title: Genotyping of intron 22 inversion of factor VIII gene for diagnosis of hemophilia A by inverse-shifting polymerase chain reaction and capillary electrophoresis.
abstract: This is the first capillary electrophoresis (CE) analysis for diagnosis of hemophilia A (HA). The intron 22 inversion of factor VIII gene (F8) causes 40-50 % of severe bleeding disorder of HA in all human populations. Consequently, identification of the disease-causing mutations is becoming increasingly important for accurate genetic counseling and prenatal diagnosis. In this study, the key steps of inverse-shifting polymerase chain reaction (IS-PCR) and of short-end injection capillary electrophoresis were used for more specific and rapid genotyping of intron 22 inversion of F8. In IS-PCR, three specific primers were used to amplify 512-bp amplicon for wild type and 584-bp amplicon for patients with intron 22 inversion. The capillary gel electrophoresis (CGE) system was performed using 1× Tris-borate-EDTA (TBE) buffer containing 0.3 % (w/v) polyethylene oxide (PEO). The PCR amplicons were electrokinetically injected at 10 kV for 10 s at a temperature of 25 °C. The optimal short-end injection CGE was applied to detect the F8 gene of HA patients and carriers within 5 min. Intron 22 inversion was indeed found on some HA patients (13/35, 37.1 %). All genotyping results showed good agreement with DNA sequencing method and long-distance polymerase chain reaction (LD-PCR). The IS-PCR combined with short-end injection CGE method was feasible and efficient for intron 22 inversion screening of F8 in the HA populations.
authors.affiliation: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan.
authors.forename: Tzu-Yu
authors.initials: TY
authors.lastname: Pan
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Wang
authors.forename: Chi-Jen
authors.initials: CJ
authors.lastname: Shih
authors.forename: Hui-Fen
authors.initials: HF
authors.lastname: Wu
authors.forename: Shyh-Shin
authors.initials: SS
authors.lastname: Chiou
authors.forename: Shou-Mei
authors.initials: SM
authors.lastname: Wu
mesh_terms: D002021:Buffers; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis; D004492:Edetic Acid / Q000737:chemistry; D019075:Electrophoresis, Capillary; D005169:Factor VIII / Q000235:genetics*; D005260:Female; D005838:Genotype; D006467:Hemophilia A / Q000175:diagnosis / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D007438:Introns; D008297:Male; D009154:Mutation; D011092:Polyethylene Glycols / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011108:Polymers / Q000737:chemistry; D013048:Specimen Handling
chemical_list: D002021:Buffers; D011108:Polymers; D011092:Polyethylene Glycols; D005169:Factor VIII; D004247:DNA; D004492:Edetic Acid