title: Newborn screening for Pompe disease by measuring acid alpha-glucosidase activity using tandem mass spectrometry.
abstract: BACKGROUND
  Pompe disease, caused by the deficiency of acid alpha-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-d(5)-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 micromol/h/L. GAA activities in routine newborn screening samples were [mean (SD)] 14.7 (7.2) micromol/h/L (n = 10,279, median 13.3, 95% CI 14.46-14.74 micromol/h/L) and in normal adult samples 9.3 (3.3) micromol/h/L (n = 229, median 9, 95% CI 8.88-9.72 micromol/h/L). GAA activity was stable for 28 days between 37 degrees C and -80 degrees C. Carryover could not be observed, whereas intrarun and interrun imprecision were <10%. The limit of detection was 0.26 micromol/h/L and limit of quantification 0.35 micromol/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.
authors.affiliation: Division of Biochemical and Paediatric Genetics, University Children's Hospital Vienna, Austria.
authors.forename: Angéla
authors.initials: A
authors.lastname: Dajnoki
authors.forename: Adolf
authors.initials: A
authors.lastname: Mühl
authors.forename: György
authors.initials: G
authors.lastname: Fekete
authors.forename: Joan
authors.initials: J
authors.lastname: Keutzer
authors.forename: Joe
authors.initials: J
authors.lastname: Orsini
authors.forename: Victor
authors.initials: V
authors.lastname: Dejesus
authors.forename: X Kate
authors.initials: XK
authors.lastname: Zhang
authors.forename: Olaf A
authors.initials: OA
authors.lastname: Bodamer
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D006009:Glycogen Storage Disease Type II / Q000175:diagnosis* / Q000201:enzymology; D006801:Humans; D007231:Infant, Newborn; D015997:Neonatal Screening; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000520:alpha-Glucosidases / Q000097:blood*
chemical_list: D000520:alpha-Glucosidases