RASSF1a Is Applicable as Universal Fetal Identifier in RQ-PCR-Based Non-Invasive Prenatal Genotyping Assays
Authors: Soussan Aicha Ait , Bossers Bernadette , Tissoudali Ahmed , Page-Christiaens Godelieve C , de Haas Masja , Van der Schoot C. Ellen 
Published: April 2020

Abstract
The existence of cell free fetal DNA, derived from apoptotic syncytiotrophoblasts, in the maternal circulation during pregnancy has opened new possibilities of non-invasive prenatal diagnosis, such as fetal RhD genotyping, prenatal diagnosis of hemoglobinopathies and fetal sexing in X-linked disorders like hemophilia. These diagnostic applications are however hampered by the lack of a generic control marker for circulating fetal DNA, which is especially cumbersome early in pregnancy. Fetal DNA concentration is extremely low and can be less then 5 genome equivalents (geq)/mL in the first trimester. It gradually increases during pregnancy, but greatly varies between women, from 15 to 150 geq/ml at 30th week of pregnancy. A negative test result can therefore be due to lack of fetal DNA in the PCR tube or to absence of the mutation/polymorphism in the fetus. Fetal DNA control assays based on Y-chromosome sequences can be used in only 50% of the pregnancies. We have previously developed a set of RQ-PCRs based on del-ins polymorphisms as fetal identifier, which has been introduced in routine diagnostics. However, in up to 5–10% of cases no suitable paternal marker can be identified and also the workload is considerable. Recently, it has been demonstrated that the promoter of RASSF1A gene is hyper-methylated in the placenta and hypo-methylated in maternal blood cells (Chiu et al., Am J Pathol.2007;170:941). This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion with BSTU1, for detecting the placental, hence fetal-derived hyper-methylated RASSF1A sequences in maternal plasma. We wanted to implement this potential fetal DNA identifier, RASSF1A, in our diagnostic tests. Initial results however showed weak positive results in virtually all male plasmas. This could be due to incomplete digestion of hypo-methylated leukocyte derived DNA or to the presence in plasma of hypermethylated DNA from other sources.
To increase the efficiency of digestion we performed a double digestion by adding another enzyme Hha1, which adds two extra restriction sites within the PCR target DNA sequence. Pilot experiments on DNA derived from male plasma showed decreased amplification signals, suggesting that the previously observed weak signals were derived from incompletely digested hypo-methylated DNA. We selected enzymes working in the same buffer. The optimal reaction time and temperature were established (1 hour at 37 °C with 10U of each enzyme). Applying this protocol, no amplification or only spurious amplification with high Ct values (mean Ct value of the positive wells: 43.3) were observed with DNA isolated from 2 ml of plasma obtained from non-pregnant women or male (n=20). In contrast, all samples obtained from pregnant women (n= 20) showed positive amplification signals in all wells tested (n=87, 3 or 5 replicates), with clearly lower Ct values (mean 41.04 ± 1.54) The beta-actin data showed in all samples complete digestion.
In conclusion: Our results indicate that a real time PCR assay on RASSF1a sequence can be used as universal fetal identifier for non-invasive fetal genotyping assays, if it is tested after double digestion with BSTU1 and Hha1. It is to be expected that the availability of this control assay will lead to wider implementation of prenatal genotyping assays based on cell-free fetal DNA.