MLDP-AS: An Optimized Next-Generation Sequencing Assay for Enhanced Detection of Technically Challenging Variants in Expanded Carrier Screening

Abstract Background Next-generation sequencing (NGS) facilitates simultaneous carrier screening for multiple single-gene disorders. However, conventional NGS methods struggle to detect complex variants, such as F8 inversions, CYP21A2 variations, and single-exon copy number variations (CNVs), resulting in residual risk. Methods We developed and validated MLDP-AS (Multiplex Long-Distance PCR followed by Amplicon Sequencing), a novel NGS assay, to screen for pathogenic variants in ten prevalent single-gene disorders in the Chinese population, including alpha- and beta-thalassemia, non-syndromic hearing loss, spinal muscular atrophy, Duchenne muscular dystrophy, phenylketonuria, 21-hydroxylase deficiency, Wilson disease, methylmalonic acidemia, and Hemophilia A. MLDP-AS detects routine variants and technically challenging types, such as F8 intron 22 inversions, CYP21A2 variations, and single-exon CNVs, in a single test. The assay was optimized using positive clinical samples, with sensitivity validated against gold-standard methods. Clinical applicability was evaluated through a prospective study of couples planning or undergoing pregnancy, with positive results confirmed by gold-standard techniques. Results MLDP-AS achieved 100% sensitivity, identifying all 255 pathogenic variants in known positive samples, including 37 technically challenging variants. In a prospective trial involving 5,209 individuals, carriers for all targeted disorders were detected, with an overall carrier rate of 22.15%. Thirty-four couples (1.09%) were identified as high-risk, spanning eight of the ten diseases. A total of 289 pathogenic variants were detected 1,290 times, including 169 technically challenging variants. The assay demonstrated a positive predictive value of 99.7% compared to gold-standard methods. MLDP-AS is rapid and cost-effective, completing testing within three days at a cost under $25. Conclusions MLDP-AS integrates detection of multiple complex variants into a single, comprehensive assay, overcoming limitations of conventional NGS. It significantly enhances variant detection and provides an efficient, cost-effective tool for carrier screening in the Chinese population.