Application of prenatal diagnosis with fetal nucleic acids in maternal blood

The discovery of cell free fetal DNA in the maternal circulation has driven developments in noninvasive prenatal diagnosis in the past decade. Detection of paternally derived alleles in cell free fetal DNA is becoming well established. Analysis of fetal DNA from maternal plasma by new techniques such HRM analysis offers great potential for noninvasive prenatal diagnosis.We examined 104 maternal plasma samples from 57 pregnant women carrier of sickle cell anemia, 32 pregnant women carrier of beta thalassemia and 15 healthy control subjects. Fetal DNA was extracted from 1 mL plasma samples. To detect male fetuses SRY and DYS14 markers specific to the Y chromosome analyzed by nested PCR and RT-PCR. DYS14 and beta globin were used for quantitation of fetal and total DNA levels respectively. Alternative to traditional methods in invasive prenatal diagnosis, we used HRM analysis for genotyping of fetal DNA in hemoglobinopathies.We detected Y chromosome specific DYS14 in maternal plasma of 100% women bearing male fetuses in early pregnancy. The value was 93.7% with SRY by nested PCR. The levels of total and fetal DNA increased with gestational age. We observed greater MoM values of fetal DNA in sickle cell anemia pregnancies compared to thalassemia and healthy groups. The MoM values were smaller in thalassemia pregnancies compared to other groups (p<0.001). In late period of pregnancy, we observed significantly increased levels of beta globin between sickle cell anemia, thalassemia and control groups. Genotyping by HRM we were able to distinguish paternal alleles of fetal DNA from maternal DNA. Problems were encountered when the fetus carries the same allele with mother.Our results show that investigation of the fetal and total DNA in early pregnancies with hemoglobinopathies is not found to be useful. HRM is an alternative method for invasive prenatal diagnosis to genotyping of fetal DNA when the fetus carries the paternal allele different from the mother?s.