Comparison of the accuracy of multiplex digital PCR versus multiplex ligation-dependent probe amplification in quantification of the survival of motor neuron genes copy numbers

•The accuracy of MLPA in SMA genetic testing was recently questioned.•Digital PCR technique has exhibited advantages in copy number variation assessment.•Digital PCR demonstrated superior accuracy in quantifying SMN1 and SMN2 copy numbers over MLPA. For over two decades, multiplex ligation-dependent probe amplification (MLPA) has served as the gold standard for genetic testing of spinal muscular atrophy. However, there is emerging evidence questioning the reliability of MLPA in determining the copy numbers (CNs) of the survival of motor neuron (SMN) gene in certain cases. Recently, digital polymerase chain reaction (dPCR) has shown potential for better performance in copy number variant detection. This study aimed to compare MLPA and dPCR in quantifying SMN1 and SMN2 CNs, identify reasons for observed discrepancies, and explore the clinical implications of false results. A total of 733 DNA samples, previously subjected to MLPA analysis, were tested using multiplex droplet dPCR assays. Samples exhibiting inconsistent results between the two methods underwent repeated dPCR assays. When inconsistencies persisted, a third method was employed for verification. Digital PCR yielded results consistent with those of MLPA in 94.4% (692/733) of samples. Forty-one cases exhibited quantitative disparities in SMN1 and/or SMN2 CNs between the two methods. Confirmatory tests revealed that 37 inaccurate results were produced by the MLPA analysis, whereas four were attributed to the dPCR method. The dPCR technique exhibits better accuracy than MLPA and is qualified for SMA genetic testing across various clinical scenarios.