Integrating LNA-qPCR and full-length SMN1 sequencing for precision SMA carrier screening: large-scale validation in 30,849 individuals.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused primarily by biallelic SMN1 loss-of-function mutations, most commonly homozygous deletions of exon 7. While large-scale carrier screening is critical for prevention, comprehensive population-level data remain limited in mainland China. METHODS: We conducted a prospective, large-scale SMA carrier screening study in Hunan Province, China, from September 2020 to September 2025. The cohort included 30,733 pre-pregnancy and early-pregnancy individuals without an SMA family history across 14 cities. SMN1 exon 7 copy number was assessed using Locked Nucleic Acid-quantitative PCR (LNA-qPCR), with MLPA validation performed in 2000 randomly selected samples. Additionally, we applied a comprehensive protocol involving full-length SMN1 amplification and Sanger sequencing to make a definitive molecular diagnosis for the suspected SMA children and fetuses from 58 high-risk couples (7 with SMA family history). RESULTS: Population screening identified 438 carriers, corresponding to a carrier frequency of 1/69 (1.43%), with regional frequencies ranging from 0.80% to 1.98%. Methodological validation demonstrated that LNA-qPCR achieved 100% sensitivity and 99.85% specificity. Notably, investigation into rare false positives revealed two previously unreported single-nucleotide polymorphisms (SNPs), c.835-44G > A and c.835-50 A > G in SMN1, that interfere with primer binding and may represent common diagnostic confounders specific to the Chinese population. Furthermore, screening among high-risk families successfully elucidated the genetic etiology for seven previously undiagnosed patients. Subsequent prenatal diagnosis for 50 at-risk pregnancies identified 16 affected fetuses, allowing for informed reproductive decision-making. CONCLUSIONS: Based on the extensive province-wide, multi-center cohort, LNA-qPCR combined with full-length SMN1 sequencing is proven to be an accurate and efficient strategy for population-level SMA carrier screening. Our identification of rare compound heterozygous genotypes that frequently elude conventional assays, alongside the successful clinical intervention for 16 affected pregnancies, underscores the clinical value of comprehensive screening programs. These findings provide critical epidemiological and technical evidence to support national SMA prevention strategies and reduce disease burden.