Digital PCR Analysis of Maternal Plasma for Noninvasive Detection of Sickle Cell Anemia
Digital PCR Analysis of Maternal Plasma for Noninvasive Detection of Sickle Cell Anemia
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DOI：
10.1373/clinchem.2011.178939
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发表时间：
2012-06-01
影响因子：
9.3
通讯作者：
Chitty, Lyn S.
中科院分区：
文献类型：
--
作者：
Barrett, Angela N.;McDonnell, Thomas C. R.;Chitty, Lyn S.
背景：无细胞胎儿DNA(CffDNA)约占母体血浆中游离DNA的10%，是非侵入性产前诊断(NIPD)适宜的胎儿遗传物质来源。为探讨数字化聚合酶链式反应在镰状细胞性贫血孕妇NIPD诊断中应用的可行性。方法：设计小沟槽结合蛋白TaqMan探针，从镰状细胞性贫血孕妇血浆中提取cffDNA，用于区分野生型血红蛋白A和由HBb基因编码的突变(血红蛋白S)等位基因。用Y染色体特异标记DYS14的数字聚合酶链式反应(DYS14)分析男性怀孕时胎儿DNA的浓度。在有女性胎儿的妊娠中，一组双等位基因插入/缺失多态(INDEL)标记被开发用于量化胎儿DNA部分。结果：82%(37/45)的男性胎儿和75%(15/20)的女性胎儿正确地确定了镰状细胞基因。在胎儿DNA浓度为7%的情况下，100%的病例(25个样本)正确地确定了突变状态。结论：数字聚合酶链式反应可用于确定镰状细胞性贫血高危胎儿的基因分型。胎儿DNA浓度分数的优化是必要的。在女性胎儿的情况下，需要更多信息量的INDELL标记物才能全面应用于这项检测。(C)2012年美国临床化学协会
BACKGROUND: Cell-free fetal DNA (cffDNA) constitutes approximately 10% of the cell-free DNA in maternal plasma and is a suitable source of fetal genetic material for noninvasive prenatal diagnosis (NIPD). The objective of this study was to determine the feasibility of using digital PCR for NIPD in pregnancies at risk of sickle cell anemia.METHODS: Minor-groove binder (MGB) TaqMan probes were designed to discriminate between wild-type hemoglobin A and mutant (hemoglobin S) alleles encoded by the HBB (hemoglobin, beta) gene in cffDNA isolated from maternal plasma samples obtained from pregnancies at risk of sickle cell anemia. The fractional fetal DNA concentration was assessed in male-bearing pregnancies with a digital PCR assay for the Y chromosome-specific marker DYS14. In pregnancies with a female fetus, a panel of biallelic insertion/deletion polymorphism (indel) markers was developed for the quantification of the fetal DNA fraction. We used digital real-time PCR to analyze the dosage of the variant encoding hemoglobin S relative to that encoding wildtype hemoglobin A.RESULTS: The sickle cell genotype was correctly determined in 82% (37 of 45) of male fetuses and 75% (15 of 20) of female fetuses. Mutation status was determined correctly in 100% of the cases (25 samples) with fractional fetal DNA concentrations >7%. The panel of indels was informative in 65% of the female-bearing pregnancies.CONCLUSIONS: Digital PCR can be used to determine the genotype of fetuses at risk for sickle cell anemia. Optimization of the fractional fetal DNA concentration is essential. More-informative indel markers are needed for this assay's comprehensive use in cases of a female fetus. (C) 2012 American Association for Clinical Chemistry