The development of novel quantification assay for mitochondrial DNA heteroplasmy aimed at preimplantation genetic diagnosis of Leigh encephalopathy
The development of novel quantification assay for mitochondrial DNA heteroplasmy aimed at preimplantation genetic diagnosis of Leigh encephalopathy
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DOI：
10.1007/s10815-007-9114-0
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发表时间：
2007-06-01
影响因子：
3.1
通讯作者：
Yoshimura, Yasunori
中科院分区：
文献类型：
--
作者：
Tajima, Hiroto;Sueoka, Kou;Yoshimura, Yasunori
目的：为了对Leigh脑病进行胚胎植入前遗传学诊断（PGD），我们建立了一种快速可靠的T8993G mtDNA突变百分比定量检测方法，并对各种标本进行了分析。方法：采用实时荧光定量PCR法测定8993T/G克隆质粒DNA序列比例，制备标准曲线，并测定(1)突变体DNA （PCR- rflp已知比例），(2)46%突变体携带者的单淋巴细胞，(3)20个异常胚胎的123个卵裂球。结果：(1)误差在-5 ~ +6%之间；(2)平均44.3%(11 ~ 70%);(3)5个胚胎携带T8993G突变（4 ~ 22%）。来自同一人的胚胎表现出不同程度的异质性，来自同一胚胎的卵裂球表现出有限的异质性分散（2-11%）。结论：(1)该方法为Leigh脑病提供了快速、可靠的PGD。(2)存在有丝分裂的可变异质性。(3) T8993G突变存在于未发育胚中，支持瓶颈理论。来自同一胚胎的卵裂球的有限异质性分散也支持了PGD对T8993G突变的可靠性。
Purpose: To perform preimplantation genetic diagnosis (PGD) of Leigh encephalopathy, we developed a rapid and reliable quantification assay for the percentage of T8993G mtDNA mutation and analyzed various specimens.Methods: We prepared the standard curve by measuring serial proportion of 8993T/G cloned plasmid DNA using real-time PCR, and measured (1) mutant DNA (known proportions by PCR-RFLP), (2) single lymphocytes from 46% mutant carrier, (3) 123 blastomeres from 20 abnormal embryos.Results: (1) These were within -5-+6% error range, (2) mean 44.3% (11-70%), (3) Five embryos harbored T8993G mutation (4-22%). Embryos from same person indicated different degrees of heteroplasmy, and blastomeres from same embryo demonstrated limited dispersion of heteroplasmy (2-11%).Conclusions: (1) This method provides rapid and reliable PGD for Leigh encephalopathy. (2) The variable heteroplasmy with somatic mitosis was suggested. (3) T8993G mutation was existed in undeveloped embryo, and the bottleneck theory was supported. The limited heteroplasmy dispersion of blastomeres from same embryo also supported reliability of PGD for T8993G mutation.