Validation on the First-Tier Fully Automated High-Throughput SMN1, SMN2, TREC, and RPP30 Quantification by Quadruplex Droplet Digital PCR for Newborn Screening for Spinal Muscular Atrophy and Severe Combined Immunodeficiency

Newborn screening (NBS) for spinal muscular atrophy (SMA) and severe combined immunodeficiency (SCID) faces challenges. Accurate and precise <i>SMN1</i> and <i>SMN2</i> copy number determination, confirmed by two orthogonal methods, are vital for SMA prognostication and treatment. Single <i>SMN1</i> copy detection also enables the further feasibility to screen for compound heterozygotes. In SCID, low-level T-cell receptor excision circle (TREC) quantification by quantitative PCR is imprecise, necessitating replicates for reliable results. An assay with enhanced accuracy, precision, and high throughput is warranted for NBS SMA and SCID. False positive of <i>SMN1</i> deletions due to allele dropout are also a potential pitfall in PCR-based methods. We evaluated a first-tier fully automated quadruplex droplet digital PCR (ddPCR) assay detecting <i>SMN1</i>, <i>SMN2</i>, TREC, and <i>RPP30</i> using dried blood spots together with a second-tier Sanger sequencing to exclude <i>SMN1</i> allele dropout. Five proficiency test samples and six patient samples with known <i>SMN1</i> and <i>SMN2</i> copy numbers confirmed by multiplex ligation-dependent probe amplification were used for accuracy evaluation with full concordance. The ddPCR assay showed high precision for <i>SMN1</i> and <i>SMN2</i> (<7% coefficient of variation (CV) for ≥0 copy) and TREC (14.6% CV at 37 copies/µL blood). Second-tier Sanger sequencing identified all SMA cases with homozygous deletions. Accuracy for TREC classification was concordant with 10 proficiency samples. The reference interval of TREC concentration was established for newborns ≥ 34 weeks (<i>n</i> = 1812) and the 2.5th percentile was 57 copies/µL blood. A two-tiered approach with fully automated quadruplex ddPCR and Sanger sequencing delivers accurate and precise quantitation for NBS SMA and SCID, enabling early treatment and counseling.