Non-Invasive Prenatal RHD Genotyping Using Cell-Free Fetal DNA from Maternal Plasma: An Italian Experience.

BACKGROUND: This study assessed the diagnostic accuracy of a non-invasive approach to fetal RHD genotyping using cell-free fetal DNA in maternal plasma and a combination of methodological strategies. METHODS: Real-time PCR (qPCR) was performed on 216 RhD-negative women between weeks 10+0 and 14+6 of gestation (1st qPCR). qPCR was repeated (2nd qPCR) to increase the amount of each sample for analysis, on 95 plasma aliquots that were available from first trimester blood collection (group 1) and on 13 samples that were collected between weeks 18+0 and 25+6 of gestation (group 2). qPCR was specific for exons 5 and 7 of the RHD gene (RHD5 and RHD7). The results were interpreted according to the number of positive replicates of both exons. RESULTS: 1st qPCR: diagnostic accuracy was of 93.3%. Diagnostic accuracy increased from 90.5% (1st qPCR) to 93.7% (2nd qPCR) in group 1 and from 84.6% (1st qPCR) to 92.3% (2nd qPCR) in group 2. These increments were not statistically significant. CONCLUSION: Our approach to RHD genotyping in early pregnancy yielded high diagnostic accuracy. Increasing the amount of DNA analyzed in each sample did not improve significantly the diagnostic accuracy of the test.

Body mass index associated to rs2021966 ENPP1 polymorphism increases the risk for gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is a condition of impaired glucose tolerance occurring in 1-14% of all pregnancies. This wide range reflects pathological involvement of single nucleotide polymorphisms (SNPs) and maternal weight as risk factors. This study evaluated the association of genetic component and maternal factors to identify women with higher risk of developing GDM. About 240 pregnant women characterized by negative Oral Glucose Tolerance Test (-OGTT) and 38 with positive OGGT (+OGTT) were enrolled. SNPs for ENPP1, NRF1, VEGFA, CEBPA, and PIK3R1 were analyzed by SNP genotyping. An association study was performed and differences in genotype and allele frequencies between cases and controls were analyzed by χ(2) test. +OGTT was associated to high values of pre-gestational body mass index (BMI) and age. SNP for ENPP1 gene was associated to +OGTT, while genetic variants for other genes did not correlate to GDM. ENPP1 homozygous for A allele and heterozygous showed altered frequencies in +OGTT when compared with -OGTT. Association of both pre-gestational BMI and age with AA homozygous genotype increased significantly the risk to +OGTT. Our results demonstrate that correlation of age and pre-gestational BMI with homozygous for A allele increased significantly the risk of impaired glucose tolerance and GDM.

The potential usefulness of free fetal DNA in maternal blood for prenatal fetal gender determination in multiple pregnancies.

We applied a noninvasive prenatal test for the determination of fetal gender in multiple pregnancies by using free fetal DNA circulating in maternal blood in order to evaluate whether the quantification of male DNA could distinguish the fetal gender and the number of male and female fetuses in multiple pregnancies. We enrolled consecutively 44 women with twin pregnancies between 11-14 weeks of gestation. Peripheral maternal blood was collected, and genomic DNA was extracted from maternal plasma and analyzed for the multicopy DYS 14 sequence by using real-time PCR to quantify male DNA. Results showed that male DNA concentration was significantly higher in twin pregnancies with at least one male fetus, compared to twin pregnancies with only female fetuses. Comparing male DNA concentration in pregnancies with two male fetuses versus pregnancies with one female fetus and one male fetus, we did not obtain a significant difference between the two groups due to a slight overlapping of the range values. Therefore, our test correctly predicted fetal gender, distinguishing twin pregnancies with at least one male fetus from twin pregnancies with only female fetuses, with a diagnostic accuracy of 100%. For distinguishing pregnancies with two male fetuses from pregnancies with both female and male fetuses, a diagnostic accuracy of 76.1% was achieved.

Identification of universal mRNA markers for noninvasive prenatal screening of trisomies.

OBJECTIVE: The discovery of placental transcripts in peripheral blood of pregnant women prompted us to investigate which was the most appropriate biological specimen, between plasma and serum, to easily detect them and to exploit hPL (human placental lactogen), betahCG (human chorionic gonadotrophin beta-subunit), LOC90625, and TFPI2 (tissue factor pathway inhibitor 2) levels in order to establish whether an abnormal variation degree of presence of these placental transcripts are likely to be associated to specific fetal trisomies. METHOD: RNA was extracted from plasma and serum samples of 255 pregnant women bearing euploid fetuses, 17 bearing fetuses affected by trisomy 21 and 10 with fetuses affected by trisomy 18. Placental transcript analysis was performed by real time RT-PCR using relative quantification. RESULTS: Results obtained from euploid samples showed that fetal transcripts were more abundant in plasma than in serum samples. Euploid samples had a placental transcript abundance distinguishable from those with trisomy 21 but not from those with trisomy 18. In particular, high betahCG abundance and advanced maternal age were significantly associated with trisomy 21 pregnancy. CONCLUSION: Plasma was the most suitable tool to be employed in the detection and dosage of placental transcripts. betahCG transcript together with maternal age could be a potential marker for noninvasive prenatal screening of fetal trisomy 21.

The best approach for early prediction of fetal gender by using free fetal DNA from maternal plasma.

OBJECTIVES: Detection of free fetal DNA (ffDNA) in maternal blood during pregnancy has given rise to the possibility of developing new noninvasive approaches for early prenatal diagnosis. On a large-scale study, two protocols of real-time polymerase chain reaction (PCR) were compared in order to establish which Y-specific locus, either multicopy DYS14 or single copy SRY sequence, was the most suitable for developing a test with high diagnostic efficiency for early fetal gender assessment. The second aim was to assess whether the combination of the two detection systems could increase the performance of the prenatal test. METHODS: We analyzed 145 plasma samples from healthy pregnant women between 11 and 12 weeks of singleton gestation. For each sample, fetal gender was determined by using both protocols (DYS14 and SRY) during the same real-time PCR run. RESULTS: The data obtained by the DYS14 and SRY assays showed an efficiency in fetal gender prediction of 97.9 and 80%, respectively. It is not advisable to combine the two protocols because this association does not help in further improvements in fetal gender prediction. CONCLUSIONS: DYS14 assay is the best approach for early fetal gender assessment because it is more sensitive, accurate, and efficient than the SRY assay.