The Molecular Genetics of Hemophilia A Stylianos

1987 ◽  
Author(s):  
E Antonarakis
Keyword(s):  
Amino Acids ◽  
Dna Sequences ◽  
Hemophilia A ◽  
Restriction Analysis ◽  
Point Mutations ◽  
Leader Peptide ◽  
Molecular Defect ◽  
Severe Hemophilia ◽  
Cpg Dinucleotides ◽  
Oligonucleotide Hybridization

Hemophilia A is a common X linked hereditary disorder of blood coagulation due to deficiency of factor 8. The gene for factor 8 has been cloned and characterized (Nature 312:326-342, 1984). It is divided into 26 exons and 25 introns and spans 186 kb of DNA. The CGNA is 9 kb and codes for 2351 amino acids. The first 19 amino acids comprise the secretory leader peptide and the mature excreted polypeptide consists of 2332 amino acids. The nucleotide sequence of the exons and the exon-intron junctions is known and the complete amino acid sequence has been deducedSeveral laboratories have used cloned factor 8 DNA sequences as probes to characterized mutations that are responsible for hemophilia A in certain pedigrees. These mutations have been characterized by restriction analysis, oligonucleotide hybridization, cloning and sequencing of DNA from appropriate patientsIn about 500 patients with hemophilia A examined, the molecular defect has been recognized in 39. Both gross alterations (mainly deletions) and point mutations of the factor 8 gene have been found.A total of 19 different deletions have been observed. No two unrelated pedigrees share the same exact deletion.The size of the deleted DNA varies from 1.5 kb to more than 210 kb. All but one of these deletions are associated with severe hemophilia A. A deletion of 6 kb that contains exon 22 only is associated with moderate hemophilia. Some deletions are present in patients with inhibitors to factor 8. No correlation of the size or the position of the deletions can be found with the presence of inhibitors to factor 8.A total of 20 point mutations have been characterized. All are recognized by restriction analysis and involve Taq I sites. All are mutations of CpG dinucleotides and generate nonsense or missence codons. Unrelated pedigrees have the same single nucleotide change because of independent origin of the same mutation. In many instances de novo occurrence of a point mutation has been observed. CpG dinucleotides are hot spots for mutation to TG or CA presumably because of spontaneous deamination of methylcytosine. Some point mutations are present in patients with inhibitors but no correlation of the site of mutation and inhibitor formation has been found. The nonsense mutations are present in patients with severe hemophilia A. A missense mutation (Arg Gin) in exon 26 was found in a patient with mild hemophilia while another Arg Gin mutation in exon 24 has been observed in a patient with severe disease. The creation of a donor splice site in IVS 4 of factor 8 gene has been observed in a patient with mild hemophilia.Few DNA polymorphisms within the factor 8 gene and two other closely linked polymorphisms have been used for carrier detection and prenatal diagnosis of hemophilia A. These DNA markers are useful in more than 90% of families at risk for hemophilia A.The author thanks Drs. Gitschier, Din, Olek, Pirastou, Lawn for communication of their data prior to publication.The hemophilia project at Johns Hopkins was supported by an Institutional grant and NIH grant to S.S.A. and Haig H. Kazazian, Jr.

Point Mutations in Four Hemophilia B Patients from China

1990 ◽  
Vol 64 (02) ◽  
pp. 302-306 ◽  
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.

scholarly journals Insertion of Mini-IS605 and Deletion of Adjacent Sequences in the Nitroreductase (rdxA) Gene Cause Metronidazole Resistance in Helicobacter pyloriNCTC11637

1999 ◽  
Vol 43 (11) ◽  
pp. 2657-2662 ◽  
Author(s):  
Yvette J. Debets-Ossenkopp ◽  
Raymond G. J. Pot ◽  
David J. van Westerloo ◽  
Avery Goodwin ◽  
Christina M. J. E. Vandenbroucke-Grauls ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Restriction Analysis ◽  
Point Mutations ◽  
Full Genome Sequence ◽  
Dna Transformation ◽  
Full Genome ◽  
Ulcer Disease ◽  
Metronidazole Resistance ◽  
H Pylori ◽  
Early Risk Factor

ABSTRACT We found that NCTC11637, the type strain of Helicobacter pylori, the causative agent of peptic ulcer disease and an early risk factor for gastric cancer, is metronidazole resistant. DNA transformation, PCR-based restriction analysis, and DNA sequencing collectively showed that the metronidazole resistance of this strain was due to mutation in rdxA (gene HP0954 in the full genome sequence of H. pylori 26695) and that resistance did not depend on mutation in any of the other genes that had previously been suggested: catalase (katA), ferredoxin (fdx), flavodoxin (fldA), pyruvate:flavodoxin oxidoreductase (porγδαβ), RecA (recA), or superoxide dismutase (sodB). This is in accord with another recent study that attributed metronidazole resistance to point mutations inrdxA. However, the mechanism of rdxAinactivation that we found in NCTC11637 is itself also novel: insertion of mini-IS605, one of the endogenous transposable elements of H. pylori, and deletion of adjacent DNA sequences including 462 bp of the 851-bp-long rdxA gene.

DETECTION OF MUTATIONS IN HEMOPHILIA A

1987 ◽  
Author(s):  
M Higuchi ◽  
L Kochhan ◽  
R Schwaab ◽  
H H Brackmann ◽  
H Egli ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Clinical Picture ◽  
Hemophilia A ◽  
Restriction Analysis ◽  
Point Mutations ◽  
Severe Form ◽  
Detection Of Mutations ◽  
Cdna Fragments

Hemophilia A (HA) is a x-linked bleeding disordercaused by lack or abnormality of factor VIII:C. Because of the heterogeneity of the clinical picture thedisease might result from many different molecular lesions.We examined 202 patients of HA from 160 families by restriction analysis with Taq I, Msp I and EcoR I using three subcloned cDNA fragments of factor VIII:C.We detected 13 mutations within the factor VI11:C gene. All of these patients suffer from the severe form of HA.Table I shows six deletions which we characterized. We also identified seven point mutations on four different exons using restriction enzyme Taq I. The results are shown in Table II.That means in approximately 3.8% of the patients we found deletions and in 4.4% we found point mutations.This confirms the results of Youssofian et al (1986):These authors consider the CpG-dimer as a mutation hotspot in the factor VI11:C gene.

scholarly journals Moderation of hemophilia A phenotype by the factor V R506Q mutation

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1183-1187 ◽  
Author(s):  
WC Nichols ◽  
K Amano ◽  
PM Cacheris ◽  
MS Figueiredo ◽  
K Michaelides ◽  
...  
Keyword(s):  
Protein C ◽  
Hemophilia A ◽  
Clinical Phenotype ◽  
Point Mutations ◽  
Molecular Genetic Analysis ◽  
Von Willebrand Disease ◽  
Clinical Severity ◽  
Molecular Defect ◽  
Factor V ◽  
Missense Mutations

Although many examples of unrelated hemophilia A patients carrying identical point mutations in the factor VIII (FVIII) gene have been reported, the clinical phenotype is not always the same among patients sharing the same molecular defect. Possible explanations for this discrepancy include undetected additional mutations in the FVIII gene or coinheritance of mutations at other genetic loci that modulate FVIII function. We report molecular genetic analysis of potential modifying genes in two sets of unrelated patients carrying common FVIII missense mutations but exhibiting different levels of clinical severity. Both mutations (FVIII R1689C and R2209Q) are associated with severe hemophilia A in some patients and mild/moderate disease in others. The common von Willebrand disease type 2N mutation (R91Q) was excluded as a modifying factor in these groups of patients. However, analysis of the recently described factor V (FV) R506Q mutation (leading to activated protein C resistance) identified a correlation of inheritance of this defect with reduced hemophilia A severity. Two moderately affected hemophilia A patients, each with either of two FVIII gene mutations, were heterozygous for FV R506Q, whereas two severely affected patients and two moderately affected patients were homozygous normal at the FV locus. Our results suggest that coinheritance of the FV R506Q mutation may be an important determinant of clinical phenotype in hemophilia A and that modification of the protein C pathway may offer a new strategy for the treatment of FVIII deficiency.

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.

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.

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.

RECURRENT MUTATIONS AND AN UNUSUAL DELETION IN HEMOPHILIA A

1987 ◽  
Author(s):  
H Youssoufiän ◽  
A Patel ◽  
D Phillips ◽  
H H Kazazian ◽  
S E Antonarakis
Keyword(s):  
Hemophilia A ◽  
De Novo ◽  
Donor Site ◽  
Point Mutations ◽  
Paternal Age ◽  
De Novo Mutations ◽  
Recurrent Point ◽  
Deletion Event ◽  
Recurrent Mutations ◽  
Oligonucleotide Hybridization

We have identified 15 mutations of the factor VIII (F8) gene from a panel of 107 patients with hemophilia A and have characterized these gene defects byrestriction analysis, oligonucleotide hybridization, cloning and DNA sequencing. Recurrent point mutations that involve CG to TG transitions were identified in exon 18, exon 22, and exon 24; a single CG to TG transition was identified in exon 23; and a CG to CA transition was identified in exon 24. In addition, a Taq I site alteration in intron 4 was identified in a patient with mild hemophilia, which arose dg. S23&in a grandpaternal germ cell. Cloning and sequencing of this region suggests the generation of a newsplice donor site. These data suggest that CG to TG transition is a prominent mechanism of mutation in hemophilia A. Six different deletions were also characterized. In one family, the deletion involved exon 26. However, the deletion endpoints in the male proband were different from those in his carrier mother, suggesting either gonadal mosaicism or a second deletion event in maternal meiosis.Of the 15 mutations, 6 occurred de novo within 2 generations: 4 in males and 2 in females. In these djg.novo mutations paternal age at conception was 35 (range = 32-38) and maternal age was 24 and 27. The ability to discover a sizable number of mutations in the F8 gene producing hemophilia A enables us to determine the frequency and nature of de novo mutations in man.

Description of a Large Duplication in F8 Gene Responsible for Severe Hemophilia A.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1149-1149
Author(s):  
Christine Vinciguerra ◽  
Jenny Goudemand ◽  
Christophe Zawadzki ◽  
Dorothee Pellecchia ◽  
Claude Negrier
Keyword(s):  
Factor Viii ◽  
Quantitative Method ◽  
Hemophilia A ◽  
High Titer ◽  
Genetic Disorders ◽  
Point Mutations ◽  
Genetic Mutation ◽  
Severe Hemophilia ◽  
Intron 1 ◽  
Large Rearrangements

Abstract Hemophilia A is a common inherited bleeding disorder, caused by factor VIII (FVIII) deficiency as a result of mutations in the factor VIII gene (F8 gene). Intron 1 and 22 inversions, small insertions/deletions and point mutations are the most common genetic defects responsible for severe hemophilia A. However, a F8 gene mutation is not found in 2–5% of patients with severe hemophilia A (FVIII:C<1IU/dL). Large rearrangements are frequent in other genetic disorders and only one case of exon 13 duplication was described in a patient with mild hemophilia (Casula et al, Blood 1990). We described here a large duplication in F8 gene in a CRM− patient with severe hemophilia A: (FVIII:C < 1 UI/dL, FVIII:Ag < 1%) who developed a high-titer inhibitor (peak 10 BU). A previous investigation did not find intron 1 and 22 inversion or exons /splice sites sequencing abnormalities. The promoter region was also sequenced, but no genetic mutation was then characterized. The duplication was detected by MP/LC (Multiplex PCR/Liquid chromatography) which is a quantitative method able to detect large rearrangements. Initially described by Dehainault et al (Nucl Acids Res 2004) in retinoblastoma patients, this method showed that a duplication affected a large part of F8 gene, e.g. the 3′ part of intron 10, exons 11 to 14 and a part of intron 14. To our knowledge, this is the first duplication responsible for severe hemophila A described so far. This finding suggests that the detection of large rearrangements with quantitative method as MP/LC, QMPSF or MLPA would be useful in hemophilic patients where the screening for inversions or genetic events in the coding regions are unsuccessful. This kind of large gene modifications may indeed account for at least some of the 2–5% cases where the coding sequence does not appeared to be altered. In this case, the truncation of the primary polypeptide sequence did not result in any secretion of the FVIII protein, that may have increased the risk of inhibitor development.

scholarly journals Wie Designer-Rekombinasen Erbkrankheiten heilen könnten

BIOspektrum ◽  
2021 ◽  
Vol 27 (2) ◽  
pp. 139-141
Author(s):  
Felix Lansing ◽  
Jenna Hoersten ◽  
Frank Buchholz
Keyword(s):  
Genome Editing ◽  
Hemophilia A ◽  
Chromosomal Rearrangements ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Severe Hemophilia ◽  
Site Specific ◽  
Large Gene ◽  
Recent Advances

AbstractRecent advances in nuclease-based genome editing allow for the correction of many point-mutations causing diseases. However, correcting genetic alterations caused by larger chromosomal rearrangements remain challenging with this approach. Designer-recombinases promise to fill this gap as demonstrated by the development of a heterodimeric Cre-based site-specific recombinase system. This system can functionally correct a large gene inversion frequently found in patients with severe Hemophilia A.

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