Supplementary Material for: Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma

Introduction: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information. Objective: Here, we report a next generation sequencing (NGS)-based assay for predicting the prenatal ABO blood group. Materials and Methods: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples. Results: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women (n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO*O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity. Conclusions: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO*O.01/ABO*O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN.

引言：孕妇与胎儿之间的ABO血型不合，作为引发胎儿或新生儿发病乃至死亡的病因，尽管相对罕见，但仍是一项不容忽视的重要风险。当孕妇体内抗A或抗B IgG抗体水平升高时，胎儿可能面临胎儿新生儿溶血病（hemolytic disease of the fetus and newborn, HDFN）的风险。产前直接测定胎儿ABO血型可提供极具价值的临床信息。
研究目的：本文报道一种基于下一代测序（next generation sequencing, NGS）的检测方法，用于产前ABO血型预测。
材料与方法：共收集26名孕周12至35周孕妇的26份血浆样本，其中20份为临床样本，6份为验证样本。提取血浆中的无细胞DNA（cell-free DNA）后，使用2组引物进行聚合酶链式反应（polymerase chain reaction, PCR）扩增，随后开展NGS检测。NGS数据分别通过FASTQ分析与grep搜索两种方法进行处理分析，以确保结果的可靠性。将胎儿ABO血型预测结果与19份样本已知的血清学婴儿ABO血型表型进行比对验证。
结果：在19份具备表型信息的可评估样本中，两种分析方法均得到了完全一致的结果。针对20名致敏孕妇，预测12例胎儿存在HDFN风险，7例无相关风险；1份临床样本结果无法判定。克隆测序发现一种新型变异体，其携带与ABO*O.01.24一致的单核苷酸变异，且额外带有c.220C>T碱基替换。6份验证样本中另有1份结果无法判定，原因为母体杂合性。2份无法判定的样本体现了本检测方法的局限性，分别由杂交ABO基因与母体杂合性导致。
结论：本研究首创了基于孕妇血浆无细胞DNA分析的NGS胎儿ABO血型预测检测方法，并在小样本队列中验证了其应用价值。通过变异频率与ABO*O.01/ABO*O.02杂合子频率的计算，我们预估在未来约95%的O型血孕妇临床样本中，可获得可靠的胎儿ABO血型分型结果。尽管ABO血型系统存在大量遗传变异，但多数变异类型较为罕见，产前ABO血型预测具备可行性，可为ABO型HDFN的预防提供宝贵的早期临床信息。