Digital droplet PCR for detection of <i>SMN1</i> deletion in low-concentration fragmented cell-free DNA: a proof of concept study

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is primarily caused by homozygous deletions in the <i>SMN1</i> gene. Although multiplex ligation-dependent probe amplification (MLPA) is the gold standard for SMA diagnosis, invasive prenatal procedures (e.g. amniocentesis or chorionic villus sampling) carry risks, including fetal loss. Non-invasive prenatal testing using digital droplet PCR (ddPCR) to detect <i>SMN1</i> deletions in cell-free fetal DNA (cffDNA) from maternal plasma presents a safer alternative. This study evaluates the feasibility of ddPCR for non-invasive prenatal detection of <i>SMN1</i> exon 7 deletions using simulated cffDNA mixtures. Blood samples were collected from two children with SMA (homozygous SMN1 deletions), their carrier mothers, and healthy controls. Genomic DNA was fragmented to simulate fetal (80-160 bp) and maternal (160-300 bp) cfDNA profiles. Simulated mixtures were prepared at varying maternal-to-fetal ratios (1:1 to 64:1). ddPCR was performed using the Bio-Rad SMN1 Copy Number Determination Kit, and *SMN1/RPP30* ratios were calculated. Linear regression analysis assessed quantitative performance. ddPCR accurately distinguished affected (mean ratio ∼0.00), carrier (∼0.46), and healthy (∼0.93) genotypes. A strong linear correlation existed between fetal DNA fraction and *SMN1/RPP30* ratio (*r* = 0.995, *p* < 0.0001). The assay detected affected fetal DNA at fractions as low as 5%, with reliable diagnostic separation at ≥10%. ddPCR exhibits high sensitivity and specificity for detecting SMN1 exon 7 deletions in simulated cffDNA, supporting its potential application in non-invasive prenatal SMA screening. Controlled evaluation under defined maternal-fetal mixture provides a reference for optimizing ddPCR-based prenatal testing for SMA.