The current applications of cell-free fetal DNA in prenatal diagnosis of single-gene diseases: A review


Prenatal diagnosis of hereditary diseases has substantially altered the way medical geneticists are helping families affected by genetic disorders. However, the risk of miscarriage and fear of invasive diagnostic procedures may discourage many couples from seeking prenatal diagnosis. With the discovery of maternal plasma cell-free fetal DNA, prenatal diagnosis has entered a new era of progress. Cell-free DNA is released during normal physiological functions as well as through the cell death programs of apoptosis and necrosis. It can be found in the plasma and other body fluids. Although this method has the advantage of being noninvasive, it is still rather expensive and requires advanced hardware and comprehensive data analysis. Promising implications of noninvasive prenatal diagnosis methods for the diagnosis of common trisomy disorders have paved the way for the development of more complicated assays of single-gene disorders. Relative mutation dosage and relative haplotype dosage are the most widely implemented assays for noninvasive prenatal diagnosis of single-gene disorders. However, each assay has its own advantages and disadvantages. Relative mutation dosage is based on the droplet digital polymerase chain reaction (PCR) technique which includes quantification features of real-time PCR assays. Relative haplotype dosage is based on next-generation sequencing that includes analysis of the maternal and paternal genome followed by sequencing of maternal plasma cell-free DNA. Co-amplification at a lower denaturation temperature PCR is another approach that is based on forming heteroduplexes between alleles to selectively amplify paternal mutations. In this review, we have described the most common noninvasive prenatal diagnosis approaches and compared their applications in genetic disorder diagnosis with different inheritance patterns.

Key words: Cell-free nucleic acids, Prenatal diagnosis, Noninvasive prenatal testing, Single-gene diseases, Non-invasive techniques.

[1] Alborelli I, Generali D, Jermann P, Cappelletti MR, Ferrero G, Scaggiante B, et al. Cell-free DNA analysis in healthy individuals by next-generation sequencing: A proof of concept and technical validation study. Cell Death Dis 2019; 10: 534.

[2] Stewart CM, Tsui DWY. Circulating cell-free DNA for non-invasive cancer management. Cancer Genet 2018; 228–229: 169–179.

[3] Fiala C, Diamandis EP. New approaches for detecting cancer with circulating cell-free DNA. BMC Med 2019; 17: 159.

[4] Shahrabi Farahani M, Shahbazi Sh, Amini Moghaddam S, Mahdian R. Evaluation of KRAS gene expression and LCS6 variant in genomic and cell-free DNA of Iranian women with endometriosis. Reprod Sci 2015; 22: 679–684.

[5] Bustamante-Aragones A, Rodriguez de Alba M, Perlado S, Trujillo-Tiebas MJ, Arranz JP, DiazRecasens J, et al. Non-invasive prenatal diagnosis of single-gene disorders from maternal blood. Gene 2012; 504: 144–149.

[6] 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: 485–487.

[7] Tounta G, Kolialexi A, Papantoniou N, Tsangaris GT, Kanavakis E, Mavrou A. Non-invasive prenatal diagnosis using cell-free fetal nucleic acids in maternal plasma: Progress overview beyond predictive and personalized diagnosis. EPMA J 2011; 2: 163–171.

[8] Benn P. Non-invasive prenatal testing using cell free DNA in maternal plasma: Recent developments and future prospects. J Clin Med 2014; 3: 537–565.

[9] Benn P, Borrell A, Chiu RW, Cuckle H, Dugoff L, Faas B, et al. Position statement from the chromosome abnormality screening committee on behalf of the board of the international society for prenatal diagnosis. Prenat Diagn 2015; 35: 725–734.

[10] Lo YMD. Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis: A review of the current state of the art. BJOG 2009; 116: 152–157.

[11] Allen S, Young E, Bowns B. Noninvasive prenatal diagnosis for single gene disorders. Curr Opin Obstet Gynecol 2017; 29: 73–79.

[12] van den Oever JME, van Minderhout IJHM, Harteveld CL, den Hollander NS, Bakker E, van der Stoep N, et al. A novel targeted approach for noninvasive detection of paternally inherited mutations in maternal plasma. J Mol Diagn 2015; 17: 590–596.

[13] Saito H, Sekizawa A, Morimoto T, Suzuki M, Yanaihara T. Prenatal DNA diagnosis of a single-gene disorder from maternal plasma. Lancet 2000; 356: 1170.

[14] Li Y, Page-Christiaens GCML, Gille JJP, Holzgreve W, Hahn S. Non-invasive prenatal detection of achondroplasia in size-fractionated cell-free DNA by MALDI-TOF MS assay. Prenat Diagn 2007; 27: 11–17.

[15] Lim JH, Kim MJ, Kim SY, Kim HO, Song MJ, Kim MH, et al. Non-invasive prenatal detection of achondroplasia using circulating fetal DNA in maternal plasma. J Assist Reprod Genet 2011; 28: 167–172.

[16] Chitty LS, Mason S, Barrett AN, McKay F, Lench N, Daley R, et al. Non-invasive prenatal diagnosis of achondroplasia and thanatophoric dysplasia: Next-generation sequencing allows for a safer, more accurate, and comprehensive approach. Prenat Diagn 2015; 35: 656–662.

[17] 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: 397–406.

[18] Vivanti AJ, Costa JM, Rosefort A, Kleinfinger P, Lohmann L, Cordier AG, et al. Optimal noninvasive diagnosis of fetal achondroplasia combining ultrasonography with circulating cell-free fetal DNA analysis. Ultrasound Obstet Gynecol 2019; 53: 87–94.

[19] Gonzalez-Gonzalez MC, Trujillo MJ, Rodriguez de Alba M, Garcia-Hoyos M, Lorda-Sanchez I, DiazRecasens J, et al. Huntington disease-unaffected fetus diagnosed from maternal plasma using QF-PCR. Prenat Diagn 2003; 23: 232–234.

20] Gonzalez-Gonzalez MC, Garcia-Hoyos M, TrujilloTiebas MJ, Bustamante Aragones A, Rodriguez de Alba M, Diego Alvarez D, et al. Improvement in strategies for the non-invasive prenatal diagnosis of Huntington disease. J Assist Reprod Genet 2008; 25: 477–481.

[21] Amicucci P, Gennarelli M, Novelli G, Dallapiccola B. Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem 2000; 46: 301–302.

[22] 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: 336–345.

[23] Hill M, Twiss P, Verhoef TI, Drury S, McKay F. Non-invasive prenatal diagnosis for cystic fibrosis: Detection of paternal mutations, exploration of patient preferences and cost analysis. Prenat Diagn 2015; 35: 950–958.

[24] Nasis O, Thompson S, Hong T, Sherwood M, Radcliffe S, Jackson L, et al. Improvement in sensitivity of allelespecific PCR facilitates reliable noninvasive prenatal detection of cystic fibrosis. Clin Chem 2004; 50: 694–701.

[25] Bustamante-Aragones A, Gallego-Merlo J, TrujilloTiebas MJ, de Alba MR, Gonzalez-Gonzalez C, Glover G, et al. New strategy for the prenatal detection/exclusion of paternal cystic fibrosis mutations in maternal plasma. J Cyst Fibros 2008; 7: 505–510.

[26] Guissart C, Debant V, Desgeorges M, Bareil C, Raynal C, Toga C, et al. Non-invasive prenatal diagnosis of monogenic disorders: An optimized protocol using MEMO qPCR with miniSTR as internal control. Clin Chem Lab Med 2015; 53: 205–215.

[27] Kanagal-Shamanna R. Digital PCR: Principles and applications. Methods Mol Biol 2016; 1392: 43–50.

[28] Shahbazi Sh, Baniahmad F, Zakiani-Roudsari M, Raigani M, Mahdian R. Nonsense-mediated decay of VWF mRNA subsequent to c.7674-7675insC mutation in type3 VWD patients. Blood Cells Mol Dis 2012; 49: 48–52.

[29] Lam KWG, Jiang P, Liao GJW, Chan KCA, Leung TY, Chiu RWK, et al. Noninvasive prenatal diagnosis of monogenic diseases by targeted massively parallel sequencing of maternal plasma: Application to betathalassemia. Clin Chem 2012; 58: 1467–1475.

[30] Wong FCK, Lo YMD. Prenatal diagnosis innovation: Genome sequencing of maternal plasma. Annu Rev Med 2016; 67: 419–432.

[31] Vermeulen C, Geeven G, de Wit E, Verstegen M, Jansen RPM, van Kranenburg M, et al. Sensitive monogenic noninvasive prenatal diagnosis by targeted haplotyping. Am J Hum Genet 2017; 101: 326–339.

[32] Jang SS, Lim BC, Yoo SK, Shin JY, Seo JS, Hwang D, et al. Development of a common platform for the noninvasive prenatal diagnosis of X-linked diseases. Prenat Diagn 2018; 38: 835–840.

[33] Kolialexi A, Tounta G, Apostolou P, Vrettou C, Papantoniou N, Kanavakis E, et al. Early non-invasive detection of fetal Y chromosome sequences in maternal plasma using multiplex PCR. Eur J Obstet Gynecol Reprod Biol 2012; 161: 34–37.

[34] Rong Y, Gao J, Jiang X, Zheng F. Multiplex PCR for 17 Y-chromosome specific short tandem repeats (STR) to enhance the reliability of fetal sex determination in maternal plasma. Int J Mol Sci 2012; 13: 5972–5981.

[35] D’Aversa E, Breveglieri G, Pellegatti P, Guerra G, Gambari R, Borgatti M. Non-invasive fetal sex diagnosis in plasma of early weeks pregnants using droplet digital PCR. Mol Med 2018; 24: 14.

[36] Colmant C, Morin-Surroca M, Fuchs F, Fernandez H, Senat MV. Non-invasive prenatal testing for fetal sex determination: Is ultrasound still relevant? Eur J Obstet Gynecol Reprod Biol 2013; 171: 197–204.

[37] Perlado S, Bustamante-Aragones A, Donas M, LordaSanchez 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: e0153258.

[38] Byrou S, Makrigiorgos GM, Christofides A, Kallikas I, Papasavva T, Kleanthous M. Fast temperature-gradient COLD PCR for the enrichment of the paternally inherited SNPs in cell-free fetal DNA; an application to non-invasive prenatal diagnosis of betathalassaemia. PLoS One 2018; 13: e0200348.

[39] Galbiati S, Monguzzi A, Damin F, Soriani N, Passiu M, Castellani C, et al. COLD-PCR and microarray: Two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma. J Med Genet 2016; 53: 481–487.

[40] Maryami F, Mahdian R, Jamali S, Karimi Arzanani M, Khatami Sh, Maryami F, et al. Comparisons between RT-PCR, Real-time PCR, and in vitro globin chain synthesis by α/β ratio calculation for diagnosis of αfrom β thalassemia carriers. Arch Iran Med 2013; 16: 217–220.

[41] Babashah S, Jamali S, Mahdian R, Hayat Nosaeid M, Karimipoor M, Alimohammadi R, et al. Detection of unknown deletions in beta-globin gene cluster using relative quantitative PCR methods. Eur J Haematol 2009; 83: 261–269.

[42] Nouri Inanlou D, Yakhchali B, Khanahmad H, Gardaneh M, Movassagh H, Ahangari Cohan R, et al. Towards β-globin gene-targeting with integrasedefective lentiviral vectors. Biotechnol Lett 2010; 32: 1615–1621.

[43] Hudecova I, Chiu RWK. Non-invasive prenatal diagnosis of thalassemias using maternal plasma cell free DNA. Best Pract Res Clin Obstet Gynaecol 2017; 39: 63–73.

[44] Lun FMF, Tsui NBY, Chan KCA, Leung TY, Lau TK, Charoenkwan P, et al. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma. Proc Natl Acad Sci USA 2008; 105: 19920–19925.

[45] Wang W, Yuan Y, Zheng H, Wang Y, Zeng D, Yang Y, et al. A pilot study of noninvasive prenatal diagnosis of alpha- and beta-thalassemia with target capture sequencing of cell-free fetal DNA in maternal blood. Genet Test Mol Biomarkers 2017; 21: 433–439.

[46] Yenilmez ED, Tuli A, Evruke IC. Noninvasive prenatal diagnosis experience in the Cukurova region of southern Turkey: Detecting paternal mutations of sickle cell anemia and beta-thalassemia in cell-free fetal DNA using high-resolution melting analysis. Prenat Diagn 2013; 33: 1054–1062.

[47] Saba L, Masala M, Capponi V, Marceddu G, Massidda M, Rosatelli MC. Non-invasive prenatal diagnosis of beta-thalassemia by semiconductor sequencing: A feasibility study in the sardinian population. Eur J Hum Genet 2017; 25: 600–607.

[48] Mortazavipour MM, Shahbazi Sh, Mahdian R. Detection of paternal IVS-II-1 (G>A) (HBB: c.315+1G>A) mutation in cell-free fetal DNA using COLD-PCR assay. Hemoglobin 2020; 44: 168–173.

[49] Li Y, Di Naro E, Vitucci A, Zimmermann B, Holzgreve W, Hahn S. Detection of paternally inherited fetal point mutations for beta-thalassemia using sizefractionated cell-free DNA in maternal plasma. JAMA 2005; 293: 843–849.

[50] Phylipsen M, Yamsri S, Treffers EE, Jansen DT, Kanhai WA, Boon EM, et al. Non-invasive prenatal diagnosis of beta-thalassemia and sickle-cell disease using pyrophosphorolysis-activated polymerization and melting curve analysis. Prenat Diagn 2012; 32: 578–587.

[51] Papasavva Th, Kalikas I, Kyrri A, Kleanthous M. Arrayed primer extension for the noninvasive prenatal diagnosis of beta-thalassemia based on detection of single nucleotide polymorphisms. Ann N Y Acad Sci 2008; 1137: 302–308.

[52] Yi P, Chen Zh, Yu L, Zheng Y, Liu G, Xie H, et al. Development of a PCR/LDR/capillary electrophoresis assay with potential for the detection of a betathalassemia fetal mutation in maternal plasma. J Matern Fetal Neonatal Med 2010; 23: 920–927.

[53] Yi P, Chen Zh, Yu L, Zheng Y, Xie H, Zheng X, et al. Prenatal detection of beta-thalassemia CD17 (A– >T) mutation by polymerase chain reaction/ligase detection reaction/capillary electrophoresis for fetal DNA in maternal plasma: A case report. Fetal Diagn Ther 2010; 27: 25-31.

[54] Chiu RWK, Lau TK, Leung TN, Chow KCK, Chui DHK, Lo YMD. Prenatal exclusion of beta thalassaemia major by examination of maternal plasma. Lancet 2002; 360: 998-1000.

[55] Li Y, Di Naro E, Vitucci A, Grill S, Zhong XY, Holzgreve W, et al. Size fractionation of cell-free DNA in maternal plasma improves the detection of a paternally inherited beta-thalassemia point mutation by MALDI-TOF mass spectrometry. Fetal Diagn Ther 2009; 25: 246–249.

[56] Ramezanzadeh M, Salehi M, Farajzadegan Z, Kamali S, Salehi R. Detection of paternally inherited fetal point mutations for beta-thalassemia in maternal plasma using simple fetal DNA enrichment protocol with or without whole genome amplification: an accuracy assessment. J Matern Fetal Neonatal Med 2016; 29: 2645–2649.

[57] Chan K, Yam I, Leung KY, Tang M, Chan TK, Chan V. Detection of paternal alleles in maternal plasma for non-invasive prenatal diagnosis of beta-thalassemia: A feasibility study in southern Chinese. Eur J Obstet Gynecol Reprod Biol 2010; 150: 28–33.