Detecting hypermethylated fetal RASSF1A sequences in maternal plasma: implications for noninvasive paternity testing in pregnancy

Up to now, prenatal paternity analysis can be routinely performed only after invasive sampling of chorionic villi or amniotic fluid, which carries a 1% risk of miscarriage. The existence of circulating cell-free fetal DNA in maternal plasma allows the noninvasive fetal Y-chromosome analysis; however, such sex-dependent assay is applicable only to pregnancies carrying a male fetus. Alternatively, an epigenetic approach could be explored. Recent studies have demonstrated that the promoter region of RASSF1A gene was hypermethylated in fetal placental tissue while hypomethylated in the maternal blood cells and thus used as a universal fetal epigenetic marker in maternal plasma. The purpose of this investigation is to develop a conventional polymerase chain reaction (PCR)based assay to detect and sequence the fetal-derived hypermethylated RASSF1A in maternal plasma, using methylation-sensitive restriction enzyme to specifically cut the hypomethylated maternal DNA background. Six milliliters of maternal peripheral blood samples from 45 pregnant women with singleton pregnancies at 11 to 41 weeks of gestation (five in the first trimester, 20 in the second trimester, and 20 in the third trimester) and 10 nonpregnant women were collected into EDTAcontaining tubes after informed consent. These patients were undergoing prenatal diagnosis or cesarean operation. The median gestational ages for first-, second-, and third-trimester pregnant women were 12 weeks (11-13 weeks), 19 weeks (17-24 weeks), and 39 weeks (37-41 weeks), respectively. Plasma was prepared by an initial centrifugation of whole blood sample at 1600 ¥ g for 10 minutes at 4°C and then a recentrifugation at 16,000 ¥ g for 10 minutes at 4°C. Fetal tissues including firsttrimester chorionic villus samples (CVSs), secondtrimester amniotic fluids, and third-trimester placental tissues were also collected. DNA extraction was performed with a commercial kit (QIAamp DNA Mini Kit, Qiagen, Chatsworth, CA, for fetal tissues; and QIAamp MinElute Virus Spin Kit, Qiagen, for plasma samples) according to the manufacturer’s instructions. Fifteen microliters of extracted plasma DNA was digested by 40 units of BstUI in 1¥ Buffer 4 (New England Biolabs, Ipswich, MA) in a total reaction volume of 25 mL. The reaction was incubated for 16 hours at 60°C. Eight microliters of digested DNA was added in a total reaction volume of 25 mL containing 12.5 mL of master mix (GoTaq, Promega, Madison, WI). The sequences of primers for RASSF1A were 5′-AGC CTGAGCTCATTGAGCTG-3′ and 5′-ACCAGCTGCCGTG TGG-3′. In addition, the ACTB gene was detected as a control to confirm complete enzyme digestion and the primer sequences were 5′-GCGCCGTTCCGAAAGTT-3′ and 5′-CGGCGGATCGGCAAA-3′. Thermal cycling conditions were 95°C for 2 minutes and then 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds for 40 cycles. All PCR products were separated by nondenaturing polyacrylamide gel electrophoresis (T = 6%, C = 3.3%) and subsequent silver staining. Direct sequencing with a cycle sequencing kit (BigDye Terminator v3.1, Applied Biosystems, Foster City, CA) was performed to determine the single-nucleotide polymorphism (SNP; id rs4688725) marker located within the RASSF1A amplicons (Fig. 1). After BstUI digestion, RASSF1A sequences were detectable in all plasma samples from 45 pregnant women but absent in those who were not pregnant, suggesting that the hypermethylated RASSF1A molecules are fetal derived. Meanwhile, ACTB amplicons were absent in all digested plasma samples, which indicates complete enzyme digestion since CpG sites in the ACTB sequence have been revealed to be unmethylated in both CVS or placenta and maternal blood. Direct sequencing of the resulted RASSF1A amplicons in 20 cases selected from the 45 studied subjects and two parentage confirmed paternity cases revealed that the genotypes of the maternal plasma DNA samples after enzyme digestion were the same as the paired fetal tissues (Table 1). These findings further confirmed that circulating enzyme digestion– resistant RASSF1A sequences were fetal-derived. By investigating the fetal methylated RASSF1A sequences in maternal plasma, this study demonstrates the feasibility of an epigenetic approach for noninvasive fetal SNP genotyping. This described assay is fast, reliable, and could be run in most forensic biology laboratories. The allele frequencies of rs 4688725 in these tested 22 unrelated maternal DNA samples are 0.3182 (A) and 0.6818 (C), respectively. The power of discrimination is 0.4339 and the power of exclusion is 0.1934. It has been reported that approximately 50 SNPs with allele frequencies of 0.2 to 0.8 can give the same power of exclusion as 12 STRs in routine paternity testing. Other fetal epigenetic markers (e.g., the gene promoter of HLCS and VAPAAPCDD1 intergenic region) have been developed and more SNP-containing markers are expected to be characterized with the help of modern high-throughput screening techniques.