Deng Jie, Zhuang Guang-Lun, Peng Wen-Lin, Zhou Can-Quan, Li Jie, Liang Xiao-Yan, Deng Ming-Fen, Zeng Yan-Hong, Sun Hong-Yu
Reproductive Medical Center, 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
Zhonghua Yi Xue Za Zhi. 2005 Mar 30;85(12):811-5.
To develop single-cell multiplex nested polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia.
Primers were designed and synthesized according to the documented mutation sites common among Chinese. Venous blood was collected from 4 pairs of husband and wife, all heterozygotes for beta-thalassemia, and underwent multiple nested PCR. Intraooplasmic sperm injection and mechanical bio psy was used to obtain single blastomere. Multiplex nested PCR was used to detect the CD41-42 mutation and the closely linked polymorphic marker, HumTHO1 gene or CD41-42, CD41-28, IVSII654 mutation and HumTHO1 gene in the single blastomeres from four clinical PGD cycles. The normal embryos with high scores capable of continuing to divide were transplanted into the uteri. The process of gestation was observed.
200 lymphocytes were amplified by nested PCR. The average amplification rate of the most common 16 beta-thalassemia mutations in Chinese population was 91.3% and the average rate of allele drop out for different sites was 17.0% without differences between any 2 sites. During the 4 PGD cycles 33 embryos underwent bioassay with a success rate of 100%. 33 blastomeres were obtained to undergo PCR, of which 30 were successfully amplified with an amplification rate of 90.9%. Explicit diagnosis was obtained in 26 of the 30 embryos: 7 normal homozygotes, 11 heterozygotes, and 8 abnormal or complex heterozygotes. One or more embryos were transferred back into the uteri of the 4 women and clinical pregnancy occurred in one woman. Five weeks after the implantation B-mode ultrasonography showed monocyesis, and in the 17th week of gestational period paracentesis of cord blood showed normal homozygote. At last a normal female infant confirming the PGD result had been born, which was the first reported unaffected pregnancy resulting from PGD using multiplex nested PCR for couples as beta-thalassemia gene carriers. The results of diagnosis for embryo all corresponded to those for blastomere. The average ADO rate of blastomere was 13.3% (4/30).
PGD using multiplex nested PCR, as an alternative to prenatal diagnosis, is a reliable and effective way to help couples-carriers of pathogenetic genes to get a healthy baby.
为有生育β地中海贫血患儿风险的夫妇开发用于植入前遗传学诊断（PGD）的单细胞多重巢式聚合酶链反应（PCR）检测方法。
根据中国人群中常见的已记录突变位点设计并合成引物。从4对夫妻（均为β地中海贫血杂合子）采集静脉血，进行多重巢式PCR。采用卵胞浆内单精子注射和机械活检获取单个卵裂球。运用多重巢式PCR检测4个临床PGD周期单个卵裂球中的CD41-42突变及紧密连锁的多态性标记HumTHO1基因，或CD41-42、CD41-28、IVSII654突变及HumTHO1基因。将能够继续分裂的高分正常胚胎移植入子宫，观察妊娠过程。
通过巢式PCR扩增出200个淋巴细胞。中国人群中最常见的16种β地中海贫血突变的平均扩增率为91.3%，不同位点的平均等位基因脱扣率为17.0%，任意两个位点之间无差异。在4个PGD周期中，33个胚胎接受活检，成功率为100%。获取33个卵裂球进行PCR，其中30个成功扩增，扩增率为90.9%。30个胚胎中有26个得到明确诊断：7个正常纯合子，11个杂合子，8个异常或复杂杂合子。将一个或多个胚胎移植回4名女性子宫，1名女性临床妊娠。着床后5周B超显示单胎妊娠，妊娠17周时脐带血穿刺显示正常纯合子。最终一名正常女婴出生，证实了PGD结果，这是首次报道的使用多重巢式PCR对β地中海贫血基因携带者夫妇进行PGD获得的未受影响妊娠。胚胎诊断结果与卵裂球诊断结果一致。卵裂球的平均ADO率为13.3%（4/30）。
使用多重巢式PCR的PGD作为产前诊断的替代方法，是帮助致病基因携带者夫妇获得健康婴儿的可靠且有效途径。
