis: a family study

La hemofilia A se caracteriza por ser una enfermedad congénita del trastorno de la coagulación y constituye un desorden recesivo ligado al cromosoma X. El estudio molecular se realiza por estudio indirecto, por ser causada por mutaciones heterogéneas en el gen del factor VIII. Se estudió una familia afectada, para la detección de portadora de la gestante y posteriormente se realizó el diagnóstico prenatal. La extracción de ADN se hizo por el método de precipitación salina Salting Out a tres muestras de sangre y una de líquido amniótico. Se efectuó el análisis de los polimorfismos St14, Bcl I y Hind III. Para la determinación de sexo fetal, se estudió el gen AMXY. La técnica empleada fue la reacción en cadena de la polimerasa. El análisis del marcador Bcl I arrojó que la gestante era portadora de hemofilia A, pero al ser homocigótica no era informativa; el polimorfismo St14 por sí solo no brindaba la información de la condición de la gestante, pero al ser heterocigótica para el mismo y conociendo de antemano la información de ser portadora, se pudo realizar diagnóstico prenatal, gracias al análisis conjunto de los marcadores. El polimorfismo Hind III no fue informativo. El feto resultó ser varón sano (AU)

Hemophilia A is a coagulation disorder congenital disease which consist in a recessive disorder linked to X chromosome. HA is cause by heterogeneous mutations in factor VIII gen, that's why, the study was carry out by indirect studies. We studied one family afected; we were determinated of carrier status of pregnancy woman and later we relizated of prenatal diagnosis. The DNA extraction from the three blood samples and one amniotic fluid was obtained by the saline precipitation procedure (Salting Out). We studied the polymorphisms St14, Hind III and Bcl1. The determination of fetal sex was studied of AMXY gen. The technique used was Polymerase Chain Reaction. The Bcl I marker analysis showed that the mother was a carrier of haemophilia A but being homozygous was not informative, ST14 polymorphism alone did not provide information on the condition of the mother but to be heterozygous for it and knowing beforehand information of being a carrier prenatal diagnosis was possible thanks to joint analysis of the markers. Hind III polymorphism was not informative. The male fetus was found to be healthy (AU)

Hemophilia a and congenital Hypofibrinogenemia: A rare association in same family.

Hemophilia A is the commonest inherited coagulation defect in human beings, whereas congenital hypofibrinogenemia is a much rarer disease. Occurrence of these two inherited diseases in the same family has not been reported so far. Younger sibling of a known case of Hemophilia A presented with recurrent, spontaneously occurring echymotic spots having prolonged PT, APTT, TT and very low absolute fibrinogen level with normal factor VIII level ultimately diagnosed as a case of congenital hypofibrinogenemia.

Caracterização Molecular da Deficiência de Fator VIII em Pacientes Hemofílicos de Minas Gerais

Hemofilia A, uma doença hemorrágica ligada ao cromossomo X, é causada pela deficiência ou disfunção do fator VIII da coagulação (FVIII). A doença resulta de alterações genéticas no gene do fator VIII (F8). Clinicamente, a hemofilia A é caracterizada por hemorragias em diferentes partes do corpo e o tratamento recomendado é a reposição do fator FVIII deficiente. O desenvolvimento de anticorpos inbidores para o FVIII é a principal complicação do tratamento. Estes anticorpos neutralizam a atividade do FVIII e, portanto invalidam a terapia. Os riscos para o desenvolvimento destes anticorpos são multifatoriais e envolvem fatores genéticos e ambientais. O objetivo deste estudo foi determinar as bases moleculares da hemofilia A grave e sua correlação com o desenvolvimento de inibidores em associação com o perfil sorológico par doenças virais. As análises moleculares consistiram da determinação da inversão do íntron 1 e do íntron 22 (inv1 e inv 22), além da determinação de outras alterações genéticas presentes na região codificadora de F8.

Inicialmente, 150 pacientes, com hemofilia A grave (níveis de atividade de fator VIII inferior a 1%) registrados no Hemominas, foram selecionados para este estudo. Nós caracterizamos 50 pacientes, dos quais, 8 tinham inv1, 29 tinham inv22 e os pacientes restantes apresentaram mutações sem sentido, mutações de sentido trocado e mutações de mudança de fase de leitura. Posteriormente, um grupo de 40 pacientes, registrados no hemocentro de Campinas e com diagnóstico molecular (32 tinham inv 22 e os pacientes restantes apresentaram mutações sem sentido, mutações de sentido trocado e mutações de mudança de fase de leitura) foram arrolados em nosso estudo. Informações a cerca de inibidores e perfil sorológico de 90 pacientes foram coletadas a partir de prontuários médicos. Dados moleculares e clínicos foram comparados por meio de análises estatísticas. As análises estatísticas não revelaram uma correlação positiva entre as diferentes categorias de mutação e a presença/ausência de inibidores sob influência do perfil sorológico.

Caracterização molecular da deficiência de fator VIII em pacientes hemofílicos de Minas Gerais / Molecular characterization of factor VIII deficiency in patients with Hemophilia Minas Gerais

Hemofilia A, uma doença hemorrágica ligada ao cromossomo X, é causada pela deficiência ou disfunção do fator VIII da coagulação (FVIII). A doença resulta de alterações genéticas no gene do fator VIII (F8). Clinicamente, a hemofilia A é caracterizada por hemorragias em diferentes partes do corpo e o tratamento recomendado é a reposição do fator FVIII deficiente. O desenvolvimento de anticorpos inbidores para o FVIII é a principal complicação do tratamento. Estes anticorpos neutralizam a atividade do FVIII e, portanto invalidam a terapia. Os riscos para o desenvolvimento destes anticorpos são multifatoriais e envolvem fatores genéticos e ambientais. O objetivo deste estudo foi determinar as bases moleculares da hemofilia A grave e sua correlação com o desenvolvimento de inibidores em associação com o perfil sorológico par doenças virais. As análises moleculares consistiram da determinação da inversão do íntron 1 e do íntron 22 (inv1 e inv 22), além da determinação de outras alterações genéticas presentes na região codificadora de F8.

Inicialmente, 150 pacientes, com hemofilia A grave (níveis de atividade de fator VIII inferior a 1%) registrados no Hemominas, foram selecionados para este estudo. Nós caracterizamos 50 pacientes, dos quais, 8 tinham inv1, 29 tinham inv22 e os pacientes restantes apresentaram mutações sem sentido, mutações de sentido trocado e mutações de mudança de fase de leitura. Posteriormente, um grupo de 40 pacientes, registrados no hemocentro de Campinas e com diagnóstico molecular (32 tinham inv 22 e os pacientes restantes apresentaram mutações sem sentido, mutações de sentido trocado e mutações de mudança de fase de leitura) foram arrolados em nosso estudo. Informações a cerca de inibidores e perfil sorológico de 90 pacientes foram coletadas a partir de prontuários médicos. Dados moleculares e clínicos foram comparados por meio de análises estatísticas. As análises estatísticas não revelaram uma correlação positiva entre as diferentes categorias de mutação e a presença/ausência de inibidores sob influência do perfil sorológico.

As bases moleculares da hemofilia A

As hemofilias são doenças hemorrágicas resultantes da deficiência de fator VIII (hemofilia A) ou de fator IX (hemofilia B) da coagulação, decorrentes de mutações nos genes que codificam os fatores VIII ou IX, respectivamente. A hemofilia A é mais frequente que a hemofilia B e acomete aproximadamente 1:10.000 nascimentos masculinos. A gravidade e frequência dos episódios hemorrágicos está relacionado ao nível residual de atividade de fator VIII presente no plasma e este relaciona-se ao tipo de mutação associada à doença. A clonagem do gene do fator VIII tornou possível o conhecimento das bases moleculares da hemofilia A, sendo hoje conhecidas mais de 1.000 mutações associadas à doença. O conhecimento das bases moleculares da hemofilia A permite uma melhor compreensão da relação genótipo-fenótipo da doença, tomada de condutas clínicas diferenciadas em casos de mutações associadas a um maior risco de desenvolvimento de inibidor, determinação da condição de portadora de hemofilia em mulheres relacionadas aos pacientes, implementação de programa de aconselhamento genético/orientação familiar e melhor compreensão das relações estruturais-funcionais do gene-proteína. Este artigo propõe revisar as bases moleculares da hemofilia A, os métodos laboratoriais utilizados para a caracterização das mutações e as implicações clínicas envolvidas no diagnóstico molecular da hemofilia A.

Hemophilias are bleeding disorders due to deficiency of the blood coagulation factor VIII (hemophilia A) or factor IX (hemophilia B), resulting from mutation on the gene coding for factor VIII or factor IX. Hemophilia A is more frequent than hemophilia B and affects 1:10,000 male newborns. The severity and frequency of hemorrhagic episodes is related to residual activity of factor VIII present in the plasma and relates to the type of mutation associated with the disorder. Cloning of the factor VIII gene has enabled researchers to better understand the molecular basis of hemophilia A, accounting to date, for more than 1,000 mutations associated with the disease. This comprehensive knowledge permits an improved comprehension of the genotype-phenotype relation, establishment of clinical policies when mutations related to higher risk of inhibitors development are known, identification of hemophilia carriers in case of women related to patients, implementation of a program of genetic counseling and discovery of structural-functional relationship between gene-protein. This article aims to review the molecular basis of hemophilia A, laboratory techniques used to characterize mutations and clinical implications involved in the molecular diagnosis of hemophilia A.

Evaluación del patrón de inactivación del cromosoma X en portadoras sintomáticas y mujeres con hemofilia / Assessing the inactivation pattern in chromosome X among symptomatic carriers and women with haemophilia

La inactivación del cromosoma X es un fenómeno estocástico que ocurre en la embriogénesis temprana femenina para lograr una compensación de dosis génica respecto a los varones. Ciertos mecanismos genéticos afectan el proceso normal, propiciando una inactivación sesgada con efectos clínicos relevantes en portadoras de trastornos recesivos ligados al cromosoma X, como la hemofilia. La herramienta molecular mayormente utilizada para la evaluación del patrón de inactivación del cromosoma X es la amplificación por PCR del gen del receptor de andrógenos humano (HUMARA). El empleo de esta técnica en portadoras sintomáticas y mujeres con hemofilia permite esclarecer si las manifestaciones de la enfermedad se deben a una lyonización desfavorable. Estos estudios, además, son importantes para la comprensión del proceso de inactivación del cromosoma X en humanos.

X chromosome inactivation is a stochastic event that occurs early in female embryo development to achieve dosage compensation with males. Certain genetic mechanisms affect the normal process causing a skewed X inactivation pattern which has clinical relevance in female carriers of X-linked recessive disorders, like haemophilia. The most commonly used assay to evaluate the X inactivation pattern is the PCR amplification of the human androgen receptor gene (HUMARA). The use of this technique in bleeding carriers and women with haemophilia allows identifying if their hemorrhagic symptoms are due to an unfavourable lyonization. Furthermore, these studies are important for understanding the X chromosome inactivation process in humans.

Efficiency of BclI restriction fragment length polymorphism for detection of hemophilia a carriers in Sistan and Baluchestan Province, Southeast of Iran

Indirect genetic diagnosis using polymorphic DNA markers can detect carriers of hemophilia A. This technique is preferable in developing countries because of its simplicity and cost effectiveness compared to direct mutation analysis. In the present study, we examined usefulness of intragenic marker BclI restriction fragment length polymorphism [RFLP] at intron 18, for carrier detection. How this marker is informative was tested in 102 members of 16 hemophiliac families from Sistan and Baluchestan province, Southeast of Iran. Blood samples were obtained from 29 hemophilia A patients and 73 of their relatives, after taking informed consents. DNA was extracted using proteinase K digestion followed by DNA precipitation. Factor VIII gene polymorphism was identified by the polymerase chain reaction/RFLP which is both sensitive and economical. Our results showed that almost 69.8% of Xchromosomes had restriction site for BclI enzyme. The heterozygosity rate for BclI polymorphism in tested women was 61.4%, signifying the usefulness of this marker in carrier detection. The informative rate respecting this polymorphism was 43.7% meaning that a remarkable percent of families from the target population could be diagnosed using this marker alone. In Sistan and Baluchestan province where there is limited access to sophisticated facilities of molecular diagnosis, use of PCR-based analysis of DNA polymorphism in the BclI locus can be used to identify a remarkable percentage of the carriers and even for prenatal diagnosis. Meanwhile, it is necessary to evaluate the effectiveness of other polymorphic DNA markers to enhance the informative rate