High precision newborn screening for mucopolysaccharidosis type I by enzymatic activity followed by endogenous, non-reducing end glycosaminoglycan analysis.

Measurement of enzymatic activity in newborn dried blood spots (DBS) is the preferred first-tier method in newborn screening (NBS) for mucopolysaccharidosis (MPS) disorders. However, false positives are observed due mainly to the presence of pseudodeficiencies. Our previous publications on glycosaminoglycan (GAG) biomarker levels in dried blood spots (DBS) for mucopolysaccharidoses demonstrated that second-tier GAG biomarker analysis can dramatically reduce the false positive rate in NBS. In the present study, we extend this approach to the analysis of a large number of false positives for MPS-I obtained from the Illinois, New York, and Tennessee NBS programs and from Greenwood Genetics Center. Results show that GAG levels measured by the Endogenous-Non-Reducing End method (Endogenous-NRE) are in the normal reference range for all samples. In a second study, we analyzed 166 samples that showed below-cutoff MPS-I enzymatic activity level after testing 384,144 newborns in the Ontario, Canada NBS program. Both genotype and Endogenous-NRE GAG levels were determined for all 166 samples. Newborns at high risk for MPS-I based on genotype also showed elevated GAG levels and were clinically confirmed to be symptomatic for MPS-I. All newborns with pseudodeficiency or carrier status by genotyping all showed normal levels of the appropriate GAG biomarker. Samples found to be inconclusive based on one or more variants of unknown significance (VUS) all showed normal GAG biomarker levels and were found to be clinically normal during follow-up. These studies show that the Endogenous-NRE GAG second-tier NBS method is preferred over second-tier DNA analysis for the NBS of MPS-I with minimal false positives.