反转录巢式PCR检测FⅧ基因内含子22倒位

Detection of intron 22 inversion in FⅧgene by reverse transcription nested PCR

目的建立凝血因子Ⅷ(FⅧ)基因内含子22倒位的反转录巢氏PCR检测技术,与长链PCR进行比较。方法选取45例已在我院确诊为重型血友病A男性患者及3例血友病患者母亲,应用改良的反转录巢氏PCR检测和长链PCR方法检测患者是否存在内含子22倒位,对22倒位阴性患者进一步做内含子1倒位和二代基因测序验证。结果长链PCR检测45例患者中,内含子22倒位阳性20例,阴性25例。反转录巢氏PCR结果与长链-PCR一致。两种方法检测均阴性患者经二代测序及内含子1倒位检测,证实均存在FⅧ基因非22倒位的突变,其中4例巢式PCR产物凝胶电泳未见扩增条带判读为阴性的样本,2例为大片段缺失,2例为无义突变。结论反转录巢式PCR与长链-PCR对内含子22倒位的检测结果完全一致,有望作为一种快速准确的内含子22倒位检测方法在血友病基因诊断中予以应用。

Objective To establish a reverse transcription nesting PCR technique for detectionof the intron 22(INV22)of the coagulation factorⅧ(FⅧ)gene in patients with hemophilia A,and compare it with long-distance PCR(LD-PCR)technology.Methods Forty-five male patients diagnosed with severe hemophilia A and three mothers were randomly selected from our hospital.Improved reverse transcription nested PCR and LD-PCR were applied to detect the presence of INV22 in patients.INV22-negative patients were further verified by intron 1 inversion detection and next-generation gene sequencing.Results Both methods have recognized the 20 INV22-positive and 25 INV22-negative cases.INV22-negative patients detected by the two methods were confirmed by second-generation sequencing and intron 1 inversiontest.Four INV22-negative samples which showed no band in reverse transcription nested PCRwere verified by DNA next-generation gene sequencing.Two cases had large fragment deletions,and the others had nonsense mutations.Conclusions Reverse transcription nested PCR have exactly the same detective results of intron 22 inversion compared with LD-PCR.It has the potential to be used as a rapid and accurate intron 22 inversion detection method in the diagnosis of hemophilia.