Hemofilia: diagnóstico molecular y alternativas de tratamiento
TL;DR: In this paper, otras terapias alternas, aunque estan en fase de investigación, permitirian obtener una produccion de proteina a largo termino and que se han desarrollado gracias al entendimiento de la naturaleza molecular de los factores de la coagulacion.
Abstract: La hemofilia es una enfermedad recesiva ligada al cromosoma X que generalmente padecen los hombres. El diagnostico genetico preimplantacion (DGP), el diagnostico prenatal y el diagnostico molecular de las mutaciones que causan hemofilia, se realizan en investigaciones aisladas con el fin de hacer prevencion primaria, asesorar a las portadoras y a sus familias, lo que ha permitido traer al mundo ninos libres de esta enfermedad y tambien mejorar la calidad de vida de los afectados. Los esperanzadores procedimientos en terapia genica (TG) han mostrado gran efectividad, se pretende con ella la produccion normal de la proteina que esta ausente o alterada en los afectados, pero en el momento los ensayos que se llevan a cabo en seres humanos estan detenidos. Aqui se muestran otras terapias alternas que aunque estan en fase de investigacion, permitirian obtener una produccion de proteina a largo termino y que se han desarrollado gracias al entendimiento de la naturaleza molecular de los factores de la coagulacion.
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TL;DR: The results establish a general overview of population exposure and can be a scientific tool to improve environmental health policies in the country and could strengthen Colombia’s efforts to increase the practice of breastfeeding.
4 citations
TL;DR: In this paper , the authors applied species richness estimation methods from ecology to estimate "variant richness" and determine how many germline pathogenic BRCA1/2 variants have yet to be identified and the frequency of these missing variants in different populations.
Abstract: There have been many surveys of genetic variation in BRCA1 and BRCA2to identify variant prevalence and catalogue population specific variants, yet none have evaluated the magnitude of unobserved variation. We applied species richness estimation methods from ecology to estimate "variant richness" and determine how many germline pathogenic BRCA1/2 variants have yet to be identified and the frequency of these missing variants in different populations. We also estimated the prevalence of germline pathogenic BRCA1/2 variants and identified those expected to be most common. Data was obtained from a literature search including studies conducted globally that tested the entirety of BRCA1/2 for pathogenic variation. Across countries, 45% to 88% of variants were estimated to be missing, i.e., present in the population but not observed in study data. Estimated variant frequencies in each country showed a higher proportion of rare variants compared to recurrent variants. The median prevalence estimate of BRCA1/2 pathogenic variant carriers was 0.64%. BRCA1 c.68_69del is likely the most recurrent BRCA1/2 variant globally due to its estimated prevalence in India. Modeling variant richness using ecology methods may assist in evaluating clinical targeted assays by providing a picture of what is observed with estimates of what is still unknown.
2 citations
Journal Article•
TL;DR: A Colombian family that apparently suffered the two diseases according to clinical diagnosis and the lack of a genetic study to verify and contrast the diagnosis made by health institutions can lead to a wrong classification of von Willebrand disease as a type of mild Hemophilia.
Abstract: El Factor von Willebrand circula en el plasma formando un complejo con el Factor VIII de coagulacion por enlaces no covalentes. Esta interaccion evita la degradacion enzimatica del Factor VIII y asegura su transporte al lugar de formacion del coagulo de fibrina. Debido a su estrecha relacion, la disminucion de la actividad de un factor puede afectar la actividad del otro, lo que genera un diagnostico clinico equivocado en cuanto a que enfermedad se padece, si Hemofilia A o Enfermedad de von Willebrand. Este estudio reporta el caso de una familia colombiana que segun diagnostico clinico de su fenotipo, padecia las dos enfermedades. Sin embargo, dicha familia carecia de un estudio genetico que permitiera verificar y contrastar el diagnostico que hacen las entidades de salud. Por tal razon, se realizo un diagnostico genetico por pruebas moleculares que detectan mutaciones, como las inversiones en los intrones 1 y 22 por PCR de fragmentos largos y la secuenciacion del gen del Factor VIII, esta ultima no aplicada y publicada en Colombia hasta el momento. Se encontraron dos mutaciones sinonimas en los exones 14 y 26 que no alteran la secuencia de aminoacidos en la proteina; por tanto, se descarta la presencia de Hemofilia A en la familia. Se plantea la posibilidad de un caso de Enfermedad de von Willebrand unicamente. El estudio demuestra la necesidad que hay en el pais de ampliar las pruebas clinicas y de incluir el diagnostico genetico en casos de ambiguedad en el diagnostico de estas coagulopatias. Palabras clave: Hemofilia A, Factor VIII, Enfermedad de von Willebrand. MOLECULAR ANALYSIS OF HEMOPHILIA A IN A COLOMBIAN FAMILY WITH DIAGNOSIS OF HEMOPHILIA A AND VON WILLEBRAND DISEASE ABSTRACT Von Willebrand Factor circulates in plasma in a protein complex together with coagulation Factor VIII, joined by noncovalent bonds. This interaction prevents enzymatic degradation of Factor VIII and ensures its transport to the place of the fibrin clot formation. Because of their close relationship, decrease activity of one factor may affect the other one. The late generates a clinical diagnosis not very accurate for Hemophilia A or von Willebrand disease. We report here a Colombian family that apparently suffered the two diseases according to clinical diagnosis. However, the lack of a genetic study to verify and contrast the diagnosis made by health institutions, which is based on phenotype, only, can lead to a wrong classification of von Willebrand disease as a type of mild Hemophilia. The aim of this study was to confirm the clinical diagnosis in this family by molecular analysis. To achieve this, we identify the presence of Factor VIII gene inversions in introns 22 and 1 by LD - PCR and a general scan of the gene for frame shift mutations or stop codons through three family generations. Two synonymous mutations were found in exons 14 and 26, so no changes were observed in the aminoacid sequences, fact that disregards the presence of Hemophilia A. This family could be a case of von Willebrand disease only. The use of molecular techniques to confirm the clinical diagnosis for bleeding disorders will improve adequate treatment and patient prognosis in Colombia. Key words: Hemophilia A, Factor VIII, von Willebrand disease.
2 citations
Cites background from "Hemofilia: diagnóstico molecular y ..."
...Frecuencia de los alelos de referencia y alterno en poblaciones reportadas en NCBI para la variación rs1800292....
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...Frecuencia de los alelos de referencia y alterno en poblaciones reportadas en NCBI para la variación rs1050705....
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...Con ayuda del programa CLC Main Workbench 7 se alinearon las secuencias de cada individuo con la secuencia consenso proporcionada por NCBI (24)....
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...De acuerdo con los resultados arrojados por SIFT y PROVEAN, así como en el reporte de NCBI la variación no afecta la proteína, pues es de tipo sinónima y en consecuencia se mantiene el aminoácido serina (NP_000123....
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...Los alineamientos realizados con el programa CLC Main workbench 7 demostraron la presencia de solo dos variaciones en algunos miembros de la familia, rs1800292 y rs1050705, ya reportadas en bases de datos como NCBI, Kaviar y HAMSTeRS....
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TL;DR: In this paper , a cohort of 165 South African women of self-identified African ancestry diagnosed with breast cancer, who were unselected for family history of cancer, were analyzed using the Illumina TruSight cancer panel for targeted sequencing of 94 cancer susceptibility genes.
Abstract: Abstract Since the discovery of the breast cancer susceptibility genes, BRCA1 and BRCA2, various other genes conferring an increased risk for breast cancer have been identified. Studies to evaluate sequence variants in cancer predisposition genes among women of African ancestry are limited and mostly focused on BRCA1 and BRCA2 . To characterize germline sequence variants in cancer susceptibility genes, we analysed a cohort of 165 South African women of self-identified African ancestry diagnosed with breast cancer, who were unselected for family history of cancer. With the exception of four cases, all others were previously investigated for BRCA1 and BRCA2 deleterious variants, and were negative for pathogenic variants. We utilized the Illumina TruSight cancer panel for targeted sequencing of 94 cancer susceptibility genes. A total of 3.6% of patients carried a pathogenic/likely pathogenic variant in a known breast cancer susceptibility gene: 1.2% in BRCA1 , 0.6% in each of BRCA2 , ATM, CHEK2 and PALB, none of whom had any family history of breast cancer. The mean age of patients who carried deleterious variant in BRCA1/BRCA2 was 39 years and 8 months compared to 47 years and 3 months among women who carried a deleterious variant in other breast cancer susceptibility genes.
1 citations
29 Sep 2020
TL;DR: Hemophilia B or Christmas disease was first differentiated from hemophilia A in 1947 and is linked to the X chromosome; women are carriers, but it manifests clinically in men, although cases of symptomatic women carriers have been described.
Abstract: La hemofilia B o enfermedad de Christmas se diferencio por primera vez de la hemofilia A en 1947. Su forma clasica consiste en un trastorno hereditario de la coagulacion causado por mutaciones en el gen F9, que codifica para el factor IX de la coagulacion. Su herencia esta ligada al cromosoma X; las mujeres son portadoras, pero se manifiesta clinicamente en hombres, aunque se han descrito casos de mujeres portadoras sintomaticas. El factor IX activado es una proteina dependiente de vitamina K, sintetizada en el higado, que forma parte del complejo tenasa, cuya funcion es formar la mayor cantidad de trombina en el nuevo modelo de la coagulacion basado en celulas. De acuerdo a la actividad del factor IX, su deficiencia se puede clasificar en leve (5% a 40%), moderada (1% a 5%), o severa (<1%). Su diagnostico se realiza con la presencia de un TPT alargado que corrige con plasma normal y con la determinacion del nivel funcional del factor IX, y se confirma con el estudio molecular que demuestra la mutacion en el gen F9. Su diagnostico diferencial incluye otras patologias como la hemofilia A. El tratamiento con factor IX recombinante es el mas utilizado en la actualidad, pero se vienen desarrollando nuevas terapias con virus adeno-asociados recombinantes que prometen mejorar la calidad de vida para algunos pacientes afectados. La profilaxis juega un papel fundamental, en particular en los casos de enfermedad moderada y severa
1 citations
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TL;DR: In this paper, the authors reviewed the aforementioned areas of progress and discuss unresolved problems (such as treatment of patients with antibodies, the risk of new infectious complications, and the issue of secondary tumors). Hopes and expectations for further improvement in the third millennium and particularly the prospects of hemophilia cure though gene replacement therapy are also mentioned.
Abstract: Known since the beginning of the first millennium, the hemophilias are among the most frequent inherited disorders of blood coagulation and definitely the most severe. In the 1970s, with the availability of concentrated preparations of the deficient coagulation factors VIII and IX and with the large-scale adoption of home treatment, hemophilia care became one of the most gratifying examples of successful secondary prevention of a chronic disease. Unfortunately, in the early 1980s it was recognized that factor concentrates prepared from plasma pooled from thousands of donors transmitted the hepatitis and the human immunodeficiency viruses. The scientific community reacted promptly to the devastation brought about by hepatitis and AIDS. The last 15 years of the second millennium have witnessed the development of methods that, when applied during concentrate manufacturing, inactivate viruses escaping the screening procedures. The adoption of these measures has reduced dramatically the risk of transmission of bloodborne infections. The production of recombinant factors and their availability for patients' treatment epitomize progress in hemophilia care through DNA technology. Methods based on the polymerase chain reaction (PCR) have unraveled an array of gene lesions associated with hemophilia, permitting improved secondary control of the disease through carrier detection in women from affected families and prenatal termination of their affected male infants. This article will review the aforementioned areas of progress and discuss unresolved problems (such as treatment of patients with antibodies, the risk of new infectious complications, and the issue of secondary tumors). Hopes and expectations for further improvement in the third millennium and particularly the prospects of hemophilia cure though gene replacement therapy will also be mentioned.
68 citations
TL;DR: In conclusion, recombinant FVIII induces a high rate of immune tolerance even in carriers of null F8 mutations, as well as other null mutations and two with small deletion/insertions in the F8 gene.
Abstract: Immune tolerance induction (ITI) eradicates inhibitors in patients with hemophilia A. This study was designed to investigate the success rate of ITI in high-responding inhibitor patients with severe hemophilia A using recombinant factor VIII (rFVIII). Twenty-six patients received different ITI regimens until a normal recovery (>66%) and half-life (>6 h) of infused FVIII was achieved. In order to maximize the chance of success, the initiation of ITI was deferred in the majority of patients until the inhibitor declined to <10 BU. Twenty-two patients (85%) had baseline inhibitor levels <10 BU (median 2.3 BU) when ITI began. Within a median of 6 months, immune tolerance was achieved in 19 of 26 patients (73%) including 12/17 (70%) with intron 22 inversion, 5/7 (71%) with other null mutations and two with small deletion/insertions in the F8 gene. In conclusion, recombinant FVIII induces a high rate of immune tolerance even in carriers of null F8 mutations.
59 citations
TL;DR: Although hemophilia A and B appear to be ideal diseases to target with gene therapy approaches, the promise of this therapy remains to be realized.
Abstract: A variety of factor concentrates are currently available for replacement therapy for patients with hemophilia. These differ by several parameters, including source (pooled from pooled blood vs recombinant), purity, pathogen inactivation, and by the presence or absence of extraneous proteins such as albumin. The choice of replacement product reflects both safety issues of pathogen transmission or inhibitor development, and personal preferences of the patient and the physician. In general, currently available products are viral pathogen-free, although there is debate about the risk of transmission of parvovirus B19 and prion pathogens. Because of this very small risk, recombinant factor is the treatment of choice in previously untreated patients. In addition, a subset of concentrates contain factor that is activated during manufacture, yielding activated products that can be used in the treatment of patients with inhibitors. Such activated products, especially recombinant factor VIIa (rFVIIa), have also acquired several off-label indications in the management of bleeding in non-hemophiliac patients. The management of hemophilia patients with inhibitors is an ongoing challenge. Immune tolerance induction using a desensitization technique is successful in up to 90% of patients with alloantibodies against factor VIII, with greatest success seen in patients with low titer inhibitors who are treated soon after detection of an alloantibody and in whom treatment includes administration of immunosuppression along with repeated infusions of high titer concentrates. Such therapy is less successful in patients with factor IX alloantibodies. Non-hemophiliac patients with acquired inhibitors represent a unique patient population that requires special management. These patients have a mortality rate that approaches 25% because of the association of acquired inhibitors with severe bleeding complications, occurrence in a largely elderly population, and the frequent presence of an underlying, often serious, primary medical condition. Treatment consists of immunosuppression with steroids, chemotherapy, or intravenous immunoglobulin. Recent studies using rituximab for selective B-cell depletion in these patients have been very promising, although prospective controlled studies have not yet been performed. Finally, although hemophilia A and B appear to be ideal diseases to target with gene therapy approaches, the promise of this therapy remains to be realized.
55 citations
TL;DR: Using the FVIII or FIX gene coding region by conformation sensitive gel electrophoresis and DNA sequencing, this approach is able to offer accurate genetic analysis to virtually all families with hemophilia.
Abstract: BACKGROUND AND OBJECTIVE: We describe our three year experience in genetic counseling at the Castelfranco Veneto Hemophilia Center, Italy. DESIGN AND METHODS: A total of 258 individuals were involved in the study of 142 females. These formed 40 families with hemophilia A and 6 families with hemophilia B. Following pedigree analysis, the FVIII inversion was first examined in severe hemophilia A patients by polymerase chain reaction (PCR) analysis. DNA polymorphisms were used to track the affected gene through the remaining families. In uninformative cases, we initiated analysis of the FVIII or FIX gene coding region by conformation sensitive gel electrophoresis and DNA sequencing to identify the mutation responsible for the disease. RESULTS: The FVIII gene inversion was present in 16 of the 32 patients (50%) affected by severe hemophilia A and was informative for 44 females. For hemophilia A, 45 cases (55%) were informative by linkage analysis, however 37 (45%) were uninformative because of lack of key individuals, homozygosity, or sporadic disease. Information from extragenic linked polymorphisms alone was present in 9 cases (6%). For hemophilia B, linkage analysis was informative in only 50% of females (8 out of 16). To date, nine mutations have been identified in patients with hemophilia A and three in patients with hemophilia B. Six novel missense mutations in hemophilia A are discussed briefly. INTERPRETATION AND CONCLUSIONS: Using this approach we are now able to offer accurate genetic analysis to virtually all families with hemophilia.
47 citations
TL;DR: The results of mutation analysis on 89 hemophiliac males showed presence of a disease‐causing mutation in 80 individuals, and thirty‐four novel mutations and three novel substitutions for previously reported amino acid residues were identified in this series of 80 mutations.
Abstract: Hemophilia A (HEMA) is an X-linked bleeding disorder caused by mutations in the factor VIII gene (F8C). Molecular genetic testing for the factor VIII gene is challenging due to its large size. Here we present results of high throughput mutation scanning based on Southern blot analysis and direct sequencing of all PCR amplified coding exons and the exon-intron boundaries of the factor VIII gene. The results of mutation analysis on 89 hemophiliac males showed presence of a disease-causing mutation in 80 individuals (90%, 95% CI of 82%-95%). Seven out of nine mutation-negative individuals were severe cases of hemophilia A with < 1% factor VIII protein in the blood. The correlation of phenotype with genotype as observed in this study was not absolute. This finding is supported by similar observations in the international database for hemophilia A mutations (HAMSTeRS). This issue raises the importance of genotypes at other loci that can act as modifiers for the phenotype. Thirty-four novel mutations and three novel substitutions for previously reported amino acid residues were identified in this series of 80 mutations. The mutations cover the full spectrum including rearrangements, deletions, frameshift, and point mutations. The novel missense mutations require careful evaluation. Prediction of a mutation as the disease-causing allele was made from the nature of the substitution and the degree of conservation of the mutated amino acid among species that have diverged in evolution. In some cases segregation analysis of the mutation with disease condition was performed when other family members were available.
46 citations