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Molecular Diagnostics and Genetics |
1 Department of Chemical Pathology, 2
Centre for Emerging Infectious Diseases, 4
Department of Obstetrics and Gynaecology, and 5
Li Ka Shing Institute of Health Sciences, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR.
3 Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, London, United Kingdom.
aAddress correspondence to this author at: Department of Chemical Pathology, Rm. 38023, 1/F, Clinical Sciences Bldg., Prince of Wales Hospital, 30-32 Ngan Shing St., Shatin, Hong Kong SAR. Fax 852-2636-5090; e-mail loym{at}cuhk.edu.hk.
Background: We recently demonstrated that the promoter of the RASSF1A gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion for detecting the placental-derived hypermethylated RASSF1A sequences in maternal plasma.
Methods: We performed real-time PCR after methylation-sensitive restriction enzyme digestion to detect placental-derived RASSF1A sequences in the plasma of 28 1st-trimester and 43 3rd-trimester pregnant women. We used maternal plasma to perform prenatal fetal rhesus D (RhD) blood group typing for 54 early-gestation RhD-negative women, with hypermethylated RASSF1A as the positive control for fetal DNA detection.
Results: Hypermethylated RASSF1A sequences were detectable in the plasma of all 71 pregnant women. The genotype of plasma RASSF1A after enzyme digestion was identical to the fetal genotype in each case, thus confirming its fetal origin. Nineteen of the 54 pregnant women undergoing prenatal fetal RhD genotyping showed undetectable RHD sequences in their plasma DNA samples. The fetal DNA control, RASSF1A, was not detectable in 4 of the 19 women. Subsequent chorionic villus sample analysis revealed that 2 of these 4 women with negative RHD and RASSF1A signals were in fact carrying RhD-positive fetuses.
Conclusions: Hypermethylated RASSF1A is a universal marker for fetal DNA and is readily detectable in maternal plasma. When applied to prenatal RhD genotyping, this marker allows the detection of false-negative results caused by low fetal DNA concentrations in maternal plasma. This new marker can also be applied to many other prenatal diagnostic and monitoring scenarios.
The following articles in journals at HighWire Press have cited this article:
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  T. Chu, K. Bunce, W. A. Hogge, and D. G. Peters Statistical model for whole genome sequencing and its application to minimally invasive diagnosis of fetal genetic disease Bioinformatics, May 15, 2009; 25(10): 1244 - 1250. [Abstract] [Full Text] [PDF]
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 E C W Hung, R W K Chiu, and Y M D Lo Detection of circulating fetal nucleic acids: a review of methods and applications J. Clin. Pathol., April 1, 2009; 62(4): 308 - 313. [Abstract] [Full Text] [PDF]
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 C. F. Wright and H. Burton The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis Hum. Reprod. Update, January 1, 2009; 15(1): 139 - 151. [Abstract] [Full Text] [PDF]
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 R. W. K. Chiu, K. C. A. Chan, Y. Gao, V. Y. M. Lau, W. Zheng, T. Y. Leung, C. H. F. Foo, B. Xie, N. B. Y. Tsui, F. M. F. Lun, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma PNAS, December 23, 2008; 105(51): 20458 - 20463. [Abstract] [Full Text] [PDF]
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F. M. F. Lun, N. B. Y. Tsui, K. C. A. Chan, T. Y. Leung, T. K. Lau, P. Charoenkwan, K. C. K. Chow, W. Y. W. Lo, C. Wanapirak, T. Sanguansermsri, et al. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma PNAS, December 16, 2008; 105(50): 19920 - 19925. [Abstract] [Full Text] [PDF]
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 K. C. A. Chan, P. B. S. Lai, T. S. K. Mok, H. L. Y. Chan, C. Ding, S. W. Yeung, and Y. M. D. Lo Quantitative Analysis of Circulating Methylated DNA as a Biomarker for Hepatocellular Carcinoma Clin. Chem., September 1, 2008; 54(9): 1528 - 1536. [Abstract] [Full Text] [PDF]
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S. S.C. Chim, S. Jin, T. Y.H. Lee, F. M.F. Lun, W. S. Lee, L. Y.S. Chan, Y. Jin, N. Yang, Y. K. Tong, T. Y. Leung, et al. Systematic Search for Placental DNA-Methylation Markers on Chromosome 21: Toward a Maternal Plasma-Based Epigenetic Test for Fetal Trisomy 21 Clin. Chem., March 1, 2008; 54(3): 500 - 511. [Abstract] [Full Text] [PDF]
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Y. M. Dennis Lo and R. W. K. Chiu Noninvasive Prenatal Diagnosis of Fetal Chromosomal Aneuploidies by Maternal Plasma Nucleic Acid Analysis Clin. Chem., March 1, 2008; 54(3): 461 - 466. [Abstract] [Full Text] [PDF]
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S. S.C. Chim, T. K.F. Shing, E. C.W. Hung, T.-y. Leung, T.-k. Lau, R. W.K. Chiu, and Y.M. Dennis Lo Detection and Characterization of Placental MicroRNAs in Maternal Plasma Clin. Chem., March 1, 2008; 54(3): 482 - 490. [Abstract] [Full Text] [PDF]
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Y. K. Tong, R. W.K. Chiu, T. Y. Leung, C. Ding, T. K. Lau, T. N. Leung, and Y.M. D. Lo Detection of Restriction Enzyme Digested Target DNA by PCR Amplification Using a Stem-Loop Primer: Application to the Detection of Hypomethylated Fetal DNA in Maternal Plasma Clin. Chem., November 1, 2007; 53(11): 1906 - 1914. [Abstract] [Full Text] [PDF]
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Y. M. D. Lo, F. M. F. Lun, K. C. A. Chan, N. B. Y. Tsui, K. C. Chong, T. K. Lau, T. Y. Leung, B. C. Y. Zee, C. R. Cantor, and R. W. K. Chiu From the Cover: Digital PCR for the molecular detection of fetal chromosomal aneuploidy PNAS, August 7, 2007; 104(32): 13116 - 13121. [Abstract] [Full Text] [PDF]
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F. M.F. Lun, R. W.K. Chiu, T. Y. Leung, T. N. Leung, T. K. Lau, and Y.M. D. Lo Epigenetic Analysis of RASSF1A Gene in Cell-Free DNA in Amniotic Fluid Clin. Chem., April 1, 2007; 53(4): 796 - 798. [Full Text] [PDF]
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 R. W.K. Chiu, S. S.C. Chim, I. H.N. Wong, C. S.C. Wong, W.-S. Lee, K. F. To, J. H.M. Tong, R. K.C. Yuen, A. S.W. Shum, J. K.C. Chan, et al. Hypermethylation of RASSF1A in Human and Rhesus Placentas Am. J. Pathol., March 1, 2007; 170(3): 941 - 950. [Abstract] [Full Text] [PDF]
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