EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples.

OBJETIVES: The extracellular DNA occurring in plasma-EDTA and serum is a biomarker of growing interest, especially in prenatal diagnosis and oncology. The objectives of the present study were to compare the DNase activity in these specimens and to investigate its ex-vivo impact over the circulating cell-free DNA yield (ccfDNA), using the circulating cell-free fetal DNA (ccffDNA) as a tool. DESIGN AND METHODS: EDTA-plasma and serum from women bearing male fetus were submitted to an endogenous DNase activity assay based on qPCR hydrolysis probe degradation, they were treated with DNAse I to investigate the action of an exogenous nuclease and also submitted to different temperature conditions to investigate the temperature-dependent degradation of the ccffDNA. In all instances, all male ccffDNA were quantified by qPCR targeting the Y chromosome-specific sequence DYS-14. Moreover, a serial dilution of EDTA was added to nonanticoagulated plasma and serum before the endogenous DNAse activity assay, to investigate the EDTA-mediated inhibition of the blood's DNase. RESULTS: The endogenous nuclease activity was 14.9-fold higher in serum compared to EDTA-plasma. The DNAse I treatment did not alter the ccffDNA yields in EDTA-plasma, but completely degraded it in serum. The addition of increasing doses of EDTA to nonanticoagulated plasma and serum resulted in a stepwise inhibition of their nucleases activity. Finally, we observed a much more pronounced temperature-mediated decrease on the ccffDNA amount in serum compared to EDTA-plasma. CONCLUSION: The exogenous and endogenous DNases are more active in serum, the anticoagulant EDTA indirectly inhibits blood DNases, and consequently ccfDNA is protected from the blood's DNase preanalytical impact in EDTA-plasma.

Fetal male lineage determination by analysis of Y-chromosome STR haplotype in maternal plasma.

The aim of this study is to determine the fetus Y-STR haplotype in maternal plasma during pregnancy and estimate, non-invasively, if the alleged father and fetus belong to the same male lineage. The study enrolled couples with singleton pregnancies and known paternity. All participants signed informed consent and the local ethics committee approved the study. Peripheral blood was collected in EDTA tubes (mother) and in FTA paper (father). Maternal plasma DNA was extracted by using NucliSens EasyMAG. Fetal gender was determined by qPCR targeting DYS-14 in maternal plasma and it was also confirmed after the delivery. From all included volunteers, the first consecutive 20 mothers bearing male fetuses and 10 mothers bearing female fetuses were selected for the Y-STR analysis. The median gestational age was 12 weeks (range 12-36). All DNA samples were subjected to PCR amplification by PowerPlex Y23, ampFLSTR Yfiler, and two in-house multiplexes, which together accounts for 27 different Y-STR. The PCR products were detected with 3500 Genetic Analyzer and they were analyzed using GeneMapper-IDX. Fetuses' haplotypes (Yfiler format) were compared to other 5328 Brazilian haplotypes available on Y-chromosome haplotypes reference database (YHRD). As a result, between 22 and 27 loci were successfully amplified from maternal plasma in all 20 cases of male fetuses. None of the women bearing female fetuses had a falsely amplified Y-STR haplotype. The haplotype detected in maternal plasma completely matched the alleged father haplotype in 16 out of the 20 cases. Four cases showed single mismatches and they did not configure exclusions; 1 case showed a mutation in the DYS 458 locus due to the loss of one repeat unit and 3 cases showed one DYS 385I/II locus dropout. All mismatches were confirmed after the delivery. Seventeen fetuses' haplotypes were not found in YHRD and one of them had a mutation, which corresponded to the paternity probability of 99.9812% and 95.7028%, respectively. Three fetuses' haplotypes occurred twice in YHRD, which corresponded to paternity probability of 99.9437%. In conclusion, high discriminatory fetal Y-STR haplotype could be determined from maternal plasma during pregnancy starting at 12 weeks of gestation. All male fetuses could be attributed to the alleged father male lineage early in pregnancy. The high probability of paternity associated with each case suggests that the relationship is not random and this strategy can be use as an alternative for male fetal kinship analysis.

Haplotype diversity of 23 Y-chromosomal STRs in a population sample from the Federal District (Brazil)--a territory that arose from nothing.

In this study, the genetic variations of 23 short tandem repeats on the Y-chromosome were analyzed in a sample of 201 males from the Federal District (Brazil). The Federal District (Brazil) was built in 1960 in Brazil's Central West region, where there was no previous population. In 2010, the population of this artificially founded district consisted of 2,500,000 inhabitants. We observed 200 different haplotypes, 199 of which were unique and one of which occurred two times. The haplotype diversity was 0.9999, and the discrimination capacity was 0.995. The data are available in the Y chromosome haplotype reference database under accession number YA003843. The results were compared to the haplotypes from other Brazilian macroregions.

Allele frequencies for 15 autosomal STRs in a population sample from the Federal District (Brazil)-a territory that arose from nothing.

The Federal District (Brazil) was created in 1960 in the Central-West Region of Brazil in a previously unpopulated area. In 2010, this artificially founded district was populated by 2,562,963 inhabitants. In this study, the genetic variations of the 15 Next Generation Multiplex (NGM(TM)) short tandem repeat loci were analyzed. The results indicate that the NGM(TM) is a highly informative genetic system in this population, which is more similar to the southeastern, northeastern, and overall Brazil populations. This conclusion agrees with the population composition reported in the 2010 National Survey Inquiries, in which most of the immigrants were from the northeast and the southeast.