Liquid Chromatography–Tandem Mass Spectrometry Assay of Leukocyte Acid α-Glucosidase for Post-Newborn Screening Evaluation of Pompe Disease

Abstract

BACKGROUND Pompe disease (PD) is the first lysosomal storage disorder to be added to the Recommended Uniform Screening Panel for newborn screening. This condition has a broad phenotypic spectrum, ranging from an infantile form (IOPD), with severe morbidity and mortality in infancy, to a late-onset form (LOPD) with variable onset and progressive weakness and respiratory failure. Because the prognosis and treatment options are different for IOPD and LOPD, it is important to accurately determine an individual’s phenotype. To date, no enzyme assay of acid α-glucosidase (GAA) has been described that can differentiate IOPD vs LOPD using blood samples. METHODS We incubated 10 μL leukocyte lysate and 25 μL GAA substrate and internal standard (IS) assay cocktail for 1 h. The reaction was purified by a liquid–liquid extraction. The extracts were evaporated and reconstituted in 200 μL methanol and analyzed by LC-MS/MS for GAA activity. RESULTS A 700-fold higher analytical range was observed with the LC-MS/MS assay compared to the fluorometric method. When GAA-null and GAA-containing fibroblast lysates were mixed, GAA activity could be measured accurately even in the range of 0%–1% of normal. The leukocyte GAA activity in IOPD (n = 4) and LOPD (n = 19) was 0.44–1.75 nmol · h−1 · mg−1 and 2.0–6.5 nmol · h−1 · mg−1, respectively, with no overlap. The GAA activity of pseudodeficiency patients ranged from 3.0–28.1 nmol · h−1 · mg−1, showing substantial but incomplete separation from the LOPD group. CONCLUSIONS This assay allows determination of low residual GAA activity in leukocytes. IOPD, LOPD, and pseudodeficiency patients can be partially differentiated by measuring GAA using blood samples.

Contribution of tandem mass spectrometry to the diagnosis of lysosomal storage disorders

“…For example, precise measurement of acid α-glucosidase activity distinguishes infantile-and late-onset PD from acid α-glucosidase pseudodeficiency (Liao et al 2017a). This has also been reported for leukocytes when confirming PD (Lin et al 2017). MS/MS also gives more accurate results than the radiometric method for measuring β-D-galactocerebrosidase activity in lymphocytes (Liao et al 2017b).…”

Multiplex Tandem Mass Spectrometry Enzymatic Activity Assay for Newborn Screening of the Mucopolysaccharidoses and Type 2 Neuronal Ceroid Lipofuscinosis

“…High analytical ranges of the MS/MS assays are predicted to yield a lower number of false positives compared to fluorometric assays when applied to newborn screening of LSDs as has been observed from large scale pilot studies and live newborn screening reports (7). Also, it is not possible with the fluorometric assays to accurately measure relatively low amounts of residual lysosomal enzymes for post-newborn screening diagnosis and prognosis studies, whereas MS/MS assays provide clear stratification of these samples (35-37). The relatively low analytical ranges for the fluorometric assays are due to the fact that the substrate itself is fluorescent and thus significantly contributes to the blank (8).…”

Moonlighting newborn screening markers: the incidental discovery of a second-tier test for Pompe disease

“…Figure 2b reported as likely benign or pseudodeficient, 9 and these cases were not included in a summary of Pompe disease in a recent report of post-newborn screening evaluation of Pompe disease in New York State. 32 For these reasons, we are considering these cases to be false positives properly recognized by the CLIR tools. The genotypes of the final two cases resolved by CLIR as Pompe disease were p.E721Rfs//p.…”