The current study demonstrated excellent performance of the RT-PCR-based NIPT in screening for common fetal trisomies. The overall performance of the NIPT using PNA probe-based RT-PCR in screening for fetal trisomies 21, 13, and 18 showed 95.45% sensitivity, 98.60% specificity, and no test failures.
PNAs, which are DNA analogs, are artificially synthesized with uncharged backbone and, therefore, have more favorable hybridization properties as well as chemical, thermal, and biological stability parameters [10, 14]. The PNA probe composed of dual-labeled (quencher and fluorophore) to improve the resolution of detection, causes a large difference in melting temperature between specific hybridization and partial hybridization. PNAs are becoming increasingly used in different molecular biology applications [7,8,9], e.g., in the detection of clarithromycin resistance in Helicobacter pylori or demonstration of microsatellite instability in colorectal carcinoma [7, 10,11,12].
In obstetrics, we have reported that PNA probes could be used for rapid determination of aneuploidy in amniotic fluid samples, as an alternative to fluorescence in situ hybridization, quantitative fluorescence PCR, and multiplex ligation-dependent probe amplification [7]. In the current study, we also demonstrated that PNA probe-based RT-PCR NIPT performed superbly in the screening for common fetal aneuploidy conditions.
The NIPT based on the analysis of cell-free maternal plasma DNA is an innovative approach to screening for common fetal aneuploidies [3, 4]. Numerous studies have shown that NIPT detects common fetal trisomies with high sensitivity and specificity. However, the NIPT analyses the placental DNA, not real fetal DNA, and might detect vanishing twin, maternal malignancy and maternal mosaicism, etc. [15]. In addition, until now, most NIPT studies have been based on NGS and therefore, reported frequent test failures up to 5% of the -when cell-free DNA concentration or fetal fraction was low [16,17,18,19]. RT-PCR-based NIPT can report the result with smaller amounts of maternal blood samples than conventional methods. For example, most existing NIPT methods require a minimum of 10 mL of maternal blood sample to perform the test, whereas just 6 mL is sufficient with the method used in our study. Both RT-PCR- and NGS-based NIPTs include PCR amplification of cell free DNA; however, the RT-PCR-based method has a single amplification step with fewer amplified region than the NGS-based method. Therefore, RT-PCR-based NIPT enables a relatively more stable amplification process with a smaller blood sample volume.
After the discovery of cffDNA, non-invasive prenatal testing using NGS methods has rapidly developed. However, the NGS approach requires expensive equipment, reagents, and software, and has limited throughput [5]. In addition, the result turnaround time is long.
Several other techniques, e.g., identification of the methylated regions, or plasma microRNAs, have been suggested for the NIPT [20,21,22]. The methylated region approaches needed pretreatment with sodium bisulfite or methylation-sensitive restriction enzymes. The former process has a low reproducibility, and the latter process requires long processing. Moreover, the plasma microRNAs approaches might be difficult to perform at early pregnant due to very low levels of microRNAs. Because of such limitations, these methods are not commonly used.
Compared with the features of the NGS-based approach, RT-PCR-based NIPT has rapid, easy-handling, and low-cost procedures for screening of common fetal trisomies. The RT-PCR-based NIPT provides the results within 6 h, i.e., much easier, and faster than other molecular methods, such as NGS, which require long turnaround time and labor-intensive experiments.
In this study, 15 out of 1,023 samples were misclassified (Additional file 1: Table 3). Fourteen cases were classified as false positives. No significant differences in the characteristics between the correctly classified and falsely classified groups in normal karyotype cases were observed (data not shown). However, one case was misclassified as false negative for trisomy 21, gestational age at sampling of false negative case was 12+4 weeks and BMI at sampling was 34.9. There may be several reasons for misclassification by the NIPT based on cell-free DNA, including confined placental or real fetal mosaicism, vanishing twins, maternal somatic mosaicism, maternal copy number variants, or undetected maternal cancer [23]. In addition, after visually inspecting the dotplots of 330 maternal plasma samples of normal fetuses and 22 trisomy cell line samples, the cut-off value for classification was determined as the value best separating the two groups. However, validating this cut-off value to our larger dataset yielded a few overlapped samples resulting in false positives and negatives. To be used in clinical practice, more larger studies may be needed to estimate actual performance in real-world.
To the best of our knowledge, this is the first study that utilized PNA probe-based RT-PCR for a NIPT. However, our study has limitations in that it was a retrospective examination of stored maternal samples and electronic medical records from five different institutions. Furthermore, only pregnant women of the Asian race (mostly Korean) were included. Due to the retrospective study design, the performance of RT-PCR-based NIPT presented in the current study may not represent the real-world data both in low risk and high risk pregnancies. Therefore, additional prospective studies with larger study cohorts, including participants of various races, are needed for the validation of this NIPT in real clinical practice. In addition, a comparative analysis of the sensitivity and cost-effectiveness of RT-PCR-based and NGS-based NIPTs is needed. While the NIPT only provides information on common trisomies such as trisomy 18, 13, and 21, the first trimester screening test (FTS) conducted in the same period may provide additional information on various genetic abnormalities. Therefore, further study is needed to compare the performance between NIPT and FTS for various genetic diseases. The novel method could technically distinguish the microdeletion/microduplication syndrome from normal control to create PNA probes for targeting subchromosomes; however, the incidence of subchromosomal abnormality is very low, and the actual practicality and applicability of its efficacy would be difficult to prove. Therefore, it is necessary to extend the range of NIPTs from trisomies of chromosome 21, 18, and 13 to other aneuploidy conditions and subchromosomal abnormalities.