Non-invasive prenatal diagnosis of trisomy 21 by dosage ratio of fetal chromosome-specific epigenetic markers in maternal plasma

Abstract

This study examined the methylation difference in AIRE and RASSF1A between maternal and placental DNA, and the implication of this difference in the identification of free fetal DNA in maternal plasma and in prenatal diagnosis of trisomy 21. Maternal plasma samples were collected from 388 singleton pregnancies, and placental or chorionic villus tissues from 112 of them. Methylation-specific PCR (MSP) and methylation-sensitive restriction enzyme digestion followed by fluorescent quantitative PCR (MSRE + PCR) were employed to detect the maternal-fetal methylation difference in AIRE and RASSF1A. Diagnosis of trisomy 21 was established according to the ratio of fetal-specific AIRE to RASSF1A in maternal plasma. Both methods confirmed that AIRE and RASSF1A were hypomethylated in maternal blood cells but hypermethylated in placental or chorionic villus tissues. Moreover, the differential methylation for each locus could be seen during the whole pregnant period. The positive rates of fetal AIRE and RASSF1A in maternal plasma were found to be 78.1% and 82.1% by MSP and 94.8% and 96.9% by MSRE + PCR. MSRE + PCR was superior to MSP in the identification of fetal-specific hypermethylated sequences (P<0.05). Based on the data from 266 euploidy pregnancies, the 95% reference interval of the fetal AIRE/RASSF1A ratio in maternal plasma was 0.33–1.77, which was taken as the reference value for determining the numbers of fetal chromosome 21 in 102 pregnancies. The accuracy rate in 98 euploidy pregnancies was 96.9% (95/98). Three of the four trisomy 21 pregnancies were confirmed with this method. It was concluded that hypermethylated AIRE and RASSF1A may serve as fetal-specific markers for the identification of fetal DNA in maternal plasma and may be used for noninvasive prenatal diagnosis of trisomy 21.

Keywords

fetal DNA / differential methylation / AIRE / RASSF1A / non-invasive prenatal diagnosis

References

[1] PapageorgiouE.A., FieglerH., RakyanV., et al.. Sites of differential DNA methylation between placenta and peripheral blood: molecular markers for noninvasive prenatal diagnosis of aneuploidies. Am J Pathol, 2009, 174(5): 1609-1618

[2] DanielsG., FinningK., MartinP.. Noninvasive fetal blood grouping: present and future. Clin Lab Med, 2010, 30(2): 431-442

[3] VlkováB., SzemesT., MinárikG., et al.. Advances in the research of fetal DNA in maternal plasma for noninvasive prenatal diagnostics. Med Sci Monit, 2010, 16(4): RA85-91

[4] TsuiN.B., KadirR.A., ChanK.C., et al.. Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA. Blood, 2011, 117(13): 3684-3691

[5] TsuiD.W., ChiuR.W., LoY.D.. Epigenetic approaches for the detection of fetal DNA in maternal plasma. Chimerism, 2010, 1(1): 30-35

[6] NygrenA.O., DeanJ., JensenT.J., et al.. Quantification of fetal DNA by use of methylation-based DNA discrimination. Clin Chem, 2010, 56(10): 1627-1635

[7] Della RagioneF., MastrovitoP., CampanileC., et al.. Differential DNA methylation as a tool for noninvasive prenatal diagnosis (NIPD) of X chromosome aneuploidies. J Mol Diagn, 2010, 12(6): 797-807

[8] ChanK.C., DingC., GerovassiliA., et al.. Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem, 2006, 52(12): 2211-2218

[9] Bustamante-AragonesA., Gonzalez-GonzalezC., de AlbaM.R., et al.. Noninvasive prenatal diagnosis using ccffDNA in maternal blood: state of the art. Expert Rev Mol Diagn, 2010, 10(2): 197-205

[10] ZimmermannB.G., MaddocksD.G., AventN.D.. Quantification of circulatory fetal DNA in the plasma of pregnant women. Methods Mol Biol, 2008, 444: 219-229

[11] PuszykW.M., CreaF., OldR.W.. Noninvasive prenatal diagnosis of aneuploidy using cell-free nucleic acids in maternal blood: promises and unanswered questions. Prenat Diagn, 2008, 28(1): 1-6

[12] Bustamante-AragonesA., Rodriguez de AlbaM., Gonzalez-GonzalezC., et al.. Foetal sex determination in maternal blood from the seventh week of gestation and its role in diagnosing haemophilia in the foetuses of female carriers. Haemophilia, 2008, 14(3): 593-598

[13] GutensohnK., MüllerS.P., ThomannK., et al.. Diagnostic accuracy of noninvasive polymerase chain reaction testing for the determination of fetal rhesus C, c and E status in early pregnancy. BJOG, 2010, 117(6): 722-729

[14] LoY.M., ChiuR.W.. Noninvasive approaches to prenatal diagnosis of hemoglobinopathies using fetal DNA in maternal plasma. Hematol Oncol Clin North Am, 2010, 24(6): 1179-1186

[15] LunF.M., TsuiN.B., ChanK.C., 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(50): 19920-19925

[16] BellidoM.L., RadpourR., LapaireO., et al.. MALDI-TOF mass array analysis of RASSF1A and SERPINB5 methylation patterns in human placenta and plasma. Biol Reprod, 2010, 82(4): 745-750

[17] ChuT., BurkeB., BunceK., et al.. A microarray-based approach for the identification of epigenetic biomarkers for the noninvasive diagnosis of fetal disease. Prenat Diagn, 2009, 29(11): 1020-1030

[18] TsuiD.W., LamY.M., LeeW.S., et al.. Systematic identification of placental epigenetic signatures for the noninvasive prenatal detection of Edwards syndrome. PLoS One, 2010, 5(11): e15069

[19] LoY.M.. 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(2): 152-157

[20] AventN.D., MadgettT.E., MaddocksD.G., et al.. Cell-free fetal DNA in the maternal serum and plasma: current and evolving applications. Curr Opin Obstet Gynecol, 2009, 21(2): 175-179

[21] ChiuR.W., ChimS.S., WongI.H., et al.. Hypermethylation of RASSF1A in human and rhesus placentas. Am J Pathol, 2007, 170(3): 941-950

[22] OldR.W., CreaF., PuszykW., et al.. Candidate epigenetic biomarkers for non-invasive prenatal diagnosis of Down syndrome. Reprod Biomed Online, 2007, 15(2): 227-235

[23] AlberryM., MaddocksD., JonesM., et al.. Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast. Prenat Diagn, 2007, 27(5): 415-418

[24] HuangQ., BaumL., HuangJ.F., et al.. Isolation and enrichment of human genomic CpG islands by methylation-sensitive mirror orientation selection. Anal Biochem, 2007, 365(2): 153-164

[25] TangD.L., LiY., ZhouX., et al.. Multiplex fluorescent PCR for noninvasive prenatal detection of fetal-derived paternally inherited diseases using circulatory fetal DNA in maternal plasma. Eur J Obstet Gynecol Reprod Biol, 2009, 144(1): 35-39