Science and Technology, BGI-Shenzhen, Shenzhen, China.
PLoS One. 2013 Jun 10;8(6):e65050. doi: 10.1371/journal.pone.0065050. Print 2013.
Currently very few noninvasive molecular genetic approaches are available to determine zygosity for twin pregnancies in clinical laboratories. This study aimed to develop a novel method to determine zygosity by using maternal plasma target region sequencing.
We constructed a statistic model to calculate the possibility of each zygosity type using likelihood ratios ( Li ) and empirical dynamic thresholds targeting at 4,524 single nucleotide polymorphisms (SNPs) loci on 22 autosomes. Then two dizygotic (DZ) twin pregnancies,two monozygotic (MZ) twin pregnancies and two singletons were recruited to evaluate the performance of our novel method. Finally we estimated the sensitivity and specificity of the model in silico under different cell-free fetal DNA (cff-DNA) concentration and sequence depth.
RESULTS/CONCLUSIONS: We obtained 8.90 Gbp sequencing data on average for six clinical samples. Two samples were classified as DZ with L values of 1.891 and 1.554, higher than the dynamic DZ cut-off values of 1.162 and 1.172, respectively. Another two samples were judged as MZ with 0.763 and 0.784 of L values, lower than the MZ cut-off values of 0.903 and 0.918. And the rest two singleton samples were regarded as MZ twins, with L values of 0.639 and 0.757, lower than the MZ cut-off values of 0.921 and 0.799. In silico, the estimated sensitivity of our noninvasive zygosity determination was 99.90% under 10% total cff-DNA concentration with 2 Gbp sequence data. As the cff-DNA concentration increased to 15%, the specificity was as high as 97% with 3.50 Gbp sequence data, much higher than 80% with 10% cff-DNA concentration.
This study presents the feasibility to noninvasively determine zygosity of twin pregnancy using target region sequencing, and illustrates the sensitivity and specificity under various detecting condition. Our method can act as an alternative approach for zygosity determination of twin pregnancies in clinical practice.
目前，临床实验室中用于确定双胞胎妊娠合子性的非侵入性分子遗传学方法非常有限。本研究旨在开发一种新的方法，通过使用母体血浆靶区域测序来确定合子性。
我们构建了一个统计模型，使用似然比（Li）和针对 22 条常染色体上的 4524 个单核苷酸多态性（SNP）位点的经验动态阈值来计算每种合子性类型的可能性。然后招募了两个双胎妊娠（DZ）、两个单卵双胎妊娠（MZ）和两个单胎妊娠来评估我们新方法的性能。最后，我们在不同的游离胎儿 DNA（cff-DNA）浓度和测序深度下，对模型的敏感性和特异性进行了计算机模拟。
结果/结论：我们平均获得了 6 个临床样本 8.90 Gbp 的测序数据。两个样本被归类为 DZ，Li 值分别为 1.891 和 1.554，高于动态 DZ 截断值 1.162 和 1.172。另外两个样本被判定为 MZ，Li 值分别为 0.763 和 0.784，低于 MZ 截断值 0.903 和 0.918。其余两个单胎妊娠样本被认为是 MZ 双胞胎，Li 值分别为 0.639 和 0.757，低于 MZ 截断值 0.921 和 0.799。在计算机模拟中，当总游离胎儿 DNA（cff-DNA）浓度为 10%时，我们的非侵入性合子性确定方法的估计敏感性为 99.90%，当 cff-DNA 浓度增加到 15%时，当使用 3.50 Gbp 测序数据时，特异性高达 97%，而当 cff-DNA 浓度为 10%时，特异性为 80%。
本研究提出了使用靶区域测序无创性确定双胞胎妊娠合子性的可行性，并说明了在各种检测条件下的敏感性和特异性。我们的方法可以作为临床实践中双胞胎妊娠合子性确定的替代方法。
