Barrett Angela N, Xiong Li, Tan Tuan Z, Advani Henna V, Hua Rui, Laureano-Asibal Cecille, Soong Richie, Biswas Arijit, Nagarajan Niranjan, Choolani Mahesh
Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Department of Gynecology & Obstetrics, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
PLoS One. 2017 Oct 30;12(10):e0186771. doi: 10.1371/journal.pone.0186771. eCollection 2017.
Cell-free DNA from maternal plasma can be used for non-invasive prenatal testing for aneuploidies and single gene disorders, and also has applications as a biomarker for monitoring high-risk pregnancies, such as those at risk of pre-eclampsia. On average, the fractional cell-free fetal DNA concentration in plasma is approximately 15%, but can vary from less than 4% to greater than 30%. Although quantification of cell-free fetal DNA is straightforward in the case of a male fetus, there is no universal fetal marker; in a female fetus measurement is more challenging. We have developed a panel of multiplexed insertion/deletion polymorphisms that can measure fetal fraction in all pregnancies in a simple, targeted sequencing reaction.
A multiplex panel of primers was designed for 35 indels plus a ZFX/ZFY amplicon. cfDNA was extracted from plasma from 157 pregnant women, and maternal genomic DNA was extracted for 20 of these samples for panel validation. Sixty-one samples from pregnancies with a male fetus were subjected to whole genome sequencing on the Ion Proton sequencing platform, and fetal fraction derived from Y chromosome counts was compared to fetal fraction measured using the indel panel. A total of 157 cell-free DNA samples were sequenced using the indel panel, and informativity was assessed, along with the proportion of fetal DNA.
Using gDNA we optimised the indel panel, removing amplicons giving rise to PCR bias. Good correlation was found between fetal fraction using indels and using whole genome sequencing of the Y chromosome (Spearmans r = 0.69). A median of 12 indels were informative per sample. The indel panel was informative in 157/157 cases (mean fetal fraction 14.4% (±0.58%)).
Using our targeted next generation sequencing panel we can readily assess the fetal DNA percentage in male and female pregnancies.
母体血浆中的游离DNA可用于非整倍体和单基因疾病的无创产前检测，也可作为监测高危妊娠（如先兆子痫风险妊娠）的生物标志物。平均而言，血浆中游离胎儿DNA的比例约为15%，但可在小于4%至大于30%之间变化。虽然对于男性胎儿，游离胎儿DNA的定量很简单，但没有通用的胎儿标志物；对于女性胎儿，测量更具挑战性。我们开发了一组多重插入/缺失多态性，可通过简单的靶向测序反应测量所有妊娠中的胎儿比例。
设计了一个包含35个插入/缺失位点以及一个ZFX/ZFY扩增子的多重引物组。从157名孕妇的血浆中提取游离DNA，并从其中20个样本中提取母体基因组DNA用于引物组验证。对61例男性胎儿妊娠的样本在Ion Proton测序平台上进行全基因组测序，并将Y染色体计数得出的胎儿比例与使用插入/缺失引物组测量的胎儿比例进行比较。使用插入/缺失引物组对总共157个游离DNA样本进行测序，并评估信息性以及胎儿DNA的比例。
使用基因组DNA对插入/缺失引物组进行了优化，去除了导致PCR偏差的扩增子。发现使用插入/缺失位点得出的胎儿比例与使用Y染色体全基因组测序得出的胎儿比例之间具有良好的相关性（斯皮尔曼相关系数r = 0.69）。每个样本中中位数为12个插入/缺失位点具有信息性。插入/缺失引物组在157/157例样本中具有信息性（平均胎儿比例14.4%（±0.58%））。
使用我们的靶向新一代测序引物组，我们可以轻松评估男性和女性妊娠中胎儿DNA的百分比。
