Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis.
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.
Chan
K C Allen
KC
Department of Chemical Pathology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR.
Ding
Chunming
C
Gerovassili
Ageliki
A
Yeung
Sze W
SW
Chiu
Rossa W K
RW
Leung
Tse N
TN
Lau
Tze K
TK
Chim
Stephen S C
SS
Chung
Grace T Y
GT
Nicolaides
Kypros H
KH
Lo
Y M Dennis
YM
D000199:Actins / Q000032:analysis / Q000097:blood / Q000235:genetics; D004247:DNA / Q000032:analysis / Q000097:blood*; D019175:DNA Methylation; D005188:False Negative Reactions; D005260:Female; D005333:Fetus; D005819:Genetic Markers; D005838:Genotype; D006801:Humans; D010920:Placenta / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D011263:Pregnancy Trimester, Third; D011296:Prenatal Diagnosis / Q000379:methods*; D015203:Reproducibility of Results; D012204:Rh-Hr Blood-Group System / Q000032:analysis; D025521:Tumor Suppressor Proteins / Q000097:blood / Q000235:genetics*
D000199:Actins; D005819:Genetic Markers; C411762:RASSF1 protein, human; D012204:Rh-Hr Blood-Group System; D025521:Tumor Suppressor Proteins; D004247:DNA