Improved Reagents for Newborn Screening of Mucopolysaccharidosis Types I, II, and VI by Tandem Mass Spectrometry

Abstract

Tandem mass spectrometry for the multiplex and quantitative analysis of enzyme activities in dried blood spots on newborn screening cards has emerged as a powerful technique for early assessment of lysosomal storage diseases. Here we report the design and process-scale synthesis of substrates for the enzymes α-l-iduronidase, iduronate-2-sulfatase, and N-acetylgalactosamine-4-sulfatase that are used for newborn screening of mucopolysaccharidosis types I, II, and VI. The products contain a bisamide unit that is hypothesized to readily protonate in the gas phase, which improves detection sensitivity by tandem mass spectrometry. The products contain a benzoyl group, which provides a useful site for inexpensive deuteration, thus facilitating the preparation of internal standards for the accurate quantification of enzymatic products. Finally, the reagents are designed with ease of synthesis in mind, thus permitting scale-up preparation to support worldwide newborn screening of lysosomal storage diseases. The new reagents provide the most sensitive assay for the three lysosomal enzymes reported to date as shown by their performance in reactions using dried blood spots as the enzyme source. Also, the ratio of assay signal to that measured in the absence of blood (background) is superior to all previously reported mucopolysaccharidosis types I, II, and VI assays.

Tandem Mass Spectrometry Has a Larger Analytical Range than Fluorescence Assays of Lysosomal Enzymes: Application to Newborn Screening and Diagnosis of Mucopolysaccharidoses Types II, IVA, and VI

“…Thus, we explored the consequence of replacing Gal-6-sulfate in our previous substrate with GalNAc-6-sulfate. We also replaced the 4MU-based aglycone in our original GALNS substrate with an aglycone containing the 4-acetamido-phenol moiety because this aglycone change results in an improved assay MS/MS response per mole for the product derived from our new I2S substrate (6). The structure of our new GALNS substrate, GalNAc-6-S-C6/C6-benzoyl group (Bz), is shown in Fig.…”

“…For mucopolysaccharidosis-II (MPS-II), MPS-IVA, and MPS-V, the deficient enzymes are, respectively, iduronide-2-sulfatase (I2S), N -acetylgalactosamine-6-sulfatase (GALNS), and N -acetylgalactosamine-4-sulfatase (ARSB). I2S can be assayed fluorometrically with the 4MU glycoside of iduronic acid-2-sulfate using human α -L-iduronidase (IDUA) to liberate 4MU after the 2-sulfate is removed (5) or by MS/MS (6). GALNS can be assayed with the 4MU glycoside of galactose-6-sulfate using bacterial β -galactosidase to release the 4MU after the sulfatase acts (7) or by MS/MS (8).…”

Precision newborn screening for lysosomal disorders

“…19 Enzyme substrates were purchased from PerkinElmer Life Sciences (Shelton, CT). 20 Testing showed adequate stability and reproducibility over time (coefficient of variation range: 7-15%; Supplementary Figure 2). Two of the biochemical methods used for secondtier tests (Krabbe disease: psychosine concentration, MPS I: dermatan sulfate and heparan sulfate concentrations) have been described previously.…”

Mucopolysaccharidosis Type I Newborn Screening: Best Practices for Diagnosis and Management

“…Available screening assays include fluorometric, 15,18 digital microfluidics, 15 and tandem mass spectrometry-based analyses. [38][39][40][41] MPS I screening may be included in multiplex assays that include screens for multiple lysosomal storage disorders in a single DBS sample. 40 If IDUA levels are below established cut-off values, the process outlined in the algorithm in the Figure is proposed to confirm and delineate disease severity, and guide appropriate follow-up and treatment.…”