摘要:
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[Abstract/Free Full Text]). 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 identi