NE Thames Regional Genetics Service Laboratories, Great Ormond Street Hospital for Children, London, UK.
Clin Chem. 2012 Jun;58(6):1026-32. doi: 10.1373/clinchem.2011.178939. Epub 2012 Mar 26.
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.
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 wild-type hemoglobin A.
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.
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.
游离胎儿 DNA（cffDNA）约占母体血浆中游离 DNA 的 10%，是用于非侵入性产前诊断（NIPD）的胎儿遗传物质的合适来源。本研究旨在确定使用数字 PCR 进行 NIPD 在镰状细胞贫血风险妊娠中的可行性。
设计了小沟结合物（MGB）TaqMan 探针，以区分从镰状细胞贫血风险妊娠的母体血浆样本中分离的 cffDNA 中野生型血红蛋白 A 和突变（血红蛋白 S）等位基因。使用数字 PCR 检测 Y 染色体特异性标记 DYS14 评估雄性胎儿的胎儿 DNA 分数浓度。对于女性胎儿，开发了双等位基因插入/缺失多态性（indel）标记物的面板，用于定量胎儿 DNA 分数。我们使用数字实时 PCR 分析编码血红蛋白 S 的变体相对于编码野生型血红蛋白 A 的变体的剂量。
在 82%（37 例中的 37 例）的男性胎儿和 75%（20 例中的 15 例）的女性胎儿中正确确定了镰状细胞基因型。在胎儿 DNA 浓度>7%的 100%（25 个样本）的情况下，突变状态被正确确定。在 65%的女性胎儿中，indel 标记物的信息是可利用的。
数字 PCR 可用于确定镰状细胞贫血风险胎儿的基因型。优化胎儿 DNA 分数浓度至关重要。需要更具信息量的 indel 标记物来全面应用于女性胎儿的检测。
