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Assays that measure lysosomal enzyme activity are important tools for the screening and diagnosis of lysosomal storage disorders (LSDs). They are often ordered in combination with urine oligosaccharide and glycosaminoglycan analysis, additional biomarker assays, and/or DNA sequencing when an LSD is suspected. Enzyme testing in whole blood/leukocytes, serum/plasma, cultured fibroblasts, or dried blood spots demonstrating deficient enzyme activity remains a key component of LSD diagnosis and is often prompted by characteristic clinical findings, abnormal newborn screening, abnormal biochemical findings (eg, elevated glycosaminoglycans), or molecular results indicating pathogenic variants or variants of uncertain significance in a gene associated with an LSD. This document, which focuses on clinical enzyme testing for LSDs, provides a resource for laboratories to develop and implement clinical testing, to describe variables that can influence test performance and interpretation of results, and to delineate situations for which follow-up molecular testing is warranted., Competing Interests: Conflict of Interest E.T.S., K.C.-O., T.W., and C.Y. direct clinical biochemical genetics laboratories that run the tests discussed in the current standard on a fee-for-service basis. C.Y. is an employee of Sema4 and has equity ownership in the form of incentive stock options and restricted stock units., (Copyright © 2021 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Multiplex tandem mass spectrometry (MS/MS)-based enzyme activity assays for newborn screening (NBS) and diagnosis of lysosomal storage diseases (LSDs) in newborns, using dried blood spots (DBS) on newborn screening cards, have garnered much attention due to its sensitivity, high precision, and the capability to screen for an unprecedented number of diseases in a single assay. Herein we report the development of MS/MS-based enzyme assays for the diagnosis of α-mannosidosis and fucosidosis. These new protocols are able to distinguish untreated patients from random newborns, carriers and a post-bone marrow transplant patient. We have successfully multiplexed the α-mannosidosis assay with a multiplex MS/MS assay for the screening and diagnosis of other LSDs, namely Fabry, Pompe, MPS I, Gaucher, Niemann-Pick-A/B, and Krabbe diseases. Additionally, we also multiplexed the fucosidosis NBS assay with a 5-plex assay that tests for MPS-II, MPS-IIIB, MPS-IVA, MPS-VI and MPS-VII., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl - /H + exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and -genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH., (Published by Elsevier Inc.)

Background: The glycoproteinoses are a subgroup of lysosomal storage diseases (LSDs) resulting from impaired degradation of N-linked oligosaccharide side chains of glycoproteins, which are commonly screened by detecting the accumulated free oligosaccharides (FOSs) in urine via thin layer chromatography (TLC). The traditional TLC method suffers from limited analytical sensitivity and specificity and lacks quantification capability. Therefore, we developed an analytically sensitive and relatively specific assay using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for urinary FOS analysis and validated its use for urine screening of glycoproteinoses and other LSDs., Methods: Urine volumes equivalent to 30 μg of creatinine were derivatized with butyl-4-aminobenzoate and then purified through a solid-phase extraction cartridge. A 7-min UPLC-MS/MS analysis was performed on a triple quadrupole mass spectrometer using an amide column for separation of derivatized FOS. Urine samples from >100 unaffected controls and 37 patients with various LSDs were studied., Results: Relative quantification was conducted on 7 selected FOSs using a single internal standard, which allowed the identification of patients with 1 of 8 different LSDs: aspartylglucosaminuria, α-fucosidosis, α-mannosidosis, β-mannosidosis, β-galactosidase deficiency, Sandhoff disease, sialidosis, and galactosialidosis. Patients treated with hematopoietic stem cell transplant show decreased FOS responses compared with untreated patients., Conclusions: This UPLC-MS/MS assay offers a valuable tool for screening of glycoproteinoses and other LSDs, with potential use for future treatment monitoring., (© 2018 American Association for Clinical Chemistry.)

Abstract: The oligosaccharidoses are a group of metabolic disorders resulting from a deficiency in enzymes responsible for the catabolism of protein bound oligosaccharides and are typified by the accumulation of corresponding sugars in the urine. Screening is typically accomplished using thin layer chromatography. However, analyte specificity can be a problem and thus complicate interpretation of results. For this reason we developed a mixed mode liquid chromatography tandem mass spectrometry assay for the screening of the oligosaccharidoses which potentially mitigates many of the problems associated with thin layer chromatography. Samples from patients previously diagnosed with I-Cell disease, mannosidosis, Pompe, galactosialidosis, and fucosidosis were derivatized with 3-methyl-1-phenyl-2-pyrazolin-5-one and subjected to analysis by liquid chromatography tandem mass spectrometry. Results were compared to normal control samples. Preliminary results suggest that each oligosaccharidoses produces a unique selected reaction monitoring fingerprint and that the developed method may be an effective screening and diagnostic tool for these disorders. [ABSTRACT FROM AUTHOR]

Alpha-mannosidosis (AMS) is an autosomal recessive lysosomal storage disorder which results from a deficiency of lysosomal alpha-mannosidosis activity and displays a wide range of clinical phenotypes. Patients have traditionally been divided into type I, a more severe form that presents in infancy, and type II, a milder form that typically presents in later childhood. We describe three Hispanic males who presented in infancy with relatively mild forms of AMS. They were aged between 6 and 24 years at their last assessment. Homozygous mutations in the MAN2B1 gene were found in all three patients, one of which is a newly reported mutation. Two of the patients were brothers who were homozygous for the same MAN2B1 mutation. Despite being homozygous for the same mutation, the older brother had more severe developmental delay, hearing loss, and growth retardation. This report illustrates the difficulty in determining a strict genotype-phenotype correlation in AMS, and supports screening for oligosaccharides in children with neurodevelopmental delay with mild phenotypic signs and symptoms. [ABSTRACT FROM AUTHOR]

Enzyme analysis is the gold standard for the diagnosis of lysosomal storage disorders (LSDs). However, enzyme analysis in leukocytes requires that whole blood samples arrive to the testing laboratory within 24-48 hours of collection, and requires a relatively large volume of blood. This is problematic for the international shipment of specimens and for testing infants. The adaptation of these enzyme assays for use in dried blood spots (DBS) has ameliorated these issues for a large number of LSDs. Initially enzyme analysis in DBS exclusively utilized 4-methylumbelliferone (4-MU) fluorogenic substrates. The limitation of this methodology is that each enzyme must be measured individually. The development of tandem mass spectrometry (MS/MS) substrates has allowed multiple enzyme reactions to be combined in a single reaction, increasing efficiency. We report validation results for a new 6-plex assay for the diagnosis of five Mucopolysaccharidosis (MPS) disorders (MPS II, IIIB, IVA, VI and VII) and neuronal ceroid lipofuscinosis type 2 (CLN2) in DBS using UPLC-MS/MS. Normal reference ranges were developed using a minimum of 236 DBS samples from unaffected controls or patients with an alternate diagnosis, and 100% clinical sensitivity was established using 99 samples from patients affected with one of the six disorders. Both intra-day and inter-day precision were acceptable (< 20% CV) and 6 month long term sample stability has been verified. Our laboratory can now analyze 18 enzymes in DBS (12 utilizing MS/MS substrates and 6 utilizing 4-MU substrates) to diagnose 20 different LSDs (including Multiple Sulfatase Deficiency and Mucolipidosis II/III). Furthermore, we can now offer a 7 enzyme MPS panel in DBS, which will significantly enhance the worldwide diagnosis of MPS patients. Finally, the development of this 6-plex has significant implications for newborn screening, as FDA approved therapies are available for five of the six included conditions. [ABSTRACT FROM AUTHOR]

Abstract: Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disease that is caused by a deficiency of the lysosomal enzyme α-N-acetylglucosaminidase (NAGLU). Over 100 different mutations in the NAGLU gene have been identified in Sanfilippo syndrome type B patients; however, no large deletions have been reported. Here we present the first case of a large homozygous intragenic NAGLU gene deletion identified in an affected child of consanguineous parents. Long range and multiplex PCR methods were used to characterize this deletion which encompasses exons 3 and 4 and is 1146 base pairs long. We propose that Alu element-mediated unequal homologous recombination between an Alu–Y in intron 2 and an Alu–Sx in intron 4 is the likely mechanism for this deletion, thereby contributing further insight into the molecular etiology of this disorder and providing additional evidence of its allelic heterogeneity. [Copyright &y& Elsevier]