Aplicació de la tecnologia de seqüenciació massiva a la determinació del genotip Kell Fetal a partir del plasma matern

Abstract

Knowledge of the fetal Kell genotype in pregnancies of sensitized women with anti-K is of great value to determine if the fetus is at risk of haemolytic anaemia and allows a better management of such, otherwise, high‐risk pregnancies. Several non-invasive approaches for fetal Kell genotyping have been developed ages earlier than 20 weeks. The next-generation sequencing (NGS) Technology offers unprecedented possibilities of massive parallel analysis of gene-targets amplified sequences from maternal plasma cell-free (cf) DNA. Initial proof of concept studies have shown promising results. Objectives: 1) To design a NGS approach for the fetal Kell genotype analysis in plasma cfDNA and 2) To evaluate its sensitivity and the reliability of the fetus in a validation study with a panel of clinical samples from sensitized pregnant women. Materials and Methods: A total of 17 plasma samples from pregnant women with anti‐K have been prospectively collected during the past three years. Cell‐free DNA has been extracted from 2 ml of these plasma samples as well as from artificial chimeric mixtures of plasma from individuals with known genotype (major KEL2/2, minor KEL1/2) using the QIAsymphony extractor. A 125b bp KEL gene fragment encompassing the KEL1/2 SNP has been amplified using gene-specific primers with NGS suitable adaptors. The resulting amplification products have been pooled with other amplicons and sequenced in a MiSeq Desktop Sequencer. The NGS pipeline output paired sequence files have been used as input for the analysis with CLC Genomics Workbench software. Results: Initial tests were performed with plasma DNA from the artificial chimeric mixtures, having a 1 to 20% minor component represented. Parallel amplification of the AMEL gene and the SRY locus has been optimized as well to include a fetal sex marker. Optimal amplicon size and input plasma DNA have been assessed with a restricted panel of clinical samples representing the real scenario of application. Conclusion: This NGS method for non-invasive fetal Kell genotyping has shown specific and sensitive detection of the fetal KEL1 allele in clinical samples of sensitized pregnant women. It is also compatible with the simultaneous sequence analysis of multiple amplicons from different loci. In this sense, further developments may include other loci of blood group polymorphisms, potentially involved in haemolytic disease of the newborn