Evaluation of a Novel Assay for Detection of the Fetal Marker RASSF1A: Facilitating Improved Diagnostic Reliability of Noninvasive Prenatal Diagnosis

Background: Analysis of cell free fetal (cff) DNA in maternal plasma is used routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination, fetal rhesus D status and some single gene disorders. True positive results rely on detection of the fetal target being analysed. No amplification of the target may be interpreted either as a true negative result or a false negative result due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A promoter has been reported as a universal fetal marker to confirm the presence of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated maternal sequences are digested leaving hypermethylated fetal sequences detectable. Complete digestion of maternal sequences is required to eliminate false positive results. Methods: cfDNA was extracted from maternal plasma (n = 90) and digested with methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A, SRY and DYS14 was performed by real-time PCR. Results: Hypermethylated RASSF1A was amplified for 79 samples (88%) indicating the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were 100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of these samples were male at delivery, five had inconclusive results for SRY analysis and one sample had no amplifiable SRY. Conclusion: Use of this assay for the detection of hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.