School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Clin Chem. 2020 Feb 1;66(2):373-378. doi: 10.1093/clinchem/hvz021.
An inversion of intron 22 in the Factor VIII gene (Inv22) is the causative mutation for 45% of severe hemophilia A cases. Available methods for molecular diagnosis of Inv22 are generally tedious and not ideal for routine clinical use.
We report here a new method using a single closed-tube nested quantitative PCR (CN-qPCR) for rapid detection of Inv22. This method combines a 12-cycle long-distance PCR (LD-PCR) amplifying the int22h regions, followed by a duplex qPCR targeting two specific regions close to the int22h regions. All reagents were added to a single PCR mixture for the closed-tube assay. Sequential LD-PCR and qPCR was achieved by designing primers at substantially different melting temperatures and optimizing PCR conditions.
Seventy-nine male hemophilia A patients of different disease severity were tested by both the CN-qPCR assay and the standard LD-PCR assay. CN-qPCR successfully made calls for all samples, whereas LD-PCR failed in eight samples. For the 71 samples where both methods made calls, the concordance was 100%. Inv22 was detected in 17 out of the 79 samples. Additionally, CN-qPCR achieved clear separation for 10 female carriers and 10 non-Inv22 females, suggesting the assay may also be useful for molecular diagnosis of female carriers.
This new CN-qPCR method may provide a convenient and accurate F8 Inv22 test suitable for clinical use.
凝血因子 VIII 基因（Inv22）内含子 22 的倒位是导致 45%严重血友病 A 病例的致病突变。现有的 Inv22 分子诊断方法通常繁琐，不适合常规临床使用。
我们在此报告一种使用单管巢式定量 PCR（CN-qPCR）快速检测 Inv22 的新方法。该方法结合了 12 个循环的长距离 PCR（LD-PCR）扩增 intr22h 区域，然后进行双管 qPCR 靶向 intr22h 区域附近的两个特定区域。所有试剂均添加到单个 PCR 混合物中进行闭管检测。通过设计在显著不同的熔点温度下的引物和优化 PCR 条件来实现顺序 LD-PCR 和 qPCR。
用 CN-qPCR 检测和标准 LD-PCR 检测了 79 名不同疾病严重程度的男性血友病 A 患者。CN-qPCR 成功地对所有样本进行了检测，而 LD-PCR 在 8 个样本中失败。对于两种方法均进行了检测的 71 个样本，一致性为 100%。在 79 个样本中检测到 Inv22。此外，CN-qPCR 为 10 名女性携带者和 10 名非 Inv22 女性提供了清晰的分离，表明该检测方法也可能有助于女性携带者的分子诊断。
这种新的 CN-qPCR 方法可能提供一种方便、准确的适合临床使用的 F8 Inv22 检测方法。
