Valoración de una reacción en cadena de la polimerasa cuantitativa y fluorescente (QF-PCR) para el diagnóstico rápido de aneuploidías

Abstract

Introduction Quantitative fluorescence polymerase chain reaction (QF-PCR) identifies in less than 24 h the most common chromosomal anomalies looked for in conventional prenatal karyotyping: numerical alterations of chromosomes 13, 18, 21, X and Y. Several polymorphic markers are used in QF-PCR for each chromosome, allowing a high reliability of results, speed, low cost and automation. Nevertheless, QF-PCR can not detect structural abnormalities, low-level mosaicism or marker chromosomes. Objective To develop and evaluate a fast, reliable and feasible method for detecting aneuploidies of chromosomes 13, 18, 21, X and Y, in the samples received for prenatal karyotyping in a cytogenetics department. Materials and methods In addition to the 465 amniotic fluids received during last year, stored DNA samples in which a disease was detected in the previous two years were assayed (corresponding to the study of around 1,000 prenatal samples). DNA extraction is automated. The STR polymorphic markers used, with high heterozygosity, are accepted internationally (Association for Clinical Cytogenetics. QF-PCR for the diagnosis of aneuploidy best practice guidelines (2007). Available at http://www.cytogenetics.org.uk/prof_standards/professional_standards.htm). The test has the same PCR amplification conditions for all four multiplex mixtures used. In each of them All the necessary components were included in each of them, except the sample, which was added at the time of the assay. Each amplified mixture is analyzed into the ABI3130 Genetic Analyzer, Applied Biosystems. Fragments analysis is achieved following the published recommendations. Results The results of all the tested samples, normal or pathological, matched the outcome of the conventional karyotype. In total the QF-PCR assay detected: 12 trisomy 21; 5 trisomy 18; 3 women 45,X ; 2 trisomy 47,XXY ; 2 trisomy 13 ; 2 Triple XXX ; 2 men XYY; and 2 triploid pregnancies. In each of these cases the proposed QF-PCR was diagnostic. A partial monosomy of chromosome 18 could not be detected, but a partial trisomy of chromosome 18 was identified. Conclusions The evaluated QF-PCR is a useful tool for the rapid diagnosis of aneuploidies (24 h). It is extremely valuable when the amniocyte cultures fail to grow, or suffer microbiological contamination. It serves to reduce maternal anxiety until the conventional karyotype is reported, and to take quick decisions when ultrasound anomalies are found in the second trimester of pregnancy.