[Rapid preimplantation genetic diagnosis of α-thalassemia SEA deletion with blastocyst cell whole genome amplification and short fragment Gap-PCR method]

The method developed in this study is a complete testing process for 4-6 blastocyst biopsy cells to allow rapid, accurate, and cost-effective PGD genotyping of α-thalassemia SEA deletion using short fragment gap-PCR. 目的: 建立一种基于囊胚细胞全基因组扩增（WGA）结合目的序列短片段Gap-PCR基因分型技术的α-地中海贫血东南亚型（SEA）缺失突变快速植入前遗传学诊断新方法。 方法: 以多重置换扩增（MDA）WGA技术为基础，建立以管家基因GAPDH和β-actin为目的序列的WGA产物双重荧光PCR质检体系，以及突变和正常序列短片段Gap-PCR的α-地中海贫血SEA缺失基因分型体系，通过对不同细胞数SEA携带者淋巴细胞样本的检测进行灵敏度与准确性评价，对12例囊胚活检样本的检测进行应用评价。 结果: 应用本研究建立的方法，不同淋巴细胞数样本的结果显示3细胞时即未出现等位基因脱扣，4细胞时目标模板的WGA产物量趋于稳定；10例WGA成功的囊胚活检样本的基因分型结果分别为--SEA/αα（5例）和αα/αα（5例），与验证基因型相符。 结论: 本研究建立的方法为一个完整检测流程，即以4~6个囊胚活检细胞为检材，对MDA产物进行WGA质检及相对浓度评估后，以短片段Gap-PCR体系而实现α-地贫SEA缺失的PGD快速基因分型；此方法简单快速、结果准确、检测成本低而适合临床常规应用。

Using multiple displacement amplification (MDA) WGA technique, we established a double-fluorescent PCR system of the housekeeping genes GAPDH and β-actin for WGA quality testing, and a genotyping PCR system of mutant and normal short sequences for α-thalassemia SEA deletion. The sensitivity and accuracy of this method for diagnosis of α-thalassemia SEA deletion were evaluated by detecting lymphocyte samples containing different cell numbers from carriers of SEA deletion. The applicability of this method was evaluated by testing of 12 blastocyst biopsy samples.

To develop a rapid preimplantation genetic diagnosis method for α-thalassemia SEA deletion based on blastocyst cell whole genome amplification (WGA) combined with short fragment Gap-PCR.

Detection of lymphocyte samples with different cell numbers using the method developed in this study revealed no ADO in 3-cell samples, and the product quantity of WGA became stable for 4-cell samples. Genotyping of the 10 blastocyst biopsy samples with successful WGA showed a genotype of --SEA/αα in 5 samples and αα/αα in the other 5 samples, which were consistent with the verification results. Conclusions: The method developed in this study is a complete testing process for 4-6 blastocyst biopsy cells to allow rapid, accurate, and cost-effective PGD genotyping of α-thalassemia SEA deletion using short fragment gap-PCR. 目的: 建立一种基于囊胚细胞全基因组扩增（WGA）结合目的序列短片段Gap-PCR基因分型技术的α-地中海贫血东南亚型（SEA）缺失突变快速植入前遗传学诊断新方法。 方法: 以多重置换扩增（MDA）WGA技术为基础，建立以管家基因GAPDH和β-actin为目的序列的WGA产物双重荧光PCR质检体系，以及突变和正常序列短片段Gap-PCR的α-地中海贫血SEA缺失基因分型体系，通过对不同细胞数SEA携带者淋巴细胞样本的检测进行灵敏度与准确性评价，对12例囊胚活检样本的检测进行应用评价。 结果: 应用本研究建立的方法，不同淋巴细胞数样本的结果显示3细胞时即未出现等位基因脱扣，4细胞时目标模板的WGA产物量趋于稳定；10例WGA成功的囊胚活检样本的基因分型结果分别为--SEA/αα（5例）和αα/αα（5例），与验证基因型相符。 结论: 本研究建立的方法为一个完整检测流程，即以4~6个囊胚活检细胞为检材，对MDA产物进行WGA质检及相对浓度评估后，以短片段Gap-PCR体系而实现α-地贫SEA缺失的PGD快速基因分型；此方法简单快速、结果准确、检测成本低而适合临床常规应用。