Abstract
This study was carried out to establish a non-invasive prenatal diagnosis method for phenylketonuria (PKU) based on droplet digital PCR (ddPCR) and to evaluate its accuracy by comparison with conventional invasive diagnostic methods. A total of 24 PKU pedigrees that required prenatal diagnosis were studied, in which the genetic mutations in the probands and parents were unambiguous. Prenatal diagnosis of sibling fetuses was performed using traditional invasive prenatal diagnostic methods as a standard. At the same time, cell-free DNA (cfDNA) was extracted from maternal plasma and the fetal genes contained within were typed and quantified using ddPCR method. Invasive prenatal diagnosis determined that 3 of the 24 fetuses were affected, 8 of them were normal, and 13 were heterozygous carriers of pathogenic mutations. Successful non-invasive prenatal diagnosis analysis of PAH gene mutations was performed for 8 of the families using ddPCR method. Non-invasive prenatal diagnosis results were consistent with the results of the invasive prenatal diagnoses and no false positive or false negative results were found. In conclusion, this study is the first to establish non-invasive prenatal diagnosis of PKU based on ddPCR. The method showed high sensitivity and specificity from cfDNA, indicating that ddPCR is a reliable non-invasive prenatal diagnosis tool for PKU diagnosis.
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References
Evans MI, Wapner RJ. Invasive prenatal diagnostic procedures 2005. Semin Perinatol. 2005;29(4):215–8. https://doi.org/10.1053/j.semperi.2005.06.004.
Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997;350(9076):485–7. https://doi.org/10.1016/S0140-6736(97)02174-0.
Daley R, Hill M, Chitty LS. Non-invasive prenatal diagnosis: progress and potential. Arch Dis Child Fetal Neonatal Ed. 2014;99(5):F426–30. https://doi.org/10.1136/archdischild-2013-304828.
Norton ME, Jacobsson B, Swamy GK, Laurent LC, Ranzini AC, Brar H, et al. Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med. 2015;372(17):1589–97. https://doi.org/10.1056/NEJMoa1407349.
Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem. 2011;83(22):8604–10. https://doi.org/10.1021/ac202028g.
Pekin D, Skhiri Y, Baret JC, Le Corre D, Mazutis L, Salem CB, et al. Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. Lab Chip. 2011;11(13):2156–66. https://doi.org/10.1039/c1lc20128j.
Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA. Evaluation of digital PCR for absolute DNA quantification. Anal Chem. 2011;83(17):6474–84. https://doi.org/10.1021/ac103230c.
Whale AS, Huggett JF, Cowen S, Speirs V, Shaw J, Ellison S, et al. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res. 2012;40(11):e82. https://doi.org/10.1093/nar/gks203.
Castellanos-Rizaldos E, Paweletz C, Song C, Oxnard GR, Mamon H, Janne PA, et al. Enhanced ratio of signals enables digital mutation scanning for rare allele detection. J Mol Diagn. 2015;17(3):284–92. https://doi.org/10.1016/j.jmoldx.2014.12.003.
Sanmamed MF, Fernandez-Landazuri S, Rodriguez C, Zarate R, Lozano MD, Zubiri L, et al. Quantitative cell-free circulating BRAFV600E mutation analysis by use of droplet digital PCR in the follow-up of patients with melanoma being treated with BRAF inhibitors. Clin Chem. 2015;61(1):297–304. https://doi.org/10.1373/clinchem.2014.230235.
Debrand E, Lykoudi A, Bradshaw E, Allen SK. A non-invasive droplet digital PCR (ddPCR) assay to detect paternal CFTR mutations in the cell-free fetal DNA (cffDNA) of three pregnancies at risk of cystic fibrosis via compound heterozygosity. PLoS One. 2015;10(11):e0142729. https://doi.org/10.1371/journal.pone.0142729.
Camunas-Soler J, Lee H, Hudgins L, Hintz SR, Blumenfeld YJ, El-Sayed YY, et al. Noninvasive prenatal diagnosis of single-gene disorders by use of droplet digital PCR. Clin Chem. 2018;64(2):336–45. https://doi.org/10.1373/clinchem.2017.278101.
Lv W, Wei X, Guo R, Liu Q, Zheng Y, Chang J, et al. Noninvasive prenatal testing for Wilson disease by use of circulating single-molecule amplification and resequencing technology (cSMART). Clin Chem. 2015;61(1):172–81. https://doi.org/10.1373/clinchem.2014.229328.
Orhant L, Anselem O, Fradin M, Becker PH, Beugnet C, Deburgrave N, et al. Droplet digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia. Prenat Diagn. 2016;36(5):397–406. https://doi.org/10.1002/pd.4790.
Perlado S, Bustamante-Aragones A, Donas M, Lorda-Sanchez I, Plaza J, Rodriguez de Alba M. Fetal genotyping in maternal blood by digital PCR: towards NIPD of monogenic disorders independently of parental origin. PLoS One. 2016;11(4):e0153258. https://doi.org/10.1371/journal.pone.0153258.
Li N, Jia H, Liu Z, Tao J, Chen S, Li X, et al. Molecular characterisation of phenylketonuria in a Chinese mainland population using next-generation sequencing. Sci Rep. 2015;5:15769. https://doi.org/10.1038/srep15769.
Manokhina I, Singh TK, Penaherrera MS, Robinson WP. Quantification of cell-free DNA in normal and complicated pregnancies: overcoming biological and technical issues. PLoS One. 2014;9(7):e101500. https://doi.org/10.1371/journal.pone.0101500.
Lun FM, Chiu RW, Chan KC, Leung TY, Lau TK, Lo YM. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem. 2008;54(10):1664–72. https://doi.org/10.1373/clinchem.2008.111385.
Illanes S, Denbow M, Kailasam C, Finning K, Soothill PW. Early detection of cell-free fetal DNA in maternal plasma. Early Hum Dev. 2007;83(9):563–6. https://doi.org/10.1016/j.earlhumdev.2006.11.001.
Andersen RF, Spindler KL, Brandslund I, Jakobsen A, Pallisgaard N. Improved sensitivity of circulating tumor DNA measurement using short PCR amplicons. Clin Chim Acta. 2015;439:97–101. https://doi.org/10.1016/j.cca.2014.10.011.
Funding
This study was supported by the National Key Research and Development Program of China (Grant Nos. 2016YFC1000307, 2018YFC1002201) and Natural Science Foundation of Gansu Province (Grant No. 18JR3RA036).
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Yan, Y., Wang, F., Zhang, C. et al. Evaluation of droplet digital PCR for non-invasive prenatal diagnosis of phenylketonuria. Anal Bioanal Chem 411, 7115–7126 (2019). https://doi.org/10.1007/s00216-019-02087-4
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DOI: https://doi.org/10.1007/s00216-019-02087-4