Newborn screening and diagnosis of mucopolysaccharidoses

Abstract

Mucopolysaccharidoses (MPS) are caused by deficiency of lysosomal enzyme activities needed to degrade glycosaminoglycans (GAGs), which are long unbranched polysaccharides consisting of repeating disaccharides. GAGs include: chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and hyaluronan. Their catabolism may be blocked singly or in combination depending on the specific enzyme deficiency. There are 11 known enzyme deficiencies, resulting in seven distinct forms of MPS with a collective incidence of higher than 1 in 25,000 live births. Accumulation of undegraded metabolites in lysosomes gives rise to distinct clinical syndromes. Generally, the clinical conditions progress if untreated, leading to developmental delay, systemic skeletal deformities, and early death. MPS disorders are potentially treatable with enzyme replacement therapy or hematopoietic stem cell transplantation. For maximum benefit of available therapies, early detection and intervention are critical. We recently developed a novel high-throughput multiplex method to assay DS, HS, and KS simultaneously in blood samples by using high performance liquid chromatography/tandem mass spectrometry for MPS. The overall performance metrics of HS and DS values on MPS I, II, and VII patients vs. healthy controls at newborns were as follows using a given set of cut-off values: sensitivity, 100%; specificity, 98.5-99.4%; positive predictive value, 54.5-75%; false positive rate, 0.62-1.54%; and false negative rate, 0%. These findings show that the combined measurements of these three GAGs are sensitive and specific for detecting all types of MPS with acceptable false negative/positive rates. In addition, this method will also be used for monitoring therapeutic efficacy. We review the history of GAG assay and application to diagnosis for MPS.

Keywords: CF; CNS; CPC; CS; DBS; DMB; DS; ERT; ESI-MS/MS; GAG; GALNS; GUS; Glycosaminoglycans; HPLC; HS; HSCT; KS; LC–MS/MS; MPS; Monitoring; Mucopolysaccharidoses; N-acetylgalactosamine-6-sulfate sulfatase; NBS; Newborn screening; SRT; Tandem mass spectrometry; central nervous system; cetylpyridinium chloride; chondroitin sulfate; cystic fibrosis; dermatan sulfate; dimethylmethylene blue; dried blood spot; electrospray ionization tandem mass spectrometry; enzyme replacement therapy; glycosaminoglycans; hematopoietic stem cell transplantation; heparan sulfate; high performance liquid chromatography; high performance liquid chromatography tandem mass spectrometry; keratan sulfate; mucopolysaccharidoses; newborn screening; substrate reduction therapy; β-glucuronidase.

Fig. 1

Production of disaccharides digested by the enzymes.

Fig. 2

Sample preparation and apparatus with automated procedure.

Fig. 3

Chromatograms for KS-I (bovine cornea), ΔDi-4S (DS), ΔDiHS-NS, ΔDiHS-0S, and chondrosine (IS) disaccharides by using Hypercarb (2.0 mm × 50 mm, 5 μm; Thermo Electron Corp.) with a solvent gradient of 0% to 100% 5 mM ammonium acetate (pH 10):acetonitrile. Polymer KS was separated with mono-sulfated KS [Galβ1-4GlcNAc(6S)] and di-sulfated KS [Gal(6S)β1-4GlcNAc(6S)] after digestion by keratanase II. Equipment: 6460 Triple Quad MS/MS with 1260 infinity LC (Agilent Technologies). IS: internal standard.

Fig. 4

Chromatograms of extracts from control (left) and MPS II (right) blood spots. Equipment; HP1100 system/API-4000. The arrows show the peaks of each disaccharide. KS has two peaks: mono-sulfated KS [Galβ1-4GlcNAc(6S)] (a major peak) and di-sulfated KS [Gal(6S)β1-4GlcNAc(6S)].

Fig. 5

Total DS and HS levels in blood of MPS patients. Equipment; HP1100 system/API-4000.

Fig. 6

Age-dependent KS in MPS IVA patients. White square; controls, red rhombus; MPS IVA patients with a severe form, green circle; MPS IVA patients with an attenuated form. Equipment; HP1100 system/API-4000.

Fig. 7

Distribution of each GAG level in DBS of normal newborns by birth weight. Average of ΔDiHS-0S, ΔDiHS-NS, and ΔDi-4S was 347 ng/ml (range: 282–502), 85 ng/ml (range: 64–159), and 96 ng/ml (range: 57–163), respectively. The average of KS was 1185 ng/ml (range: 684–1511). Yellow: ΔDiHS-0S, green: ΔDiHS-NS, pink: ΔDi-4S, blue line: KS. Equipment; HP1100 system/API-4000.

Fig. 8

Frequency distribution of A) ΔDiHS-NS, B) ΔDi-4S, and C) ΔDiHS-0S for DBS from 326 controls to 6 MPS cases. Values for 6 cases are marked with arrows. Bars represent controls with frequency. Equipment; HP1100 system/API-4000.

Fig. 9

Proposed two-tier strategy of newborn screening for mucopolysaccharidoses.