MassARRAY-based targeted detection of SLC22A5 mutations: A feasibility study for secondary screening of primary...

This study aimed to evaluate the feasibility of using MassARRAY molecular mass spectrometry to detect SLC22A5 gene mutations in second-tier screening for primary carnitine deficiency (PCD) among newborns. The goal is to provide technical evidence to support improvements in PCD screening and to reduce diagnostic delays. We first reviewed literature and databases to identify commonly reported pathogenic variants in the SLC22A5 gene among the Chinese population and established a hotspot mutation panel. MassARRAY analysis was conducted on dried blood spot samples using multiplex PCR, single-base extension, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The constructed panel included 29 mutations with an estimated allele coverage of 87.3%, and the assay achieved a detection success rate of 99.75%. A total of 1200 neonates suspected of having PCD, identified via tandem mass spectrometry (MS/MS) between January 2023 and December 2024 in Beijing, were consecutively enrolled in this study. Out of the 1200 newborns, 125 (10.42%) carried at least one mutation: five cases (0.42%) had two variants and 120 cases (10%) had a single variant. A total of 991 samples (82.58%) were mutation-negative, though one case was a confirmed false negative. Incomplete mutation data were observed in 84 samples (7%). Among the 29 targeted sites, 19 mutations were detected (65.52%), with the most frequent being c.1400C > G (p.S467C), followed by c.51C > G (p.F17L), c.92C > T (p.P31L), and c.428C > T (p.P143L). Eight PCD cases were clinically confirmed; five were identified as having two mutations via MassARRAY, two had single mutations, and one showed no detectable mutation. MassARRAY-based detection of SLC22A5 mutations offers a viable method for second-tier PCD screening. It can reduce recall rates and false positives in initial MS/MS screening and shorten the diagnostic process. Further validation in larger cohorts and continued expansion of the mutation panel are recommended to enhance the accuracy of this method.