Testování DHPLC analýzy pro detekci mutací u hemofilie A

Evaluation of DHPLC Analysis for Mutation Detection in Haemophilia A

Background.
Haemophilia A is one of the most prevalent inherited bleeding disorders. Causal mutations in the factor VIII gene are detected to facilitate the genetic counselling and to estimate the risk of serious complication associated with standard treatment (factor VIII inhibitor). Wide range of mutations located across the entire length of the factor VIII gene underlies the factor VIII deficiency of variable severity. The only two common recurrent mutations in the factor VIII gene are intron 22 and intron 1 inversions. In the remaining cases it is necessary to screen all 26 exons encoding 9kb mRNA together with adjacent nonncoding sequences. In order to speed up genotyping in haemophilia A families in the Czech Republic we evaluated DHPLC-based screening technique. 

Methods and Results.
We tested sensitivity of the analysis on a panel of DNA samples containing 49 different sequence variations distributed over 21 exons. All the genetic alterations were readily detected. Analysis of family members has shown good reproducibility of the respective elution patterns. DHPLC analysis detected mutations in 4 out of 5 samples from apparently unrelated haemophilia patients, where previously applied multiplex CSGE was not successful. 

Conclusions.
Establishing of DHPLC analysis will substantially speed up the genotyping of haemophilia A families in the Czech Republic.

Key words:
haemophilia A, factor VIII gene, mutation detection, DHPLC.