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. 2025 Aug 18;18(1):40.
doi: 10.1007/s12308-025-00655-5.

Application of capillary gel electrophoresis in detection of Factor VIII gene intron 22 inversion of hemophilia A

Liyan Li  1   2 , Wenlong Luo  1 , Limin Huang  3 , Qiang Li  4 , Wangjie Jin  2 , Qianni Liang  5 , Xu Yang  1 , Yihong Li  2 , Kun Li  1 , Yuming Liu  1 , Xuexi Yang  6 , Juanjuan Chen  7
Affiliations

Application of capillary gel electrophoresis in detection of Factor VIII gene intron 22 inversion of hemophilia A

Liyan Li et al. J Hematop. .

Abstract

Intron 22 inversion (Inv22) of the factor VIII gene (F8) accounts for approximately 45% of severe hemophilia A (HA) cases. Detecting Inv22 has become the primary screening method for severe HA. Currently, agarose gel electrophoresis (AGE) following long-distance polymerase chain reaction (LD-PCR) is commonly used in clinical settings to separate the amplified fragments of Inv22. However, AGE is hindered by lengthy processing times, instability, and inaccuracies in quantifying DNA content and assessing fragment sizes. We combined LD-PCR with capillary gel electrophoresis (CGE) for the identification of Inv22 in HA. Three primers were designed for LD-PCR to differentiate between Inv22, carriers, and wild types. We optimized the reaction system and conditions for CGE to effectively separate the amplified fragments. The optimal dilution ratio and buffer conditions for detecting Inv22 using CGE were 300 × and 0.1 × TE buffer. The ideal voltage and duration were 5.0 kV for 80 min. Under these conditions, the amplified fragments could be effectively separated, allowing for the direct measurement of concentration and size of the target fragments using ProSize data analysis software. The LD-PCR combined with the CGE assay for detecting Inv22 in F8 within HA populations has been successfully established. This method reduces both the time and labor required for detecting Inv22 in clinical practice, thereby advancing genetic diagnostic technology for hemophilia.

Keywords: Capillary gel electrophoresis; Factor VIII; Hemophilia A; Intron 22 inversion; LD-PCR.

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Conflict of interest statement

Declarations. Funding: This study was funded by the Guangdong Basic and Applied Basic Research Foundation (Grant No.2022A1515110801), the Guangdong Medical Science and Technology Research Foundation (Grant No. A2024354), the Science and Technology Program of Guangzhou (Grant No.2025A04J4085), and the Guangdong-Hong Kong-Macao Special Project of the President's Fund of Nanfang Hospital, Southern Medical University (Grant No.2021Y001). Conflict of Interest: The authors declare no competing interests. Ethical approval: This study was approved by the Medical Ethics Committee of Nanfang Hospital (Approval no. NFEC-2016-035) and was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent: Patients or their legal representatives were informed about the study and provided consent to participate. Consent for publication: For this type of study consent for publication is not required.

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