Extended Non-Invasive Prenatal Testing for Chromosomal and Monogenic Disorders by Cell-Free DNA Sequencing: A Blinded...

To determine whether molecular integrated non-invasive fetal exome sequencing (iNIFS), using clinical exome sequencing of maternal-plasma cell-free DNA, concords per pregnancy with paired amniocentesis.

Prospective, blinded, single center, paired sample diagnostic accuracy study in 641 consecutive pregnancies referred for clinically indicated amniocentesis at 16-18 weeks. Plasma cell-free DNA underwent low-pass whole-genome sequencing for aneuploidies and CNVs and, after short-fragment enrichment, clinical exome sequencing within a parent fetus trio design. The reference standard comprised QF-PCR, karyotyping, array-comparative genomic hybridization and, where indicated, trio clinical exome sequencing on amniotic-fluid DNA.

iNIFS was reportable in 624/641 pregnancies (97.3%); 612 (95.5%) had complete paired data and formed the analysis population. The reference established a pathogenic or likely pathogenic finding in 107/612 (17.5%). NIPT gave 106 true positives, 503 true negatives, 2 discordant positives and 1 discordant negative: sensitivity 99.1% (95% CI 94.9-99.8), specificity 99.6% (98.6-99.9), PPV 98.1% (93.5-99.5), NPV 99.8% (98.9-100.0), agreement 99.5% and Cohen's κ 0.98. The discordant negative was a maternally inherited variant, the class for which distinguishing maternal from fetal contributions is most challenging.

Molecular iNIFS showed high concordance with paired amniocentesis across chromosomal, copy-number and monogenic findings, while retaining an explicit no call rate. Multicentre validation and invasive confirmation of positive findings remain necessary.