High Precision Newborn Screening for Mucopolysaccharidosis Type I by Enzymatic Activity followed by Endogenous, Non-Reducing End Glycosaminoglycan Analysis

Abstract

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.

1. Introduction

Newborn screening (NBS) for the lysosomal storage disease mucopolysaccharidosis type I (MPS-I) started about a decade ago and is now occurring in about 45 NBS laboratories worldwide (USA, Taiwan, The Netherlands, Canada). All NBS laboratories first measure the activity of the relevant enzyme (α-iduronidase) in dried blood spots (DBS). Most of the first-tier abnormal results are false positives often due to the presence of pseudodeficiency alleles. To reduce false positives, some NBS laboratories carry out DNA sequencing to look for variants in the IDUA gene [1], while other laboratories measure glycosaminoglycan (GAG)-derived biomarkers by tandem mass spectrometry [2][3]. Available data shows that GAG analysis is a more powerful second-tier test to reduce false positives than DNA analysis since many of the observed genotypes are inconclusive, examples of which are discussed in this publication.

We have studied in detail two different GAG analysis methods in DBS for NBS of the full set of MPSs including MPS-I [2][4][5][6]. The Internal Disaccharide method involves the in vitro enzymatic digestion of GAG polymers into several different disaccharides that form the internal repeating unit of the GAG polymer. These species are detected by combined liquid chromatography-tandem mass spectrometry (LC-MS/MS). The second GAG analysis method is called the Endogenous Non-Reducing End method (Endogenous-NRE) which is based on LC-MS/MS detection of short oligosaccharide fragments that come from the non-reducing end of the GAG polymer by one or more enzymes endogenously present in humans. A comparison of the two GAG analysis methods has shown that the Endogenous-NRE method leads to a much bigger separation compared to the Internal Disaccharide method between biomarker levels in affected patients versus the normal reference range [4]-[6]. Also, false positives remain with the Internal Disaccharide method, but so far none are detected with the Endogenous-NRE method [4]-[6]. In this paper, we extended our study of GAG analysis for MPS-I by analyzing DBS from samples from MPS-I carriers as well as newborns that showed pseudodeficiency variants in the IDUA gene. We also carried out the Endogenous-NRE method on 166 (of 187 total) newborn DBS identified with below-cutoff IDUA activity by the Ontario NBS laboratory from 384,144 newborns screened between August 2020 and April 2023. These studies show that the Endogenous-NRE GAG method is helpful in resolving cases where enzymatic activity is low and the genotype is ambiguous.

2. Materials and Methods

All patient samples were de-identified and analyzed as part of a quality assurance project. This study received IRB approval by the University of Washington. The source of DBS samples is provided as Supplemental Material along with genotypes for all MPS-I pseudodeficiency and MPS-I carrier samples. The Endogenous-NRE GAG analysis was applied as described in detail previously [2]. Quality control DBS samples containing authentic GAG marker for MPS-I were purchased from GelbChem, LLC. The QC DBS were run in all separate batch runs of DBS samples and all datasets were normalized to the initial dataset (shown in Figure 1). This is important because the internal standard is not chemically identical to the MPS-I GAG biomarker, and the relative MS/MS response factor changes over time and on different MS/MS instruments.

Figure 1.

Endogenous-NRE GAG biomarker in newborn DBS from clinically-confirmed MPS-I patients and obtained from state NBS laboratories. See maintext for full description.

3. Results

Figure 1 shows the level of the Endogenous-NRE MPS-I GAG biomarker (UA-HNAc(1S)-Early) in different groups of newborn DBS. The exact chemical structure of this marker is not known; thus, UA refers to uronic acid (iduronic or glucuronic acid), HNAc is either N-acetyl-galactosamine or N-acetyl-glucosamine, and (1S) means there is a single sulfate group somewhere in the disaccharide. The "Early" designation refers to the earlier-eluting peak from the LC column that is isobaric with a later-eluting species [2]. Included in Figure 1 are newborn DBS from 16 patients clinically diagnosed with early-onset MPS-I and 3 with attenuated (later-onset) MPS-I. These are the same previous samples [2] along with one additional attenuated MPS-I sample not previously reported. Age of onset of symptoms and genotype information for all DBS presented in this study are given in Supplemental Material Tables S1 and S2. These DBS were obtained from NBS laboratories that store residual newborn DBS collected at birth [2]. As reported previously [2], all of the MPS-I DBS show levels of UA-HNAc(1S)-Early GAG biomarker well above the normal reference range defined by 231 random newborn DBS (Figure 1). Note also that two attenuated MPS-I DBS samples showed GAG marker lower than all of the severe MPS-I samples but still above the normal reference range (Figure 1).

Figure 1 shows a collection of DBS samples obtained from the Illinois, Tennessee, and New York NBS programs and from Greenwood Genetics Center from newborns that displayed low IDUA activity (below the screen positive cutoffs used in each of NBS programs), but genotype suggested a false positive (not MPS-I). These samples include presumed MPS-I carriers that included 16 newborns that were found to contain a pseudodeficiency variant combined with a Pathogenic or Likely-Pathogenic variant (defined by ACMG guidelines) as well as 1 newborn with only one Pathogenic variant. Figure 1 also includes 5 newborns with one pseudodeficiency variant and one VUS, and 28 newborn DBS that were found to contain only pseudodeficiency variants. All of these presumed false positive samples showed that the Endogenous-NRE GAG biomarker is in the normal reference range.

In a second study, we collaborated with the newborn screening program for Ontario, Canada. They screened 384,144 newborns for MPS-I by measuring IDUA enzymatic activity in DBS and found 187 (0.05%) that were below the cutoff and were sent for DNA sequencing. The Endogenous-NRE GAG biomarker UA-HNAc(1S)-Early was measured in 166 of the 187 samples, and results are summarized in Figures 2A and 2B. Genotype information for 137 of the 187 suggested no risk for MPS-I, i.e., no variant of interest (NVOI), benign or likely-benign variants (ACMG defined), only one VUS (heterozygotes), peusodeficiency variants (hetero- or homozygotes), or pseudodeficiency together with a VUS were seen. All of these gave GAG biomarker levels in the normal reference range (Figure 2A). Fourteen were suggested to be MPS-I carriers based on genotype, and all showed GAG biomarker in the normal reference range (Figure 2A). Three were suggested to have MPS-I based on the observation of two Pathogenic or Likely-Pathogenic variants, and all had greatly elevated UA-HNAc(1S)-Early biomarker (Figure 2A). One had an inconclusive genotype (heterozygous Pathogenic, Likely- Pathogenic, and pseudodeficiency variants) and this individual showed elevated GAG biomarker with clinically confirmed attenuated disease. Four had inconclusive genotypes (a Pathogenic or Likely-Pathogenic variant combined with a VUS or two VUS), and all of these displayed GAG biomarker in the normal reference range. Figure 2B shows a zoomed-in version of Figure 2A. Among these four patients with inconclusive genotypes, 1 had slightly elevated GAG marker but still well below the level seen in the patients with confirmed attenuated MPS-I (Figure 2B). This patient was determined to be unaffected and was discharged.

Figure 2.

(A) Endogenous-NRE GAG biomarker in newborn DBS from clinically-confirmed MPS-I patients and obtained from the Ottawa NBS laboratory. See maintext for full description. (B) Zoomed-in version of Figure 2A.

4. Discussion

In the first part of the study we measured the Endogenous-NRE MPS-I biomarker in newborn DBS from a large collection of samples obtained from several state NBS laboratories in which genotype information suggested the low-IDUA activity sample was a false positive. In all cases the biomarker was in the normal reference range showing the high accuracy of this biomarker to predict disease status.

In the second phase of this study we compared the Endogenous-NRE GAG second-tier analysis to genotype second-tier analysis for a large set of samples found to display IDUA activity below the screen-positive cutoff in the primary screen carried out in the Ontario NBS program (187 low enzyme samples out of 384,144 screened). For most of the samples, the GAG analysis and the genotype analysis were congruent. The key finding is that 4 newborns had inconclusive genotype. All 4 showed no significant elevation of the Endogenous-NRE MPS-I GAG biomarker and no signs of disease following clinical examination. The Endogenous-NRE GAG biomarker analysis is highly specific, leading to a reduction in false positives in NBS of MPS-I, even when the genotype is uncertain. This in turn shows that the Endogenous-NRE GAG biomarker method is more powerful than genotyping to support high-precision NBS for MPS-I.

Data Availability Statement:

Data is contained within the article or supplementary material.