Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins
Abstract
:1. Introduction
2. Materials and Methods
2.1. Subject Enrollment
2.2. Sample Processing and DNA Extraction
2.3. Quality Control of cffDNA (QuantYfeX)
2.4. Maternal Plasma DNA Sequencing and Data Analysis
3. Results and Discussion
3.1. Retrospective Study on Stored DNA Libraries
Sample | Chr13 z-score | Chr18 z-score | Chr21 z-score | Fetal fraction (%) | Gestational age (p.m.) | NIPT result | Fetus A karyotype | Fetus B karyotype | Invasive method |
---|---|---|---|---|---|---|---|---|---|
RDLN015823 | 0.0 | 0.0 | −0.4 | 37 | 10 + 6 | negative | 46,XX | 46,XY | CVS |
RDLN015835 | −0.6 | 0.9 | 0.7 | 35 | 12 + 6 | negative | 46,XY | 46,XX | CVS |
RDLN015916 | 1.8 | 1.9 | 0.8 | 24 | 16 + 2 | negative | 46,XY | 46,XY | AC |
RDLN016042 | 0.9 | 0.5 | 1.0 | 23 | 17 + 4 | negative | 46,XX | 46,XY | AC |
RDLN016047 | 1.3 | 0.7 | −1.6 | 45 | 13 + 5 | negative | 46,XY | 46,XY | CVS |
RDLN016114 | −0.9 | −0.2 | 8.4 | 29 | 14 + 4 | T21 positive | 47,XY,+21 | 46,XX | CVS |
RDLN016116 | −0.4 | 0.7 | 4.4 | 20 | 13 + 4 | T21 positive | 47,XX,+21 | 46,XX | CVS |
RDLN016450 | −0.1 | 1.4 | 8.4 | 31 | 16 + 0 | T21 positive | 47,XX,+21 | 46,XX | AC |
RDLN016457 | 1.0 | 0.9 | −0.3 | 22 | 17 + 5 | negative | 46,XX | 46,XY | AC |
RDLN016474 | 0.8 | 0.3 | 5.4 | 16 | 18 + 4 | T21 positive | 47,XX,+21 | 46,XX | AC |
RDLN016519 | 0.2 | −1.0 | 0.2 | 20 | 15 + 0 | negative | 46,XX | 46,XX | AC |
RDLN016778 | 0.2 | −0.1 | −0.1 | 12 | 16 + 0 | negative | 46,XX | 46,XX | AC |
RDLN017192 | −1.2 | −0.4 | −1.0 | 13 | 16 + 1 | negative | 46,XY | 46,XY | AC |
RDLN017624 | 1.0 | 0.9 | 0.7 | 15 | 17 + 0 | negative | 46,XY | 46,XY | AC |
RDLN017641 | −1.0 | 0.3 | 0.7 | 8 | 15 + 2 | negative | 46,XY | 46,XY | AC |
RDLN017670 | 0.8 | 0.1 | −0.9 | 24 | 17 + 1 | negative | 46,XX | 46,XX | AC |
3.2. Prospective Study on Blood Samples from Multiple Pregnancies Collected during Laboratory Routine
Sample | Chr13 z-score | Chr18 z-score | Chr21 z-score | Fetal fraction (%) | Gestational age (p.m.) | No. of fetuses, chorionicity, amnionicity | NIPT result |
---|---|---|---|---|---|---|---|
LCMPC01 | 0.8 | −0.4 | 1.0 | n.a. | 11 + 0 | 2, monochorionic, n.a. | Negative |
LCMPC02 | 0.0 | 0.3 | 0.2 | n.a. | 21 + 0 | 2, dichorionic, diamniotic | Negative |
LCMPC03 | 0.4 | 1.0 | 0.1 | n.a. | 22 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC04 | −0.3 | −0.6 | 0.0 | n.a. | n.a. | 3, n.a., n.a. | negative |
LCMPC05 | 1.3 | −1.0 | −0.8 | 16.7 | 11 + 5 | 3, trichorionic, triamniotic | negative |
LCMPC06 | −0.4 | 1.1 | 8.5 | 18.0 | 13 + 2 | 2, monochorionic, n.a. | T21 positive |
LCMPC07 | −1.0 | 0.3 | 0.9 | 7.9 | 19 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC08 | 0.7 | 1.2 | 0.0 | 16.5 | 18 + 1 | 2, dichorionic, diamniotic | negative |
LCMPC09 | 0.6 | −0.8 | 0.7 | 8.9 | 11 + 5 | 2, monochorionic, diamniotic | negative |
LCMPC10 | 0.3 | 0.7 | −0.7 | 17.6 | 20 + 4 | 2, dichorionic, diamniotic | negative |
LCMPC11 | −0.9 | −0.8 | 0.7 | 11.5 | 23 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC12 | −0.9 | −0.7 | −2.0 | 13.3 | 11 + 1 | 2, monochorionic, diamniotic | negative |
LCMPC13 | 1.3 | 0.1 | 0.3 | 21.4 | 16 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC14 | 0.2 | −0.3 | 0.0 | 6.8 | 12 + 5 | 2, n.a., n.a. | negative |
LCMPC15 | 2.2 | 0.1 | 14.7 | 24.8 | 16 + 0 | 2, dichorionic, diamniotic | T21 positive |
LCMPC16 | 1.1 | 1.7 | 0.5 | 5.4 | 12 + 5 | 2, n.a., n.a. | negative |
LCMPC17 | 0.7 | 1.4 | 0.5 | 16.5 | 14 + 2 | 2, n.a., n.a. | negative |
LCMPC18 | 0.3 | 2.6 | 0.0 | 18.5 | 18 + 3 | 2, n.a., n.a. | negative |
LCMPC19 | −0.2 | 0.8 | 0.3 | 16.6 | 14 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC20 | −0.7 | −0.9 | 0.1 | 13.1 | 15 + 4 | 2, dichorionic, diamniotic | negative |
LCMPC21 | 1.0 | −0.7 | 1.2 | 8.4 | 9 + 3 | 2, dichorionic, diamniotic | negative |
LCMPC22 | −1.1 | −0.2 | 0.3 | 5.6 | 16 + 2 | 2, monochorionic, n.a. | negative |
LCMPC23 | −2.2 | 2.2 | −0.8 | 20.6 | 19 + 5 | 2, monochorionic, n.a. | negative |
LCMPC24 | −1.6 | −0.4 | −0.5 | 14.7 | 22 + 2 | 2, monochorionic, diamniotic | negative |
LCMPC25 | −0.8 | −0.2 | −1.5 | 12.1 | 11 + 5 | 2, n.a., n.a. | negative |
LCMPC26 | −0.4 | −0.6 | −1.3 | 7.5 | 13 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC27 | 0.5 | −0.8 | −0.4 | 16.3 | 12 + 6 | 2, n.a., n.a. | negative |
LCMPC28 | −1.2 | −0.3 | −0.7 | 19.4 | 10 + 1 | 2, dichorionic, diamniotic | negative |
LCMPC29 | −0.8 | 0.7 | −0.4 | 14.2 | 13 + 2 | 2, monochorionic, n.a. | negative |
LCMPC30 | 0.7 | 0.3 | 0.9 | 14.9 | 12 + 2 | 2, monochorionic, monoamniotic | negative |
LCMPC31 | −0.2 | 0.3 | −0.9 | 19.3 | 19 + 1 | 2, dichorionic, diamniotic | negative |
LCMPC32 | −1.1 | 2.5 | −2.2 | 11.6 | 20 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC33 | 0.2 | 2.2 | −1.6 | 8.6 | 11 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC34 | −1.0 | 1.2 | 0.0 | 15.1 | 15 + 4 | 2, dichorionic, diamniotic | negative |
LCMPC35 | −0.3 | −0.8 | −0.3 | 19.2 | 12 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC36 | −1.4 | −0.5 | −0.8 | 13.9 | 12 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC37 | 1.8 | −0.7 | 0.1 | 13.8 | 17 + 6 | 2, dichorionic, diamniotic | negative |
LCMPC38 | −0.1 | 1.1 | −0.7 | 13.4 | 13 + 1 | 2, dichorionic, diamniotic | negative |
LCMPC39 | −1.9 | 0.2 | −2.2 | 15.0 | 17 + 0 | 2, dichorionic, diamniotic | negative |
LCMPC40 | 0.6 | −0.4 | 0.8 | 16.2 | 18 + 3 | 2, dichorionic, diamniotic | negative |
3.3. Minimum Fetal Fraction Needed for the Detection of Aneuploidies in Multiple Pregnancies
3.4. Case Reports of Discordant NIPT Results Due to Vanishing Twins
Sample code | Sample type | Gestational age | Total reads (×106) | Chr13 z-score | Chr18 z-score | Chr21 z-score | % cffDNA calculation by ChromRep Y | Fetal fraction (QuantYfeX) |
---|---|---|---|---|---|---|---|---|
VTA01 | maternal plasma sample | 17 + 2 | 14.94 | 1.3 | −1.5 | 13.5 | 9.2 | 20.7 |
VTA02 | Back-up sample | 17 + 2 | 17.08 | 0.4 | −1.7 | 11.1 | 9.3 | 24.8 |
VTA03 | maternal plasma sample collected prior birth | 38 + 2 | 17.76 | 0.8 | 1.9 | −0.3 | 21.7 | 21.4 |
VTB01 | initial maternal plasma sample | 13 + 2 | 16.79 | −0.2 | 0.1 | 3.4 | 3.0 | 13.4 |
VTB02 | Back-up sample | 13 + 2 | 11.97 | −0.1 | 0.3 | 2.6 | 2.7 | 10.0 |
4. Conclusions
Appendix
Component | Sequence | Concentration | Final concentration (μM) | 1× μL | |||||
---|---|---|---|---|---|---|---|---|---|
master mix | 2× | 1× | 12.5 | ||||||
RASSF1 sens/insens | primer for p0009 | ATT GAG CTG CGG GAG CTG GC | 100 μM | 1.4 | 0.35 | ||||
primer re p0010 | TGC CGT GTG GGG TTG CAC | 100 μM | 1.4 | 0.35 | |||||
probe s0001 | FAM-ACC CGG CTG GAG CGT-MGB | 100 μM | 0.14 | 0.035 | |||||
primer for p0003 | GGT CAT CCA CCA CCA AGA AC | 100 μM | 1.4 | 0.35 | |||||
primer re p0004 | TGC CCA AGG ATG CTG TCA AG | 100 μM | 1.4 | 0.35 | |||||
probe s0002 | VIC-GGG CCT CAA TGA CTT CAC GT-MGB | 100 μM | 0.14 | 0.035 | |||||
TBX3 sens/insens | primer for p0011 | GGT GCG AAC TCC TCT TTG TC | 100 μM | 1.4 | 0.35 | ||||
primer re p0012 | TTA ATC ACC CAG CGC ATG GC | 100 μM | 1.4 | 0.35 | |||||
probe s0010 | 6FAM-CCC TCC CGG TGG GTG ATA AA-MGBNFQ | 100 μM | 0.14 | 0.035 | |||||
Primer for p0021 | TGT TCA CTG GAG GAC TCA TC | 100 μM | 1.4 | 0.35 | |||||
primer re p0022 | CAG TCC ATG AGG GTG TTT G | 100 μM | 1.4 | 0.35 | |||||
probe s0011 | VIC-GAG GTC CCA TTC TCC TTT-MGBNFQ | 100 μM | 0.14 | 0.035 | |||||
general reagents | DMSO | 100% | 0.025 | 0.625 | |||||
MgCl2 | 50 mM | 2 | 1 | ||||||
template | 10 | ||||||||
water | - | ||||||||
summary | 25 |
Temperature | Time | Cycles | Analysis mode | |
---|---|---|---|---|
pre-incubation | 95 °C | 5 min | 1 | none |
denaturation | 95 °C | 10 s | 45 | quantification |
annealing | 60 °C | 10 s | none | |
elongation | 72 °C | 8 s | single | |
cooling | 40 °C | none |
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Share and Cite
Grömminger, S.; Yagmur, E.; Erkan, S.; Nagy, S.; Schöck, U.; Bonnet, J.; Smerdka, P.; Ehrich, M.; Wegner, R.-D.; Hofmann, W.; et al. Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins. J. Clin. Med. 2014, 3, 679-692. https://doi.org/10.3390/jcm3030679
Grömminger S, Yagmur E, Erkan S, Nagy S, Schöck U, Bonnet J, Smerdka P, Ehrich M, Wegner R-D, Hofmann W, et al. Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins. Journal of Clinical Medicine. 2014; 3(3):679-692. https://doi.org/10.3390/jcm3030679
Chicago/Turabian StyleGrömminger, Sebastian, Erbil Yagmur, Sanli Erkan, Sándor Nagy, Ulrike Schöck, Joachim Bonnet, Patricia Smerdka, Mathias Ehrich, Rolf-Dieter Wegner, Wera Hofmann, and et al. 2014. "Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins" Journal of Clinical Medicine 3, no. 3: 679-692. https://doi.org/10.3390/jcm3030679
APA StyleGrömminger, S., Yagmur, E., Erkan, S., Nagy, S., Schöck, U., Bonnet, J., Smerdka, P., Ehrich, M., Wegner, R.-D., Hofmann, W., & Stumm, M. (2014). Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins. Journal of Clinical Medicine, 3(3), 679-692. https://doi.org/10.3390/jcm3030679