O-45: Quantification of Cell-Free-Fetal-DNAfrom Maternal Plasma for the First Time in Pakistan:Diagnosis of Genetic Disorders

Background: Current prenatal diagnosis requires invasive testing which carries a 1-4% procedure-related-risk of miscarriage; hence, non-invasive techniques are desired. The recent demonstration of cell-free-fetal-DNA enriched from maternal plasma has opened new possibilities for non-invasive-prenatal-diagnosis of not only genetic-disorders such as β-thalassaemia and haemophilia but also chromosomal-aneuploidy such as trisomy 21. Here, we report successful isolation of cell-free-fetal- DNA from maternal plasma as early as 12 gestational weeks for the first time in Pakistan. Materials and Methods: Cell-free-fetal-DNA from 49 maternal plasma samples ranging from 12 to 41 weeks was analyzed for the SRY gene-sequence using quantitative- real-time-PCR. Male-genomic-DNA was used to prepare a four-point calibration-curve. Fetal gender confirmation was done at birth. Results: From the 10 cases between 12-24 weeks, 2 male fetuses were observed with mean fetal DNA concentration of 15.86 (range: 11.96 – 19.76) GE/mL plasma; seven were determined to be female fetuses. From the 39 cases between 25-41 weeks, 10 male fetuses were identified with mean fetal DNA concentration of 268.4 (range = 0.85 – 1790.83) GE/mL plasma; twenty eight were identified as female fetuses. There was one case from each group which was reported as female fetuses; however, male gender was determined at birth. Conclusion: We have demonstrated (1) 96% accuracy in fetal gender determination using maternal plasma as early as 12 weeks gestation; (2) that fetal DNA concenBackground: Current prenatal diagnosis requires invasive testing which carries a 1-4% procedure-related-risk of miscarriage; hence, non-invasive techniques are desired. The recent demonstration of cell-free-fetal-DNA enriched from maternal plasma has opened new possibilities for non-invasive-prenatal-diagnosis of not only genetic-disorders such as β-thalassaemia and haemophilia but also chromosomal-aneuploidy such as trisomy 21. Here, we report successful isolation of cell-free-fetal- DNA from maternal plasma as early as 12 gestational weeks for the first time in Pakistan. Materials and Methods: Cell-free-fetal-DNA from 49 maternal plasma samples ranging from 12 to 41 weeks was analyzed for the SRY gene-sequence using quantitative- real-time-PCR. Male-genomic-DNA was used to prepare a four-point calibration-curve. Fetal gender confirmation was done at birth. Results: From the 10 cases between 12-24 weeks, 2 male fetuses were observed with mean fetal DNA concentration of 15.86 (range: 11.96 – 19.76) GE/mL plasma; seven were determined to be female fetuses. From the 39 cases between 25-41 weeks, 10 male fetuses were identified with mean fetal DNA concentration of 268.4 (range = 0.85 – 1790.83) GE/mL plasma; twenty eight were identified as female fetuses. There was one case from each group which was reported as female fetuses; however, male gender was determined at birth. Conclusion: We have demonstrated (1) 96% accuracy in fetal gender determination using maternal plasma as early as 12 weeks gestation; (2) that fetal DNA concentration increased as pregnancy progressed. The lowest fetal DNA concentration quantified from maternal plasma was 0.85 GE/mL. Isolation of cell-free-fetal-DNA from maternal plasma provides an alternate access to fetal genetic material without invasive procedures. Potential genetic analysis in future includes assessment of sex-linked-disorders such as haemophilia, fetal RhD status, single-gene-disorders such as β-thalassaemia and chromosomal-aneuploidy such as trisomy 21.