Estabelecimento de metodologias de análise do DNA livre plasmático para o diagnóstico Pré-natal não invasivo: sexagem fetal
Rocha, Bruno Garcia
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In this paper we proposed and analyzed methodologies using the technology of Cell-Free Fetal Nucleic Acid-Free (cffDNA = Cell Free Fetal DNA) in noninvasive prenatal diagnosis (NIPD). Due to the modern technologies employed and their repercussion among the involved families, we sought to discuss some ethical, social and legal implications. Contrary to the popular belief that the placenta forms an impermeable barrier between mother and child, there is bidirectional traffic between the fetus and mother during pregnancy. Several studies have shown that not only intact cells but also fetal cell-free fetal nucleic acids (cffNA, ie, DNA and RNA) cross the placenta and travels in the mother's bloodstream. Four different applications of analysis technology were identified: cffDNA: a) Prenatal Sex Determination, used in pregnancies under the risk of sexual transmitted diseases and performed through the detection of the Y chromosome b) The diagnosis of certain diseases of a single gene through the detection of paternally inherited mutation c) Fetal Aneuploidy, such as Down syndrome, where chromosomal abnormalities may occur d) identifying the type of fetal blood in pregnancies under the risk of incompatibility, especially RhD. To carry out this work it was used the pre-natal determination of sex in 53 pregnant women at different gestational periods. For that it was proposed and tested methods of DNA extraction, amplification of DNA obtained by PCR (Polymerase Chain Reaction) and a new methodology called LAMP (Loop Mediated Isothermal Amplification) and the analysis of final results. Furthermore, the efficiency of LAMP and PCR was compared by amplifying different segments of the Y chromosome: DSY14 and TSPY. In three cases the samples were discarded because there was no fetal sex confirmation after molecular tests due to loss of contact with pregnant women. In two other cases the results pointed to male fetuses whilst the ultrasound confirmed these fetuses as females due to contamination. Finally it was obtained 28 male samples (58.33%) with amplification of the sequences of the Y chromosome and 20 female samples (41.67%) that did not amplify the sequence of the Y chromosome but only for the control. These results showed that the amplification lamp is more efficient than PCR, in the analysis of DSY14, the limit of detection is 10 pg and 0.1 pg for LAMP and PCR respectively. It was concluded that the amplification using the LAMP method is faster (60 min) and has a high sensitivity and specificity and does not require sophisticated equipment for reaction if compared to the PCR method. These characteristics make this methodology feaseable in laboratories with limited resources.