Detection efficiency of fetal epigenetic marker in maternal plasma

Abstract

Objective To analyze the detection efficiency of non-sex dependent Ras association domain family 1A (RASSF1A) sequences in maternal plasma as a fetal epigenetic marker,and to explore the feasibility of replacing SRY gene in quantitative detection of fetal free DNA.Methods Maternal peripheral blood samples were collected in the first,second and third trimester from 50 normal pregnant women who had their prenatal care in the First and Fifth People's Hospital in Datong,Shanxi Province,between May 1,2011 and April 30,2012.Among them,pregnant women with single male fetus (n=28) were selected as study group,and another 20 normal healthy non-pregnant women in the same period were selected as control.The methylationsensitive restriction enzymes were used specifically to cut the maternal hypomethylated RASSF1A gene,the fetal hypermethylated sequences were determined by fluorescence quantitative polymerase chain reaction,and SRY sequences in maternal plasma were determined as a reference.Shapiro-wilk,Pearson or Spearman correlation analysis and Chi-square test were applied for statistical analysis.Results Among the 28 pregnant women with male fetus,the hypermethylated RASSF1A sequences in maternal plasma was tested 84 times and identified as early as at 8+2 weeks of gestation,giving the detection rate of 97.62％ (82/84).However,no RASSF1A or SRY gene was identified in control group.The mean concentrations of hypermethylated RASSF1A and SRY gene in the first,second and third trimester group were (60.19± 14.16) copies/ml vs (58.22± 15.13) copies/ml (r=0.933,P＜0.01),(99.18±28.44) copies/ml vs (95.43±32.33) copies/ml (r=0.931,P＜0.01)and (144.93±33.51) copies/ml vs (132.96±28.24) copies/ml (r=0.654,P＜0.01),respectively.The mean concentrations of hypermethylated RASSF1A and SRY gene showed positive correlation with gestational weeks (r=0.933 and 0.896,P＜0.01,respectively).Conclusions By using hypermethylated RASSF1A gene as an epigenetic marker,the detected free fetal DNA results is highly consistent with SRY gene without gender influence.Thus,it is helpful to expand its clinical application of noninvasive prenatal diagnosis. Key words: Tumor suppressor proteins; Methylation; Epigenomics; Genes, SRY; Prenatal diagnosis; Polymerase chain reaction