Use of amplicon-based sequencing for testing fetal identity and monogenic traits with Single Circulating Trophoblast (SCT) as one form of cell-based NIPT

Abstract

A major challenge for cell-based non-invasive prenatal testing (NIPT) is to distinguish individual presumptive fetal cells from maternal cells in female pregnancies. We have sought a rapid, robust, versatile, and low-cost next-generation sequencing method to facilitate this process. Toward this goal, single isolated cells underwent whole genome amplification prior to genotyping. Multiple highly polymorphic genomic regions (including HLA-A and HLA-B) with 10–20 very informative single nucleotide polymorphisms (SNPs) within a 200 bp interval were amplified with a modified method based on other publications. To enhance the power of cell identification, approximately 40 Human Identification SNP (Applied Biosystems) test amplicons were also utilized. Using SNP results to compare to sex chromosome data from NGS as a reliable standard, the true positive rate for genotyping was 83.4%, true negative 6.6%, false positive 3.3%, and false negative 6.6%. These results would not be sufficient for clinical diagnosis, but they demonstrate the general validity of the approach and suggest that deeper genotyping of single cells could be completely reliable. A paternal DNA sample is not required using this method. The assay also successfully detected pathogenic variants causing Tay Sachs disease, cystic fibrosis, and hemoglobinopathies in single lymphoblastoid cells, and disease-causing variants in three cell-based NIPT cases. This method could be applicable for any monogenic diagnosis.

Noninvasive prenatal screening for cystic fibrosis using circulating trophoblasts: Detection of the 50 most common disease‐causing variants

“…For the cell‐based NIPT results using both fragment length analysis and NGS ( N = 27), the ADO rates were 5.4% (CI 95 : 2.5%–8.4%, median: 2.0) and 1.2% per sample (CI 95 : 0.3%–2.2%, median: 0.0%), respectively. These ADO rates are lower than what has previously been reported in similar studies using WGA‐DNA from circulating trophoblasts (27%–74%) 22,23 . In this study, the initial material for the CF analysis was WGA‐DNA pooled from multiple trophoblasts, and this may explain the lower ADO rate.…”

“…Another recent study by Zhuo et al. used a similar approach to detect pathogenic variants for Tay Sachs disease, CF and hemoglobinopathies, as well as to detect family‐specific pathogenic variants 23 . Together, these studies support the feasibility for developing cell‐based NIPT for various monogenic disorders using NGS.…”

“…This allows the extraction of placental DNA without the background of maternal DNA 20 . Circulating trophoblasts may therefore be useful for prenatal screening purposes, as direct variant analyses can be performed without the need for paternal‐ and proband samples 16,17,21–23 …”

“…20 Circulating trophoblasts may therefore be useful for prenatal screening purposes, as direct variant analyses can be performed without the need for paternal-and proband samples. 16,17,[21][22][23] This aim of this study was to establish cell-based NIPT as a screening tool for the 50 most common pathogenic CFTR variants.…”

Overview and recent developments in cell‐based noninvasive prenatal testing

“…DNA from individual trophoblasts was analyzed by low‐coverage NGS for genome‐wide copy number analysis, with correct identification of fetal aneuploidy in 11 cases and a known CNV in four cases (ranging from 1.1 Mb to 19 Mb in size). A new genotyping method confirmed the fetal origin of all analyzed cells by comparing fetal single nucleotide polymorphisms (SNPs) to the maternal (and when available, paternal) SNP profile (based on 50–60 SNPs to genome‐wide analysis, described in 86 and unpublished data). Our most recent method includes an enrichment strategy based on a combination of anti‐HLA‐G, anti‐Trop2, and anti‐EpCAM antibodies resulting in an average yield of 0.20 trophoblasts/ml from 95 blood samples 25 …”