Diagnosis of Mucopolysaccharidoses and Mucolipidosis by Assaying Multiplex Enzymes and Glycosaminoglycans

Abstract

Mucopolysaccharidoses (MPS) and mucolipidosis (ML II/III) are a group of lysosomal storage disorders (LSDs) that occur due to a dysfunction of the lysosomal hydrolases responsible for the catabolism of glycosaminoglycans (GAGs). However, ML is caused by a deficiency of the enzyme uridine-diphosphate N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. A timely diagnosis of MPS and ML can lead to appropriate therapeutic options for patients. To improve the accuracy of diagnosis for MPS and ML in a high-risk population, we propose a combination method based on known biomarkers, enzyme activities, and specific GAGs. We measured five lysosomal enzymes (α-L-iduronidase (MPS I), iduronate-2-sulfatase (MPS II), α-N-acetylglucosaminidase (MPS IIIB), N-acetylglucosamine-6-sulfatase (MPS IVA), and N-acetylglucosamine-4-sulfatase (MPS VI)) and five GAGs (two kinds of heparan sulfate (HS), dermatan sulfate (DS), and two kinds of keratan sulfate (KS)) in dried blood samples (DBS) to diagnose suspected MPS patients by five-plex enzyme and simultaneous five GAGs assays. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for both assays. These combined assays were tested for 43 patients with suspected MPS and 103 normal control subjects. We diagnosed two MPS I, thirteen MPS II, one MPS IIIB, three MPS IVA, two MPS VI, and six ML patients with this combined method, where enzymes, GAGs, and clinical manifestations were compatible. The remaining 16 patients were not diagnosed with MPS or ML. The five-plex enzyme assay successfully identified MPS patients from controls. Patients with MPS I, MPS II, and MPS IIIB had significantly elevated HS and DS levels in DBS. Compared to age-matched controls, patients with ML and MPS had significantly elevated mono-sulfated KS and di-sulfated KS levels. The results indicated that the combination method could distinguish these affected patients with MPS or ML from healthy controls. Overall, this study has shown that this combined method is effective and can be implemented in larger populations, including newborn screening.

Keywords: enzyme assay; glycosaminoglycans; mucolipidosis; mucopolysaccharidoses; newborn screening.

Figure 1

Dermatan sulfate (DS) values for control subjects and those diagnosed with MPS or ML. MPS I and MPS II had significantly increased DS values. Mean is shown with the horizontal bars. A p-value of <0.05 is denoted by * and a p-value of <0.001 is denoted by ***.

Figure 2

N-sulfated heparan sulfate (HS-NS) for control subjects and those diagnosed with MPS or ML. MPS I, II, and IIIB had significantly increased HS-NS values. Mean is shown with the horizontal bars. A p-value of <0.001 is denoted by ***.

Figure 3

Non-sulfated heparan sulfate (HS-0S) for control subjects and those diagnosed with MPS or ML. The samples from MPS I, II, and IIIB had significantly increased HS-0S values. Mean is shown with the horizontal bars. A p-value of <0.001 is denoted by ***.

Figure 4

Di-sulfated keratan sulfate (KS) values for control subjects by age. All values are in ng/mL.

Figure 5

Mono-sulfated keratan sulfate (KS) values for control subjects by age. All values are in ng/mL.

Figure 6

Mono-sulfated keratan sulfate (KS) values for control group and those diagnosed with MPS or ML. MPS IVA, VI, and ML had significantly increased mono-sulfated KS values. Mean is shown with the horizontal bars. A p-value of <0.01 is denoted by **, and a p-value of < 0.001 is denoted by ***.

Figure 7

Di-sulfated keratan sulfate (KS) for control subjects and those diagnosed with MPS or ML. MPS IVA, VI, and ML had significantly increased di-sulfated KS values. Mean is shown with the horizontal bars. A p-value of <0.001 is denoted by ***.