scholarly journals The molecular genetic analysis of hemophilia A: a directed search strategy for the detection of point mutations in the human factor VIII gene

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2242-2248
Author(s):  
JK Pattinson ◽  
DS Millar ◽  
JH McVey ◽  
CB Grundy ◽  
K Wieland ◽  
...  
Keyword(s):  
Factor Viii ◽  
Cytosine Methylation ◽  
Search Strategy ◽  
Hemophilia A ◽  
Point Mutations ◽  
Molecular Genetic Analysis ◽  
Directed Search ◽  
Factor Viii Gene ◽  
Human Factor Viii ◽  
Oligonucleotide Hybridization

A directed-search strategy for point mutations in the factor VIII gene causing hemophilia A was used to screen eight potentially hypermutable CpG dinucleotides occurring at sites deemed to be of functional importance. Polymerase chain reaction-amplified DNA samples from 793 unrelated individuals with hemophilia A were screened by discriminant oligonucleotide hybridization. Point mutations were identified in 16 patients that were consistent with a model of 5-methylcytosine (5mC) deamination. Four new examples of recurrent mutation were demonstrated at the following codons: 336 (CGA----TGA), 372 (CGC----TGC), 372 (CGC--- -CAC), and 1689 (CGC----TGC). These are functionally important cleavage sites for either activated protein C or thrombin. Further novel C----T transitions were identified in the remaining arginine codons screened (- 5, 427, 583, 795, and 1696), resulting in the creation of TGA termination codons. Differences in mutation frequency were found both within and between the CpG sites and between ethnic groups. These differences are assumed to be due to differences in the level of cytosine methylation at these sites, although direct evidence for this inference is lacking.

scholarly journals The molecular genetic analysis of hemophilia A: a directed search strategy for the detection of point mutations in the human factor VIII gene

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2242-2248 ◽  
Author(s):  
JK Pattinson ◽  
DS Millar ◽  
JH McVey ◽  
CB Grundy ◽  
K Wieland ◽  
...  
Keyword(s):  
Factor Viii ◽  
Cytosine Methylation ◽  
Search Strategy ◽  
Hemophilia A ◽  
Point Mutations ◽  
Molecular Genetic Analysis ◽  
Directed Search ◽  
Factor Viii Gene ◽  
Human Factor Viii ◽  
Oligonucleotide Hybridization

Abstract A directed-search strategy for point mutations in the factor VIII gene causing hemophilia A was used to screen eight potentially hypermutable CpG dinucleotides occurring at sites deemed to be of functional importance. Polymerase chain reaction-amplified DNA samples from 793 unrelated individuals with hemophilia A were screened by discriminant oligonucleotide hybridization. Point mutations were identified in 16 patients that were consistent with a model of 5-methylcytosine (5mC) deamination. Four new examples of recurrent mutation were demonstrated at the following codons: 336 (CGA----TGA), 372 (CGC----TGC), 372 (CGC--- -CAC), and 1689 (CGC----TGC). These are functionally important cleavage sites for either activated protein C or thrombin. Further novel C----T transitions were identified in the remaining arginine codons screened (- 5, 427, 583, 795, and 1696), resulting in the creation of TGA termination codons. Differences in mutation frequency were found both within and between the CpG sites and between ethnic groups. These differences are assumed to be due to differences in the level of cytosine methylation at these sites, although direct evidence for this inference is lacking.

Identification of a Novel Missense Mutation in Exon 4 of the Human Factor VIII Gene Associated with Sever Hemophilia a Patient

2007 ◽  
Vol 10 (23) ◽  
pp. 4299-4302 ◽  
Author(s):  
Habib Onsori ◽  
Mohammad Ali Hossein . ◽  
Sheideh Montaser-Kou . ◽  
Mohammad Asgharzadeh . ◽  
Abbas Ali Hosseinpou .
Keyword(s):  
Factor Viii ◽  
Missense Mutation ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Viii Gene ◽  
Human Factor Viii ◽  
Exon 4

scholarly journals Eleven novel mutations in the factor VIII gene from Brazilian hemophilia A patients

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 3015-3020
Author(s):  
VR Arruda ◽  
WC Pieneman ◽  
PH Reitsma ◽  
PP Deutz-Terlouw ◽  
JM Annichino-Bizzacchi ◽  
...  
Keyword(s):  
Factor Viii ◽  
Southern Blotting ◽  
Hemophilia A ◽  
Severe Disease ◽  
Point Mutations ◽  
Electrophoretic Pattern ◽  
Telomeric Region ◽  
Factor Viii Gene ◽  
Single Strand Conformational Polymorphism ◽  
Almost All

The molecular characterization of the mutations in hemophilia A patients is hampered by the large size of the factor VIII gene and the great heterogeneity of mutations. In this study, we have performed a protocol involving multiplex polymerase chain reaction in which 19 exons were amplified in four different combinations followed by nonradioactive single-strand conformational polymorphism (SSCP) to screen for mutations. Southern blotting was used to detect inversion of the factor VIII gene resulting from recombination between copies of the gene A (F8A) located in intron 22 of the factor VIII gene and two copies close telomeric region of X chromosome. Forty-two hemophilia A patients (21 with severe and 21 with mild-to-moderate disease) were studied. The inversion of factor VIII occurred in 13 of 21 patients affected by severe hemophilia A. One patient showed a large extra band in addition to the three bands observed after Southern blotting with the F8A probe. An abnormal electrophoretic pattern of SSCP was detected in 85% and 50% of the patients affected by mild-to-moderate and severe disease, respectively. Sixteen different mutations were identified. Eleven mutations were novel and comprised 9 point mutations and 2 small deletions. This study shows that the methodology used is safe and rapid and has potential for detecting almost all of the genetic defects of the studied hemophilia A patients.

Application of PCR to the Detection and Analysis of Point Mutations in the Human Factor VIII Gene

PCR Topics ◽  
1991 ◽  
pp. 23-31
Author(s):  
Lutz-Peter Berg ◽  
David S. Millar ◽  
Catherine B. Grundy ◽  
Kerstin Wieland ◽  
Jonathan K. Pattinson ◽  
...  
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Point Mutations ◽  
Factor Viii Gene ◽  
Human Factor Viii

scholarly journals Eleven novel mutations in the factor VIII gene from Brazilian hemophilia A patients

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 3015-3020 ◽  
Author(s):  
VR Arruda ◽  
WC Pieneman ◽  
PH Reitsma ◽  
PP Deutz-Terlouw ◽  
JM Annichino-Bizzacchi ◽  
...  
Keyword(s):  
Factor Viii ◽  
Southern Blotting ◽  
Hemophilia A ◽  
Severe Disease ◽  
Point Mutations ◽  
Electrophoretic Pattern ◽  
Telomeric Region ◽  
Factor Viii Gene ◽  
Single Strand Conformational Polymorphism ◽  
Almost All

Abstract The molecular characterization of the mutations in hemophilia A patients is hampered by the large size of the factor VIII gene and the great heterogeneity of mutations. In this study, we have performed a protocol involving multiplex polymerase chain reaction in which 19 exons were amplified in four different combinations followed by nonradioactive single-strand conformational polymorphism (SSCP) to screen for mutations. Southern blotting was used to detect inversion of the factor VIII gene resulting from recombination between copies of the gene A (F8A) located in intron 22 of the factor VIII gene and two copies close telomeric region of X chromosome. Forty-two hemophilia A patients (21 with severe and 21 with mild-to-moderate disease) were studied. The inversion of factor VIII occurred in 13 of 21 patients affected by severe hemophilia A. One patient showed a large extra band in addition to the three bands observed after Southern blotting with the F8A probe. An abnormal electrophoretic pattern of SSCP was detected in 85% and 50% of the patients affected by mild-to-moderate and severe disease, respectively. Sixteen different mutations were identified. Eleven mutations were novel and comprised 9 point mutations and 2 small deletions. This study shows that the methodology used is safe and rapid and has potential for detecting almost all of the genetic defects of the studied hemophilia A patients.

scholarly journals Correction of murine hemophilia A following nonmyeloablative transplantation of hematopoietic stem cells engineered to encode an enhanced human factor VIII variant using a safety-augmented retroviral vector

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 526-534 ◽  
Author(s):  
Ali Ramezani ◽  
Robert G. Hawley
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Factor Viii ◽  
Insertional Mutagenesis ◽  
Hemophilia A ◽  
Point Mutations ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Human Factor Viii ◽  
A1 Domain

Abstract Insertional mutagenesis by retroviral vectors is a major impediment to the clinical application of hematopoietic stem cell gene transfer for the treatment of hematologic disorders. We recently developed an insulated self-inactivating gammaretroviral vector, RMSinOFB, which uses a novel enhancer-blocking element that significantly decreases genotoxicity of retroviral integration. In this study, we used the RMSinOFB vector to evaluate the efficacy of a newly bioengineered factor VIII (fVIII) variant (efVIII)—containing a combination of A1 domain point mutations (L303E/F309S) and an extended partial B domain for improved secretion plus A2 domain mutations (R484A/R489A/P492A) for reduced immunogenicity—toward successful treatment of murine hemophilia A. In cell lines, efVIII was secreted at up to 6-fold higher levels than an L303E/F309S A1 domain–only fVIII variant (sfVIIIΔB). Most important, when compared with a conventional gammaretroviral vector expressing sfVIIIΔB, lower doses of RMSin-efVIII-OFB–transduced hematopoietic stem cells were needed to generate comparable curative fVIII levels in hemophilia A BALB/c mice after reduced-intensity total body irradiation or nonmyeloablative chemotherapy conditioning regimens. These data suggest that the safety-augmented RMSin-efVIII-OFB platform represents an encouraging step in the development of a clinically appropriate gene addition therapy for hemophilia A.

scholarly journals 760. Optimized AAV-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice and Cynomolgus Macaques

2016 ◽  
Vol 24 ◽  
pp. S300
Author(s):  
Jenny A. Greig ◽  
Qiang Wang ◽  
Amanda L. Reicherter ◽  
Erin Bote ◽  
Deirdre McMenamin ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Viii Gene ◽  
Human Factor Viii ◽  
Cynomolgus Macaques

Characterization of Adeno-Associated Viral Vector-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice

2017 ◽  
Vol 28 (5) ◽  
pp. 392-402 ◽  
Author(s):  
Jenny A. Greig ◽  
Qiang Wang ◽  
Amanda L. Reicherter ◽  
Shu-Jen Chen ◽  
Alexandra L. Hanlon ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Viral Vector ◽  
Factor Viii Gene ◽  
Human Factor Viii

Double Strand Conformation Polymorphism (DSCP) Detects Two Point Mutations at Codon 280 (AAC→ATC) and at Codon 431 (TAC→AAC) of the Blood Coagulation Factor VIII Gene

1993 ◽  
Vol 69 (05) ◽  
pp. 473-475 ◽  
Author(s):  
W C Pieneman ◽  
P H Reitsma ◽  
E Briët
Keyword(s):  
Factor Viii ◽  
Electrophoretic Mobility ◽  
Hemophilia A ◽  
Point Mutations ◽  
Coagulation Factor ◽  
Severe Hemophilia ◽  
Factor Viii Gene ◽  
Exon 9 ◽  
Blood Coagulation Factor Viii ◽  
Conformation Polymorphism

SummaryHemophilia A is a hereditary, X-linked, bleeding disorder that is caused by a defect in the factor VIII gene. Here, we report two novel point mutations in the factor VIII gene that result in an aberrant electrophoretic mobility of double strand PCR fragments (double strand conformation polymorphism, DSCP). In exon 9 a TAC→AAC mutation at codon 431, replacing Tyr by Asn, was observed in a family (A211) with moderately severe hemophilia A. A family with mild hemophilia A revealed an A→T mutation in codon 280 (exon 7) that results in the replacement of Asn by Ile. One of these two mutations was not detected in an analysis based on single strand conformation polymorphisms (SSCP).At present we have no explanation for the effect of the nucleotide changes on the electrophoretic mobility of double strand DNA. Although DSCP is not able to detect all mutations the combination of DSCP analysis with SSCP analysis increases the sensitivity in a screening for factor VIII mutations.

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