Newborn screening for Pompe disease by measuring acid α-glucosidase activity using tandem mass spectrometry

BACKGROUND: Pompe disease, caused by the deficiency of acid α-glucosidase (GAA), is a lysosomal storage disorder that manifests itself in its most severe form within the first months of life. Early detection by newborn screening is warranted, since prompt initiation of enzyme replacement therapy may improve morbidity and mortality. We evaluated a tandem mass spectrometry (MS/MS) method to measure GAA activity for newborn screening for Pompe disease. METHODS: We incubated 3.2-mm punches from dried blood spots (DBS) for 22 h with the substrate [7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6- hydroxymethyl-tetrahydro-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester] and internal standard [7-d₅-benzoylamino-heptyl)- [2-(4-hydroxyphenylcarbamoyl)-ethyl]-carbamic acid tertbutyl ester]. We quantified the resulting product and internal standard using MS/MS. We assessed inter- and intrarun imprecision, carryover, stability, and correlation between enzyme activities and hematocrit and punch location and generated a Pompe disease-specific cutoff value using routine newborn screening samples. RESULTS: GAA activities in DBS from 29 known Pompe patients were <2 μmol/h/L. GAA activities in routine newborn screening samples were [mean (SD)] 14.7 (7.2) μmol/h/L (n = 10 279, median 13.3, 95% CI 14.46 -14.74 μmol/h/L) and in normal adult samples 9.3 (3.3) μmol/h/L (n = 229, median 9, 95% CI 8.88-9.72 μmol/h/L). GAA activity was stable for 28 days between 37°C and -80°C. Carryover could not be observed, whereas intrarun and interrun imprecision were <10%. The limit of detection was 0.26 μmol/h/L and limit of quantification 0.35 μmol/h/L. CONCLUSIONS: The measurement of GAA activities in dry blood spots using MS/MS is suitable for high-throughput analysis and newborn screening for Pompe disease.