Simultaneous Detection of SMN1, SMN2, NAIP, H4F5, and GTF2H2 Copy Numbers and SMN1 Loss-of-Function Variants for SMA by MALDI-TOF Mass Spectrometry

Spinal muscular atrophy (SMA) is a common fatal genetic disorder with a high carrier rate. For the prevention program and treatment plan of this disease, a comprehensive assay is practically needed to gain multigenetic information, enabling effective molecular screening and accurate clinical classification. In this study, a novel single-tube matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry assay was developed to simultaneously analyze the copy numbers of survival motor neuron ( SMN ), neuronal apoptosis-inhibitory protein ( NAIP ), small EDRK-rich factor 1A ( H4F5 ), and general transcription factor IIH subunit 2 ( GTF2H2 ); identify six common loss-of-function variants in SMN1 ; and identify hybrid SMN . The results of this assay were 100% consistent with the predetermined values in 158 genotype-known samples. Furthermore, a cohort of 162 clinical samples was double-blind detected to evaluate this assay, and the results showed a 100% overall concordance with comparative methods. Herein, this novel MALDI-TOF mass spectrometry assay was found to be practical and cost-effective and to have multigenetic information available, which is suitable for large-scale molecular screening and routine clinical diagnosis of spinal muscular atrophy. © 2026 Association for Molecular Pathology and American Society for Investigative Pathology.