Newborn screening and diagnosis of mucopolysaccharidoses: application of tandem mass spectrometry

Abstract

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by the deficiency of lysosomal enzymes. The enzymes are required to break down glycosaminoglycans (GAGs) that help build bone, cartilage, tendons, corneas, skin and connective tissue. In patients with MPS, a missing enzyme leads to the accumulation of GAGs in the cells, blood, connective tissues, and multiple organs. The consequence is permanent, with progressive cellular damage affecting patients' appearance, physical abilities, organ and system function, and skeletal and mental development. The measurement of each specific GAG in a variety of specimens is required to establish the correlation between GAGs and physiological status of patients and/or prognosis and pathogenesis of the disease and to separate the patients with MPS from the healthy controls. We have developed a highly accurate, sensitive, and cost-effective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measurements of disaccharides derived from four specific GAGs [chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS)]. Disaccharides were produced by specific enzyme digestion of each GAG, and subsequently, quantified by negative ion mode of multiple reaction monitoring. Subclasses of GAGs with the same molecular weights can be separated by liquid chromatography. We have also developed another GAG assay by high-throughput mass spectrometry (HT-MS/MS). The HT-MS/MS consists of an integrated solid phase extraction robot that binds and de-salts samples from assay plates and directly injects them into a MS/MS detector, reducing sample processing time to within ten seconds. HT-MS/MS consequently yields much faster throughput than conventional LC-MS/MS-based methods; however, the HT-MS/MS system does not use a chromatographic step, and therefore, cannot separate GAGs that have the same molecular weights. Both techniques can be applied to the analysis of dried blood spots, blood, and urine specimens. In this review, we describe the assay methods for GAGs and the application to newborn screening and diagnosis of MPS.

Keywords: diagnosis; glycosaminoglycans; mucopolysaccharidoses; newborn screening; tandem mass spectrometry.

Figure 1. Disaccharides after digestion of glycosaminoglycans by enzymes

(adapted from Tomatsu et al. Assay for Glycosaminoglycans by Tandem Mass Spectrometry and its Applications. J Anal Bioanal Tech. 2014 Mar 1; 2014 (Suppl 2): 006). KS; Keratan sulfate, HS; heparan sulfate, DS; dermatan sulfate, CS; chondroitin sulfate.

Figure 2. Procedures of LC-MS/MS and RF-MS/MS

(adapted from Tomatsu et al. Assay for Glycosaminoglycans by Tandem Mass Spectrometry and its Applications. J Anal Bioanal Tech. 2014 Mar 1; 2014 (Suppl 2): 006). The same samples were analyzed by both LC-MS/MS and HT-MS/MS.

Figure 3. Chromatograms of disaccharides

(adapted from Tomatsu et al. Assay for Glycosaminoglycans by Tandem Mass Spectrometry and its Applications. J Anal Bioanal Tech. 2014 Mar 1; 2014 (Suppl 2): 006). Chromatograms for disaccharides of mono-sulfated KS, di-sulfated KS, ΔDiHS-NS, ΔDiHS-0S, ΔDiHS-6S, ΔDi-4S (DS), ΔDi-6S (C6S), and chondrosine (IS). Equipment: 6460 Triple Quad MS/MS with 1260 infinity LC (Agilent Technologies). IS: internal standard.

Figure 4. Correlation in GAG concentration between LC-MS/MS and HT-MS/MS

Correlation strength is interpreted as outlined by Johnston (27). Correlations are interpreted based on r values as follows: 0.0 to 0.2, very weak to negligible correlation; 0.2 to 0.4, weak correlation; 0.4 to 0.7, moderate correlation; 0.7 to 0.9, strong correlation. Analysis was performed using SPSS for Windows (version 17.0, SPSS Inc., Chicago, IL, USA). Rectangle: normal controls, square: MPS II, triangle: MPS IVA.

1. ΔDiHS-NS concentration. Correlation in blood (plasma or serum) ΔDiHS-NS of human subjects between LC-MS/MS and HT-MS/MS is significant (Control n = 187, MPS IVA n = 17, MPS II n = 10, p<0.0001).

2. ΔDiHS-0S concentration. Correlation in blood (plasma or serum) ΔDiHS-0S of human subjects between LC-MS/MS and HT-MS/MS is significant (Control n = 187, MPS IVA n = 17, MPS II n = 10, p<0.0001).

Figure 5. MRM of plasma samples in patients with MPS I-VII by LC-MS/MS

(adapted from Tomatsu et al Establishment of Glycosaminoglycan Assays for Mucopolysaccharidoses. Metabolites 2014, 4, 655-679). ΔDiHS-0S (left) and mono-sulfated KS (right) are shown.

Figure 6. Scheme for newborn screening and diagnosis of mucopolysaccharidoses

Aim 1. Determine the specificity and sensitivity of a new LC-MS/MS method and evaluate specific GAGs as biomarkers for MPS. Aim 2. Perform a MPS newborn screening pilot study on de-identified newborn samples.

Figure 7. Proposed mechanism of secondary elevation of blood KS in MPS

a) Elevation of synthesis of KS by storage of other GAGs. b) Inhibition of GALNS activity by the increased concentrations of HS. c) KS secretion into the circulation due to damage of cartilage and its ECM. d) Co-deposition of KS with the other accumulated GAGs. e) Altered KS resistant to degradation.