Pilot study of newborn screening for six lysosomal storage diseases using Tandem Mass Spectrometry

Abstract

Background: There is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life.

Objective: To evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases.

Methods and results: Enzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0).

Discussion: A 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform.

Keywords: Dried blood spot; Fabry disease; Gaucher disease; Hurler disease; Krabbe disease; Lysosomal storage disorders; Newborn screening; Niemann-Pick-A/B disease; Pompe disease; Tandem mass spectrometry.

Fig. 1

Substrates used for the 6-plex assay. For GLA, GAA, and IDUA the enzymatic products are the substrates with the sugar replaced with hydrogen, and the internal standards are the products with 5 deuteriums on the benzoyl group. For GBA, GALC and SMPD1, the product is the substrate with the sugar (GBA and GALC) or the phosphocholine (SMPD1) replaced with hydrogen. The internal standards are the products but with deuterium in the fatty acyl chain (CD₃CD₂CD₂ for SMPD1, and CD₃CD₂ for GALC) or in the sphingosine chain (CD₃CD₂CD₂ for GBA).

Fig. 2

Distribution of 6 enzyme activities in the 6-plex.