Supplementary Material for: Noninvasive prenatal diagnosis of beta-thalassemia disease by using digital PCR analysis of cell-free fetal DNA in maternal plasma

Introduction: Prenatal diagnosis of thalassemia disease usually based on invasive technique. Non-invasive diagnosis using cell-free fetal DNA (cff-DNA) were described with various laboratory technique. The aim of this study to identify the performance of dPCR for analyzing cff-DNA in maternal plasma to diagnose fetal beta-thalassemia diseases. Methods: Thirty-five couples at risk of fetal beta-thalassemia disease caused by four common mutations of HBB were enrolled at 12-18 weeks. The dPCR assay was designed to detect and quantify paternally-inherited beta-thalassemia allele (PIB-M) and maternally-inherited beta-thalassemia allele (MIB-M) from cff-DNA in maternal plasma. Results: Of 29 couples with different paternal/maternal mutations, all cases who inherited paternal mutation had detectable PIB-M. The MIB-mutant/wild-type (MIB-M/MIB-N) ratio in the mothers whose fetuses did not inherit maternal mutation was 0.87+/-0.07 which was significantly lower than that of the mothers whose fetuses inherited maternal mutation, 1.01+/-0.05. The sensitivity and specificity of MIB-M/MIB-N ratio >0.95 in predicting fetus inheriting maternal mutation was 100 and 92.3%, respectively. In four couples with same paternal/maternal mutation, IB-M/IB-N ratio of >0.95 correctly predicted the presence of an inheritance of at least one beta-thalassemia allele. In two couples with paternal Hb E/beta-thalassemia, the presence of PIB-M and the MIB-M/MIB-N ratio of >0.95 correctly predicted the presence of paternal/maternal mutations, respectively. Conclusions: The method of analyzing cff-DNA in maternal plasma by dPCR is efficient for prenatal diagnosis of beta-thalassemia.

引言：地中海贫血的产前诊断通常依赖侵入性检测技术。现有多种实验室技术可借助胎儿游离DNA（cell-free fetal DNA, cff-DNA）开展无创产前诊断。本研究旨在评估数字聚合酶链式反应（digital polymerase chain reaction, dPCR）分析孕妇血浆中cff-DNA以诊断胎儿β地中海贫血的诊断效能。方法：本研究纳入了35例因HBB基因4种常见突变而存在胎儿罹患β地中海贫血风险的夫妇，纳入孕周为12~18周。本研究设计了dPCR检测体系，可从孕妇血浆的cff-DNA中检测并定量父源性β地中海贫血等位基因（paternally-inherited beta-thalassemia allele, PIB-M）与母源性β地中海贫血等位基因（maternally-inherited beta-thalassemia allele, MIB-M）。结果：在29例父母突变类型存在差异的夫妇中，所有胎儿继承父源性突变的病例均能检测到PIB-M。胎儿未继承母源性突变的孕妇，其MIB-M/MIB-N比值为0.87±0.07，显著低于胎儿继承母源性突变的孕妇（1.01±0.05）。以MIB-M/MIB-N比值＞0.95作为预测胎儿继承母源性突变的判定阈值，其灵敏度与特异度分别为100%与92.3%。在4例父母突变类型一致的夫妇中，IB-M/IB-N比值＞0.95可准确预测胎儿至少继承1个β地中海贫血等位基因。在2例父亲为Hb E/β地中海贫血基因型的夫妇中，检测到PIB-M与MIB-M/MIB-N比值＞0.95，可分别准确预测父源性与母源性突变的存在。结论：依托dPCR分析孕妇血浆中cff-DNA的方法，可高效应用于β地中海贫血的产前诊断。