Molecular Analysis of Factor VIII and Factor IX Genes in Hemophilia Patients: Identification of Novel Mutations and Molecular Dynamics Studies

Background: Haemophilia A (HA) and Haemophilia B (HB) are X-linked blood disorders that are caused by various mutations in the factor VIII (F8) and factor IX (F9) genes respectively. Identification of mutations is essential as some of the mutations are associated with the development of inhibitors. This study is the first comprehensive study of the F8 mutational profile in Malaysia.Materials and methods: We analysed 100 unrelated HA and 15 unrelated HB patients for genetic alterations in the F8 and F9 genes by using the long-range PCR, DNA sequencing, and the multiplex-ligation-dependent probe amplification assays. The prediction software was used to confirm the effects of these mutations on factor VIII and IX proteins.Results: 44 (53%) of the severe HA patients were positive for F8 intron 22 inversion, and three (3.6%) were positive for intron 1 inversion. There were 22 novel mutations in F8, including missense (8), frameshift (9), splice site (3), large deletion (1) and nonsense (1) mutations. In HB patients, four novel mutations were identified including the splice site (1), small deletion (1), large deletion (1) and missense (1) mutation.Discussion: The mutational spectrum of F8 in Malaysian patients is heterogeneous, with a slightly higher frequency of intron 22 inversion in these severe HA patients when compared to other Asian populations. Identification of these mutational profiles in F8 and F9 genes among Malaysian patients will provide a useful reference for the early detection and diagnosis of HA and HB in the Malaysian population.

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Management of Haemophilia in Sweden

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647991 ◽

1976 ◽

Vol 35 (03) ◽

pp. 510-521 ◽

Cited By ~ 13

Author(s):

Inga Marie Nilsson

Keyword(s):

Factor Viii ◽

Total Population ◽

Age Groups ◽

Factor Ix ◽

Haemophilia A ◽

Severe Haemophilia ◽

Haemophilia B ◽

Human Fraction ◽

Mild Haemophilia

SummaryThe incidence of living haemophiliacs in Sweden (total population 8.1 millions) is about 1:15,000 males and about 1:30,000 of the entire population. The number of haemophiliacs born in Sweden in 5-year periods between 1931-1975 (June) has remained almost unchanged. The total number of haemophilia families in Sweden is 284 (77% haemophilia A, 23% haemophilia B) with altogether 557 (436 with A and 121 with B) living haemophiliacs. Of the haemophilia A patients 40 % have severe, 18 % moderate, and 42 % mild, haemophilia. The distribution of the haemophilia B patients is about the same. Inhibitors have been demonstrated in 8% of the patients with severe haemophilia A and in 10% of those with severe haemophilia B.There are 2 main Haemophilia Centres (Stockholm, Malmo) to which haemophiliacs from the whole of Sweden are admitted for diagnosis, follow-up and treatment for severe bleedings, joint defects and surgery. Minor bleedings are treated at local hospitals in cooperation with the Haemophilia Centres. The concentrates available for treatment in haemophilia A are human fraction 1-0 (AHF-Kabi), cryoprecipitate, Antihaemophilic Factor (Hyland 4) and Kryobulin (Immuno, Wien). AHF-Kabi is the most commonly used preparation. The concentrates available for treatment in haemophilia B are Preconativ (Kabi) and Prothromplex (Immuno). Sufficient amounts of concentrates are available. In Sweden 3.2 million units of factor VIII and 1.0 million units of factor IX are given per year. Treatment is free of charge.Only 5 patients receive domiciliary treatment, but since 1958 we in Sweden have practised prophylactic treatment of boys (4–18 years old) with severe haemophilia A. At about 5-10 days interval they receive AHF in amounts sufficient to raise the AHF level to 40–50%. This regimen has reduced severe haemophilia to moderate. The joint score is identical with that found in moderate haemophilia in the same age groups. For treatment of patients with haemophilia A and haemophilia B complicated by inhibitors we have used a large dose of antigen (factor VIII or factor IX) combined with cyclophosphamide. In most cases this treatment produced satisfactory haemostasis for 5 to 30 days and prevented the secondary antibody rise.

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The Effect of Adrenaline Infusions on Blood Coagulation in Normal and Haemophilia B Dogs

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649437 ◽

1966 ◽

Vol 15 (03/04) ◽

pp. 349-364 ◽

Cited By ~ 5

Author(s):

A.H Özge ◽

H.C Rowsell ◽

H.G Downie ◽

J.F Mustard

Keyword(s):

Body Weight ◽

Factor Viii ◽

Factor Ix ◽

Clotting Factor ◽

Blood Ph ◽

Order Of Magnitude ◽

Dose Dependent ◽

Generation Test ◽

Plasma Activity

SummaryThe addition of trace amounts of adrenaline to whole blood in plasma in vitro increased factor VIII, factor IX and whole plasma activity in the thromboplastin generation test. This was dose dependent.Adrenaline infusions less than 22 (μg/kg body weight in normal dogs accelerated clotting, increased factor IX, factor VIII and whole plasma activity in the thromboplastin generation test and caused a fall in blood pH. In a factor IX deficient dog, there was no increase in factor IX activity. After adrenaline infusions, however, the other changes occurred and were of the same order of magnitude as in the normal. Adrenaline in doses greater than 22 μg/kg body weight did not produce as great an effect on clotting in normal or factor IX deficient dogs. The platelet count in the peripheral blood was increased following the infusion of all doses of adrenaline. These observations suggest that the accelerating effect of adrenaline on clotting is not mediated through increase in activity of a specific clotting factor.

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A Survey of the Effectiveness of Prothrombin Complex Concentrates in Controlling Hemorrhage in Patients with Hemophilia and Anti-Factor VIII Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650077 ◽

1980 ◽

Vol 44 (01) ◽

pp. 039-042 ◽

Cited By ~ 14

Author(s):

Philip M Blatt ◽

Doris Ménaché ◽

Harold R Roberts

Keyword(s):

Factor Viii ◽

Ad Hoc ◽

Hemophilia A ◽

Working Party ◽

International Committee ◽

Factor Ix ◽

Prothrombin Complex Concentrates ◽

Factor Viii Concentrates

SummaryThe treatment of patients with hemophilia A and anti-Factor VIII antibodies is difficult. Between July 1977 and June 1978, a survey was carried out by an ad hoc working party of the subcommittee on Factor IX concentrates of the International Committee on Thrombosis and Hemostasis to assess the effectiveness of Prothrombin Complex Concentrates in controlling hemorrhage in these patients. The results are presented in this paper and, although subjective, support the view that these concentrates are not as effective in patients with inhibitors as Factor VIII concentrates are in patients without inhibitors.

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In Vitro Thrombogenicity Tests of Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657035 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1355-1367 ◽

Cited By ~ 8

Author(s):

C V Prowse ◽

A Chirnside ◽

R A Elton

Keyword(s):

Factor Viii ◽

Partial Thromboplastin Time ◽

Generation Time ◽

Activated Partial Thromboplastin Time ◽

Factor Ix ◽

In Vitro Tests ◽

Factor Viii Inhibitor ◽

Factor Ixa ◽

Viii Inhibitor

SummaryVarious factor IX concentrates have been examined in a number of in vitro tests of thrombogenicity. The results suggest that some tests are superfluous as in concentrates with activity in any of these tests activation is revealed by a combination of the non-activated partial thromboplastin time, the thrombin (or Xa) generation time and factor VIII inhibitor bypassing activity tests. Assay of individual coagulant enzymes revealed that most concentrates contained more factor IXa than Xa. However only a small number of concentrates, chiefly those that had been purposefully activated, contained appreciable amounts of either enzyme.

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Molecular Weights of Factor VIII (AHF) and Factor IX (PTC) by Electron Irradiation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655426 ◽

1962 ◽

Vol 08 (02) ◽

pp. 270-275 ◽

Cited By ~ 2

Author(s):

David L Aronson ◽

John W Preiss ◽

Michael W Mosesson

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

Electron Irradiation ◽

Factor Ix ◽

Molecular Weights

SummaryThe molecular weights of AHF (factor VIII) and of PTC (factor IX) have been estimated by their sensitivity to inactivation by 7 kilovolt electrons. The molecular weight of AHF was found to be 180 000 by this method and that of PTC was found to be 110 000.

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Management of Haemophilia A with Antibodies - The Effect of Combined Treatment with Factor VIII, Hydrocortisone and Cyclophosphamide

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660131 ◽

1985 ◽

Vol 54 (04) ◽

pp. 776-779 ◽

Cited By ~ 13

Author(s):

U Hedner ◽

L Tengborn

Keyword(s):

Factor Viii ◽

Combined Treatment ◽

Factor Ix ◽

Treatment Schedule ◽

High Antibody ◽

Anamnestic Response ◽

Short Intervals ◽

Low Responders ◽

Antibody Levels ◽

High Responders

SummaryImmune tolerance has by several methods been induced in haemophiliacs with antibodies. A conversion of “high responders” into “low responders” was previously reported after repeated moderate factor IX doses over periods of 7-10 days in combination with cyclophosphamide and steroids in two patients with haemophilia B and inhibitors. This paper reports similar results in a heamophilia A patient by giving factor VIII, cyclophosphamide, and steroids during relatively short periods of time (7-8 days). The anamnestic response markedly decreased already following the first treatment and never exceeded a level of 1 u/ml (˜ 3 BU/ml) even when boosted with ordinary factor VIII doses for only 3 days. It is concluded that the markedly decreased secondary antibody response is most probably the result of factor VIII given at short intervals (twice a day) for periods of up to about one week when given in combination with cyclophosphamide and steroids. The same effect may be achieved by other methods. The treatment schedule suggested in the present paper is, however, simple and avoids long periods of high antibody levels. Furthermore, the total factor VIII dose used is lower than suggested in most other treatment schedules, which makes the treatment substantially less expensive.

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Gene Therapy in Hemophilia: Recent Advances

International Journal of Molecular Sciences ◽

10.3390/ijms22147647 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7647

Author(s):

E. Carlos Rodríguez-Merchán ◽

Juan Andres De Pablo-Moreno ◽

Antonio Liras

Keyword(s):

Gene Therapy ◽

Adverse Events ◽

Factor Viii ◽

Coagulation Factor ◽

Cost Savings ◽

Factor Ix ◽

Clotting Factors ◽

Advanced Therapies ◽

Potential Benefits

Hemophilia is a monogenic mutational disease affecting coagulation factor VIII or factor IX genes. The palliative treatment of choice is based on the use of safe and effective recombinant clotting factors. Advanced therapies will be curative, ensuring stable and durable concentrations of the defective circulating factor. Results have so far been encouraging in terms of levels and times of expression using mainly adeno-associated vectors. However, these therapies are associated with immunogenicity and hepatotoxicity. Optimizing the vector serotypes and the transgene (variants) will boost clotting efficacy, thus increasing the viability of these protocols. It is essential that both physicians and patients be informed about the potential benefits and risks of the new therapies, and a register of gene therapy patients be kept with information of the efficacy and long-term adverse events associated with the treatments administered. In the context of hemophilia, gene therapy may result in (particularly indirect) cost savings and in a more equitable allocation of treatments. In the case of hemophilia A, further research is needed into how to effectively package the large factor VIII gene into the vector; and in the case of hemophilia B, the priority should be to optimize both the vector serotype, reducing its immunogenicity and hepatotoxicity, and the transgene, boosting its clotting efficacy so as to minimize the amount of vector administered and decrease the incidence of adverse events without compromising the efficacy of the protein expressed.

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Insertion Loop 256−268 in Coagulation Factor IX Restricts Enzymatic Activity in the Absence but Not in the Presence of Factor VIII†

Biochemistry ◽

10.1021/bi992735q ◽

2000 ◽

Vol 39 (25) ◽

pp. 7398-7405 ◽

Cited By ~ 23

Author(s):

Joost A. Kolkman ◽

Koen Mertens

Keyword(s):

Enzymatic Activity ◽

Factor Viii ◽

Coagulation Factor ◽

Factor Ix ◽

Coagulation Factor Ix

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TT virus contaminates first-generation recombinant factor VIII concentrates

Blood ◽

10.1182/blood.v98.8.2571 ◽

2001 ◽

Vol 98 (8) ◽

pp. 2571-2573 ◽

Cited By ~ 27

Author(s):

Alberta Azzi ◽

Riccardo De Santis ◽

Massimo Morfini ◽

Krystyna Zakrzewska ◽

Roberto Musso ◽

...

Keyword(s):

Factor Viii ◽

First Generation ◽

Second Generation ◽

Human Albumin ◽

Factor Ix ◽

Recombinant Factor Viii ◽

Tt Virus ◽

Recombinant Product ◽

Before And After ◽

Factor Viii Concentrates

Abstract Recombinant factor VIII and factor IX concentrates, human-plasma–derived albumin, and samples from previously untreated patients with hemophilia were examined for the presence of TT virus (TTV) by using polymerase chain reaction testing. Blood samples from the patients were obtained prospectively before and every 3 to 6 months after therapy was begun. TTV was detected in 23.5% of the recombinant-product lots and 55.5% of the albumin lots tested. Only first-generation factor VIII recombinant concentrates stabilized with human albumin were positive for TTV, whereas all second-generation (human protein–free) concentrates were negative for the virus. In 59% of patients treated with either first- or second-generation recombinant factor concentrates, TTV infection developed at some point after the initial infusion. Infection with TTV in these patients before and after treatment did not appear to be clinically important. Thus, first-generation recombinant factor VIII concentrates may contain TTV and the source of the viral contamination may be human albumin.

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