Mutation spectrum of 122 hemophilia A families from Taiwanese population by LD-PCR, DHPLC, multiplex PCR and evaluating the clinical application of HRM.
BACKGROUND
Hemophilia A represents the most common and severe inherited hemorrhagic disorder. It is caused by mutations in the F8 gene, which leads to a deficiency or dysfunctional factor VIII protein, an essential cofactor in the factor X activation complex.


METHODS
We used long-distance polymerase chain reaction and denaturing high performance liquid chromatography for mutation scanning of the F8 gene. We designed the competitive multiplex PCR to identify the carrier with exonal deletions. In order to facilitate throughput and minimize the cost of mutation scanning, we also evaluated a new mutation scanning technique, high resolution melting analysis (HRM), as an alternative screening method.


RESULTS
We presented the results of detailed screening of 122 Taiwanese families with hemophilia A and reported twenty-nine novel mutations. There was one family identified with whole exons deletion, and the carriers were successfully recognized by multiplex PCR. By HRM, the different melting curve patterns were easily identified in 25 out of 28 cases (89%) and 15 out of 15 (100%) carriers. The sensitivity was 93 % (40/43). The overall mutation detection rate of hemophilia A was 100% in this study.


CONCLUSIONS
We proposed a diagnostic strategy for hemophilia A genetic diagnosis. We consider HRM as a powerful screening tool that would provide us with a more cost-effective protocol for hemophilia A mutation identification.
Lin
Shin-Yu
SY
Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan. p95421002@ntu.edu.tw
Su
Yi-Ning
YN
Hung
Chia-Cheng
CC
Tsay
Woei
W
Chiou
Shyh-Shin
SS
Chang
Chieh-Ting
CT
Ho
Hong-Nerng
HN
Lee
Chien-Nan
CN
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007446:Chromosome Inversion; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D019075:Electrophoresis, Capillary; D005260:Female; D006467:Hemophilia A / Q000235:genetics*; D006801:Humans; D007438:Introns; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D013624:Taiwan
D017931:DNA Primers