Background We have expanded the use of tandem mass spectrometry combined with liquid chromatography (HPLC-MS/MS) for multiplex newborn screening of seven lysosomal enzymes in dried blood spots (DBS). The new assays are for enzymes responsible for the mucopolysaccharidoses MPS-I, -II, -IIIB, -IVA, -VI, and −VII) and type 2 neuronal ceroid lipofuscinosis (LINCL). Methods New substrates were prepared and characterized for tripeptidyl peptidase 1 (TPP1), α-N-acetylglucosaminidase (NAGLU), and lysosomal β-glucuronidase (GUSB). These assays were combined with previously developed assays to provide a multiplex HPLC-MS/MS assay of seven lysosomal storage diseases (LSDs). Multiple reaction monitoring (MRM) of ion dissociations for enzyme products and deuterium-labeled internal standards was used to quantify the enzyme activities. Results Deidentified DBS samples from 62 non-affected newborns were analyzed to simultaneously determine (run time 2 min per DBS) the activities of TPP1, NAGLU, and GUSB, along with those for α-iduronidase (IDUA) iduronate-2-sulfatase (I2S), N-acetylgalactosamine-6-sulfatase (GALNS), and N-acetylgalactosamine-4-sulfatase (ARSB). The activities measured in the 7-plex format showed analytical ranges of 102-909 that clearly separated healthy infants from affected children. Conclusions The new multiplex assay provides a robust comprehensive newborn screening assay for the mucopolysaccharidoses, which is the most rapid method reported to date. The method is shown to be expandable to include additional LSDs including neuronal ceroid lipofuscinosis. The assay is also useful for biochemical diagnosis and prognosis studies.

Newborn screening (NBS) for Krabbe disease, a rare neurodegenerative disorder caused by deficient galactocerebrosidase (GALC) enzyme activity, has recently been implemented in a number of US states. However, the spectrum of phenotypic manifestations associated with deficient GALC activity complicates the management of screen-positive newborns and underscores the need to identify clinically relevant biomarkers. Earlier studies with a small number of patients identified psychosine, a substrate of the GALC enzyme, as a potential biomarker for Krabbe disease. In this study, we provide, for the first time, longitudinal data on dried blood spot (DBS) psychosine concentrations in different Krabbe disease phenotypes for both untreated patients and those treated with hematopoietic stem cell transplantation (HSCT). Our cohort included patients previously identified by NBS to be at high risk to develop Krabbe disease. Substantially elevated DBS psychosine concentration during the newborn period was found to be a highly specific marker for infantile Krabbe disease. This finding supports the use of DBS psychosine concentration as a second-tier NBS test to aid in the identification of patients who require urgent evaluation for HSCT. In addition, longitudinal assessments showed that both natural disease progression and treatment with HSCT were associated with decreases in DBS psychosine concentrations. Based on these findings we provide recommendations for the interpretation of psychosine concentrations in DBS specimens collected during the first year of life. Future studies should aim to better delineate the relationship between DBS psychosine concentration and disease onset in patients with later-onset forms of Krabbe disease.

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Objective-To test, in a newborn screening (NBS) laboratory, the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assay 5 enzymatic activities in dried blood spots (DBS) for NBS of 5 lysosomal storage diseases (mucopolysaccharidosis [MPS]-II, MPS-IIIB, MPS-IVA, MPS-VI, and MPS-VII).Study design-Three mm punches from de-identified DBS were obtained from the Washington NBS laboratory and submitted to the 5-plex LC-MS/MS assay. Screen cut-offs were established by analyzing the enzymatic activity in patients confirmed to have the MPS disorder. DNA sequencing of the relevant gene was performed on a second DBS punch for all samples with enzyme activity below 10% of the mean daily activity. Results-(1)For MPS-II, 18 below cut-off samples, 1 pathogenic genotype, and 2 "high risk" genotypes; (2) For MPS-IIIB, no below cut-off samples; (3) For MPS-IVA, (4) For MPS-VI, 4 below cut-off samples and no high-risk genotypes; (5) MPS-VII, 1 below cut-off sample confirmed by genotype and clinical report to be affected.

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Liquid chromatography–tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.

that all APCS species with acyl groups ranging from C14 to C24 were elevated in NPC1 plasma. PPCS is also elevated in both central and peripheral tissues of the NPC1 cat model. Identification of APCS structures provide an opportunity for broader exploration of the roles of these novel lipids in NPC1 disease pathology and diagnosis.

Purpose Cerebrotendinous xanthomatosis (CTX) is a treatable hereditary disorder caused by the deficiency of sterol 27- hydroxylase, which is encoded by the CYP27A1 gene. Different newborn screening biomarkers for CTX have been described, including 7α,12α-dihydroxy-4-cholesten-3-one ( 7α12αC4 ), 5b-cholestane-3 α, 7α,12α,25-tetrol glucuronide(GlcA-tetrol), GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) ratio (GlcA-tetrol/t-CDCA), and tauro- trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol ratio (t-THCA/GlcA-tetrol ). We set out to evaluate these screening methods in a research study using 32,000–55,000 newborn dried blood spots (DBS). Method Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Results The measurement of 7α12αC4 was complicated by isobaric interferences and was discontinued after 2,033 samples. A total of 55,250 newborns were screened for the GlcA-tetrol/t-CDCA ratio, 32,737 of which had quantitative data on GlcA-tetrol. Only one newborn displayed both highly elevated GlcA-tetrol and a typical CTX biochemical profile. This newborn was interpreted as a CTX-affected patient as CYP27A1 gene sequencing identified two known pathogenic variants. Conclusion The results indicate that both GlcA-tetrol and GlcA-tetrol/t-CDCA ratio are excellent CTX biomarkers suitable for newborn screening. By characterizing the relationship of GlcA-tetrol, t-CDCA, and t-THCA as secondary markers, 100% assay specificity can be achieved.