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

Francisco Vidal; Elisenda Farssac; Carme Altisent; Lluís Puig; Dominique Gallardo

doi:10.1055/s-0037-1615637

Thrombosis and Haemostasis, Inhaltsverzeichnis

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

Autoren

Francisco Vidal

¹Unitat de Recerca del Centre de Transfusió i Banc de Teixits, Barcelona
Elisenda Farssac

¹Unitat de Recerca del Centre de Transfusió i Banc de Teixits, Barcelona
Carme Altisent

²Unitat d’Hemofília de l’Hospital General Vall d’Hebron, Barcelona, Spain
Lluís Puig

²Unitat d’Hemofília de l’Hospital General Vall d’Hebron, Barcelona, Spain
Dominique Gallardo

¹Unitat de Recerca del Centre de Transfusió i Banc de Teixits, Barcelona

Abstract

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

Volltext

Referenzen

References
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