Li Liyan, Luo Wenlong, Huang Limin, Li Qiang, Jin Wangjie, Liang Qianni, Yang Xu, Li Yihong, Li Kun, Liu Yuming, Yang Xuexi, Chen Juanjuan
Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, 510515, China.
Technology Center of Prenatal Diagnosis and Genetic Diseases Diagnosis, Department of Gynecology and Obstetrics, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
J Hematop. 2025 Aug 18;18(1):40. doi: 10.1007/s12308-025-00655-5.
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.
凝血因子 VIII 基因（F8）的内含子 22 倒位（Inv22）约占重度甲型血友病（HA）病例的 45%。检测 Inv22 已成为重度 HA 的主要筛查方法。目前，临床环境中常用长距离聚合酶链反应（LD-PCR）后的琼脂糖凝胶电泳（AGE）来分离 Inv22 的扩增片段。然而，AGE 存在处理时间长、稳定性差以及在定量 DNA 含量和评估片段大小方面不准确等问题。我们将 LD-PCR 与毛细管凝胶电泳（CGE）相结合用于 HA 中 Inv22 的鉴定。设计了三种引物用于 LD-PCR 以区分 Inv22、携带者和野生型。我们优化了 CGE 的反应体系和条件以有效分离扩增片段。使用 CGE 检测 Inv22 的最佳稀释倍数和缓冲液条件分别为 300 倍和 0.1 倍 TE 缓冲液。理想的电压和时长分别为 5.0 kV 持续 80 分钟。在这些条件下，扩增片段能够有效分离，可使用 ProSize 数据分析软件直接测量目标片段的浓度和大小。已成功建立 LD-PCR 与 CGE 检测法相结合用于检测 HA 群体中 F8 基因的 Inv22。该方法减少了临床实践中检测 Inv22 所需的时间和人力，从而推动了血友病的基因诊断技术发展。
