Development of Newborn Screening Methods for Mucopolysaccharidosis III type A and type B in Dried Blood Spot using Tandem Mass Spectrometry

Abstract

Chapter I. Development of Newborn Screening Method for Mucopolysaccharidosis III Type A in Dried Blood Spot using Tandem Mass Spectrometry Mucopolysaccharidosis III type A (MPS III A) is an autosomal recessive inherited disease with no current treatment available. It is caused by the deficiency of sulfamidase (SGSH) in the lysosome and the inability to degrade a polysaccharide called heparan sulfate. Gene therapy and enzyme replacement therapy for MPS III A are already in development and have shown promising preclinical result. To achieve the best result from the potential treatment, it is necessary to administrate the treatment before the onset of irreversible damage to the central nervous system. To fulfill this requirement, a simple, fast and robust method for screening MPS III A in newborns is in demand. The first high-throughput screening method for MPS III A that is compatible with dried blood spot (DBS) was developed. This method used a synthetic substrate to assay the enzyme activity in dried blood spots via tandem mass spectrometry (MS/MS). Preliminary data suggested that the newly developed assay was able to distinguish patients from normal population in sulfamidase activity: patient SGSH activity ranged from 0.013 – 0.056 μmol/h/L; while normal newborn SGSH activity ranged from 0.10 - 0.81 μmol/h/L. Chapter II. Development of Newborn Screening Method for Mucopolysaccharidosis III Type B in Dried Blood Spot using Tandem Mass Spectrometry Mucopolysaccharidosis III type B (MPS III B) is an autosomal recessive inherited disease with no current treatment. It is caused by the deficiency of N-acetyl glucosaminidase (NAGLU) in the lysosome and the inability to degrade a polysaccharide called heparan sulfate. NAGLU is a downstream enzyme of sulfamidase, which causes MPS III A. Sulfamidase and NAGLU are working together with several other enzymes, acetyl-CoA: α-glucosaminide acetyltransferase and N-acetylglucosamine 6-sulfatase, to cleave off the nonreducing end glucosamine residue from a heparan sulfate polysaccharide molecule. The accumulation of heparan sulfate in the lysosome will cause damage to the central nervous system which in turn will result in mild somatic and severe neurological manifestation. It is crucial to identify the affected individual as soon as possible, preferably right after birth. In order to do so, a high-throughput screening method was developed, and it had shown its power in distinguish patients from the normal people. Preliminary data suggested that a random normal person has a NAGLU activity 100 times higher than the one of the affected persons.