Novel hemophilia B mouse models exhibiting a range of mutations in the Factor IX gene

Abstract Animal models have been critical to the development of novel therapeutics in hemophilia. A deficiency of current murine models of hemophilia B is that they are all due to gene deletions, a type of mutation that is relatively rare in the human hemophilia population. We generated mice with a range of mutations in the Factor IX (F.IX) gene; these more faithfully reflect the types of mutations that cause disease in the human population. Transgenic mice expressing either wild-type human F.IX (hF.IX), or F.IX variants with premature translation termination codons, or missense mutations, under the control of the murine transthyretin promoter, were generated and crossed with mice carrying a large deletion of the murine F.IX gene. Gene copy number, F.IX transcript levels in the liver, intrahepatocyte protein expression, and circulating levels of F.IX protein in the mice were determined and compared with data generated by transient transfection assays using the same F.IX variants. Mice were injected with a viral vector expressing hF.IX and displayed a range of immune responses to the transgene product, depending on the underlying mutation. These new mouse models faithfully mimic the mutations causing human disease, and will prove useful for testing novel therapies for hemophilia. (Blood. 2004;104:2767-2774)

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Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1

Blood ◽

10.1182/blood-2003-05-1446 ◽

2004 ◽

Vol 103 (1) ◽

pp. 85-92 ◽

Cited By ~ 117

Author(s):

Valder R. Arruda ◽

Joerg Schuettrumpf ◽

Roland W. Herzog ◽

Timothy C. Nichols ◽

Nancy Robinson ◽

...

Keyword(s):

Skeletal Muscle ◽

Viral Vector ◽

Gene Copy Number ◽

Factor Ix ◽

Hemophilia B ◽

Gene Copy ◽

Immunodeficient Mice ◽

Treatment Regimens ◽

Human Factor Ix ◽

Inhibitory Antibodies

Abstract Adeno-associated viral (AAV) vectors (serotype 2) efficiently transduce skeletal muscle, and have been used as gene delivery vehicles for hemophilia B and for muscular dystrophies in experimental animals and humans. Recent reports suggest that AAV vectors based on serotypes 1, 5, and 7 transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2-fold to 1000-fold. We sought to determine whether this increased efficacy could be observed in species other than mice. In immunodeficient mice we saw 10- to 20-fold higher levels of human factor IX (hF.IX) expression at a range of doses, and in hemophilic dogs we observed approximately 50-fold higher levels of expression. The increase in transgene expression was due partly to higher gene copy number and a larger number of cells transduced at each injection site. In all immunocompetent animals injected with AAV-1, inhibitory antibodies to F.IX developed, but in immunocompetent mice treated with high doses of vector, inhibitory antibodies eventually disappeared. These studies emphasize that the increased efficacy of AAV-1 vectors carries a risk of inhibitor formation, and that further studies will be required to define doses and treatment regimens that result in tolerance rather than immunity to F.IX.

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A Novel Case of Hemophilia B Due to a Partial Duplication of the Factor 9 Gene

Blood ◽

10.1182/blood.v116.21.4423.4423 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4423-4423

Author(s):

Ruth Wheeler ◽

Jayanthi Alamelu ◽

Jacqueline Cutler

Keyword(s):

De Novo ◽

Conflicts Of Interest ◽

Direct Sequencing ◽

Gene Copy Number ◽

Factor Ix ◽

Hemophilia B ◽

Gene Copy ◽

Coagulation Disorder ◽

Severe Hemophilia ◽

Bleeding Events

Abstract Abstract 4423 Hemophilia B is an X-linked recessive coagulation disorder. The disease is caused by a deficiency of procoagulant Factor IX and is characterised by easy bruising and prolonged bleeding and oozing after injury or surgery. The severity of the disease and the frequency of bleeding events vary, depending on the FIX clotting activity, and in severe cases males suffer from spontaneous joint and muscle bleeds which significantly impact on their health and quality of life. Hemophilia B is less common than Hemophilia A, with a frequency of approximately 1 in 25000 males worldwide. The F9 gene is located on the long arm of the × chromosome at Xq27. The gene comprises 8 exons, spanning approximately 33.5 kb DNA. The molecular basis of Hemophilia B is heterogeneous and to date over a 1000 different mutations have been reported, including missense and nonsense mutations, splicing mutations and large deletions. Here we report the identification of a previously uncharacterised duplication spanning a large part of the F9 gene in a patient with severe Hemophilia B. The patient, a 6 year old male of mixed Iranian and UK origin, was referred to the centre with severe Hemophilia B. He had a FIX level of less than 1iu/dL. No family history of bleeding diathesis could be confirmed due to loss of maternal family members in an earthquake. Mutation analysis of all exons including immediate flanking regions to allow detection of splice site mutations, and the 5’ and 3’ untranslated regions was performed by direct sequencing but no changes from the normal sequence were identified. There have been several reports of Hemophilia B being associated with partial duplications of the F9 gene so we analysed gene copy number using multiplex ligation-dependant probe amplification (MLPA). A large duplication, involving exons 2 to 6 was identified in the affected male. Subsequent analyses of maternal samples were normal, indicating that the mutation arose de novo. Disclosures: No relevant conflicts of interest to declare.

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Targeting the RHOA pathway improves learning and memory in Kctd13 and 16p11.2 deletion mouse models

10.1101/2020.05.22.110098 ◽

2020 ◽

Author(s):

Sandra Martin Lorenzo ◽

Valérie Nalesso ◽

Claire Chevalier ◽

Marie-Christine Birling ◽

Yann Herault

Keyword(s):

Object Recognition ◽

Recognition Memory ◽

Mouse Models ◽

Copy Number Variants ◽

Gene Copy Number ◽

Chronic Administration ◽

Autism Spectrum ◽

Gene Copy ◽

List Type ◽

Object Recognition Memory

ABSTRACTGene copy number variants (CNV) have an important role in the appearance of neurodevelopmental disorders. Particularly, the deletion of the 16p11.2 locus is associated with autism spectrum disorder, intellectual disability, and several other features. Earlier studies highlighted the implication of Kctd13 genetic imbalance in the 16p11.2 deletion through the regulation of the RHOA pathway. Here, we target the pathway and rescue the cognitive phenotypes of the 16p11.2 deletion mouse models. We used a chronic administration of fasudil (HA1077), an inhibitor of the Rho-associated protein kinase (ROCK), in mouse models carrying a heterozygous inactivation of Kctd13, or the deletion of the entire 16p11.2 BP4-BP5 region. We focused our attention on the most robust cognitive phenotypes seen in the 16p11.2 models and we showed that a chronic fasudil treatment can restore object recognition memory in both mouse models but does not change other behavioural traits. These findings confirm KCTD13 as one target gene causing cognitive deficits in 16p11.2 deletion patients, and the pertinence of the RHOA pathway as a therapeutic path and reinforce the contribution of other gene(s) involved in cognitive defects found in the 16p11.2 CNV models.HIGHLIGHTS- Kctd13 haploinsufficiency recapitulates most of the behaviour phenotypes found in the 16p11.2 Del/+ models- Fasudil treatment restores Kctd13 and 16p11.2 Del/+ mutant phenotypes in novel location and novel object recognition memory tests- Fasudil treatment restores the RhoA pathway in Kctd13+/- and 16p11.2 Del/+ models

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Extensive gene deletions in Japanese patients with Diamond-Blackfan anemia

Blood ◽

10.1182/blood-2011-07-368662 ◽

2012 ◽

Vol 119 (10) ◽

pp. 2376-2384 ◽

Cited By ~ 37

Author(s):

Madoka Kuramitsu ◽

Aiko Sato-Otsubo ◽

Tomohiro Morio ◽

Masatoshi Takagi ◽

Tsutomu Toki ◽

...

Keyword(s):

Ribosomal Proteins ◽

Single Nucleotide Polymorphism Array ◽

Gene Copy Number ◽

Japanese Patients ◽

Large Deletion ◽

Gene Copy ◽

Easy Method ◽

Diamond Blackfan Anemia ◽

Large Deletions ◽

Novel Approach

Abstract Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.

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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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Immune deviation by mucosal antigen administration suppresses gene-transfer–induced inhibitor formation to factor IX

Blood ◽

10.1182/blood-2005-11-4668 ◽

2006 ◽

Vol 108 (2) ◽

pp. 480-486 ◽

Cited By ~ 23

Author(s):

Ou Cao ◽

Elina Armstrong ◽

Alexander Schlachterman ◽

Lixin Wang ◽

David K. Okita ◽

...

Keyword(s):

Gene Transfer ◽

Intranasal Administration ◽

Viral Vector ◽

Cell Epitope ◽

Coagulation Factor ◽

Antigen Expression ◽

Gene Replacement ◽

Factor Ix ◽

Hemophilia B ◽

Immune Deviation

Formation of inhibitory antibodies is a serious complication of protein or gene replacement therapy for hemophilias, congenital X-linked bleeding disorders. In hemophilia B (coagulation factor IX [F.IX] deficiency), lack of endogenous F.IX antigen expression and other genetic factors may increase the risk of antibody formation to functional F.IX. Here, we developed a protocol for reducing inhibitor formation in gene therapy by prior mucosal (intranasal) administration of a peptide representing a human F.IX-specific CD4+ T-cell epitope in hemophilia B mice. C3H/HeJ mice with a F.IX gene deletion produced inhibitory IgG to human F.IX after hepatic gene transfer with an adeno-associated viral vector. These animals subsequently lost systemic F.IX expression. In contrast, repeated intranasal administration of the specific peptide resulted in reduced inhibitor formation, sustained circulating F.IX levels, and sustained partial correction of coagulation following hepatic gene transfer. This was achieved through immune deviation to a T-helper–cell response with increased IL-10 and TGF-β production and activation of regulatory CD4+CD25+ T cells.

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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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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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