Evaluation of DHPLC in the analysis of hemophilia A

The manifestation of hemophilia A, a common hereditary bleeding disorder in humans, is caused by abnormalities in the factor VIII (FVIII) gene. A wide range of different mutations has been identified and provides the genetic basis for the extensive variability observed in the clinical phenotype. The knowledge of a specific mutation is of great interest as this may facilita te genetic counseling and prediction of the risk of anti-FVIII antibody dev elopment, the most serious complication in hemophilia A treatment to date. Due to its considerable size (7.2 kb of the coding sequence, represented by 26 exons). mutation detection in this gene represents a challenge that is only partially met by conventional screening methods such as denaturing gra dient gel electrophoresis (DGGE) or single stranded conformational polymorp hism (SSCP). These techniques are time consuming, require specific expertis e and are limited to detection rates of 70-85%. In contrast, the recently i ntroduced denaturing high performance liquid chromatography (dHPLC) offers a promising new method for a fast and sensitive analysis of PCR-amplified D NA fragments. To test the applicability of dHPLC in the molecular diagnosis of hemophilia A, we first assessed a cohort of 156 patients with previousl y identified mutations in the FVIII gene. Applying empirically determined e xon-specific melting profiles, a total of 150 mutations (96.2%) were readil y detected. Five mutations (3.2%) could be identified after temperatures we re optimized for the specific nucleotide change. One mutation (0.6%) failed to produce a detectable heteroduplex signal. In a second series, we analyz ed 27 hemophiliacs in whom the mutation was not identified after extensive DGGE and chemical mismatch cleavage (CMC) analysis. In 19 of these patients (70.4%), dHPLC facilitated the detection of the disease-associated nucleot ide alterations. From these findings we conclude that the dHPLC technology is a highly sensitive method well suited to the molecular analysis of hemop hilia A. (C) 2001 Elsevier Science B.V. All rights reserved.