Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.

Higuchi, Miyoko; Kazazian, Haig H.; Kasch, Laura; Warren, Tina C.; McGinniss, Matthew J.; Phillips, John A.; Kasper, Carol K.; Janco, Robert L.; Antonarakis, Stylianos E.

doi:10.1073/pnas.88.16.7405

Cited by 142 publications

(70 citation statements)

References 34 publications

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“…The amino acid sequence of factor VIII predicted from DNA sequencing of the factor VIII gene identifies 25 potential N-glycosylation sites, 19 of which are in the connecting (B) domain (16,27 (30). The other instance, fibrinogen Asahi, has impaired polymerization and delayed cross-linking of the mutant y chain, associated with a substitution of threonine for methionine-310 and consequent N-glycosylation at asparagine-308 (31).…”

Section: Discussionmentioning

confidence: 99%

Hemophilia A due to mutations that create new N-glycosylation sites.

Aly

Higuchi

Kasper

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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In studying the molecular defects responsible for cross-reacting material-positive hemophilia A, we have identified two patients in whom the nonfunctional factor VIIIlike protein has abnormal, slower-moving heavy or light chains on SDS/PAGE. Both patients have severe hemophilia A (<1% of normal factor VIII activity) with a normal plasma level of factor VIII antigen. The molecular defects were identified by denaturing gradient gel electrophoresis screening of PCRamplified products of the factor VIII-coding DNA sequence followed by nucleotide sequencing of the abnormal PCR products. In patient ARC-21, a methionine-to-threonine substitution at position 1772 in the factor VIII light chain creates a potential new N-glycosylation site at asparagine-1770. In patient ARC-22, an isoleucine-to-threonine substitution at position 566 creates a potential new N-glycosylation site at asparagine-564 in the A2 domain of the factor VIII heavy chain. The mobility of these chains on SDS/PAGE was normal after N-Glycanase digestion and procoagulant activity was generated-to a maximum of 23% and 45% of control normal plasma.

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Section: Discussionmentioning

confidence: 99%

Hemophilia A due to mutations that create new N-glycosylation sites.

Aly

Higuchi

Kasper

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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“…When an effort was made to characterize all the mutations in a series of patients with hemophilia A, an unexpected finding was obtained. Higuchi et al were able to identify mutations in almost every case of mild-to-moderate hemophilia A (3), but were unable to identify the mutation in about half of the severely affected patients (4). Subsequently, Naylor et al using RT-PCR of illegitimate transcripts of the factor Vm gene could not detect an amplification product between exons 22 and 23 of the gene in about 40% of severely affected patients (5).…”

Section: Introductionmentioning

confidence: 99%

Factor VIII gene inversions causing severe hemophilia A originate almost exclusively in male germ cells

et al. 1994

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The factor VIII gene, which Is defective In hemophilia A, Is located In the last megabase of the long arm of the X chromosome. Inversions due to intrachromosomal homologous recombination between mispaired copies of gene A located within intron 22 of the gene and about 500 kb telomerlc to it account for nearly half of all cases of severe hemophilia A. We hypothesized that pairing of Xq with its homolog inhibits the Inversion process, and that, therefore, the event originates predominantly in male germ cells. In all 20 informative cases In which the inversion originated in a maternal grandparent, DNA polymorphism analysis determined that it occurred in the male germline. In addition, all but one of 50 mothers of sporadic cases due to an inversion were carriers. Thus, these data support the hypothesis and Indicate that factor VIII gene inversions leading to severe hemophilia A occur almost exclusively In male germ cells.

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“…Higuchi et al 1,2 observed that thorough screening of all the exons of the factor VIII (F8) gene was efficient in detecting the mutations of patients with mild and moderate hemophilia A and yet failed in 50% of patients with severe disease. Traces of factor VIII messenger RNA (mRNA) from peripheral lymphocytes soon revealed that this high failure rate was largely due to mutations affecting internal regions of intron 22 of the F8 gene.…”

Section: Introductionmentioning

confidence: 99%

Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A

Bagnall

Waseem

Green

et al. 2002

Blood

367

386

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The messenger RNA (mRNA) from 5 of 69 patients with severe hemophilia A did not support amplification of complementary DNA containing the first few exons of the factor VIII (F8) gene but supported amplification of mRNA containing exon 1 of F8 plus exons of the VBP1 gene. This chimeric mRNA signals an inversion breaking intron 1 of the F8 gene. Using an inversion patient, one deleted for F8 exons 1 to 6, and cosmids mapped 70 to 100 kb telomeric of the F8 gene, this study shows that this break strictly affects a sequence (int1h-1) repeated (int1h-2) about 140 kb more telomerically, between the C6.1A and VBP1 genes. The 1041-base pair repeats differ at a single nucleotide (although int1h-2 also showed one polymorphism) and are in opposite orientation. The results demonstrate that they cause inversions by intrachromosome or intrachromatid homologous recombination. The genomic structure of the inversion region shows that transcription traverses intergenic spaces to produce the 2 chimeric mRNAs containing the F8 sequences and characteristic of the inversion. This observation prompts the suggestion that nature may use such extended transcription to test whether the addition of novel domains from neighboring genes creates desirable new genes. A rapid polymerase chain reaction test was developed for the inversion in both patients and carriers. This has identified 10 inversions, affecting F8 genes with 5 different haplotypes for the BclI, IntroductionHiguchi et al 1,2 observed that thorough screening of all the exons of the factor VIII (F8) gene was efficient in detecting the mutations of patients with mild and moderate hemophilia A and yet failed in 50% of patients with severe disease. Traces of factor VIII messenger RNA (mRNA) from peripheral lymphocytes soon revealed that this high failure rate was largely due to mutations affecting internal regions of intron 22 of the F8 gene. 3 These mutations were later shown to be inversions resulting from homologous intrachromatid or intrachromosome (ie, intranemic) recombination between a 9503-base pair (bp) sequence (int22h-1) in intron 22 of the F8 gene and one or other of 2 inverted copies of this sequence (int22h-2, int22h-3) located, respectively, 500 and 600 kb more telomeric. [4][5][6] The int22h-related inversions appeared to be sufficiently frequent to account for the shortfall in mutation detection experienced using methods based on the screening of all exons of the F8 gene. However, analysis of factor VIII mRNA continued to detect further mutations that escape detection by this approach. Base substitutions deep inside introns were found that generate novel exons disrupting the factor VIII coding sequence 7 (R. D. Bagnall, unpublished observations, January 2001). Moreover, an inversion was identified that broke the F8 gene at intron 1 and resulted in the production of 2 chimeric mRNAs. 8 One of these mRNAs, presumably under the control of the factor VIII promoter, contains the first exon of the F8 gene followed by facultative exons and then exons 2 to 6 of a gene (VBP...

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Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.

Cited by 142 publications

References 34 publications

Hemophilia A due to mutations that create new N-glycosylation sites.

Hemophilia A due to mutations that create new N-glycosylation sites.

Factor VIII gene inversions causing severe hemophilia A originate almost exclusively in male germ cells

Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A

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