Chromosomal structural abnormalities and tissue-specific mosaicism: insights into false-negative noninvasive prenatal...

To investigate the causes of false-negative results in noninvasive prenatal testing (NIPT) and provide insights for technical optimization and genetic counseling in clinical practice.

We retrospectively analyzed clinical and genetic data from three cases with false-negative NIPT results. In case 1, amniocentesis was performed due to fetal growth restriction (FGR) and a single umbilical artery (SUA). In case 2, a female infant was delivered at 35 weeks' gestation for FGR and vasa previa, with postnatal manifestations including neonatal respiratory distress syndrome, low birth weight, and hexadactyly. In case 3, amniocentesis was carried out for a high-risk NIPT result indicating a 14q duplication. Genetic analyses including karyotyping, copy number variation sequencing (CNV-seq), trio whole-exome sequencing (WES-trio), and fluorescence in situ hybridization (FISH) were performed on amniotic fluid, placental tissue, maternal peripheral blood, and buccal mucosal samples, respectively.

Ring chromosome 21, dicentric chromosome 18, and multiple chromosomal rearrangements were identified in the three cases. In Case 1, amniotic fluid analysis revealed mosaic r (21) by karyotyping and a terminal deletion of 21q by CNV-seq, which was attributed to dynamic mosaicism and potential cell culture artifacts. In Case 2, karyotyping and WES detected 42% and 65% mosaic dic (18) in peripheral blood, respectively, whereas FISH on buccal mucosal cells only identified 8% mosaicism. This substantial discrepancy reflected tissue-specific mosaic distribution between buccal cells (ectoderm) and peripheral blood (mesoderm) of dic (18). In Case 3, prenatal karyotyping and CNV-seq confirmed a pathogenic 5p deletion in the fetus. Subsequent genetic testing of placental specimens revealed complex mosaic CNVs (10%-52%), whereas umbilical cord findings were consistent with amniotic fluid results. Complex chromosomal rearrangements occurring during early embryogenesis led to widespread multi-CNV mosaicism across placental and fetal tissues. Such heterogeneous genomic alterations are incompletely represented in cffDNA, resulting in concurrent false-positive and false-negative NIPT results.

Mosaicism levels, tissue distribution variability, cellular heterogeneity, and complex chromosomal structural abnormalities are key contributors to false-negative NIPT results. Our findings highlight the importance of comprehensive clinical and genetic evaluation in managing discordant NIPT findings.