Tandem Mass Spectrometry Fingerprint Tags (TMSFT) Applied for Early Gestation Noninvasive Prenatal Detection of Single-Gene Disorders

Noninvasive prenatal testing for single-gene disorders faces significant challenges due to low fetal DNA abundance in maternal blood, particularly in early gestation. Although next-generation sequencing (NGS) is widely used, it frequently encounters false-negative outcomes and high costs when detecting low-abundance fetal variants. To address these limitations, we developed a highly sensitive and cost-effective analytical method utilizing high-resolution tandem mass spectrometry, capable of accurately detecting single nucleotide variants (SNVs) as early as 5-8 weeks gestation. It employs gas-phase enrichment combined with fragment mass spectral analysis for precise isolation and identification of fetal DNA amidst predominant maternal DNA. Analytical validation with synthetic DNA and cultured cells confirmed exceptional sensitivity, achieving single copy level detection despite significant interference. Clinically, our method accurately detected paternally inherited SNVs in 134 cell-free fetal DNA samples collected at 5-8 weeks gestation, achieving 100% accuracy. Additionally, it reliably identified pathogenic SNVs linked to phenylketonuria from 12 samples collected at 11-13 weeks gestation. This approach significantly reduces operational costs, enhances sensitivity, and eliminates false-negative results compared to traditional NGS-based methods.