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Newborn Screening for Pompe Disease by Measuring Acid -Glucosidase Activity Using Tandem Mass Spectrometry

¹ Division of Biochemical and Paediatric Genetics, University Children’s Hospital Vienna, Austria;² 2nd Department of Paediatrics, Semmelweis University Budapest, Hungary;³ Genzyme Corporation, Framingham, MA;⁴ Wadsworth Center, New York State Laboratories, Albany, NY;⁵ Newborn Screening Branch, Centers for Disease Control and Prevention, Atlanta, GA.

^aAddress correspondence to this author at: Division of Biochemical and Paediatric Genetics, Department of General Paediatrics, University Children’s Hospital Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria. Fax 0043 1 406 3484; e-mail olaf.bodamer{at}meduniwien.ac.at.

background: Pompe disease, caused by the deficiency of acid -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-hydroxy-phenylcarbamoyl)-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.

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