Korean Journal of Pediatrics 2010;53(3):397-407.
Published online March 15, 2010.
Direct detection of hemophilia B F9 gene mutation using multiplex PCR and conformation sensitive gel electrophoresis
Ki Young Yoo1, Hee Jin Kim2, Kwang Chul Lee3
1Korea Hemophilia Foundation, Seoul, Korea
2Department of Laboratory Medicine & Genetics, Sumsung Medical Center, School of Medicine, Sungkyunkwan University, Seoul, Korea
3Department of Pediatrics, College of Medicine, Korea University, Seoul, Korea
Multiplex PCR과 Conformation Sensitive Gel Electrophoresis를 이용한 혈우병B F9 유전자 돌연변이 직접 진단법
유기영1, 김희진2, 이광철3
1한국혈우재단 서울의원
2성균관의과대학 삼성의료원 진단검사유전학과
3고려대학교 의과대학 소아청소년과
Ki Young Yoo, Email: gowho@hanmail.net
Received: 19 December 2009   • Revised: 14 January 2009   • Accepted: 12 February 2010
: 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.
: 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.
: 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.
: 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.
Key Words: Hemophilia B, DNA mutational analysis, Polymerase chain reaction, Electrophoresis

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