Korean J Pediatr.  2010 Mar;53(3):397-407. 10.3345/kjp.2010.53.3.397.

Direct detection of hemophilia B F9 gene mutation using multiplex PCR and conformation sensitive gel electrophoresis

• Yoo KY
• Kim HJ
• Lee KC

• KMID: 2281322
• DOI: http://doi.org/10.3345/kjp.2010.53.3.397

Abstract

PURPOSE
The F9 gene is known to be the causative gene for hemophilia B, but unfortunately the detection rate for restriction fragment length polymorphism-based linkage analysis is only 55.6%. Direct DNA sequencing can detect 98% of mutations, but this alternative procedure is very costly. Here, we conducted multiplex polymerase chain reactions (PCRs) and conformation sensitive gel electrophoresis (CSGE) to perform a screened DNA sequencing for the F9 gene, and we compared the results with direct sequencing in terms of accuracy, cost, simplicity, and time consumption.
METHODS
A total of 27 unrelated hemophilia B patients were enrolled. Direct DNA sequencing was performed for 27 patients by a separate institute, and multiplex PCR-CSGE screened sequencing was done in our laboratory. Results of the direct DNA sequencing were used as a reference, to which the results of the multiplex PCR-CSGE screened sequencing were compared. For the patients whose mutation was not detected by the 2 methods, multiplex ligation-dependent probe amplification (MLPA) was conducted.
RESULTS
With direct sequencing, the mutations could be identified from 26 patients (96.3%), whereas for multiplex PCR-CSGE screened sequencing, the mutations could be detected in 23 (85.2%). One patient's mutation was identified by MLPA. A total of 21 different mutations were found among the 27 patients.
CONCLUSION
Multiplex PCR-CSGE screened DNA sequencing detected 88.9% of mutations and reduced costs by 55.7% compared with direct DNA sequencing. However, it was more labor-intensive and time-consuming.

Keyword

Hemophilia B; DNA mutational analysis; Polymerase chain reaction; Electrophoresis

MeSH Terms

DNA Mutational Analysis
Electrophoresis
Hemophilia A
Hemophilia B
Humans
Multiplex Polymerase Chain Reaction
Polymerase Chain Reaction
Sequence Analysis, DNA