Evaluation of Two Methods for Quantification of Glycosaminoglycan Biomarkers in Newborn Dried Blood Spots from Patients with Severe and Attenuated Mucopolysaccharidosis Type II

Abstract

All newborn screening (NBS) for mucopolysaccharidosis-I and -II (MPS-I and MPS-II) is carried out via the measurement of α-iduronidase (IDUA) and iduronate-2-sulfatase (IDS) enzymatic activity, respectively, in dried blood spots (DBS). The majority of low enzyme results are due to pseudodeficiencies, and data from recent MPS-II population screenings and studies from the Mayo Clinic show that the false positive rate can be dramatically reduced by the inclusion of a second-tier analysis of glycosaminoglycans (GAGs) in DBS as part of NBS. In the present study, which focused on MPS-II, we obtained newborn DBS from 17 patients with severe MPS-II, 1 with attenuated MPS-II, and 6 patients with various IDS pseudodeficiencies. These samples were submitted to two different GAG mass spectrometry analyses in a comparative study: (1) internal disaccharide biomarkers and (2) endogenous biomarkers. For both of these methods, the biomarker levels in six patients with pseudodeficiencies were below the range measured in MPS-II patients. One patient with attenuated MPS-II was not distinguishable from severe disease patients, but all MPS-II patients were distinguishable from the reference range using both methods. The minimal differential factor (lowest GAG marker level in MPS-II samples divided by highest level in the reference range of 60 random newborns) was 3.01-fold for the internal disaccharide method. The endogenous biomarker method demonstrated an improved minimum differential of 5.41-fold. The minimum differential factors between MPS-II patients and patients with pseudodeficiencies for the internal disaccharide and endogenous biomarker methods were 3.77-fold and 2.06-fold, respectively. This study supports use of the second-tier GAG analysis of newborn DBS, especially the endogenous disaccharide method, as part of NBS to reduce the false positive rate.

Newborn screening for genetic disorders: Current status and prospects for the future

“…Thus, genetic testing is often conducted in many NBS programs to verify the patients with low enzyme activity. In addition, recent data have shown that a second‐tier analysis of biomarkers appears to be a powerful tool to reduce high false positive rates associated with pseudo deficiencies in NBS of Krabbe disease, MPS I, and MPS II 35–37 …”

“…In addition, recent data have shown that a second‐tier analysis of biomarkers appears to be a powerful tool to reduce high false positive rates associated with pseudo deficiencies in NBS of Krabbe disease, MPS I, and MPS II. 35 , 36 , 37 …”

Newborn screening and the recommended uniform screening panel: Optimal submissions and suggested improvements based on an advocacy organization's decade‐long experience

“…Finally, it is important that screening methods minimize the potential false positives from heterozygous individuals and from pseudodeficiency alleles as well as the uncertain findings of variants of unknown significance by adopting optimal second tier evaluations of disease biomarkers using dried blood spot (DBS) samples (Clarke et al, 2020;Peck et al, 2020). Advocacy groups have a role in this effort, and can assist as the Society has, in procuring consented access to patient DBS, which facilitated the further development and validation of DBS glycosaminoglycans as biomarkers of MPS I and MPS II (Herbst et al, 2020(Herbst et al, , 2022.…”

Biochemical signatures of disease severity in multiple sulfatase deficiency

“…Specific GAG nonreducing end species (GAG-NREs) have been validated as biomarkers for diagnosis, severity classification, and response to therapy in several MPS disorders. 8,9,11,12,[14][15][16][17] In single sulfatase MPS disorders, a single enzyme is deficient leading to accumulation of that enzyme's specific GAG-NRE substrate. In MSD, multiple enzymes are deficient, so we hypothesize that several GAG-NREs will accumulate.…”

“…Multiple methods have been developed to evaluate GAG degradation products. Here, we utilize three previously reported techniques: (1) the "internal disaccharide" method, 18,19 where GAG polymers are degraded in vitro with bacterial enzymes into resultant disaccharides, which are quantified via LC-MS/MS; (2) the "Sensi-Pro ® " method, 8,11,20 which quantifies the GAG non-reducing end (GAG-NRE) fragment via LC-MS/MS after its in vitro liberation from the GAG polymer via bacterial enzymes; and (3) the "endogenous NRE" method, [15][16][17] which quantifies small endogenously produced GAG-NRE fragments from patient samples without in vitro degradation. These techniques show variable sensitivity and specificity when applied to single sulfatase disorders.…”