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Korean J Clin Pathol. 2001 Jun;21(3):231-234. Korean. Original Article.
Han JY , Kim KH , Lee YH , Lee JN , Lee SY , Choi OH , Kim IJ , Kim CM .
Department of Clinical Pathology, Dong-A University College of Medicine, Pusan, Korea.
Department of Pediatrics, Dong-A University College of Medicine, Pusan, Korea.
Department of Clinical Pathology, Inje University College of Medicine, Pusan Paik Hospital, Pusan, Korea.
Department of Pediatrics, Inje University College of Medicine, Pusan Paik Hospital, Pusan, Korea.
Department of Obstetrics and Gynecology, College of Medicine, Pusan National University, Pusan, Korea.
Department of Biochemistry, College of Medicine, Pusan National University, Pusan, Korea.
Abstract

BACKGROUND: Hemophilia A is the most common X-linked bleeding disorder with an incidence of 1/5,000 males. Inversions within the factor VIII gene cause almost half of all cases of severe hemophilia A. However, DNA-based diagnosis has previously been carried out only by linkage analysis in Korean hemophilia A families. In this study, we aimed to establish direct inversion detection using a single-tube polymerase chain reaction (PCR) assay. METHODS: We have modified a single-tube PCR assay that combines overlapping PCR with long-distance PCR; performing PCR directly from genomic DNA with four primers P, Q, A, and B that differentiate the wild type, inversion, and the carrier detected the inversion. RESULTS: Segments PQ (12 kb) and AB (10 kb) were produced in hemizygous wild-type males. Males with hemophilia A due to the inversion showed segments PB+AQ (11 kb) along with the 10 kb segment from the nonrecombined extragenic homologue. In 20 (18.7%) patients, an inversion was found. The three segments were readily identifiable and all PCR amplifications achieved uniform reproducible results. CONCLUSIONS: The PCR was successful for the direct detection of factor VIII gene inversions. The method is simple, inexpensive, and more standardized; therefore, it may be the natural starting point for ascertaining mutations in families with severe hemophilia A.

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