Characterization of missense mutations in the signal peptide and propeptide of FIX in hemophilia B by a cell-based assay

Abstract Many mutations in the signal peptide and propeptide of factor IX (FIX) cause hemophilia B. A FIX variants database reports 28 unique missense mutations in these regions that lead to FIX deficiency, but the underlying mechanism is known only for the mutations on R43 that interfere with propeptide cleavage. It remains unclear how other mutations result in FIX deficiency and why patients carrying the same mutation have different bleeding tendencies. Here, we modify a cell-based reporter assay to characterize the missense mutations in the signal peptide and propeptide of FIX. The results show that the level of secreted conformation-specific reporter (SCSR), which has a functional γ-carboxyglutamate (Gla) domain of FIX, decreases significantly in most mutations. The decreased SCSR level is consistent with FIX deficiency in hemophilia B patients. Moreover, we find that the decrease in the SCSR level is caused by several distinct mechanisms, including interfering with cotranslational translocation into the endoplasmic reticulum, protein secretion, γ-carboxylation of the Gla domain, and cleavage of the signal peptide or propeptide. Importantly, our results also show that the SCSR levels of most signal peptide and propeptide mutations increase with vitamin K concentration, suggesting that the heterogeneity of bleeding tendencies may be related to vitamin K levels in the body. Thus, oral administration of vitamin K may alleviate the severity of bleeding tendencies in patients with missense mutations in the FIX signal peptide and propeptide regions.

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The Impacts of G4S Mutation on N-glycosylation and conformation of the Human Coagulation Factor IX GLA domain: In silico and In vitro Analysis

10.21203/rs.3.rs-1071688/v1 ◽

2021 ◽

Author(s):

Fahimeh Ghasemi ◽

Mina Maddah ◽

Hourieh Kalhor ◽

Mohsen Khorashadizadeh ◽

Alireza Zomorodipour

Keyword(s):

Amino Acid ◽

Coagulation Factor ◽

Glycosylation Site ◽

Factor Ix ◽

Hemophilia B ◽

Phospholipid Bilayer ◽

Single Amino Acid ◽

Missense Mutations ◽

Coagulation Factor Ix ◽

Gla Domain

Abstract Missense mutations are the most prevalent form of mutation in hemophilia B patients. These alterations may result in the creation of novel and non-native N-glycosylation sites (Asn-X-Ser/Thr) through single amino acid substitutions. The pathogenic mechanisms of N-glycosylation mutations in hemophilia B patients have not been extensively studied yet. By survey among known missense mutations, we found only one N-glycosylation mutation in the γ-carboxyglutamic-rich (GLA) domain of the human coagulation factor IX (hFIX). This mutation that was reported in patients with mild and moderate hemophilia B, is caused by G4S amino acid substitution. To investigate the possibility of glycan attachment to the novel N-glycosylation site in G4S-mutant hFIX and the occurrence of hyperglycosylation, site-directed mutagenesis was applied to introduce the selected mutation into the coding sequence of the hFIX. The nucleotide sequences of the both native and G4S-mutant hFIX were separately cloned into the pcDNA3.1 expression plasmid and transiently expressed in HEK293T cells. Our results from gradient SDS-PAGE and western blotting analysis of the both recombinant native and mutant hFIX demonstrated no glycan attachment to the new N-glycosylation site in the G4S-mutant hFIX. Molecular dynamics (MD) simulation was also conducted to provide atomistic insights into structure and behavior of the native and G4S-mutant GLA domains in the both free and membrane-bound states. The results revealed that the mutation slightly affected the dynamic behavior of the mutant GLA domain. The conformational analysis proved that the native GLA domain had less fluctuation and more stability than the mutant GLA domain. The slight conformational changes may influence the binding capacity and interaction of the mutant GLA domain to phospholipid bilayer which is necessary for coagulation activity of the hFIX. These findings were in accordance with the nature of the G4S mutation which causes mild hemophilia B.

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Mutations which Introduce Free Cysteine Residues in the Gla-Domain of Vitamin K Dependent Proteins Result in the Formation of Complexes with α1-Microglobulin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650223 ◽

1996 ◽

Vol 75 (01) ◽

pp. 070-075 ◽

Cited By ~ 20

Author(s):

E G C Wojcik ◽

P Simioni ◽

M v d Berg ◽

A Girolami ◽

R M Bertina

Keyword(s):

Vitamin K ◽

Protein C ◽

Cysteine Residue ◽

Factor Ix ◽

Disulphide Bond ◽

Clotting Factors ◽

High Molecular Weight Complex ◽

Low Concentrations ◽

Gla Domain ◽

Formation Of Complexes

SummaryWe have previously described a genetic factor IX variant (Cys18→Arg) for which we demonstrated that it had formed a heterodimer with armicroglobulin through formation of a disulphide bond with the remaining free cysteine residue of the disrupted disulphide bond in the Gla-domain of factor IX. Recently, we observed a similar high molecular weight complex for a genetic protein C variant (Arg-1→Cys). Both the factor IX and the protein C variants have a defect in the calcium induced conformation. In this study we show that the aminoterminus of this protein C variant is prolonged with one amino acid, cysteine. This protein C variant, as well as protein C variants with Arg9→Cys and Ser12→Cys mutations which also carry a free cysteine residue, are shown to be present in plasma as a complex with α1-microglobulin. A prothrombin variant with a Tyr44→Cys mutation, had not formed such a complex. Furthermore, complexes between normal vitamin K-dependent clotting factors and α1-microglobulin were shown to be present in plasma at low concentrations. The data suggest that the presence of an unpaired cysteine residue in the propeptide or the N-terminal half of the Gla-domain has strongly promoted the formation of a complex with α1-microglobulin in the variants.

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Extravascular Administration of Factor IX: Potential for Replacement Therapy of Canine and Human Hemophilia B

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656082 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0944-0948 ◽

Cited By ~ 20

Author(s):

Darla Liles ◽

Charles N Landen ◽

Dougald M Monroe ◽

Celeste M Lindley ◽

Marjorie s Read ◽

...

Keyword(s):

Gene Therapy ◽

Vitamin K ◽

Absorption Rate ◽

Venous Access ◽

Factor Ix ◽

Hemophilia B ◽

Current Therapy ◽

Home Therapy ◽

Routes Of Administration ◽

Plasma Compartment

SummaryCurrent therapy for hemophilia B requires large intravenous doses of factor IX (F.IX) given in the clinic or at home. Although home therapy is possible for many patients, it is often complicated by factors such as the lack of good venous access. Very little is known about extravascular routes for administering proteins like F.IX (57 kD) or other vitamin K-dependent procoagulant factors into the circulation. Questions about the absorption rate from extravascular administration as well as plasma recovery and bioavailability have arisen recently with the growing availibility of highly purified procoagulant proteins and increased interest in gene therapy of hemophilia B. Therefore, a group of studies were undertaken to determine the absorption rate, plasma recovery, and bioavailability of high purity, human plasma-derived F.IX concentrates administered via extravascular routes in hemophilia B dogs and in one human hemophilia B subject. Five hemophilia B dogs were given human F.IX via either a subcutaneous (SC), intramuscular (IM), intra- peritoneal (IP) or intravenous (IV) route. In a subsequent study, a single SC administration of human F.IX was compared to an identical IV dose of F.IX in the human hemophilia B subject. All extravascular routes of F.IX administration in both the canine and human gave lower levels of circulating plasma F.IX than the IV route, however all routes resulted in measurable F.IX activity. Of the extravascular routes, the IM injection in the canine resulted in a bioavailibility of 82.8%, while the SC injection resulted in a bioavailability of 63.5%. F.IX reached the plasma compartment by all extravascular routes used, confirming that F.IX can be absorbed extravascularly. The duration of measurable F.IX activity following extravascular administration is prolonged beyond that typically seen with IV administration. These data show that significant levels of F.IX may be obtained via SC injection in canine and ‘ human hemophilia B subjects and further highlight the potential of extravascular routes of administration for future experimental and clinical uses of F.IX and other procoagulant proteins.

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Factor VII Activity and Antigen in Hemophilia B Variants

10.1055/s-0039-1684592 ◽

1979 ◽

Author(s):

M.G. Mazzucconi ◽

M. Bertina ◽

D. Orlando ◽

G. Romoli ◽

G. Avvisati ◽

...

Keyword(s):

Vitamin K ◽

Factor Ix ◽

Hemophilia B ◽

Factor Vii ◽

Normal Range ◽

Vitamin K Deficiency ◽

K Deficiency

In 23 patients with Hemophilia B (variants: II B-, 5 BR and 7 B+) factor VII Activity (VII:C) and Antigen (VII:Ag), in correlation with Thrombotest were measured. Thrombotest was found prolonged in 14 patients (in 8 > x+3SD and in 6 >x+2SD). Factor VII:C was found reduced in 1/11 B-, in 2/5 BR and in 4/7 B+ variants. Factor VII:Ag was normal in all but one patient (a 5 years old boy). The ratio VII:C/VII:Ag was abnormal in 8 patients. In these patients factors II and X activities were always within the normal range. The discrepances between VII:C and VII:Ag may be due to: 1) very minor vitamin K deficiency (presence of small amounts of PIVKA-II), 2) an inhibition of factor VII activation by a factor IX “abnormal” molecule, and 3) the synthesis of an abnormal molecule of factor VII.

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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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Six Novel and Three Recurrent Mutations in Nine Austrian Patients with Hemophilia B

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648814 ◽

1994 ◽

Vol 72 (01) ◽

pp. 074-077 ◽

Cited By ~ 2

Author(s):

J Walter ◽

I Pabinger-Fasching ◽

H H Watzke

Keyword(s):

Direct Sequencing ◽

Point Mutations ◽

Factor Ix ◽

Hemophilia B ◽

Causative Mutation ◽

Genetic Defects ◽

Missense Mutations ◽

Enzymatic Amplification ◽

Recurrent Mutations ◽

Polymerase Chain

SummaryIn this report we describe the molecular basis of the factor IX (FIX) deficiency in nine patients with severe (n = 6), moderate (n = 1) or mild (n = 2) hemophilia B. The following genetic defects were identified by enzymatic amplification with the polymerase chain reaction (PCR) and subsequent direct sequencing of all exons and exon-intron-junctions: patient B.B. (FIX “Vienna I”): deletion of nucleotides 6343 to 6362; patient M.H. and W. J. (FIX “Vienna II”): nucleotide 17704 (C to G), Gin 97 to Glu; patient L. K. (FIX “Vienna III”): nucleotide 17761 (C to T), Arg 116 to stop; patient U. A. (FIX “Vienna IV”): nucleotide 10415 (C to G), Pro 55 to Ala; patient H.G. (FIX “Vienna V”): nucleotide 6488 (C to T), Thr 38 to lie; patient H. M. (FIX “Vienna VI”): nucleotide 31276 (G to C), Trp 385 to Cys; patient L. C. (FIX “Vienna VII”): deletion of nucleotide 6700; patient S.F. (FIX “Vienna VIII”): nucleotide 10392 (A to T), Asp 47 to Val. The causative mutation was detected in the FIX gene in each of the nine patients with hemophilia B. There was one small deletion, one point deletion and seven point mutations. The latter include six missense mutations and one nonsense mutation. The mutations in Vienna III, IV and V have already been described in previous studies. The two deletions, Vienna I and Vienna VII have not been reported previously. The genetic defects observed in Vienna II, VI and VIII are novel missense mutations which result in amino acid changes at residues 97,47 and 385, respectively.

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Detection of Ten New Mutations by Screening the Gene Encoding Factor IX of Danish Hemophilia B Patients

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653867 ◽

1995 ◽

Vol 73 (05) ◽

pp. 774-778 ◽

Cited By ~ 7

Author(s):

Marianne Schwartz ◽

Jørgen Ingerslev ◽

Elma Scheibel ◽

Lise Rud Nielsen

Keyword(s):

Point Mutations ◽

Splice Site Mutation ◽

Factor Ix ◽

Hemophilia B ◽

Missense Mutations ◽

Coding Region ◽

Site Mutation ◽

Gene Encoding ◽

Base Pair Deletion ◽

Wide Range

SummaryHemophilia B is caused by a wide range of mutations. In order to characterize the mutations among patients in Denmark, we have systematically screened the entire coding region, the promoter region and exon flanking sequences of the gene encoding factor IX using single strand conformation and heteroduplex analyses. Patients from 32 different families were examined, and point mutations (23 different) were found in all of them. Ten of the mutations have not been reported by others; they include a splice site mutation, a single base pair deletion, and missense mutations. Notably, the study contains a female patient and a previously described Leyden mutation. In ten families with sporadic cases of hemophilia B, all 10 mothers were found to be carriers. The origin of two of these mutations was established.

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Purified factor IX using monoclonal immunoaffinity technique: clinical trials in hemophilia B and comparison to prothrombin complex concentrates

Blood ◽

10.1182/blood.v79.3.568.568 ◽

1992 ◽

Vol 79 (3) ◽

pp. 568-575 ◽

Cited By ~ 55

Author(s):

HC Kim ◽

CW McMillan ◽

GC White ◽

GE Bergman ◽

MW Horton ◽

...

Keyword(s):

Monoclonal Antibody ◽

Vitamin K ◽

Half Life ◽

Viral Infections ◽

Factor Ix ◽

Hemophilia B ◽

Terminal Phase ◽

Factor Ix Deficiency ◽

In Vivo Recovery

Abstract Replacement therapy for hemophilia B (factor IX deficiency) using prothrombin complex concentrate (PCC) has been associated with serious complications of thromboembolic events and transmission of viral infections. Monoclonal antibody-purified factor IX (Mononine) provides a highly purified factor IX concentrate, while eliminating other vitamin K-dependent factors (II, VII, and X). Mononine was evaluated for in vivo recovery, half-life, and for its safety and efficacy in 10 patients with hemophilia B. The in vivo recovery of factor IX with Mononine was a 0.67 +/- 0.14 U/dL (mean +/- SD) increase per 1U/kg of infused factor IX, and the biologic half-life (t1/2), determined using the terminal phase of elimination, was 22.6 +/- 8.1 hours. Comparison of in vivo recovery of other vitamin K-dependent factors following a single infusion of either Mononine or PCC showed that, whereas Mononine infusion caused no changes in other vitamin K-dependent factors or in prothrombin activation fragment (F1+2), PCC infusion was associated with significant increases of factors II (2.7 U/dL per 1 U/dL of IX increase) and X (2.2 U/dL for 1 U/dL for 1 U/dL of IX). Patients who used Mononine as their sole therapeutic material during the 12-month period showed an excellent response in hemostasis for their bleeding episodes. Their experience with long-term use of Mononine was at least equivalent to their previous experience with PCC in the frequency and amount of factor usage. No patients developed antibody against mouse IgG or an increase in IX inhibitor during the 12-month period. These results indicate that monoclonal antibody-purified factor IX concentrate provides hemostatically effective factor IX replacement while avoiding extraneous thrombogenic substances.

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Molecular analysis of hemophilia B in Poland: 12 novel mutations of the factor IX gene.

Acta Biochimica Polonica ◽

10.18388/abp.1999_4144 ◽

1999 ◽

Vol 46 (3) ◽

pp. 721-726 ◽

Cited By ~ 9

Author(s):

K Wulff ◽

K Bykowska ◽

S Lopaciuk ◽

F H Herrmann

Keyword(s):

Direct Sequencing ◽

Point Mutations ◽

Factor Ix ◽

Hemophilia B ◽

Heteroduplex Analysis ◽

Missense Mutations ◽

Nonsense Mutations ◽

Pcr Products ◽

Factor Ix Deficiency ◽

High Degree

We examined the molecular basis of factor IX deficiency in 53 unrelated Polish patients with hemophilia B. Heteroduplex analysis and direct sequencing of polymerase chain reaction (PCR) products were applied to identify the gene defect. Forty-three different point mutations were detected in the factor IX gene of 47 patients. There were 29 missense mutations, 9 nonsense mutations, 4 splice site mutations and 1 point mutation in the promoter region. Twelve mutations were novel. The results of this study emphasize a very high degree of heterogeneity of hemophilia B.

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