Direct detection of hemophilia B F9 gene mutation using multiplex PCR and conformation sensitive gel electrophoresis.
10.3345/kjp.2010.53.3.397
- Author:
Ki Young YOO
1
;
Hee Jin KIM
;
Kwang Chul LEE
Author Information
1. Korea Hemophilia Foundation, Seoul, Korea. gowho@hanmail.net
- Publication Type:Original Article
- Keywords:
Hemophilia B;
DNA mutational analysis;
Polymerase chain reaction;
Electrophoresis
- MeSH:
DNA Mutational Analysis;
Electrophoresis;
Hemophilia A;
Hemophilia B;
Humans;
Multiplex Polymerase Chain Reaction;
Polymerase Chain Reaction;
Sequence Analysis, DNA
- From:Korean Journal of Pediatrics
2010;53(3):397-407
- CountryRepublic of Korea
- Language:Korean
-
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.
