Kumar Praveen, Husain Nuzhat, Soni Priyanka, Faridi Nuzhat Jahan, Goel Sudhir Kumar
Department of Pathology, Genetics Lab, Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh, India.
Department of Pathology, Genetics Lab, Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Clin Appl Thromb Hemost. 2015 Apr;21(3):255-9. doi: 10.1177/1076029613498817. Epub 2013 Sep 19.
Hemophilia A is a X-linked recessive bleeding disorder characterized by qualitative and quantitative deficiency of factor VIII resulting from heterogeneous mutations in the factor VIII gene located in the Xq28 region. Intron 22 inversion (Inv22) mutation is one of the major causes of the protein alteration in factor VIII; its frequency is 40% to 50% in severe patients. Long polymerase chain reaction (PCR) and inverse PCR (I-PCR) have been used for the detection of Inv22 mutation.
Development of new protocol for detection of Inv22 mutation.
We have designed a new method for the detection of Inv22 mutation in complementary DNA (cDNA) of patients. Real-time PCR targeting exons 21 to 22, 22 to 23, and 23 to 24 of factor VIII gene were used in cases with hemophilia A. Samples that were inversion positive by this new method were cross-checked by the conventional I-PCR method. We observed that region between exons 22 and 23 could not be amplified, while in negative cases and controls a 480 bp product is obtained.
The method was validated in 20 cases with severe hemophilia A by the new cDNA method, and 8 cases were inversion positive, whereas 12 were negative cases. The findings were confirmed by standard I-PCR method. Complete correlation was observed.
Conventional long PCR and I-PCR methods are work intensive, prolonged, and sometimes difficult to be standardize. The cDNA method is short, involves 3 short-segment amplifications, and is easy to reproduce.
甲型血友病是一种X连锁隐性出血性疾病，其特征是位于Xq28区域的凝血因子VIII基因发生异质性突变，导致凝血因子VIII在质量和数量上缺乏。内含子22倒位（Inv22）突变是凝血因子VIII蛋白改变的主要原因之一；在重症患者中其发生率为40%至50%。长聚合酶链反应（PCR）和反向PCR（I-PCR）已用于检测Inv22突变。
开发检测Inv22突变的新方法。
我们设计了一种检测患者互补DNA（cDNA）中Inv22突变的新方法。对甲型血友病患者使用针对凝血因子VIII基因外显子21至22、22至23和23至24的实时PCR。通过这种新方法检测为倒位阳性的样本用传统I-PCR方法进行交叉核对。我们观察到外显子22和23之间的区域无法扩增，而在阴性病例和对照中可获得一个480 bp的产物。
通过新的cDNA方法在20例重症甲型血友病患者中验证了该方法，8例为倒位阳性，12例为阴性。结果通过标准I-PCR方法得到证实。观察到完全相关性。
传统的长PCR和I-PCR方法工作量大、耗时，有时难以标准化。cDNA方法耗时短，涉及3次短片段扩增，且易于重复。
