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Newborn screening for Pompe disease by measuring acid alpha-glucosidase activity using tandem mass spectrometry
Angéla Dajnoki1, Adolf Mühl, György Fekete
1Division of Biochemical and Paediatric Genetics, University Children's Hospital Vienna, Austria.
Insights
Newborn screening for Pompe disease is feasible using a tandem mass spectrometry (MS/MS) method to measure acid alpha-glucosidase (GAA) activity in dried blood spots. This assay is accurate and reliable for early detection, enabling timely treatment.
Area of Science:
- Biochemistry
- Genetics
- Pediatrics
Background:
- Pompe disease is a severe lysosomal storage disorder caused by acid alpha-glucosidase (GAA) deficiency.
- Early diagnosis and treatment are crucial for improving outcomes in infants with Pompe disease.
- Newborn screening is essential for identifying affected infants promptly.
Purpose of the Study:
- To evaluate a tandem mass spectrometry (MS/MS) method for measuring GAA activity in dried blood spots (DBS).
- To assess the suitability of this method for newborn screening of Pompe disease.
- To establish a reliable diagnostic cutoff for early detection.
Main Methods:
- Incubation of DBS punches with a specific substrate and internal standard.
- Quantification of enzyme product and standard using MS/MS.
- Assessment of assay precision, stability, carryover, and correlation with hematocrit and punch location.
Main Results:
- GAA activity in DBS from known Pompe patients was <2 micromol/h/L.
- Mean GAA activity in 10,279 newborn screening samples was 14.7 (7.2) micromol/h/L.
- The MS/MS assay demonstrated stability, minimal carryover, and high precision (<10% imprecision).
Conclusions:
- The MS/MS method for measuring GAA activity in DBS is suitable for high-throughput newborn screening.
- This assay enables accurate and reliable detection of Pompe disease in newborns.
- Early identification through newborn screening facilitates timely enzyme replacement therapy.
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.
