应用荧光聚合酶链反应对α地中海贫血进行植入前遗传学诊断

目的探讨应用跨越断裂点荧光聚合酶链反应（PCR）技术在α地中海贫血（简称α地贫）植入前遗传学诊断（PGD）中的应用。方法获取Ot地贫东南亚缺失型携带者单个淋巴细胞,建立了稳定的单细胞跨越断裂点荧光PCR检测技术,并对4对夫妇双方均为α地贫——SEA缺失型杂合子应用荧光PCR进行了α地贫的PGD。结果单个淋巴细胞平均扩增效率为90．0％（72／80）,平均等位基因脱扣（ADO）率为8．3％（6／72）。对4对夫妇进行4个周期PGO,共检测38个胚胎,获得38个卵裂球,其中34个卵裂球扩增成功,扩增效率为89．5％（34／38）,ADO率为5．9％（2／34）。经PCR分析,共获得11个正常胚胎,8个杂合子胚胎,15个重型地贫胚胎。移植了11个胚胎,获得2例临床妊娠。孕17周时经脐带血穿刺,分别证实为完全正常胚胎和杂合子胚胎,现已出生两名健康男婴。结论应用单细胞荧光PCR技术可对α地贫进行植入前遗传学诊断,达到优生的目的。

Successful preimplantation genetic diagnosis for alpha-thalassemia using fluorescent polymerase chain reaction

OBJECTIVE: To develop single-cell fluorescent gap polymerase chain reaction (PCR) assay for preimplantation genetic diagnosis (PGD) in couples at risk of having child with alpha-thalassemia. METHODS: Single cell fluorescent gap PCR which can detect the alpha-thalassemia Southeast Asia deletion (-SEA deletion), was applied to single lymphocytes and blastomeres which coming from four clinical PGD cycles. RESULTS: The Single cell fluorescent gap PCR can detect the alpha-thalassemia -SEA deletion, which account for 94% of hydrop fetalis in Chinese population with an amplification efficiency of 90.0% (72/80) and allele drop-out (ADO) rate of 8.3% (6/72) in lymphocytes. In four clinical PGD cycles, a total 38 embryos were detected and 38 blastomeres were obtained. Thirty-four blastomeres were amplified with the amplification efficiency of 89.5% (34/38) and ADO rate of 5.9% (2/34). Eleven embryos were shown to be normal homozygous, eight embryos were shown to be heterozygous and 15 embryos were shown to be affected homozygous. Eleven embryos were transferred back to the uterus of the patients. Two pregnancy achieved, resulting in two live healthy births, which confirmed the results of PGD. CONCLUSION: This first reported unaffected pregnancy resulting from PGD using fluorescent gap PCR for alpha-thalassemia demonstrates that this technique, as an alternative to prenatal diagnosis, is a reliable and effective way to help those carrier couples to get a healthy baby.