Locked nucleic acid (LNA) based PCR approach for the diagnosis and screening of spinal muscular atrophy

Background and objectives Spinal muscular atrophy (SMA) is an autosomal recessive disorder most often caused by homozygous deletion of exon 7 in the SMN1 gene. Current guidelines recommend initial testing of SMN1 exon 7, with additional analyses if no deletions are found. Commercial kits are typically probe-based; however, LNA (locked nucleic acid)-modified primers provide high sensitivity and specificity at lower cost. We aimed to develop an LNA-based assay for the diagnosis and screening of SMA. Methods Peripheral blood samples (2 mL) were collected from 31 patients diagnosed with SMA, 37 confirmed healthy controls, and 47 carrier parents between September and December 2021. 3' LNA-modified primers were designed for the SMN1, SMN2, and β-actin genes. For each PCR reaction, 20 ng of template DNA was used in a final volume of 20 µL. Products were validated by Sanger sequencing, and ΔCt values were compared with multiplex ligation-dependent probe amplification (MLPA). Results The LNA primers for SMN1, SMN2, and β-actin were designed and optimised to amplify efficiently under uniform polymerase chain reaction (PCR) conditions. PCR results and ΔCt values were fully concordant with MLPA results. Interpretation and conclusions Using only LNA-modified primers, we accurately detected SMN1 and SMN2 presence, absence, and copy numbers, without probes. This assay reliably identified both SMA patients and carriers, supporting its potential for diagnostic and screening purposes.