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Thromb Haemost 2001; 85(04): 580-583
DOI: 10.1055/s-0037-1615637
Rapid Communication
Schattauer GmbH

Rapid Hemophilia A Molecular Diagnosis by a Simple DNA Sequencing Procedure: Identification of 14 Novel Mutations

Francisco Vidal
1   Unitat de Recerca del Centre de Transfusió i Banc de Teixits, Barcelona
,
Elisenda Farssac
1   Unitat de Recerca del Centre de Transfusió i Banc de Teixits, Barcelona
,
Carme Altisent
2   Unitat d’Hemofília de l’Hospital General Vall d’Hebron, Barcelona, Spain
,
Lluís Puig
2   Unitat d’Hemofília de l’Hospital General Vall d’Hebron, Barcelona, Spain
,
Dominique Gallardo
1   Unitat de Recerca del Centre de Transfusió i Banc de Teixits, Barcelona
› Author Affiliations
Further Information

Publication History

Received 23 August 2000

Accepted after revision 27 November 2000

Publication Date:
08 December 2017 (online)

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Summary

We here describe a simple, efficient DNA sequencing procedure for hemophilia A molecular diagnosis. In severe patients we first test for the presence of factor VIII gene intron 22 inversion using a recently described single-tube PCR method. In moderate, mild, or inversion-negative severe patients we systematically sequence the promoter, all exons and splice junctions of factor VIII gene. Specially designed primers allow amplification of 23 PCR products under the same salt conditions and thermocycling parameters. The whole sequencing procedure, from blood extraction to mutation identification, can be readily done within 42 h when using regular instruments or in just 14 h when using a high-throughput sequencer. Thus, this is a versatile and cost-effective strategy with little hands-on time requirements. Since its implementation we have identified mutations in 45/46 hemophilia A patients, 14 of which are novel. Once the genetic defect has been identified, accurate genetic counseling is then easily performed.

Keywords

Factor VIII - hemophilia A - mutations - automated DNA sequencing - F8c
 

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