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

Yoo, Ki Young; Kim, Hee Jin; Lee, Kwang Chul

Original Article

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

유기영^¹^, 김희진^²^, 이광철^³

^¹한국혈우재단 서울의원
^²성균관의과대학 삼성의료원 진단검사유전학과
^³고려대학교 의과대학 소아청소년과

Correspondence:
Ki Young Yoo, Email: gowho@hanmail.net

Received: 19 December 2009 • Revised: 14 January 2009 • Accepted: 12 February 2010

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.

Key Words: Hemophilia B, DNA mutational analysis, Polymerase chain reaction, Electrophoresis