Detection of three novel mutations in two haemophilia A patients by rapid screening of whole essential region of factor VIII gene

Naylor, J.A.; Green, P.M.; Montandon, A. J.; Giannelli, F.; Rizza, C. R.

doi:10.1016/0140-6736(91)92450-g

Cited by 106 publications

(72 citation statements)

References 29 publications

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“…This approach permits scanning of the complete coding sequence of a complex tissue-specific gene for a comprehensive range of mutations, including coding sequence changes, splice site mutations, and gross gene rearrangements, using a venous blood sample as its starting material. The use of mismatch detection to detect point mutations in amplified products of ectopic transcripts can also be applied to other diseases, as has been shown for example in hemophilia A (34) and Hunter syndrome (R. H. Flomen, P. M. Green, D.R.B., F. Giannelli, and E. P. Green, unpublished data). Although its application to autosomal disorders may be complicated by the additional presence of a normal gene, there is evidence that ectopic dystrophin transcripts in female lymphocytes originate from both copies of the gene (ref.…”

Section: Discussionmentioning

confidence: 99%

Point mutations in the dystrophin gene.

Roberts

Bobrow

Bentley

1992

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

168

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Derming the range of mutations in genes that cause human disease is essential to determine the mechanisms of genetic variation and the function of gene domains and to perform precise carrier and prenatal diagnosis. The mutations in one-third of Duchenne muscular dystrophy patients remain unknown as they do not involve gross rearrangements of the dystrophin gene. The size and complexity of the gene have prohibited the systematic definition of point mutations. We have developed a method for the identification of these mutations by nested amplification, chemical mismatch detection, and sequencing of reverse transcripts of trace amounts of dystrophin mRNA from peripheral blood lymphocytes. Analysis of the entire coding region (11 kilobases) in seven patients has resulted in detection of a sequence change in each case that is clearly sufficient to cause the disease. All mutations should cause premature translational termination, and the resulting phenotypes are thus equivalent to those caused by frameshifting deletions. The results support a particular functional importance for the C-terminal region of dystrophin. Application of this approach to mutation detection will extend direct carrier and prenatal diagnosis to virtually every affected family.

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Section: Discussionmentioning

confidence: 99%

Point mutations in the dystrophin gene.

Roberts

Bobrow

Bentley

1992

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

168

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“…A nested PCR reaction was performed (Naylor et al, 1991) to amplify all essential sequences of factor VIII gene, except exon 14, which was amplified genomically and analyzed as described by Arruda et al (1993, from cDNA obtained by reverse transcription of the ectopic factor VIII transcripts in peripheral lymphocytes. The final PCR products were digested with restriction enzymes in order to get fragc ments of 70 to 300 bp length, which were subsequently screened for mutations using the method of nonradioactive single-strand conformation polymorphism; the enzymes and primer sequences used for this screening are available from the authors on request, SSCP conditions (according to Arruda et al, 1995).…”

Section: Methodsmentioning

confidence: 99%

Identification of four novel mutations in the factor VIII gene: Three missense mutations (E1875G, G2088S, I2185T) and a 2-bp deletion (1780delTC)

et al. 1998

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“…An inhibitor to this epitope may interfere with the interaction between the FVIII subunits or the A3-C1-C2 sub unit and phospholipids. Mutations in hemophilia A patients have been found in both the Cl [46,62] and C2 domains [39,40,46,59,[61][62][63]. Epitopes for allo-, auto-or monoclonal antibodies have been also identified for residues 2189-2346 [39,40,59].…”

Section: Concurrence Of Fviii Inhibitor Specificitiesmentioning

confidence: 99%

Identification of Novel Factor VIII Inhibitor Epitopes using Synthetic Peptide Arrays

Palmer¹,

Dudani²,

Drouin³

et al. 1997

Vox Sanguinis

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Objectives: Mapping the antibody-binding sites on the factor VIII (FVIII) protein opens the prospect of studying the development of FVIII inhibitors and the alteration of inhibitor specificities over time. This paper describes a novel approach to the mapping of FVIII antibody-binding sites. Methods: Immobilized synthetic peptide arrays covering 80% of the complete 2351 amino acid sequence of factor VIII (FVIII) were used to determine epitope specificity of 6 alloantibodies and 3 autoantibodies inhibitory to FVIII activity. This detailed assessment was carried out using a modified enzyme-linked immunosorbent assay with plasma from normal persons or hemophilia A patients without inhibitors as negative controls. Results: Antibody-combining sites could be differentiated in both a qualitative and quantitative manner and were patient-specific. Highly reactive peptides were restricted to specific sites in the A1-A3 and C1-C2 domains and were not proximal to known proteolytic cleavage sites. Free peptides incubated in vitro with the plasmas of 3 patients significantly reduced residual inhibitor titers in a dose-dependent manner. Conclusion: This technique permits the study of the development and specificity of FVIII inhibitors, can detect and differentiate between inhibitory and noninhibitory antibodies using immobilized or free peptides respectively, permits correlation of antibody-combining sites with inhibition of FVIII activity and provides a basis for the development of inhibitor adsorption or neutralization technology.

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Detection of three novel mutations in two haemophilia A patients by rapid screening of whole essential region of factor VIII gene

Cited by 106 publications

References 29 publications

Point mutations in the dystrophin gene.

Point mutations in the dystrophin gene.

Identification of four novel mutations in the factor VIII gene: Three missense mutations (E1875G, G2088S, I2185T) and a 2-bp deletion (1780delTC)

Identification of Novel Factor VIII Inhibitor Epitopes using Synthetic Peptide Arrays

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