Separation of intron 22 inversion type 1 and 2 of hemophilia A by modified inverse-shifting polymerase chain reaction and capillary gel electrophoresis

• An IS-PCR-CGE was used for genotyping of intron 22 inversion Type 1 and 2 of HA.
• The amplicons (bp) were 405, 457 and 512 for wild type.
• 333, 457 and 584 for Inv22-1/385, 405 and 584 for Inv22-2/Fast resolution of intron 22 inversion of F8 was within 5 min.
• Total of 50 hemophilia A patients and 7 carriers were diagnosed in the application.

An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45–50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512 bp were recognized for wild type; 333, 457 and 584 bp for Inv22-1; 385, 405 and 584 bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584 bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584 bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333 bp for Inv22-1 carrier and 385 bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5 min. The separation voltage was set at 8 kV (cathode at detector), and the temperature was kept at 25 °C. The sieving matrix was 89 mM Tris, 89 mM boric acid, 2 mM EDTA containing 0.4% (w/v) HPMC and 1 μM of YO-PRO®-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR.

Schematic of IS-PCR for genotyping intron 22-related rearrangements. (A) non-Inv22 (WT); (B) intron 22 inversion type 1 (Inv22-1); (C) intron 22 inversion type 2 (Inv22-2). Figure optionsDownload as PowerPoint slide