The combined use of enzyme activity and metabolite assays as a strategy for newborn screening of mucopolysaccharidosis type I.

Giulia Polo,Daniela Gueraldi,Alessandro P Burlina,Laura Rubert,Alberto B Burlina,Leonardo Salviati,Antonio Marzollo,Antonella Giuliani,Chiara Cazzorla,Alessandra Biffi

doi:10.1515/cclm-2020-0064

Abstract

Objectives Mucopolysaccharidosis type I (MPS I) was added to our expanded screening panel in 2015. Since then, 127,869 newborns were screened by measuring α-L-iduronidase (IDUA) enzyme activity with liquid chromatography tandem mass spectrometry (LC-MS/MS). High false positives due to frequent pseudodeficiency alleles prompted us to develop a second-tier test to quantify glycosaminoglycan (GAG) levels in dried blood spot (DBS). Methods Heparan-sulfate (HS) and dermatan-sulfate (DS) were measured with LC-MS/MS after methanolysis. DBSs were incubated with methanolic-HCl 3 N at 65 °C for 45 min. Chromatographic separation used an amide column with a gradient of acetonitrile and water with 10 mM ammonium acetate in a 9-min run. The method was validated for specificity, linearity, lower limit of quantification (LOQ), accuracy and precision. Results Intra- and inter-day coefficients of variation were <15% for both metabolites. Reference values in 40 healthy newborns were: HS mean 1.0 mg/L, 0-3.2; DS mean 1.5 mg/L, 0.5-2.7). The two confirmed newborn MPS I patients had elevated HS (4.9-10.4 mg/L, n.v. <3.2) and DS (7.4-8.8 mg/L, n.v. <2.7). Since its introduction in February 2019, the second-tier test reduced the recall rate from 0.046% to 0.006%. Among 127,869 specimens screened, the incidence was 1:63,935 live births. Both patients started enzyme replacement therapy (ERT) within 15 days of birth and one of them received allogenic hematopoietic stem cell transplantation (HSCT) at ht age of 6 months. Conclusions GAGs in DBS increased the specificity of newborn screening for MPS I by reducing false-positives due to heterozygosity or pseudodeficiency. Early diagnosis and therapeutical approach has improved the outcome of our patients with MPS I.

Highlights

IntroductionMucopolysaccharidosis type I (MPS I) involves a deficiency of the lysosomal enzyme α-L-iduronidase (IDUA) resulting in progressive lysosomal accumulation of dermatan and heparan sulfates with clinical manifestations characteristic of the MPS phenotype [2]
Mucopolysaccharidoses (MPSs) are a group of rare diseases caused by a reduction or lack of the lysosomal enzymes that catalyze the stepwise degradation of glycosaminoglycans (GAGs) [1].Mucopolysaccharidosis type I (MPS I) involves a deficiency of the lysosomal enzyme α-L-iduronidase (IDUA) resulting in progressive lysosomal accumulation of dermatan and heparan sulfates with clinical manifestations characteristic of the MPS phenotype [2]
To improve the performance of newborn screening (NBS) for MPS I and reduce the large number of recalls, we recently developed a second-tier test based on quantification of GAG levels in DBSs by methanolysis followed by LC-MS/MS analysis

Summary

Introduction

Mucopolysaccharidosis type I (MPS I) involves a deficiency of the lysosomal enzyme α-L-iduronidase (IDUA) resulting in progressive lysosomal accumulation of dermatan and heparan sulfates with clinical manifestations characteristic of the MPS phenotype [2]. Polo et al.: Second-tier test for mucopolysaccharidosis type I screening syndromes represent phenotypes at the severe and mild extremes of the clinical spectrum, respectively, whereas the Hurler-Scheie syndrome has an intermediate phenotype. Therapeutic approaches for MPS I include enzyme replacement therapy (ERT) with L-iduronidase, allogeneic hematopoietic stem cell transplantation (HSCT) and recently, ex vivo gene therapy in autologous hematopoietic stem cells [3, 4]. It has been demonstrated that newborn screening (NBS) for MPS I allows timely initiation of therapy, potentially changing its natural history [5]

Objectives

Methods

Conclusion