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.

Expression study of Krabbe Disease missense variants - Insights from quantification profiles of residual enzyme activity, secretion and psychosine levels.

Krabbe disease (KD) is an autosomal recessive lysosomal storage disorder caused by loss-of-function mutations in the gene, which encodes for the enzyme galactosylceramidase (GALC). GALC is crucial for myelin metabolism. Functional deficiency of GALC leads to toxic accumulation of psychosine, dysfunction and death of oligodendrocytes, and eventual brain demyelination.

Sequence-specific targeting of intrinsically disordered protein regions.

A general approach to design proteins that bind tightly and specifically to intrinsically disordered regions (IDRs) of proteins and flexible peptides would have wide application in biological research, therapeutics, and diagnosis. However, the lack of defined structures and the high variability in sequence and conformational preferences has complicated such efforts. We sought to develop a method combining biophysical principles with deep learning to readily generate binders for any disordered sequence.

Tandem mass spectrometric enzyme assay for simultaneous detection of Tay-Sachs and Sandhoff diseases in dried blood spots for newborn screening.

GM2 gangliosidosis is a group of rare lysosomal storage disorders (LSDs) including Tay-Sachs disease (TSD) and Sandhoff disease (SD), caused by deficiency in activity of either β-hexosaminidase A (HexA) or both β-hexosaminidase A and β-hexosaminidase B (HexB). Methods for screening and diagnosis of TSD and SD include measurement and comparison of the activity of these two enzymes. Here we report a novel method for duplex screening of dried blood spots (DBS) for TSD and SD by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Perinatal loss of galactosylceramidase in both oligodendrocytes and microglia is crucial for the pathogenesis of Krabbe disease in mice.

Lysosomal galactosylceramidase (GALC) is expressed in all brain cells, including oligodendrocytes (OLs), microglia, and astrocytes, although the cell-specific function of GALC is largely unknown. Mutations in GALC cause Krabbe disease (KD), a fatal neurological lysosomal disorder that usually affects infants. To study how Galc ablation in each glial cell type contributes to Krabbe pathogenesis, we used conditional Galc-floxed mice.

Quantification of N-acetyl-l-aspartate in dried blood spots: A simple and fast LC-MS/MS neonatal screening method for the diagnosis of Canavan disease.

Background: Canavan disease is a devastating neurometabolic disorder caused by accumulation of N acetylaspartate in brain and body fluids due to genetic defects in the aspartoacylase gene (ASPA). New gene therapies are on the horizon but will require early presymptomatic diagnosis to be fully effective.

Methods: We therefore developed a fast and highly sensitive liquid chromatography mass spectrometry (LC-MS/MS)-based method for quantification of N-acetylaspartate in dried blood spots and established reference ranges for neonates and older controls.

Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Analysis of Urinary Oligosaccharides and Glycoamino Acids for the Diagnosis of Mucopolysaccharidosis and Glycoproteinosis.

Background: Mucopolysaccharidosis (MPS) and glycoproteinosis are 2 groups of heterogenous lysosomal storage disorders (LSDs) caused by defective degradation of glycosaminoglycans (GAGs) and glycoproteins, respectively. Oligosaccharides and glycoamino acids have been recognized as biomarkers for MPS and glycoproteinosis. Given that both groups of LSDs have overlapping clinical features, a multiplexed assay capable of unambiguous subtyping is desired for accurate diagnosis, and potentially for severity stratification and treatment monitoring.

Higher precision, first tier newborn screening for metachromatic leukodystrophy using 16:1-OH-sulfatide.

Newborn screening (NBS) for metachromatic leukodystrophy (MLD) is based on first-tier measurement of sulfatides in dried blood spots (DBS) followed by second-tier measurement of arylsulfatase A in the same DBS. This approach is very precise with 0-1 false positives per ∼30,000 newborns tested. Recent data reported here shows that the sulfatide molecular species with an α-hydroxyl, 16‑carbon, mono-unsaturated fatty acyl group (16:1-OH-sulfatide) is superior to the original biomarker 16:0-sulfatide in reducing the number of first-tier false positives.

ScreenPlus: A comprehensive, multi-disorder newborn screening program.

The increasing availability of novel therapies highlights the importance of screening newborns for rare genetic disorders so that they may benefit from early therapy, when it is most likely to be effective. Pilot newborn screening (NBS) studies are a way to gather objective evidence about the feasibility and utility of screening, the accuracy of screening assays, and the incidence of disease. They are also an optimal way to evaluate the complex ethical, legal and social implications (ELSI) that accompany NBS expansion for disorders.

Tandem mass spectrometric assay of N-acetylglucosamine-6-sulfatase for multiplex analysis of mucopolysaccharidosis-IIID in dried blood spots.

Previously we developed a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay using dried blood spots for all subtypes of mucopolysaccharidoses (MPS) except MPS-IIID. Here we show that the MPS-IIID enzyme N-acetylglucosamine-6-sulfatase (GNS) is inhibited in dried blood spot (DBS) extracts, but activity can be recovered if the extract is diluted to reduce the concentrations of endogenous inhibitors. The new GNS assay displays acceptable characteristics including linearity in product formation with incubation time and amount of enzyme, low variability, and ability to distinguish MPS-IIID-affected from healthy patients using DBS.

Biochemical signatures of disease severity in multiple sulfatase deficiency.

Sulfatases catalyze essential cellular reactions, including degradation of glycosaminoglycans (GAGs). All sulfatases are post-translationally activated by the formylglycine generating enzyme (FGE) which is deficient in multiple sulfatase deficiency (MSD), a neurodegenerative lysosomal storage disease. Historically, patients were presumed to be deficient of all sulfatase activities; however, a more nuanced relationship is emerging.

Newborn screening for the full set of mucopolysaccharidoses in dried blood spots based on first-tier enzymatic assay followed by second-tier analysis of glycosaminoglycans.

Newborn screening (NBS) for the full set of mucopolysaccharidoses (MPSs) is now possible by either measuring all of the relevant enzymatic activities in dried blood spots (DBS) using tandem mass spectrometry followed by measurement of accumulated glycosaminoglycans (GAGs) or the vice-versa approach. In this study we considered multiple factors in detail including reagent costs, time per analysis, false positive rates, instrumentation requirements, and multiplexing capability. Both NBS approaches are found to provide acceptable solutions for comprehensive MPS NBS, but the enzyme-first approach allows for better multiplexing to include numerous additional diseases that are appropriate for NBS expansion.

Endogenous, non-reducing end glycosaminoglycan biomarkers for the mucopolysaccharidoses: Accurate diagnosis and elimination of false positive newborn screening results.

The mucopolysaccharidoses (MPS) are a family of inborn errors of metabolism resulting from a deficiency in a lysosomal hydrolase responsible for the degradation of glycosaminoglycans (GAG). From a biochemical standpoint, excessive urinary excretion of GAG has afforded first-tier laboratory investigations for diagnosis whereas newborn screening programs employ lysosomal hydrolase measurements. Given false positives are not uncommon, second-tier diagnostic testing relies on lysosomal hydrolase measurements following elevated urinary GAG, and newborn screening results are often corroborated with GAG determinations.

Predicting disease severity in metachromatic leukodystrophy using protein activity and a patient phenotype matrix.

Background: Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by mutations in the arylsulfatase A gene (ARSA) and categorized into three subtypes according to age of onset. The functional effect of most ARSA mutants remains unknown; better understanding of the genotype-phenotype relationship is required to support newborn screening (NBS) and guide treatment.

Results: We collected a patient data set from the literature that relates disease severity to ARSA genotype in 489 individuals with MLD.

Endogenous, non-reducing end glycosaminoglycan biomarkers are superior to internal disaccharide glycosaminoglycan biomarkers for newborn screening of mucopolysaccharidoses and GM1 gangliosidosis.

Measurement of enzymatic activity in newborn dried blood spots (DBS) is the preferred first-tier method in newborn screening (NBS) for mucopolysaccharidoses (MPSs). Our previous publications on glycosaminoglycan (GAG) biomarker levels in DBS for mucopolysaccharidosis type 1 (MPS-I) and MPS-II demonstrated that second-tier GAG biomarker analysis can dramatically reduce the false positive rate in NBS. In the present study, we evaluate two methods for measuring GAG biomarkers in seven MPS types and GM1 gangliosidosis.

Multiplex tandem mass spectrometry enzymatic activity assay for the screening and diagnosis of Mucolipidosis type II and III.

Mucolipidosis type II and III (MLII/III) is caused by defects in the mannose-6-phosphate system, which is essential to target most of the lysosomal hydrolases to the lysosome. MLII/III patients present with marked elevations in the activities of most lysosomal enzymes in plasma, but their profiles in dried blood spots (DBS) have not been well described. In the current study, we measured the activities of 12 lysosomal enzymes in DBS, among which acid sphingomyelinase, iduronate-2-sulfatase, and alpha--acetylglucosaminidase were significantly elevated in MLII/III patients when compared to random newborns.

Drug screening identifies tazarotene and bexarotene as therapeutic agents in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD, MIM #272200) results from pathogenic variants in the SUMF1 gene that impair proper function of the formylglycine-generating enzyme (FGE). FGE is essential for the posttranslational activation of cellular sulfatases. MSD patients display reduced or absent sulfatase activities and, as a result, clinical signs of single sulfatase disorders in a unique combination.

Liquid Chromatography-Tandem Mass Spectrometry in Newborn Screening Laboratories.

Tandem mass spectrometry (MS/MS) is the most universal platform currently available for the analysis of enzymatic activities and biomarkers in dried blood spots (DBS) for applications in newborn screening (NBS). Among the MS/MS applications in NBS, the most common is flow-injection analysis (FIA-) MS/MS, where the sample is introduced as a bolus injection into the mass spectrometer without the prior fractionation of analytes. Liquid chromatography combined with MS/MS (LC-MS/MS) has been employed for second-tier tests to reduce the false-positive rate associated with several nonspecific screening markers, beginning two decades ago.

Newborn screening for Cerebrotendinous Xanthomatosis: A retrospective biomarker study using both flow-injection and UPLC-MS/MS analysis in 20,000 newborns.

Background And Aims: Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1- leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t-CDCA) and tauro-trihydroxycholestanoic acid (t-THCA).

Materials And Methods: To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories.