Three Distinct Point Mutations in the Factor IX Gene of Three Japanese CRM+ Hemophilia B Patients (Factor IX BMNagoya 2, Factor IX Nagoya 3 and 4)

SummaryEnzymatic DNA amplification and complete sequence analysis were used to investigate human factor IX coding sequences in three CRM+ hemophilia B patients. In a patient with severe hemophilia B and a markedly prolonged ox-brain prothrombin time, a C to T transition in exon VI changed the codon for Argl80 to Trp (factor IX BMNagoya 2). This mutation would impair the cleavage by factor XIa required for activation of the zymogen. In a patient with mild hemophilia B, a G to A transition in exon VI changed the codon for Argl45 to His (factor IX Nagoya 3). This substitution also would be predicted to preclude the cleavage of factor IX by factor XIa at this peptide bond (Argl45-Alal46). Furthermore, this point mutation creates a new NlaIII restriction site which provides a quick and reliable method for carrier detection in the affected family members. A patient with severe hemophilia B (factor IX Nagoya 4) had a G to A transition in exon II changing the codon for Glu21 to Lys. This novel point mutation is assumed to impair the function of factor IX by disrupting the calcium binding of factor IX.

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Molecular defect in factor IXHilo, a hemophilia Bm variant: Arg----Gln at the carboxyterminal cleavage site of the activation peptide

Blood ◽

10.1182/blood.v73.3.718.718 ◽

1989 ◽

Vol 73 (3) ◽

pp. 718-721 ◽

Cited By ~ 14

Author(s):

MN Huang ◽

CK Kasper ◽

HR Roberts ◽

DW Stafford ◽

KA High

Keyword(s):

Amino Acid ◽

Point Mutation ◽

Cleavage Site ◽

Human Factor ◽

Dna Amplification ◽

Factor Ix ◽

Molecular Defect ◽

Human Factor Ix ◽

Amplification Technique ◽

Activation Peptide

Abstract A genomic DNA library and the enzymatic DNA amplification technique were used to isolate human factor IX coding sequences of a hemophilia Bm variant, factor IXHilo. A point mutation that resulted in the substitution of a glutamine (CAG) for an arginine (CGG) at amino acid 180 was found in exon VI of the factor IX gene (G----A at nucleotide 20519). This mutation alters the carboxy terminal cleavage site for the activation peptide at Arg180-Val181. The arginine residue at the activation peptide cleavage site is conserved in mouse, canine, bovine, and human factor IX, suggesting that the arginine at amino acid 180 is important for normal cleavage. Sequencing of all of the coding regions of factor IXHilo revealed no other mutations. We have also shown that the point mutation in exon VI creates a new Dde I restriction site, which, in combination with the enzymatic DNA amplification technique, provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first report of the molecular defect in a hemophilia Bm patient with a markedly prolonged ox brain prothrombin time.

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Factor IX New London: substitution of proline for glutamine at position 50 causes severe hemophilia B

Blood ◽

10.1182/blood.v75.5.1097.1097 ◽

1990 ◽

Vol 75 (5) ◽

pp. 1097-1104 ◽

Cited By ~ 19

Author(s):

JN Lozier ◽

DM Monroe ◽

S Stanfield-Oakley ◽

SW Lin ◽

KJ Smith ◽

...

Keyword(s):

Translation Termination ◽

Factor Ix ◽

Hemophilia B ◽

Molar Ratio ◽

Carrier Status ◽

Severe Hemophilia ◽

Base Pairs ◽

Human Factor Ix ◽

Restriction Endonuclease Cleavage Site ◽

Status Analysis

Abstract We describe a novel point mutation in the fourth exon of human factor IX (encoding the first EGF-like domain) in which cytosine is substituted for adenosine at position 10,401, resulting in the substitution of proline for glutamine at position 50 in the polypeptide chain. Sequence analysis of all eight exons, all exon-intron junctions, 160 base pairs (bp) of DNA 5′ to the proposed translation start site, and 60 bp 3′ to the translation termination site shows no other difference from the normal factor IX gene, with the exception of a previously described benign polymorphism at position 148 in the protein (Ala----Thr). The affected subject has severe hemophilia B with no detectable factor IX activity despite normal factor IX antigen levels. We purified the abnormal factor IX by immunoaffinity chromatography and demonstrated that its activation by factor Xla is markedly delayed compared with normal factor lX. Once activated, the abnormal factor lX binds antithrombin III in a 1:1 molar ratio, and the activated protein demonstrates catalytic activity, suggesting an intact active site. The mutation creates a new Bst Yl restriction endonuclease cleavage site. Restriction with Bst Yl shows the mutation in maternal DNA and offers the possibility of direct carrier status analysis and prenatal diagnosis in kindreds with this mutation. We designate this new mutation factor lXNew London. This is the only reported mutation in the first EGF-like domain that causes severe hemophilia B.

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Factor IX Chongqing: a New Mutation in the Calcium-Binding Domain of Factor IX Resulting in Severe Hemophilia B

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645679 ◽

1990 ◽

Vol 63 (01) ◽

pp. 024-026 ◽

Cited By ~ 7

Author(s):

N S Wang ◽

M Zhang ◽

A R Thompson ◽

S-H Chen

Keyword(s):

Calcium Binding ◽

Somatic Mosaicism ◽

Chinese Patient ◽

Factor Ix ◽

Hemophilia B ◽

Severe Hemophilia ◽

New Mutation ◽

Exon 2 ◽

Calcium Dependent ◽

Gla Domain

SummaryA Chinese patient with sporadic, severe hemophilia B was found to have a low level of total factor IX antigen (3.5 U/dl), but less apparent antigen in an assay using a calcium-dependent antibody fraction (1.1 U/dl). This suggested a defect in the factor IX Gla domain coded mainly by exon 2 of the factor IX gene. Exon 2 was therefore amplified and sequenced. An A to T substitution was found at nucleotide 6455 of the patient’s factor IX gene. This transversion changes the codon for Glu 27 in normal factor IX to a codon for Val. Since Glu 27 becomes an essential Gla residue, the defect should result in altered calcium-binding or calcium-dependent conformation of the patient’s factor IX. The introduction of a hydrophobic side chain also appears to affect the hemophilic protein’s stability.In leukocyte DNA from the patient’s mother, the nucleotide sequence of exon 2 was entirely normal. Thus, barring somatic mosaicism within her germ cells, the new mutation occurred in oogenesis of her ovary.

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Two Mutations of the Factor IX Gene Including a Donor Splice Consensus Deletion and a Point Mutation in a Dutch Patient with Severe Hemophilia B

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647323 ◽

1990 ◽

Vol 64 (03) ◽

pp. 379-384 ◽

Cited By ~ 7

Author(s):

S R Poort ◽

E Briët ◽

R M Bertina ◽

P H Reitsma

Keyword(s):

Point Mutation ◽

Splice Site ◽

Normal Population ◽

Factor Ix ◽

Hemophilia B ◽

Donor Splice Site ◽

Severe Hemophilia ◽

Exon 2 ◽

Donor Splice ◽

Site Consensus

SummaryThe abnormal factor IX gene of a patient with severe hemophilia B (hemophilia B Ursem) was selected for study. All of the coding and their flanking regions and parts of the 5′- and 3′-untranslated regions of the factor IX gene were amplified from the patient’s genomic DNA by using the polymerase chain reaction (PCR). By analyzing the nucleotide sequence of the PCR products we have identified two mutations in the patient’s factor IX gene, viz. a tetranucleotide deletion (GAGT, nt 6492 to 6495) or (TGAG, nt 6491 to 6494) in the 5′-donor splice site consensus at the exon 2-intron B boundary, and a point mutation at nucleotide 31103 in the catalytic domain (exon 8) of factor IXa, which changes the codon for valine 328 (GTT) to one for isoleucine (ATT). PCR-amplified exon 8 from 45 normal males and 55 normal females had the codon for valine-328. We propose that the deletion within the donor splice-site consensus is the cause of the disease in this individual, whereas the substitution of valine-328 by isoleucine may be a neutral variant which is, at least, very rare in the normal population. In a family study the DNA sequence of the patient’s mother shows both the G to A transition in exon 8 and the 5′-donor splice consensus deletion in intron B in one allele.

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Point Mutations in Four Hemophilia B Patients from China

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647306 ◽

1990 ◽

Vol 64 (02) ◽

pp. 302-306 ◽

Cited By ~ 4

Author(s):

N S Wang ◽

S H Chen ◽

A R Thompson

Keyword(s):

Dna Sequences ◽

Genomic Dna ◽

Stop Codon ◽

Direct Sequencing ◽

Point Mutations ◽

Factor Ix ◽

Hemophilia B ◽

Chinese Patients ◽

Severe Hemophilia ◽

Plasma Factor

SummaryPoint mutations in factor IX genes of four unrelated Chinese patients with hemophilia B have been identified by direct sequencing of amplified genomic DNA fragments. These four mutations occur in exon 8 of the factor IX gene. A C to T transition at nucleotide 30,863 changes codon 248 from Arg (CGA) to a new Stop codon (TGA), described in a previous family as factor IXMalmo3 (Green P M et al., EMBO J 1989; 8: 1067). A G to A transition, at nucleotide 31,051 changes codon 310 from Trp (TGG) to a nonsense or Stop codon (TGA; factor IXchongqing2)- A G to A transition at nucleotide 31,119 changes codon 333 which is for Arg (CGA) in normal factor IX, to one for Gin (CAA) in the variant previously described as factor IXLondon2 (Tsang T C et al., EMBO J 1988; 7: 3009) in a patient with moderately severe hemophilia B. The fourth patient has a novel C to A transversion at nucleotide 31,290, which corresponds to replacement of codon 390 which is for Ala (GCA) in normal factor IX, to one for Glu (GAA) in a patient with moderately severe hemophilia B (factor IXChongqing3)- DNA sequences of amplified fragments from mothers of three showed both their son’s variant and a normal nucleotide at the appropriate position, indicating that they are carriers. The fourth patient’s (factor IXMalmo3) mother, whose DNA was not evaluable, was most probably a carrier because of her low plasma factor IX levels.

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Hemophilia B: Genetic Variants and Carrier Detection

10.1055/s-0039-1682645 ◽

1977 ◽

Author(s):

C.K. Kasper ◽

B. Østerud ◽

S.I. Rapaport

Keyword(s):

Prothrombin Time ◽

Genetic Variants ◽

Human Factor ◽

Factor Ix ◽

Hemophilia B ◽

Carrier Detection ◽

Severe Hemophilia ◽

Human Factor Ix ◽

Normal Women

In 92 males with hemophilia B from 71 kindreds, we measured factor IX activity, prothrombin time using bovine thromboplastin (bovine tpln time), and factor IX antigen both by inhibitor neutralization using a human factor IX inhibitor and by electroimmunoassay using a precipitating rabbit anti-human-factor IX antiserum. Eighty patients with 3% or less factor IX activity could be divided into 4 groups:(1) 7 patients with greatly prolonged bovine tpln times and normal levels of factor IX antigen;(2) 17 patients with mildly prolonged bovine tpln times and factor IX antigen levels between about 25% and normal;(3) 8 patients with normal bovine tpln times and antigen levels between about 25% and normal; (4) 48 patients with normal bovine tpln times and no measureable antigen excess. Some of the latter group were also tested with a chicken anti-human-factor IX antiserum and no antigen was found. None of 12 patients with mild hemophilia B (factor IX activity of 4 to 22%) had a prolonged bovine tpln time although 4 patients had excess factor IX antigen over activity. Thus, about 1/3 of these 92 hemophilia B patients had evidence of an abnormal factor IX molecule. Factor IX activity was also measured in 48 normal women and in 51 definite carriers of severe hemophilia B. Probability curves were derived to estimate the chance of a woman being a carrier based upon her factor IX level and her degree of kinship to a definite carrier. The relation between factor IX activity and antigen was also delineated for normal women and for carriers. In kindreds in which affected males had excess antigen, some carriers could be distinguished from normal women on the basis of excess antigen over activity. In appropriate kindreds, prolonged bovine tpln times helped distinguish some carriers.

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Pharmacokinetics of Human Factor IX in a dog with Severe Hemophilia B.

10.1055/s-0039-1682760 ◽

1977 ◽

Author(s):

P.A. Gentry ◽

A.R. Thompson ◽

A.W. Forrey

Keyword(s):

Human Factor ◽

Numerical Procedure ◽

Factor Ix ◽

Hemophilia B ◽

High Yield ◽

Severe Hemophilia ◽

Activity Data ◽

Human Factor Ix

In preparing a factor IX concentrate with a high yield and low hepatitis and thromboembolic risks, we have tested this material for survival in an in vivo system, the hemophiliac dog. By following the disappearance of radiolabeled, isolated factor IX in addition to the classic clotting assays, data on protein survival and more accurate kinetic parameters were obtained.Crude factor IX concentrate was prepared by batchwise adsorption-elution with DEAE-Sephadex using cryoprecipitate-poor human plasma. Isolated human factor IX was radiolabeled with 125I by chloramine-T without in vitro loss of clotting activity (Thompson, J Clin Invest, in press, 1977). A preparation containing both crude and isolated factor IX was then subjected to filtration (0.22 μm) and lyophilization; clotting and radioactivity were not altered by these steps.Following infusion of the combined preparation into a dog with severe hemophilia B (0% baseline factor IX) 10 post infusion samples were taken over 96 h for determination of radioactivity and factor IX clotting activity. These data were then analyzed by fitting to a two exponential expression using a Marquart non-linear least squares numerical procedure for a two compartment open model. The central volume was 14.5% of the animal’s body weight; the total volume of distribution was 28% with a t 1/2 distribution of 114 min. The t 1/2 elimination was 20 h; the slower phase of elimination (β, or that affected by redistribution) had a t 1/2 of 40 h. Factor IX clotting activity from the crude concentrate closely paralleled radioactivity from the isolated factor IX throughout the 96 h; t 1/2 β was slightly longer from the clotting activity data.

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Molecular defect in factor IXHilo, a hemophilia Bm variant: Arg----Gln at the carboxyterminal cleavage site of the activation peptide

Blood ◽

10.1182/blood.v73.3.718.bloodjournal733718 ◽

1989 ◽

Vol 73 (3) ◽

pp. 718-721

Author(s):

MN Huang ◽

CK Kasper ◽

HR Roberts ◽

DW Stafford ◽

KA High

Keyword(s):

Amino Acid ◽

Point Mutation ◽

Cleavage Site ◽

Human Factor ◽

Dna Amplification ◽

Factor Ix ◽

Molecular Defect ◽

Human Factor Ix ◽

Amplification Technique ◽

Activation Peptide

A genomic DNA library and the enzymatic DNA amplification technique were used to isolate human factor IX coding sequences of a hemophilia Bm variant, factor IXHilo. A point mutation that resulted in the substitution of a glutamine (CAG) for an arginine (CGG) at amino acid 180 was found in exon VI of the factor IX gene (G----A at nucleotide 20519). This mutation alters the carboxy terminal cleavage site for the activation peptide at Arg180-Val181. The arginine residue at the activation peptide cleavage site is conserved in mouse, canine, bovine, and human factor IX, suggesting that the arginine at amino acid 180 is important for normal cleavage. Sequencing of all of the coding regions of factor IXHilo revealed no other mutations. We have also shown that the point mutation in exon VI creates a new Dde I restriction site, which, in combination with the enzymatic DNA amplification technique, provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first report of the molecular defect in a hemophilia Bm patient with a markedly prolonged ox brain prothrombin time.

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Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer

Blood ◽

10.1182/blood-2011-09-382317 ◽

2012 ◽

Vol 119 (13) ◽

pp. 3038-3041 ◽

Cited By ~ 117

Author(s):

George Buchlis ◽

Gregory M. Podsakoff ◽

Antonetta Radu ◽

Sarah M. Hawk ◽

Alan W. Flake ◽

...

Keyword(s):

Skeletal Muscle ◽

Gene Transfer ◽

Muscle Tissue ◽

Viral Vector ◽

Factor Ix ◽

Hemophilia B ◽

Immunofluorescent Staining ◽

Severe Hemophilia ◽

Aav Vector ◽

Human Factor Ix

AbstractIn previous work we transferred a human factor IX–encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.

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Genetic Variants of Hemophilia B.

10.1055/s-0039-1682644 ◽

1977 ◽

Author(s):

H.R. Roberts ◽

K.S. Chung ◽

J.C. Goldsmith

Keyword(s):

Genetic Variants ◽

Biochemical Characterization ◽

Cross Reactivity ◽

Cross Reaction ◽

Factor Ix ◽

Hemophilia B ◽

Clinical Severity ◽

Antigenic Activity ◽

Human Factor Ix ◽

Factor Xia

The purpose of this report is to describe genetic variants of hemophilia B. Most variants have been distinguished on the basis of clinical severity of their disease, as well as immunological, functional, and biochemical characterization of the Factor IX molecule. They have been classified according to the degree of cross-reactivity of the Factor IX molecule with specific homologous and heterologous anti-Factor IX antibodies using both inhibitor neutralization and radioimmunoassay techniques. Some hemophilia B variants have a Factor IX molecule that cross-reacts completely with anti-Factor IX antibodies. Other hemophilia B patients have a Factor IX molecule that has no detectable cross-reaction with anti-Factor IX antibodies. Still other hemophilia B patients show reduced levels of cross-reacting material. We have extensively studied one hemophilia B variant with a Factor IX molecule that shows complete cross-reaction with anti-Factor IX antibody. The Factor IX from this patient has been isolated and has been shown to have 5% clotting activity and 100% antigenic activity. This variant Factor IX molecule shows delayed activation in the presence of partially purified Factor XIa and Ca2+ although it is otherwise similar to normal human Factor IX in terms of molecular weight and the number of α-carboxyglutamic acid residues per molecule. Other Factor IX variants have also been characterized and will be discussed. For example, hemophilia BM variants, unlike the usual type of hemophilia B, show prolonged ox-brain prothrombin times. These patients have an abnormal Factor IX molecule that is recognized antigenically, but not functionally.

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