P-542 Accuracy of Rapid Prenatal Testing by 24-chromosome analysis using Next Generation Sequencing

Abstract

Abstract Study question What is the accuracy of NGS versus QF-PCR in the Rapid Prenatal Testing? Summary answer NGS seems to be a reliable alternative for Rapid Prenatal Testing and expand the aneuploidy screening to all 24 chromosomes. What is known already As part of the prenatal care, whenever an invasive testing is needed, rapid prenatal analysis is performed to rule out the most common aneuploidies (i.e., trisomy for chromosomes 13, 18 and 21), using FISH or QF-PCR. Some studies describe the use of the NGS as an alternative to detect genetic alterations in the DNA from amniocytes or chorionic villus sampling (CVS) in Prenatal Genetic Testing. Study design, size, duration This research study evaluates the accuracy of NGS analysis for Rapid Prenatal Testing comparing the results with gold standard techniques. A total of 32 amniotic fluid samples clinically analysed by karyotype (n = 3) or QF-PCR (n = 29) were used as reference resulting in: normal male (n = 3); normal female (n = 9); trisomy 13 (n = 3); trisomy 18 (n = 5; one by karyotype); trisomy 21 (n = 10; one by karyotype); partial trisomy 3q26q26/partial monosomy 5p13p15.33 (n = 1; by karyotype); and triploid XXY (n = 1). Participants/materials, setting, methods All 32 samples were analysed using an NGS platform validated for PGT-A (Preimplantation Genetic Testing for aneuploidy). Ion ReproSeq™ PGS kit was used for library preparation, Ion Chef™ and Ion S5 System instruments for sequencing and Ion Reporter software for data analysis (Thermo Fisher Scientific, USA), with a proprietary bioinformatics pipeline (v2.0) for the analysis of 24-chromosome aneuploidies and partial duplication/deletions (≥10Mb). NGS results were compared with the reference QF-PCR/Karyotype results for accuracy evaluation. Main results and the role of chance When comparing NGS versus QF-PCR/Karyotype results, we observed a 100% concordance for the detection of trisomy 13 (3 out of 3 samples), trisomy 18 (5 out of 5 samples), trisomy 21 (10 out of 10 sample) and partial trisomy 3q/partial monosomy 5p (1 out of 1 sample). We did not identify false positives for any of the chromosomes in which comparison could be done as they were analysed in the reference samples (chromosomes 13, 18, 21, X and Y for QF-PCR reference samples, and all 24 chromosomes for Karyotype reference samples). In one of the samples, the triploid XXY, the NGS profile for the sex chromosomes was compatible either with a normal male result with maternal cell contamination or with a triploid XXY result, and was finally classified as non-informative for NGS. Limitations, reasons for caution The NGS cannot identify deletions/duplications <10Mb and mosaicism <30%. The platform used cannot identify some types of polyploidies, therefore, additional analysis (Short Tandem Repeats, karyotype, or SNP-arrays) when normal results are needed. However, improvements in technology that are currently underway will solve this limitation. Wider implications of the findings Based on the experience gained since the implantation of the NGS method for the characterization of the chromosomal status of DNA samples, the use of the NGS platform and protocols for the rapid prenatal test samples has as benefit to expand the screening analysis to all 24 chromosomes. Trial registration number Not applicable