Rapid and Modular Assembly of Click Substrates To Assay Enzyme Activity in the Newborn Screening of Lysosomal Storage Disorders
- Philipp SkrinjarPhilipp SkrinjarInstitute of Applied Synthetic Chemistry, Vienna University of Technology (TU Wien), 1060 Vienna, AustriaMore by Philipp Skrinjar
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- Markus SchwarzMarkus SchwarzInstitute of Applied Synthetic Chemistry, Vienna University of Technology (TU Wien), 1060 Vienna, AustriaARCHIMED Life Science GmbH, 1110 Vienna, AustriaMore by Markus Schwarz
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- Stefan LexmüllerStefan LexmüllerInstitute of Applied Synthetic Chemistry, Vienna University of Technology (TU Wien), 1060 Vienna, AustriaMore by Stefan Lexmüller
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- Thomas P. Mechtler
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- Maximilian ZeydaMaximilian ZeydaDepartment of Pediatrics and Adolescent Medicine, Medical University of Vienna, 1090 Vienna, AustriaMore by Maximilian Zeyda
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- Susanne Greber-PlatzerSusanne Greber-PlatzerDepartment of Pediatrics and Adolescent Medicine, Medical University of Vienna, 1090 Vienna, AustriaMore by Susanne Greber-Platzer
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- Joe TrometerJoe TrometerPerkinElmer, Diagnostics, Waltham, Massachusetts 02451, United StatesMore by Joe Trometer
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- David C. Kasper
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- Hannes Mikula*Hannes Mikula*E-mail: [email protected]Institute of Applied Synthetic Chemistry, Vienna University of Technology (TU Wien), 1060 Vienna, AustriaMore by Hannes Mikula
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Abstract
Synthetic substrates play a pivotal role in the development of enzyme assays for medical diagnostics. However, the preparation of these chemical tools often requires multistep synthetic procedures complicating structural optimization and limiting versatility. In particular, substrates for enzyme assays based on tandem mass spectrometry need to be designed and optimized to fulfill the requirements to finally enable the development of robust diagnostic assays. In addition, isotope-labeled standards need to be prepared to facilitate accurate quantification of enzyme assay products. Here we report the development of a building block strategy for rapid and modular assembly of enzyme substrates using click chemistry as a key step. These click substrates are made up of a sugar moiety as enzyme responsive unit, a linker that can easily be isotope-labeled for the synthesis of internal standards, and a modifier compound that can readily be exchanged for structural optimization and analytical/diagnostic tuning. Moreover, the building block assembly eliminates the need for extensive optimization of different glycosylation reactions as it enables the divergent synthesis of substrates using a clickable enzyme responsive unit. The outlined strategy has been applied to obtain a series of synthetic α-l-iduronates and sulfated β-d-galactosides as substrates for assaying α-l-iduronidase and N-acetylgalactosamine-6-sulfate sulfatase, enzymes related to the lysosomal storage disorders mucopolysaccharidosis type I and type IVa, respectively. Selected click substrates were finally shown to be suitable to assay enzyme activities in dried blood spot samples from affected patients and random newborns.
Synopsis
Clickable building blocks can be used to rapidly access enzyme substrates and isotope-labeled standards for the LC-MS/MS-based assaying of enzyme activities in dried blood spot samples.
Introduction
ARTICLE SECTIONS
Lysosomal storage disorders (LSDs) are severe diseases, caused by genetic defects leading to a deficiency of lysosomal enzyme activity. The resulting progressive accumulation of macromolecular substrates is specific to each disorder and causes a gradual deterioration of cellular and tissue function. (1−3) Individual LSDs are considered as rare diseases with a round combined prevalence estimated at 1 per 7700 live births. (4) In contrast to general treatments for the symptoms of LSDs various therapeutic options have been developed dealing with the cause of the disease: (i) enzyme replacement therapy, (ii) chaperone-mediated enzyme enhancement, (iii) substrate reduction therapy, (iv) stem cell transplantation, and (v) hematopoietic stem cell gene therapy. (5−9) The development of those treatment options has led to an intense interest into newborn screening (NBS) of LSDs as early initiation of treatment often leads to a better outcome. (10) Different methods for NBS have been developed including (i) direct analysis of enzymatic activity, (ii) gene sequencing, (iii) direct measurement of enzyme abundance, and (iv) biomarker quantification. (10) While measurements of enzyme abundance and biomarker analysis are still at an early stage of development, and gene sequencing is limited by the lack of a complete list of pathogenic mutations, direct enzyme activity analysis has been extensively developed in recent years. (10) In their pioneering studies, Chamoles and co-workers assayed enzyme activities in rehydrated dried blood spots (DBS) using fluorometric and radiometric assays. (10−13) DBS analysis has several advantages over other methods as it requires only a few drops of blood, and samples can easily be sent in plastic envelopes at room temperature to other cities, countries, or specialized reference laboratories. (14)
Tandem mass spectrometry (MS/MS) has been developed as a diagnostic platform for early detection and screening of genetic disorders and many countries have implemented newborn screening using MS/MS. (15) Pioneered by Gelb and co-workers, MS/MS was applied for the newborn screening of several LSDs. (16−24) Most importantly, MS/MS changed the paradigm of analyzing one analyte per disorder as it allows the analysis of multiple enzymes in a single DBS punch (multiplexing). Several multiplex assays have been developed in recent years. (17,19,22) To simplify sample cleanup before flow injection into the MS/MS instrument several groups interfaced ultra-high-pressure liquid chromatography (UHPLC) with MS/MS. Two-dimensional chromatography (applying perfusion columns or turboflow online sample cleanup) was used to avoid the need for offline liquid–liquid and solid-phase extraction. (25−31)
In addition, fluorogenic substrates have been developed and used to assay several lysosomal enzymes. (11−14,32−41) Fluorometric methods that make use of digital microfluidics represent an emerging technology that is considered to be at least as effective as MS/MS for high-throughput screening of multiple LSDs. (42−44)
A key drawback in the development of enzyme assays (in particular MS/MS-based ones) is that the preparation of substrates often requires more than 10 synthetic steps. (10) Furthermore, as required for MS/MS methods, corresponding internal standards (in most cases isotope-labeled reference compounds of the assay products) need to be prepared. The labor- and cost-intensive development of substrates and internal standards impedes fast structural optimization (considering substrate stability, enzyme kinetics, MS ionization, in-source fragmentation, etc.) (26) with the aim to develop a robust assay. For instance, the development of improved substrates for the newborn screening of mucopolysaccharidosis (MPS) types I, II, and VI has been reported in 2014, (17) thus almost a decade after the first described synthetic substrate for MPS I, (23) and 6 years after the first structural optimization (13 synthetic steps and additional 2 steps to obtain substrate and internal standard, respectively). (16)
Deficiency of glycosidases is related to several LSDs and thus receiving increasing interest. (1,2,45) The preparation of substrates for the development of glycosidase assays (Figure 1a) involves glycosylation as a key step, a reaction that is known to be notoriously difficult and thus requiring extensive optimization. (46) Hence, this further complicates the screening and optimization of substrate structures when using a linear (or convergent) synthetic sequence.
Figure 1
Figure 1. (a) General concept of LC-MS/MS-based glycosidase assays. Glycosylated substrates are cleaved by the enzyme yielding the product (P) as analyte for subsequent analysis. An isotope-labeled internal standard (IS; here shown as deuterated product, P*) is required and used for quantification. (b) Divergent building block assembly of substrates using a single optimized glycosylation reaction and click chemistry to access a library of click substrates (CS). (c) Corresponding internal standards (IS) can easily be prepared using a single deuterated linker in combination with other building blocks.
In this light, we herein report the design of a strategy for the building block assembly of substrates using a click reaction as key step. A click tag is attached to a linker that can subsequently be conjugated to a library of compounds (modifiers) to tune the MS/MS properties of the final substrate. The sugar moiety is connected to a click aglycone using a single optimized glycosylation reaction, and the resulting building block is reacted with the clickable linker–modifier conjugates. This approach enables the divergent synthesis of click substrates (Figure 1b). Furthermore, a single deuterated linker can be used for facile preparation of the corresponding isotope-labeled internal standards (Figure 1c). While the click aglycone can be exchanged to optimize enzyme kinetics, variation of the sugar moiety leads to substrates for different enzyme assays using the same linker–modifier building blocks, which we finally show by the development of click substrates to assay a clinically relevant sulfatase. The versatility of this approach thus enables the synthesis, screening, and optimization of numerous substrates in short time. Individual building blocks can easily be exchanged without having to start a multistep synthesis from the beginning.
Results and Discussion
ARTICLE SECTIONS
To develop and test the outlined strategy we started by focusing on substrates to assay α-L-iduronidase (IDUA). Deficiency of this lysosomal enzyme causes mucopolysaccharidosis type I (MPS I). As discussed above several substrates to assay MPS I have been developed including fluorogenic compounds and substrates optimized for MS/MS. (11,13,16,17,23) It is noteworthy that we did not aim for structural optimization of MPS I substrates to improve currently used assays that have been gradually optimized in the past decade, but to evaluate the general applicability of our click strategy for the divergent synthesis of enzyme substrates. IDUA is a lysosomal hydrolase responsible for the degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate by cleaving an iduronic acid unit. (47) Hence, synthetic α-L-iduronidates are used to assay IDUA (Figure 2). Chemical glycosylation to afford α-l-iduronidates represents a challenging, not well-studied reaction, which makes these compounds an ideal target for the outlined click strategy.
Figure 2
Figure 2. Cleavage of α-l-iduronates by IDUA.
For the synthesis of a readily accessible linker methyl 4-formylbenzoate (1) was reduced with NaBH4 to afford 2a, which was converted to 3a in an Appel reaction. Reduction with LiAlH4 yielding 4a and subsequent introduction of an azide moiety via nucleophilic substitution afforded clickable linker 5a(Figure 3a). This synthesis could easily be modified by using deuteride reagents to obtain the isotope-labeled linker 5b. A library of modifier compounds was prepared and used in this study for the synthesis of selected click markers (CM1–CM5) as shown in Figure 3a. Propargyl alcohol was used as a simple click aglycone and glycosylated by reaction with the known fluoroiduronyl donor 6 (17) to afford the clickable iduronate 7. Copper-catalyzed azide alkyne cycloaddition (click chemistry) (48−51) was used for the divergent synthesis of click substrates. Selected compounds CS1–CS5 have been prepared via 8–12 (Figure 3b) by click reaction of 7 with CM1a–CM5a. The corresponding internal standards IS1–IS5 were prepared by reacting isotope-labeled click markers CM1b–CM5b with propargyl alcohol to obtain different analytical sets (CS, IS) for further investigations and the development of LC-MS/MS-based enzyme assays. The same synthetic procedures were used to obtain the products (P1–P5) of the enzymatic reactions (click of nonlabeled markers CM1a–CM5a with propargyl alcohol). These compounds were used for the development and tuning of analytical methods (synthesis not shown; for detailed description see the Supporting Information).
Figure 3
Figure 3. (a) Synthesis of nonlabeled and isotope-labeled azide-modified linkers 5a and 5b, respectively, and subsequent conjugation to modifier compounds (M) to obtain a library of click markers (CM). (b) Glycosylation of propargyl alcohol using iduronyl donor 6 and click assembly with CM1–CM5 to afford click substrates CS1–CS5. (c) Analytical sets of click substrates (CS) and corresponding internal standards (IS) for the development of LC-MS/MS-based α-l-iduronidase assays.
We have first used the click approach for the preparation of various substrates and internal standards applying chemically different modifier compounds. Selected compounds, including t-butylcarbonates (set 1), diethylcarbamates (set 2), and t-butyloxycarbonyl-protected (boc-protected) bis-carbamates (set 3), are shown in Figure 3c. These first generation sets were tested using recombinant human IDUA. Briefly, the substrates were reacted at a concentration of 1 mg/mL with increasing concentrations of pure IDUA (0, 25, 50, and 100 ng/mL) in IDUA assay buffer at pH 4.04 at 22 °C for 20 min. The isotope-labeled internal standards (IS) were added to the assay cocktail and used to quantify the product of the enzymatic cleavage of the click substrate. Enzymatic reactions were quenched by the addition of acetonitrile. Samples were centrifuged, and supernatants were diluted and analyzed by UHPLC-MS/MS, wherein mass spectrometric detection was performed using a triple quadrupole system operated in positive electrospray ionization (ESI) mode (for details see the Supporting Information). In these assays, we could show that the enzyme degrades all three substrates (CS1–CS3) as indicated by a linear correlation of IDUA enzyme activity and concentration (Figure 4a).
Figure 4
Figure 4. (a) Enzyme assays using recombinant human α-l-iduronidase (IDUA) and sets 1–5 of click substrate (CS1–CS5) and corresponding internal standard (IS1–IS5) (calculated specific activities are shown in μmol per min and mg enzyme). (b) Analysis of dried blood spots (DBS) using CDC control cards (QCL, QCM, QCH = quality control low, medium, high). (c) Analysis of DBS of confirmed MPS I patients (n = 9, anonymized) and random newborns (n = 88, anonymized). [****p < 0.0001.]
Subsequently, analyses of dried blood spots (DBS) were performed using quality control cards provided by the CDC (Center for Disease Control and Prevention, USA). Briefly, DBS cards were punched (3.2 mm diameter), and each spot was extracted with PBS buffer. The extracts were incubated with the click substrates in assay buffer (pH 4.04) for 22 h at 37 °C followed by quenching with acetonitrile and centrifugation after complete precipitation. The supernatants were diluted and analyzed by UHPLC-MS/MS using the same method as for IDUA assays as described above (for details see the Supporting Information). Data for selected CS/IS sets is shown in Figure 4b. In contrast to assays using recombinant human IDUA, sets 1 and 2 gave only poor results in the DBS assay, while set 3 was shown to be applicable to discriminate between low (QCL), medium (QCM), and high (QCH) enzyme concentration in DBS (Figure 4b). In general, DBS analyses differ from enzyme assays in terms of complexity and, most importantly, incubation time due to the much lower enzyme concentration in DBS. While IDUA assays were used to test and show enzyme activity, DBS analyses were performed to test our substrates in a clinically more relevant setup. The poor performance of sets 1 and 2 clearly indicates the need for a robust, rapid, and modular method for substrate synthesis as it is difficult to predict the performance in DBS assays and LC-MS/MS in general (considering solubility, stability, ionization efficiency, chromatographic performance, etc.). Based on these results sets 4 and 5 have been prepared that differ from set 3 only in the carbon-chain length of the modifier (Figure 3c, second generation sets). UHPLC-MS/MS assays have been performed using IDUA (Figure 4a) and DBS controls (Figure 4b) affording results similar to those of set 3.
Analysis of enzyme activity in DBS obtained from randomly selected newborns and 9 confirmed MPS I patients using CS/IS sets 3, 4, and 5 shows that the activity of IDUA in the patient samples is below the activity observed for random newborns (Figure 4c). Similar results were obtained for all three sets (except a higher variation in IDUA activity when using set 5). Even though these substrates do not achieve activities as described for recently developed optimized substrates for MPS I screening, (17) the building block approach can be used for further optimization, e.g., by exchanging the click aglycone to improve the kinetics of the enzymatic cleavage reaction.
In general, the major advantage of the click approach is that many substrates can be prepared by changing a single building block without the need for new synthetic strategies or optimized protocols. In addition, the corresponding internal standards can easily be prepared by using a single isotope-labeled linker. Hence, a group of substrates and IS that differ only in the carbon-chain length of the modifier can be prepared by exchanging the modifier building block as shown for sets 3–5. To investigate if such a group of similar substrates can be applied for the simultaneous analysis of different DBS (different patients) using a single UHPLC run, we performed a triplex assay, wherein “triplex” stands for 3 different samples screened for the activity of the same enzyme. DBS analyses were carried out as described above using the CS/IS sets 3, 4, and 5. Subsequently, the three separately incubated samples were combined and analyzed using a single injection (Figure 5a). All substrates and the corresponding internal standards could be separated in a 5 min UHPLC run (Figure 5b). To enable direct and better comparison of the data we have analyzed each DBS using the three different CS/IS sets and combined these samples before LC-MS/MS analysis. As shown in Figure 5c, we have been able to achieve results almost equivalent to those of the singleplex assays (Figure 4c), even though a higher variation from single- to triplex was observed for set 5 (Figure 5d). These results clearly indicate the feasibility of the approach that can potentially be extended by using additional substrates.
Figure 5
Figure 5. (a) Simultaneous analysis of three different samples using a single UHPLC-MS/MS run. (b) Chromatographic separation of CS/IS sets 3, 4, and 5 (including the products P3–P5 of the corresponding enzyme assays) using a 5 min gradient. (c) Triplex assay of three combined DBS samples using CS/IS sets 3, 4, and 5 (affected, n = 8; random, n = 23; ****p < 0.0001). (d) Analyzed DBS samples (random) in triplex vs singleplex assays (n = 23). [S = substrate, P* = IS, P = product.]
In addition to structural tuning and divergent synthesis of substrate libraries, a variety of substrates to assay different enzymes can be prepared by exchanging the clickable sugar moiety (enzyme responsive unit) and using the same synthetic protocols for rapid click assembly with already available linker–modifier building blocks. To demonstrate this key advantage, we focused on the development of substrates to assay N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Deficiency of GALNS leads to accumulation of chondroitin-6-sulfate and keratan sulfate causing the lysosomal storage disorder mucopolysaccharidosis type IVa (Morquio syndrome, Figure 6a). (52,53) GALNS has been assayed in dried blood spots to screen for MPS IVa using fluorometric and LC-MS/MS-based methods. (20,26,54−56)
Figure 6
Figure 6. (a) Deficiency of GALNS leads to accumulation of chondroitin-6-sulfate and keratan sulfate causing MPS IVa. (b) Click assembly of GALNS substrates and internal standards using clickable galactose derivatives and already available linker–modifier building blocks. (c) Synthesis of GALNS sets 6–8 of click substrates (CS6–CS8) and corresponding internal standards (IS6–IS8) using click markers CM3a–CM5a and isotope-labeled CM3b–CM5b, respectively. (d) Analysis of dried blood spots (DBS) using CDC control cards. (e) Analysis of DBS of confirmed affected patients (n = 9, anonymized) and random newborns (n = 116, anonymized). [****p < 0.0001.]
Similar to the overall strategy as outlined in Figure 1, we aimed to use clickable galactose building blocks to prepare sulfated click substrates and the respective nonsulfated internal standards by using already available click marker (CM) building blocks (linker, deuterated linker, modifier; Figure 6b). Starting from 1-O-propargyl-β-d-galactose (13, click galactose) we have prepared the sulfated galactose building block 14 by protecting group manipulations (TIPS-protection, acetylation, removal of TIPS) and chemical sulfation on C-6. Click assembly of the alkyne-modified galactose moieties 14 and 13 with click markers CM3a–CM5a and CM3b–CM5b, respectively, afforded click substrates CS6–CS8, internal standards IS6–IS8 (Figure 6c), and the non-isotope-labeled products P6–P8 as reference compounds for tuning the MS instrument (see the Supporting Information).
These GALNS substrates were tested in DBS analyses using CDC control cards (similar to assaying IDUA in DBS as described above) showing that all three sets can be used to discriminate between low (QCL), medium (QCM), and high (QCH) enzyme concentration (Figure 6d). Moreover, sets 6–8 gave highly similar results showing independence of the assay from the length of the modifier. As the observed activities of >10 μM/h are significantly higher than or at least similar to previously reported data using MS/MS-based (20,26) or fluorometric methods, (54,55) we aimed to test our GALNS click substrates in a clinically more relevant setup. Therefore, we have analyzed DBS of 9 confirmed MPS IVa patients in comparison to randomly selected newborns (all anonymized). DBS cards were punched, and each spot was incubated in assay buffer (pH 4.04) containing substrate and internal standard for 22 h at 37 °C followed by quenching with acetonitrile and centrifugation. The supernatants were diluted and analyzed by UHPLC-MS/MS (see the Supporting Information). We obtained mean activities of 0.76 μM/h (affected patients) and 13.5 μM/h (random newborns), and thus an activity ratio (normal/affected) of >17 with a mean absolute difference of 12.7 μM/h between normal and affected newborns.
Not only could we thus show the modularity of the click approach by designing substrates for a different enzyme by changing a single building block, but we also expanded our strategy to the assaying of sulfatases by using a clickable sulfated sugar moiety.
Conclusions
ARTICLE SECTIONS
The presented approach for building block assembly of click substrates (CS) and corresponding internal standards (IS) was shown to be suitable for the divergent, versatile, and modular synthesis of chemical tools and the development of diagnostic LC-MS/MS-based enzyme assays. A library of substrates to screen for MPS I was prepared and investigated in enzyme assays and DBS analyses. Selected substrates have been shown to be suitable to determine the enzymatic activity in patient samples. By changing a single building block, we were able to show (i) rapid synthesis of a group of similar CS/IS sets for the simultaneous analysis of DBS using a single chromatographic run, and (ii) the development of MPS IVa substrates using a sulfated clickable galactose derivative and already available linker–modifier building blocks. These GALNS click substrates were evaluated using quality control cards and shown to be suitable to assay enzyme activities in dried blood spot samples from affected patients and random newborns.
Overall, we were able to demonstrate the advantages and further potential of the click approach and are thus convinced that this strategy will accelerate and contribute to the development of diagnostic tools, LC-MS/MS-based enzyme assays, and screening methods.
Supporting Information
ARTICLE SECTIONS
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscentsci.8b00668.
Synthesis, compound characterization (including copies of NMR spectra), detailed analytical methods, and materials (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Author Information
ARTICLE SECTIONS
- Hannes Mikula - Institute of Applied Synthetic Chemistry, Vienna University of Technology (TU Wien), 1060 Vienna, Austria;
http://orcid.org/0000-0002-9218-9722;
Acknowledgments
ARTICLE SECTIONS
We thank the Austrian Research Promotion Agency (FFG) for funding (Grant 838557). DBS quality control cards were kindly provided by the CDC (Center for Disease Control and Prevention, USA), including permission for their use in this and related studies. DBS samples of newborns were provided by the Medical University of Vienna and the Vienna General Hospital (Newborn Screening Program), and anonymized DBS samples of affected patients (non-newborns) were kindly provided by the University Medical Center of the Johannes Gutenberg-University Mainz (Germany). The institutional ethics committee approved the study (EK 478/2009 and EK 1687/2014).
References
ARTICLE SECTIONS
This article references 56 other publications.
- 1Futerman, A. H.; van Meer, G. The cell biology of lysosomal storage disorders. Nat. Rev. Mol. Cell Biol. 2004, 5, 554– 565, DOI: 10.1038/nrm1423Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlt1Ortb8%253D&md5=929c8b53bf57c04520cdd01d7b8974edThe cell biology of lysosomal storage disordersFuterman, Anthony H.; van Meer, GerritNature Reviews Molecular Cell Biology (2004), 5 (7), 554-565CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Lysosomal storage disorders, of which more than 40 are known, are caused by the defective activity of lysosomal proteins, which results in the intra-lysosomal accumulation of undegraded metabolites. Despite years of study of the genetic and mol. bases of lysosomal storage disorders, little is known about the events that lead from this intra-lysosomal accumulation to pathol. Here, we summarize the biochem. of lysosomal storage disorders. We then discuss downstream cellular pathways that are potentially affected in these disorders and that might help us to delineate their pathol. mechanisms.
- 2Vellodi, A. Lysosomal storage disorders. Br. J. Haematol. 2005, 128, 413– 431, DOI: 10.1111/j.1365-2141.2004.05293.xGoogle Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXit1Wmur8%253D&md5=665737194c681c93b01e9a0555074c69Lysosomal storage disordersVellodi, AshokBritish Journal of Haematology (2005), 128 (4), 413-431CODEN: BJHEAL; ISSN:0007-1048. (Blackwell Publishing Ltd.)A review. Although the first description of a lysosomal storage disorder was that of Tay-Sachs disease in 1881, the lysosome was not discovered until 1955, by Christian De Duve. The first demonstration by Hers in 1963 of a link between an enzyme deficiency and a storage disorder (Pompe's disease) paved the way for a series of seminal discoveries about the intracellular biol. of these enzymes and their substrates, culminating in the successful treatment of Gaucher's disease with β-glucosidase in the early 1990s. It is now recognized that these disorders are not simply a consequence of pure storage, but result from perturbation of complex cell signalling mechanisms. These in turn give rise to secondary structural and biochem. changes, which have important implications for therapy. Significant challenges remain, particularly the treatment of central nervous system disease. It is hoped that recent advances in our understanding of lysosomal biol. will enable successful therapies to be developed.
- 3Wang, R. Y.; Bodamer, O. A.; Watson, M. S.; Wilcox, W. R. Lysosomal storage diseases: diagnostic confirmation and management of presymptomatic individuals. Genet. Med. 2011, 13, 457– 484, DOI: 10.1097/GIM.0b013e318211a7e1Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3Mnjt1ygsw%253D%253D&md5=e80dbf16cb613bf7fe369f956fbaf031Lysosomal storage diseases: diagnostic confirmation and management of presymptomatic individualsWang Raymond Y; Bodamer Olaf A; Watson Michael S; Wilcox William RGenetics in medicine : official journal of the American College of Medical Genetics (2011), 13 (5), 457-84 ISSN:.PURPOSE: To develop educational guidelines for the diagnostic confirmation and management of individuals identified by newborn screening, family-based testing after proband identification, or carrier testing in at-risk populations, and subsequent prenatal or postnatal testing of those who are presymptomatic for a lysosomal storage disease. METHODS: Review of English language literature and discussions in a consensus development panel comprised an international group of experts in the clinical and laboratory diagnosis, treatment and management, newborn screening, and genetic aspects of lysosomal storage diseases. RESULTS: Although clinical trial and longitudinal data were used when available, the evidence in the literature is limited and consequently the recommendations must be considered as expert opinion. Guidelines were developed for Fabry, Gaucher, and Niemann-Pick A/B diseases, glycogen storage type II (Pompe disease), globoid cell leukodystrophy (Krabbe disease), metachromatic leukodystrophy, and mucopolysaccharidoses types I, II, and VI. CONCLUSION: These guidelines serve as an educational resource for confirmatory testing and subsequent clinical management of presymptomatic individuals suspected to have a lysosomal storage disease; they also help to define a research agenda for longitudinal studies such as the American College of Medical Genetics/National Institutes of Health Newborn Screening Translational Research Network.
- 4Meikle, P. J.; Hopwood, J. J.; Clague, A. E.; Carey, W. F. Prevalence of lysosomal storage disorders. JAMA 1999, 281, 249– 254, DOI: 10.1001/jama.281.3.249Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK1M7hsFSguw%253D%253D&md5=26f325562b3ff5e300adf6e5660c0a04Prevalence of lysosomal storage disordersMeikle P J; Hopwood J J; Clague A E; Carey W FJAMA (1999), 281 (3), 249-54 ISSN:0098-7484.CONTEXT: Lysosomal storage disorders represent a group of at least 41 genetically distinct, biochemically related, inherited diseases. Individually, these disorders are considered rare, although high prevalence values have been reported in some populations. These disorders are devastating for individuals and their families and result in considerable use of resources from health care systems; however, the magnitude of the problem is not well defined. To date, no comprehensive study has been performed on the prevalence of these disorders as a group. OBJECTIVE: To determine the prevalence of lysosomal storage disorders individually and as a group in the Australian population. DESIGN: Retrospective case studies. SETTING: Australia, from January 1, 1980, through December 31, 1996. MAIN OUTCOME MEASURE: Enzymatic diagnosis of a lysosomal storage disorder. RESULTS: Twenty-seven different lysosomal storage disorders were diagnosed in 545 individuals. The prevalence ranged from 1 per 57000 live births for Gaucher disease to 1 per 4.2 million live births for sialidosis. Eighteen of 27 disorders had more than 10 diagnosed cases. As a group of disorders, the combined prevalence was 1 per 7700 live births. There was no significant increase in the rate of either clinical diagnoses or prenatal diagnoses of lysosomal storage disorders during the study period. CONCLUSIONS: Individually, lysosomal storage disorders are rare genetic diseases. However, as a group, they are relatively common and represent an important health problem in Australia.
- 5Pastores, G. M. Therapeutic approaches for lysosomal storage diseases. Ther. Adv. Endocrinol. Metab. 2010, 1, 177– 188, DOI: 10.1177/2042018810384429Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVKisLzK&md5=6ddf6c6f96172aee21e75b79c7d5febaTherapeutic approaches for lysosomal storage diseasesPastores, Gregory M.Therapeutic Advances in Endocrinology and Metabolism (2010), 1 (4), 177-188CODEN: TAEMBU; ISSN:2042-0188. (Sage Publications Ltd.)A review. The lysosomal storage disorders (LSDs) comprise a heterogeneous group of inborn errors of metab. characterized by tissue substrate deposits, most often caused by a deficiency of the enzyme normally responsible for catabolism of various byproducts of cellular turnover. Individual entities are typified by involvement of multiple body organs, in a pattern reflecting the sites of substrate storage. It is increasingly recognized that one or more processes, such as aberrant inflammation, dysregulation of apoptosis and/or defects of autophagy, may mediate organ dysfunction or failure. Several therapeutic options for various LSDs have been introduced, including hematopoietic stem cell transplantation, enzyme replacement therapy and substrate redn. therapy. Addnl. strategies are being explored, including the use of pharmacol. chaperones and gene therapy. Most LSDs include a variant characterized by primary central nervous system (CNS) involvement. At present, therapy of the CNS manifestations remains a major challenge because of the inability to deliver therapeutic agents across the intact blood-brain barrier. With improved understanding of underlying disease mechanisms, addnl. therapeutic options may be developed, complemented by various strategies to deliver the therapeutic agent(s) to recalcitrant sites of pathol. such as brain, bones and lungs.
- 6Desnick, R. J.; Schuchman, E. H. Enzyme replacement and enhancement therapies: lessons from lysosomal disorders. Nat. Rev. Genet. 2002, 3, 954– 966, DOI: 10.1038/nrg963Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XptFCjtL8%253D&md5=9fde9e7a61c0f781b9e6a3d0b76e0bb1Enzyme replacement and enhancement therapies: lessons from lysosomal disordersDesnick, Robert J.; Schuchman, Edward H.Nature Reviews Genetics (2002), 3 (12), 954-966CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A review. The past decade has witnessed remarkable advances in our ability to treat inherited metabolic disorders, esp. the lysosomal storage diseases, a group of more than 40 disorders, each of which is caused by the deficiency of a lysosomal enzyme or protein. During the past few years, both enzyme replacement and enhancement therapies have been developed to treat these disorders. This review discusses the successes and shortcomings of these therapeutic strategies, and the contributions that they have made to treating lysosomal storage diseases.
- 7Coutinho, M. F.; Santos, J. I.; Alves, S. Less is more: substrate reduction therapy for lysosomal storage disorders. Int. J. Mol. Sci. 2016, 17, 1065, DOI: 10.3390/ijms17071065Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVGntLjI&md5=c36d29878ee3883ad97972199e079220Less is more: substrate reduction therapy for lysosomal storage disordersCoutinho, Maria Francisca; Santos, Juliana Ines; Alves, SandraInternational Journal of Molecular Sciences (2016), 17 (7), 1065/1-1065/22CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)Lysosomal storage diseases (LSDs) are a group of rare, life-threatening genetic disorders, usually caused by a dysfunction in one of the many enzymes responsible for intralysosomal digestion. Even though no cure is available for any LSD, a few treatment strategies do exist. Traditionally, efforts have been mainly targeting the functional loss of the enzyme, by injection of a recombinant formulation, in a process called enzyme replacement therapy (ERT), with no impact on neuropathol. This ineffectiveness, together with its high cost and lifelong dependence is amongst the main reasons why addnl. therapeutic approaches are being (and have to be) investigated: chaperone therapy; gene enhancement; gene therapy; and, alternatively, substrate redn. therapy (SRT), whose aim is to prevent storage not by correcting the original enzymic defect but, instead, by decreasing the levels of biosynthesis of the accumulating substrate(s). Here we review the concept of substrate redn., highlighting the major breakthroughs in the field and discussing the future of SRT, not only as a monotherapy but also, esp., as complementary approach for LSDs.
- 8Biffi, A. Hematopoietic stem cell gene therapy for storage disease: current and new indications. Mol. Ther. 2017, 25, 1155– 1162, DOI: 10.1016/j.ymthe.2017.03.025Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXntFelt7c%253D&md5=eb2da5f3983ae056908ad9d132769efeHematopoietic Stem Cell Gene Therapy for Storage Disease: Current and New IndicationsBiffi, AlessandraMolecular Therapy (2017), 25 (5), 1155-1162CODEN: MTOHCK; ISSN:1525-0024. (Cell Press)Lysosomal storage disorders (LSDs) are a broad class of monogenic diseases with an overall incidence of 1:7,000 newborns, due to the defective activity of one or more lysosomal hydrolases or related proteins resulting in storage of un-degraded substrates in the lysosomes. The over 40 different known LSDs share a life-threatening nature, but they are present with extremely variable clin. manifestations, detd. by the characteristics and tissue distribution of the material accumulating due to the lysosomal dysfunction. The majority of LSDs lack a curative treatment. This is particularly true for LSDs severely affecting the CNS. Based on current preclin. and clin. evidences, among other treatment modalities, hematopoietic stem cell gene therapy could potentially result in robust therapeutic benefit for LSD patients, with particular indication for those characterized by severe brain damage. Optimization of current approaches and technol., as well as implementation of clin. trials for novel indications, and prolonged and more extensive follow-up of the already treated patients will allow translating this promise into new medicinal products.
- 9Shihabuddin, L. S.; Cheng, S. H. Neural stem cell transplantation as a therapeutic approach for treating lysosomal storage diseases. Neurotherapeutics 2011, 8, 659– 667, DOI: 10.1007/s13311-011-0067-8Google ScholarThere is no corresponding record for this reference.
- 10Gelb, M. H.; Scott, C. R.; Turecek, F. Newborn screening for lysosomal storage diseases. Clin. Chem. 2015, 61, 335– 346, DOI: 10.1373/clinchem.2014.225771Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXislGnsrg%253D&md5=7e4232da6432b577779e8d18de6ed7ffNewborn screening for lysosomal storage diseasesGelb, Michael H.; Scott, C. Ronald; Turecek, FrantisekClinical Chemistry (Washington, DC, United States) (2015), 61 (2), 335-346CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)A review. Background: There is worldwide interest in newborn screening for lysosomal storage diseases because of the development of treatment options that give better results when carried out early in life. Screens with high differentiation between affected and nonaffected individuals are crit. because of the large no. of potential false positives. Content: This review summarizes 3 screening methods: (a) direct assay of enzymic activities using tandem mass spectrometry or fluorometry, (b) immunocapture-based measurement of lysosomal enzyme abundance, and (c) measurement of biomarkers. Assay performance is compared on the basis of small-scale studies as well as on large-scale pilot studies of mass spectrometric and fluorometric screens. Summary: Tandem mass spectrometry and fluorometry techniques for direct assay of lysosomal enzymic activity in dried blood spots have emerged as the most studied approaches. Comparative mass spectrometry vs fluorometry studies show that the former better differentiates between nonaffected vs affected individuals. This in turn leads to a manageable no. of screen positives that can be further evaluated with second-tier methods.
- 11Chamoles, N. A.; Blanco, M.; Gaggioli, D. Diagnosis of α-l-iduronidase deficiency in dried blood spots on filter paper: the possibility of newborn diagnosis. Clin. Chem. 2001, 47, 780– 781Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXis1Kis7s%253D&md5=c986557f2b6616968e2af7ad9eb379c4Diagnosis of α-L-iduronidase deficiency in dried blood spots on filter paper: the possibility of newborn diagnosisChamoles, Nestor A.; Blanco, Mariana; Gaggioli, DanielaClinical Chemistry (Washington, DC, United States) (2001), 47 (4), 780-781CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Mucopolysaccharidosis type I (MPS I), produced by deficiency of a-L-iduronidase. In the last few years treatment of MPS I became possible by bone marrow transplantation (2), enzyme replacement The filter paper need not be removed during the therapy (3), and gene transfer or gene modification. The effectiveness of these therapies, particularly for MPS tube was run for each sample of the assay. The presymptomatic detection of MPS I can be achieved only by newborn screening. In this case, a simple technique suitable for dried blood spots on filter paper (DBFP) is needed. The present methodol. is easier, faster, and less expensive than the leukocyte assay. A drop of blood obtained through heel prick is sufficient to perform the assay in duplicate plus one blank. Sample transportation is safe. Minimal activity loss occurs during storage at room temp. up to 20 days. The iduronidase activity test in DBFP samples appears to be a reasonable approach for the initial diagnosis of MPS I.
- 12Chamoles, N. A.; Blanco, M.; Gaggioli, D.; Casentini, C. Gaucher and Niemann–Pick diseases—enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards. Clin. Chim. Acta 2002, 317, 191– 197, DOI: 10.1016/S0009-8981(01)00798-7Google ScholarThere is no corresponding record for this reference.
- 13Chamoles, N. A.; Blanco, M. B.; Gaggioli, D.; Casentini, C. Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paper. Clin. Chem. 2001, 47, 2098– 2102Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXovVOmsrs%253D&md5=16560c7cd5ce59dad07cc45fd9976d03Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paperChamoles, Nestor A.; Blanco, Mariana B.; Gaggioli, Daniela; Casentini, CarinaClinical Chemistry (Washington, DC, United States) (2001), 47 (12), 2098-2102CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Background: Clin. differentiation among mucopolysaccharidosis, oligosaccharidosis, and mucolipidosis II and III is difficult. We describe methods for the assay of 8 lysosomal enzymes in dried blood spots on filter paper that allow screening for 12 lysosomal storage diseases that present with a Hurler-like phenotype. Methods: To test tubes contg. 3-mm blood spots, we added elution liq. and fluorescent or radioactive substrate soln. After incubation at 37°, the reaction was terminated by the addn. of a stop buffer. The amt. of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. Sample stability was studied during storage for 21 days and during shipment of samples. We measured enzyme activities in 85 healthy controls (35 newborn, 50 adult), 57 patients suffering from 11 lysosomal storage diseases, and 46 obligate carriers. Results: Intra- and interassay CVs were <9% and <15%, resp. Mean activity losses during transportation or storage for up to 21 days at 4° were ≤27%. Enzyme activities in all patients were outside the ranges of values seen for carriers and controls. Conclusions: The described methodol. distinguishes between patients and controls with samples that are sufficiently stable to be mailed to the testing lab.
- 14Ceci, R.; Francesco, P.; Mucci, J.; Cancelarich, L.; Fossati, C.; Rozenfeld, P. Reliability of enzyme assays in dried blood spots for diagnosis of 4 lysosomal storage disorders. Adv. Biol. Chem. 2011, 1, 58– 64, DOI: 10.4236/abc.2011.13008Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XltFKht7k%253D&md5=6805941b51f8b416bb27817b0aac4cc9Reliability of enzyme assays in dried blood spots for diagnosis of 4 lysosomal storage disordersCeci, Romina; de Francesco, Pablo N.; Mucci, Juan M.; Cancelarich, Lorena N.; Fossati, Carlos A.; Rozenfeld, Paula A.Advances in Biological Chemistry (2011), 1 (3), 58-64CODEN: ABCDAS; ISSN:2162-2183. (Scientific Research Publishing, Inc.)Lysosomal storage disorders (LSD) are inherited diseases caused, in the majority of them, by the deficiency of lysosomal enzymic activities. We aimed to analyze the usefulness of DBS samples for diagnosis of 4 LSDs, with the availability of a large quantity of patient samples. Blood samples from previously diagnosed patients with Fabry, Gaucher, Hunter, and Maroteaux-Lamy syndromes and normal control individuals, were collected and dispensed in filter paper, and used for enzymic activity detn. Diagnosis of hemi/homo-zygous patients with Fabry, Hunter and Maroteaux-Lamy diseases using DBS samples showed ideal parameters of 100% sensitivity and specificity. DBS assay for Gaucher disease would need a posterior confirmatory step. Leukocyte measurement is the only reliable way to diagnose Gaucher disease. For Hunter, Fabry and Maroteaux-Lamy disorders discrimination between patients and controls seems adequate by DBS.
- 15Garg, U.; Dasouki, M. Expanded newborn screening of inherited metabolic disorders by tandem mass spectrometry: Clinical and laboratory aspects. Clin. Biochem. 2006, 39, 315– 332, DOI: 10.1016/j.clinbiochem.2005.12.009Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjvVGisLg%253D&md5=aa3ec45e117e16efe534286ccead4db7Expanded newborn screening of inherited metabolic disorders by tandem mass spectrometry: clinical and laboratory aspectsGarg, Uttam; Dasouki, MajedClinical Biochemistry (2006), 39 (4), 315-332CODEN: CLBIAS; ISSN:0009-9120. (Elsevier)A review. Newborn screening started in the 1960 s for the purpose of identifying phenylketonuric patients to begin early intervention and to prevent mental retardation in these patients. Soon thereafter, screening programs expanded to include addnl. genetic disorders added individually one at a time. In the 1980 s, tandem mass spectrometry (MS/MS) was introduced in clin. labs., and in the 1990 s, the technique was used for newborn screening. Unlike measuring one analyte at a time, MS/MS allows measurement of > 40 analytes, in a few minutes with the use of a single assay. Currently, MS/MS is being used for the identification of several amino acid, org. acid and fatty acid disorders. Several states in the United States and many other countries are using MS/MS in newborn screening. However, there is a significant disparity among different newborn screening programs for disorders being screened by MS/MS and many other challenges are faced by the expanded newborn screening. It is anticipated that in the future the use of MS/MS in newborn screening will expand both at the analyte and geog. levels. Clinicians and lab. scientists should become familiar with MS/MS, disorders being screened in their patients' population and the future of this emerging technol.
- 16Blanchard, S.; Sadilek, M.; Scott, C. R.; Turecek, F.; Gelb, M. H. Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis I. Clin. Chem. 2008, 54, 2067– 2070, DOI: 10.1373/clinchem.2008.115410Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjtFeitg%253D%253D&md5=10a78214da663c38b4339aa0be6c91d1Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis IBlanchard, Sophie; Sadilek, Martin; Scott, C. Ronald; Turecek, Frantisek; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2008), 54 (12), 2067-2070CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)BACKGROUND: Treatments now available for mucopolysaccharidosis I require early detection for optimum therapy. Therefore, we have developed an assay appropriate for newborn screening of the activity of the relevant enzyme, α-L-iduronidase. METHODS: We synthesized a new α-L-iduronidase substrate that can be used to assay the enzyme by use of tandem mass spectrometry together with an internal std. or by fluorometry. The assay uses a dried blood spot on a newborn screening card as the enzyme source. The assay protocol uses a simple liq.-liq. extn. step before mass spectrometry. We optimized enzyme reaction conditions and procedures for the assay, including the concn. of substrate, the reaction pH, the incubation time, and mass spectrometer operation. We also assessed inter- and intraassay imprecision. RESULTS: When the assay was tested on dried blood spots, the α-L-iduronidase activity measured for 5 patients with mucopolysaccharidosis I was well below the interval found for 10 randomly chosen newborns. Inter- and intraassay imprecision were <10%. The synthesis of the α-L-iduronidase substrate is practical for use on a scale needed to support newborn screening demands. CONCLUSIONS: This newly developed tandem mass spectrometry assay has the potential to be adopted for newborn screening of mucopolysaccharidosis I. This assay has advantages over a previously reported assay also developed in this lab. and has the potential to be performed in a multiplex fashion to measure several lysosomal enzymes relevant to treatable lysosomal storage diseases.
- 17Chennamaneni, N. K.; Kumar, A. B.; Barcenas, M.; Spacil, Z.; Scott, C. R.; Turecek, F.; Gelb, M. H. Improved reagents for newborn screening of mucopolysaccharidosis types I, II, and VI by tandem mass spectrometry. Anal. Chem. 2014, 86, 4508– 4514, DOI: 10.1021/ac5004135Google ScholarThere is no corresponding record for this reference.
- 18Duffey, T. A.; Khaliq, T.; Scott, C. R.; Turecek, F.; Gelb, M. H. Design and synthesis of substrates for newborn screening of Maroteaux-Lamy and Morquio A syndromes. Bioorg. Med. Chem. Lett. 2010, 20, 5994– 5996, DOI: 10.1016/j.bmcl.2010.08.080Google ScholarThere is no corresponding record for this reference.
- 19Gelb, M. H.; Turecek, F.; Scott, C. R.; Chamoles, N. A. Direct multiplex assay of enzymes in dried blood spots by tandem mass spectrometry for the newborn screening of lysosomal storage disorders. J. Inherited Metab. Dis. 2006, 29, 397– 404, DOI: 10.1007/s10545-006-0265-4Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlsV2ltbc%253D&md5=891bb8799620949e228a8088a137c6c1Direct multiplex assay of enzymes in dried blood spots by tandem mass spectrometry for the newborn screening of lysosomal storage disordersGelb, Michael H.; Turecek, Frantisek; Scott, C. Ron; Chamoles, Nestor A.Journal of Inherited Metabolic Disease (2006), 29 (2/3), 397-404CODEN: JIMDDP; ISSN:0141-8955. (Springer)A review. Tandem mass spectrometry is currently used in newborn screening programs to quantify the level of amino acids and acylcarnitines in dried blood spots for detection of metabolites assocd. with treatable diseases. We have developed assays for lysosomal enzymes in rehydrated dried blood spots in which a set of substrates is added and the set of corresponding enzymic products are quantified using tandem mass spectrometry with the aid of mass-differentiated internal stds. We have developed a multiplex assay of the set of enzymes that, when deficient, cause the lysosomal storage disorders Fabry, Gaucher, Hurler, Krabbe, Niemann-Pick A/B and Pompe diseases. These diseases were selected because treatments are now available or expected to emerge shortly. The discovery that acarbose is a selective inhibitor of maltase glucoamylase allows the Pompe disease enzyme, acid α-glucosidase, to be selectively assayed in white blood cells and dried blood spots. When tested with dried blood spots from 40 unaffected individuals and 10-12 individuals with the lysosomal storage disorder, the tandem mass spectrometry assay led to the correct identification of the affected individuals with 100% sensitivity. Many of the reagents needed for the new assays are com. available, and those that are not are being prepd. under Good Manufg. Procedures for approval by the FDA. Our newborn screening assay for Krabbe disease is currently being put in place at the Wadsworth Center in New York State for the anal. of ∼1000 dried blood spots per day. Summary We have developed tandem mass spectrometry for the direct assay of lysosomal enzymes in rehydrated dried blood spots that can be implemented for newborn screening of lysosomal storage disorders. Several enzymes can be analyzed by a single method (multiplex anal.) and in a high-throughput manner appropriate for newborn screening labs.
- 20Khaliq, T.; Sadilek, M.; Scott, C. R.; Turecek, F.; Gelb, M. H. Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis IVA. Clin. Chem. 2011, 57, 128– 131, DOI: 10.1373/clinchem.2010.149880Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXitFSksro%253D&md5=a44d9eb0fda8bb9f807704fd35212f69Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis IVAKhaliq, Tanvir; Sadilek, Martin; Scott, C. Ronald; Turecek, Frantisek; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2011), 57 (1), 128-131CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Treatments are being developed for an increasing no. of mucopolysaccharidoses, and early diagnosis is expected to be necessary to maximize the benefits of therapy. Therefore, we developed an assay for N-acetylgalactosamine-6-sulfate sulfatase (GALNS), the enzyme deficient in mucopolysaccharidosis IVA (Morquio A syndrome), that is applicable for clin. diagnosis. A novel substrate for GALNS was synthesized for a new enzyme activity assay that is based on tandem mass spectrometry and uses dried blood spots (DBSs) as the enzyme source. We optimized the assay conditions, including the substrate concn., reaction pH, lead formate concn., incubation time, punch size of the DBS, and mass spectrometer conditions. We also assessed inter- and intraassay variation. The assay uses either solid-phase or liq.-phase extn. before anal. by mass spectrometry. An evaluation of blood spots from 90 randomly chosen healthy newborns and 9 patients with Morquio A syndrome showed a well-defined interval between their resp. enzyme activities. Inter- and intraassay imprecision was <10%. This tandem mass spectrometry assay requires a minimal no. of sample-prepn. steps, thus making it easy to implement. The assay has the potential to be adopted for early diagnosis of Morquio A syndrome. We believe this assay could be performed in a multiplex fashion with assays for other lysosomal enzymes.
- 21Li, Y.; Brockmann, K.; Turecek, F.; Scott, C. R.; Gelb, M. H. Tandem mass spectrometry for the direct assay of enzymes in dried blood spots: application to newborn screening for Krabbe disease. Clin. Chem. 2004, 50, 638– 640, DOI: 10.1373/clinchem.2003.028381Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhvVChs7g%253D&md5=7dcd950e60f9765fdcbb3357a038c90eTandem mass spectrometry for the direct assay of enzymes in dried blood spots: Application to newborn screening for Krabbe diseaseLi, Yijun; Brockmann, Knut; Turecek, Frantisek; Scott, C. Ronald; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2004), 50 (3), 638-640CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)A study was conducted to investigate whether MS/MS can be used to directly assay enzymes in dried blood spots, in this case, galactocerebroside β-galactosidase (GALC) for the detection of Krabbe disease. Results suggest that an assay for GALC in dried blood spots from newborns can be implemented and evaluated for the early detection of Krabbe disease. Measurement of GALC activity with a series of substrates that differed in the lengths of their fatty acyl groups revealed that C8:0 ceramide gives fivefold higher activity than substrates contg. ceramides with the longer chain fatty acids found in natural ceramides, a factor that was important in the ability to detect GALC in dried blood spots. The use of β-Gal-C8-Cer also has an advantage in that it generates the unnatural product C8-Cer, thus avoiding interference from the natural ceramides present in biol. specimens.
- 22Li, Y.; Scott, C. R.; Chamoles, N. A.; Ghavami, A.; Pinto, B. M.; Turecek, F.; Gelb, M. H. Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin. Chem. 2004, 50, 1785– 1796, DOI: 10.1373/clinchem.2004.035907Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXotVylt7s%253D&md5=f6fb0eb04e31d75bc440b1223e82c589Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screeningLi, Yijun; Scott, C. Ronald; Chamoles, Nestor A.; Ghavami, Ahmad; Pinto, B. Mario; Turecek, Frantisek; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2004), 50 (10), 1785-1796CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Background: Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards contg. dried blood spots as the enzyme source. Methods: We used a cassette of substrates and internal stds. to directly quantify the enzymic activities, and tandem mass spectrometry for enzymic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liq.-liq. extn. followed by solid-phase extn. with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid α-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed. Results: We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann-Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the min. activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5-9 heterozygous carriers were approx. one-half those measured with 15-32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals. Conclusion: For all five diseases, the affected individuals were detected. The assay can be readily automated, and the anticipated reagent and supply costs are well within the budget limits of newborn-screening centers.
- 23Wang, D.; Eadala, B.; Sadilek, M.; Chamoles, N. A.; Turecek, F.; Scott, C. R.; Gelb, M. H. Tandem mass spectrometric analysis of dried blood spots for screening of mucopolysaccharidosis I in newborns. Clin. Chem. 2005, 51, 898– 900, DOI: 10.1373/clinchem.2004.047167Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjvVKjsrk%253D&md5=49a454b7ad48bf47fa6af79ee30cb5cdTandem mass spectrometric analysis of dried blood spots for screening of mucopolysaccharidosis I in newbornsWang, Ding; Eadala, Bhramara; Sadilek, Martin; Chamoles, Nestor A.; Turecek, Frantisek; Scott, C. Ronald; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2005), 51 (5), 898-900CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)An electrospray ionization tandem mass spectrometry (ESI-MS/MS) assay that directly measures the reaction velocity of α-L-iduronidase (IDUA) in rehydrated dried blood spots (DBS) for the newborn screening of mucopolysaccharidosis type I (MPS-I) is described. The assay can be combined with ESI-MS/MS assays of Niemann-Pick type A/B, Krabbe, Gaucher, Pompe, and Fabry diseases for the simultaneous anal. of six lysosomal storage diseases. The assay is compatible with microtiter plate and multichannel pipetting techniques. Each IDUA assay requires only 13.3 μg of substrate, which can be readily prepd. from com. available heparin and 0.1 μg of internal std.
- 24Wolfe, B. J.; Ghomashchi, F.; Kim, T.; Abam, C. A.; Sadilek, M.; Jack, R.; Thompson, J. N.; Scott, C. R.; Gelb, M. H.; Turecek, F. New substrates and enzyme assays for the detection of mucopolysaccharidosis III (Sanfilippo Syndrome) types A, B, C, and D by tandem mass spectrometry. Bioconjugate Chem. 2012, 23, 557– 564, DOI: 10.1021/bc200609xGoogle Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtVWiur8%253D&md5=21afdbb74116b48f6531ecaed0094d8fNew Substrates and Enzyme Assays for the Detection of Mucopolysaccharidosis III (Sanfilippo Syndrome) Types A, B, C, and D by Tandem Mass SpectrometryWolfe, Brian J.; Ghomashchi, Farideh; Kim, Tim; Abam, Cynthia A.; Sadilek, Martin; Jack, Rhona; Thompson, Jerry N.; Scott, C. Ronald; Gelb, Michael H.; Turecek, FrantisekBioconjugate Chemistry (2012), 23 (3), 557-564CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)The clin. phenotype of Sanfilippo Syndrome is caused by one of four enzyme deficiencies that are assocd. with a defect in mucopolysaccharide metab. The four subtypes (A, B, C, and D) are each caused by an enzyme deficiency involved in the degrdn. of heparan sulfate. A highly efficient synthesis of the substrates and internal stds. required for the enzymic assay of each of the four enzymes was developed. The synthesis of the substrates involves chem. modification of a common intermediate. The substrates and internal stds. allow the measurement of the enzymes relevant to heparan N-sulfatase (type A); N-acetyl-α-glucosaminidase (type B); acetyl-CoA:α-glucosamide N-acetyltransferase (type C); and N-acetylglucosamine 6-sulfatase (type D). The internal stds. are similar to the substrates and allow for the accurate quantification of the enzyme assays using tandem mass spectrometry. The synthetic substrates incorporate a coumarin moiety and can also be used in fluorometric enzyme assays. It was confirmed that all four substrates can detect the appropriate Sanfilippo Syndrome in fibroblast lysates, and the measured enzyme activities are distinctly lower by a factor of 10 when compared to fibroblast lysates from unaffected persons.
- 25Spáčil, Z.; Elliott, S.; Reeber, S. L.; Gelb, M. H.; Scott, C. R.; Tureček, F. Comparative triplex tandem mass spectrometry assays of lysosomal enzyme activities in dried blood spots using fast liquid chromatography: application to newborn screening of Pompe, Fabry, and Hurler diseases. Anal. Chem. 2011, 83, 4822– 4828, DOI: 10.1021/ac200417uGoogle ScholarThere is no corresponding record for this reference.
- 26Spacil, Z.; Tatipaka, H.; Barcenas, M.; Scott, C. R.; Turecek, F.; Gelb, M. H. High-throughput assay of 9 lysosomal enzymes for newborn screening. Clin. Chem. 2013, 59, 502– 511, DOI: 10.1373/clinchem.2012.189936Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjvFOhu7g%253D&md5=9f66f6221142df65473ea4350333aa5fHigh-throughput assay of 9 lysosomal enzymes for newborn screeningSpacil, Zdenek; Tatipaka, Haribabu; Barcenas, Mariana; Scott, C. Ronald; Turecek, Frantisek; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2013), 59 (3), 502-511CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymic assays that uses HPLC-tandem mass spectrometry (MS/MS). The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal stds. and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre-HPLC-MS/MS sample prepn. required only 4 liq. transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to sepn. and column equilibration. Product-specific and internal std.-specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode. Anal. of blood spots from 58 random newborns and lysosomal storage disease-affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex anal. (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening labs. HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the no. of sep. incubations necessary.
- 27la Marca, G.; Casetta, B.; Malvagia, S.; Guerrini, R.; Zammarchi, E. New strategy for the screening of lysosomal storage disorders: the use of the online trapping-and-cleanup liquid chromatography/mass spectrometry. Anal. Chem. 2009, 81, 6113– 6121, DOI: 10.1021/ac900504sGoogle ScholarThere is no corresponding record for this reference.
- 28Kasper, D. C.; Herman, J.; De Jesus, V. R.; Mechtler, T. P.; Metz, T. F.; Shushan, B. The application of multiplexed, multi-dimensional ultra-high-performance liquid chromatography/tandem mass spectrometry to the high-throughput screening of lysosomal storage disorders in newborn dried bloodspots. Rapid Commun. Mass Spectrom. 2010, 24, 986– 994, DOI: 10.1002/rcm.4496Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjt1Gmtbw%253D&md5=2163c3dfc20bd7bd9c46d73f2bd7f5a3The application of multiplexed, multi-dimensional ultra-high-performance liquid chromatography/tandem mass spectrometry to the high-throughput screening of lysosomal storage disorders in newborn dried bloodspotsKasper, David C.; Herman, Joseph; De Jesus, Victor R.; Mechtler, Thomas P.; Metz, Thomas F.; Shushan, BoriRapid Communications in Mass Spectrometry (2010), 24 (7), 986-994CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)Lysosomal storage disorders are just beginning to be routinely screened using enzyme activity assays involving dried blood spots and tandem mass spectrometry (MS/MS). This paper discusses some of the anal. challenges assocd. with published assays including complex sample prepn. and potential interference from excess residual substrate. Solns. to these challenges are presented in the form of online two-dimensional chromatog. to eliminate off-line liq.-liq. extn. (LLE) and solid-phase extn. (SPE), the use of ultra-HPLC (UHPLC) to sep. excess substrate from all other analytes and multiplexed sample introduction for higher throughput required of a population screening assay. High sensitivity, specificity and throughput were demonstrated using this novel method. Copyright © 2010 John Wiley & Sons, Ltd.
- 29Mechtler, T. P.; Stary, S.; Metz, T. F.; De Jesus, V. R.; Greber-Platzer, S.; Pollak, A.; Herkner, K. R.; Streubel, B.; Kasper, D. C. Neonatal screening for lysosomal storage disorders: feasibility and incidence from a nationwide study in Austria. Lancet 2012, 379, 335– 341, DOI: 10.1016/S0140-6736(11)61266-XGoogle ScholarThere is no corresponding record for this reference.
- 30Metz, T. F.; Mechtler, T. P.; Orsini, J. J.; Martin, M.; Shushan, B.; Herman, J. L.; Ratschmann, R.; Item, C. B.; Streubel, B.; Herkner, K. R.; Kasper, D. C. Simplified newborn screening protocol for lysosomal storage disorders. Clin. Chem. 2011, 57, 1286– 1294, DOI: 10.1373/clinchem.2011.164640Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFOjtbrJ&md5=af79f7ce24b6387ea71c9320b7069596Simplified newborn screening protocol for lysosomal storage disordersMetz, Thomas F.; Mechtler, Thomas P.; Orsini, Joseph J.; Martin, Monica; Shushan, Bori; Herman, Joseph L.; Ratschmann, Rene; Item, Chike B.; Streubel, Berthold; Herkner, Kurt R.; Kasper, David C.Clinical Chemistry (Washington, DC, United States) (2011), 57 (9), 1286-1294CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Interest in lysosomal storage disorders, a collection of more than 40 inherited metabolic disorders, has increased because of new therapy options such as enzyme replacement, stem cell transplantation, and substrate redn. therapy. We developed a high-throughput protocol that simplifies anal. challenges such as complex sample prepn. and potential interference from excess residual substrate assocd. with previously reported assays. After overnight incubation (16-20 h) of dried blood spots with a cassette of substrates and deuterated internal stds., we used a TLX-2 system to quantify 6 lysosomal enzyme activities for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and mucopolysaccharidosis I disease. This multiplexed, multidimensional ultra-HPLC-tandem mass spectrometry assay included Cyclone P Turbo Flow and Hypersil Gold C8 columns. The method did not require offline sample prepn. such as liq.-liq. and solid-phase extn., or hazardous reagents such as Et acetate. Obviating the offline sample prepn. steps led to substantial savings in anal. time (approx. 70%) and reagent costs (approx. 50%). In a pilot study, lysosomal enzyme activities of 8586 newborns were measured, including 51 pos. controls, and the results demonstrated 100% diagnostic sensitivity and high specificity. The results for Krabbe disease were validated with parallel measurements by the New York State Screening Lab. Turboflow online sample cleanup and the use of an addnl. anal. column enabled the implementation of lysosomal storage disorder testing in a nationwide screening program while keeping the total anal. time to <2 min per sample.
- 31Gucciardi, A.; Legnini, E.; Di Gangi, I. M.; Corbetta, C.; Tomanin, R.; Scarpa, M.; Giordano, G. A column-switching HPLC-MS/MS method for mucopolysaccharidosis type I analysis in a multiplex assay for the simultaneous newborn screening of six lysosomal storage disorders. Biomed. Chromatogr. 2014, 28, 1131– 1139, DOI: 10.1002/bmc.3133Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXps1yqug%253D%253D&md5=b04eba1bdfc8c819db44dac8be1e6ca9A column-switching HPLC-MS/MS method for mucopolysaccharidosis type I analysis in a multiplex assay for the simultaneous newborn screening of six lysosomal storage disordersGucciardi, Antonina; Legnini, Elisa; Di Gangi, Iole Maria; Corbetta, Carlo; Tomanin, Rosella; Scarpa, Maurizio; Giordano, GiuseppeBiomedical Chromatography (2014), 28 (8), 1131-1139CODEN: BICHE2; ISSN:0269-3879. (John Wiley & Sons Ltd.)Lysosomal storage disorders comprise a group of rare genetic diseases in which a deficit of specific hydrolases leads to the storage of undegraded substrates in lysosomes. Impaired enzyme activities can be assessed by MS/MS quantification of the reaction products obtained after incubation with specific substrates. In this study, a column-switching HPLC-MS/MS method for multiplex screening in dried blood spot of the lysosomal enzymes activities was developed. Mucopolysaccharidosis type I, Fabry, Gaucher, Krabbe, Niemann-Pick A/B and Pompe diseases were simultaneously assayed. Dried blood spots were incubated with substrates and internal stds.; thereafter, supernatants were collected with minor manipulations. Samples were injected, trapped into an online perfusion column and, by a six-port valve, switched online through the C18 anal. column to perform sepn. of metabolites followed by MS/MS anal. A total of 1136 de-identified newborn screening samples were analyzed to det. refs. for enzymes activity values. As pos. controls, we analyzed dried blood spots from three patients with Pompe, one with Fabry, one with Krabbe disease and two with MPS I, and in all cases the enzyme activities were below the cutoff values measured for newborns, except for an MPS I patient after successful hematopoietic stem cell transplantation. Copyright © 2014 John Wiley & Sons, Ltd.
- 32He, W.; Voznyi, Y. V.; Boer, A. M.; Kleijer, W. J.; van Diggelen, O. P. A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type D (MPS IIID). J. Inherited Metab. Dis. 1993, 16, 935– 941, DOI: 10.1007/BF00711508Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXis1Chsbo%253D&md5=4180310d828c6e95f2526cf4431c115fA fluorometric enzyme assay for the diagnosis of Sanfilippo disease type D (MPS IIID)He, Wang; Voznyi, Ya. V.; Boer, A. M.; Kleijer, W. J.; van Diggelen, O. P.Journal of Inherited Metabolic Disease (1993), 16 (6), 935-41CODEN: JIMDDP; ISSN:0141-8955.4-Methylumbelliferyl-α-N-acetylglucosamine 6-sulfate was synthesized and shown to be a substrate for the lysosomal N-acetylglucosamine-6-sulfate sulfatase (GlcNAc-6S sulfatase). Fibroblasts and leukocytes from 3 different Sanfilippo D patients showed <1% of mean normal GlcNAc-6S sulfatase activity. The enzymic liberation of the fluorochrome from 4-methylumbelliferyl-α-N-acetylglucosamine 6-sulfate requires the sequential action of the GlcNAc-6S sulfatase and α-N-acetylglucosaminidase. A normal level of α-N-acetylglucosaminidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl-α-N-acetylglucosaminide formed by the GlcNAc-6S sulfatase. A second incubation in the presence of excess α-N-acetylglucosaminidase is needed to avoid underestimation of the GlcNAc-6S sulfatase activity.
- 33Karpova, E. A.; Voznyi, Y. V.; Keulemans, J. L. M.; Hoogeveen, A. T.; Winchester, B.; Tsvetkova, I. V.; van Diggelen, O. P. A fluorimetric enzyme assay for the diagnosis of sanfilippo disease type A (MPS IIIA). J. Inherited Metab. Dis. 1996, 19, 278– 285, DOI: 10.1007/BF01799255Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XksFentLg%253D&md5=5700f006001507b1011dee9b4e1afcd6A fluorometric enzyme assay for the diagnosis of Sanfilippo disease type A (MPS IIIA)Karpova, E. A.; Voznyi, Ya. V.; Keulemans, J. L. M.; Hoogeveen, A. T.; Winchester, B.; Tsvetkova, I. V.; Van Diggelen, O. P.Journal of Inherited Metabolic Disease (1996), 19 (3), 278-285CODEN: JIMDDP; ISSN:0141-8955. (Kluwer)4-Methylumbelliferyl-α-D-N-sulfoglucosaminide (MU-α-GlcNS) was synthesized and shown to be a substrate for the lysosomal heparin sulfamidase. Sanfilippo A patients' fibroblasts and lymphocytes showed 0-3% of mean normal heparin sulfamidase activity; in total leukocytes from patients sulfamidase activity was clearly deficient. In fibroblasts from obligate heterozygotes for Sanfilippo A, the sulfamidase activity was reduced in 9 out of 10 cases. Heparin sulfamidase desulfates MU-αGlcNS to MU-αGlcNH2 and further hydrolysis during a second incubation is required to liberate 4-methylumbelliferone, which can be measured. Yeast α-glucosidase, which has low but sufficient α-glucosaminidase activity, was used to hydrolyze the reaction intermediate MU-αGlcNH2 to release 4-methylumbelliferone and free glucosamine.
- 34Voznyi, Y. V.; Karpova, E. A.; Dudukina, T. V.; Tsvetkova, I. V.; Boer, A. M.; Janse, H. C.; van Diggelen, O. P. A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease C (MPS III C). J. Inherited Metab. Dis. 1993, 16, 465– 472, DOI: 10.1007/BF00710299Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXjtFyjtbY%253D&md5=f72815d38131e1d593ff14573b5a2948A fluorometric enzyme assay for the diagnosis of Sanfilippo disease C (MPS III C)Voznyi, Ya. V.; Karpova, E. A.; Dudukina, T. V.; Tsvetkova, I. V.; Boer, A. M.; Janse, H. C.; van Diggelen, O. P.Journal of Inherited Metabolic Disease (1993), 16 (2), 465-72CODEN: JIMDDP; ISSN:0141-8955.Both the α- and β-anomers of 4-methylumbelliferyl-D-glucosaminide were synthesized and shown to be substrates for the lysosomal acetyl-CoA:glucosaminide N-acetyltransferase. Using the β-anomer, fibroblasts and leukocytes from 11 different Sanfilippo C patients showed < 1% of mean normal N-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymic liberation of the fluorochrome from 4-methylumbelliferyl-β-D-glucosaminide requires the sequential action of the N-acetyltransferase and β-hexosaminidase. Normal β-hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl-β-D-N-acetylglucosaminide formed by the N-acetyltransferase. In cell exts. with a β-hexosaminidase deficiency, however, a second incubation in the presence of excess β-hexosaminidase is needed to avoid underestimation of the N-acetyltransferase activity.
- 35Voznyi, Y. V.; Keulemans, J. L. M.; van Diggelen, O. P. A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease). J. Inherited Metab. Dis. 2001, 24, 675– 680, DOI: 10.1023/A:1012763026526Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XltVSisA%253D%253D&md5=a06782de1b11dafe842ccc5d9b61c9e9A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease)Voznyi, Ya. V.; Keulemans, J. L. M.; van Diggelen, O. P.Journal of Inherited Metabolic Disease (2001), 24 (6), 675-680CODEN: JIMDDP; ISSN:0141-8955. (Kluwer Academic Publishers)4-Methylumbelliferyl-α-iduronate 2-sulfate was synthesized and shown to be a specific substrate for the lysosomal iduronate-2-sulfate sulfatase (IDS). Fibroblasts (n = 17), leukocytes (n = 3) and plasmas (n = 9) from different MPS II patients showed < 5% of mean normal IDS activity. The enzymic liberation of the fluorochrome from 4-methylumbelliferyl-α-iduronate 2-sulfate requires the sequential action of IDS and α-iduronidase. A normal level of α-iduronidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl-α-iduronide formed by IDS. A second incubation step in the presence of excess purified α-iduronidase is needed to avoid underestimation of the IDS activity.
- 36van Diggelen, O. P.; Voznyi, Y. V.; Keulemans, J. L. M.; Schoonderwoerd, K.; Ledvinova, J.; Mengel, E.; Zschiesche, M.; Santer, R.; Harzer, K. A new fluorimetric enzyme assay for the diagnosis of Niemann–Pick A/B, with specificity of natural sphingomyelinase substrate. J. Inherited Metab. Dis. 2005, 28, 733– 741, DOI: 10.1007/s10545-005-0105-yGoogle Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2MvotFSgug%253D%253D&md5=b8625aa93b719824525ec8739e9af75cA new fluorimetric enzyme assay for the diagnosis of Niemann-Pick A/B, with specificity of natural sphingomyelinase substratevan Diggelen O P; Voznyi Ya V; Keulemans J L M; Schoonderwoerd K; Ledvinova J; Mengel E; Zschiesche M; Santer R; Harzer KJournal of inherited metabolic disease (2005), 28 (5), 733-41 ISSN:0141-8955.6-Hexadecanoylamino-4-methylumbelliferylphosphorylcholine (HMUPC) was shown to be a specific substrate for the determination of acid (lysosomal) sphingomyelinase (ASM; gene SMPD1). Fibroblasts (n = 27) and leukocytes (n = 8) from both the A and B types of Niemann-Pick disease showed < 6% and < 10% of mean normal ASM activity, respectively. Niemann-Pick A or B patients bearing the Q292K mutation had apparently normal ASM activity with our new artificial substrate. These patients with false-normal sphingomyelinase activity, however, could readily be detected by determining the extent of inhibition of enzymatic hydrolysis of the artificial substrate HMU-PC by an unlabelled natural substrate, in particular lysosphingomyelin. This approach is generally applicable. Our novel assay for ASM combines the ease of a rapid and robust enzyme assay using a fluorogenic substrate with the specificity of an ASM assay using a natural substrate. Such assays are obviously more convenient to the diagnostic laboratory, since radiolabelled substrates are not required.
- 37van Diggelen, O. P.; Zhao, H.; Kleijer, W. J.; Janse, H. C.; Poorthuis, B. J. H. M.; van Pelt, J.; Kamerling, J. P.; Galjaard, H. A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A). Clin. Chim. Acta 1990, 187, 131– 139, DOI: 10.1016/0009-8981(90)90339-TGoogle ScholarThere is no corresponding record for this reference.
- 38Gasparotto, N.; Tomanin, R.; Frigo, A. C.; Niizawa, G.; Pasquini, E.; Blanco, M.; Donati, M. A.; Keutzer, J.; Zacchello, F.; Scarpa, M. Rapid diagnostic testing procedures for lysosomal storage disorders: alpha-glucosidase and beta-galactosidase assays on dried blood spots. Clin. Chim. Acta 2009, 402, 38– 41, DOI: 10.1016/j.cca.2008.12.006Google ScholarThere is no corresponding record for this reference.
- 39Chien, Y.-H.; Chiang, S.-C.; Zhang, X. K.; Keutzer, J.; Lee, N.-C.; Huang, A.-C.; Chen, C.-A.; Wu, M.-H.; Huang, P.-H.; Tsai, F.-J.; Chen, Y.-T.; Hwu, W.-L. Early detection of Pompe disease by newborn screening is feasible: results from the Taiwan screening program. Pediatrics 2008, 122, e39– 45, DOI: 10.1542/peds.2007-2222Google ScholarThere is no corresponding record for this reference.
- 40Sista, R.; Eckhardt, A. E.; Wang, T.; Sellos-Moura, M.; Pamula, V. K. Rapid, single-step assay for Hunter syndrome in dried blood spots using digital microfluidics. Clin. Chim. Acta 2011, 412, 1895– 1897, DOI: 10.1016/j.cca.2011.06.015Google ScholarThere is no corresponding record for this reference.
- 41Sista, R. S.; Eckhardt, A. E.; Wang, T.; Graham, C.; Rouse, J. L.; Norton, S. M.; Srinivasan, V.; Pollack, M. G.; Tolun, A. A.; Bali, D.; Millington, D. S.; Pamula, V. K. Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns. Clin. Chem. 2011, 57, 1444– 1451, DOI: 10.1373/clinchem.2011.163139Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlSht7vE&md5=525b2fc48d2157574e36fa05034d3d1dDigital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newbornsSista, Ramakrishna S.; Eckhardt, Allen E.; Wang, Tong; Graham, Carrie; Rouse, Jeremy L.; Norton, Scott M.; Srinivasan, Vijay; Pollack, Michael G.; Tolun, Adviye A.; Bali, Deeksha; Millington, David S.; Pamula, Vamsee K.Clinical Chemistry (Washington, DC, United States) (2011), 57 (10), 1444-1451CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymic anal. of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using std. fluorometric methods. We performed bench-based, fluorometric enzymic anal. on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochem. Genetics Lab. using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymic assays at Advanced Liq. Logic Inc. using ext. from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h. Assay results were generally comparable, although mean enzymic activity for GAA using microfluidics was approx. 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concn. Clear sepn. was obsd. between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics. A digital microfluidic platform compared favorably with a clin. ref. lab. to perform enzymic anal. in DBSs for Pompe and Fabry disorders. This platform presents a new technol. for a newborn screening lab. to screen LSDs by fully automating all the liq.-handling operations in an inexpensive system, providing rapid results.
- 42Millington, D. S.; Sista, R.; Eckhardt, A.; Rouse, J.; Bali, D.; Goldberg, R.; Cotten, M.; Buckley, R.; Pamula, V. Digital Microfluidics: A Future technology in the newborn screening laboratory?. Semin. Perinatol 2010, 34, 163– 169, DOI: 10.1053/j.semperi.2009.12.008Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3c7osVGmug%253D%253D&md5=7b2cf37b11a1d466399d362f40eaecb2Digital microfluidics: a future technology in the newborn screening laboratory?Millington David S; Sista Ramakrishna; Eckhardt Allen; Rouse Jeremy; Bali Deeksha; Goldberg Ronald; Cotten Michael; Buckley Rebecca; Pamula VamseeSeminars in perinatology (2010), 34 (2), 163-9 ISSN:.Expansion of newborn screening for inherited metabolic disorders using tandem mass spectrometry has generated interest in screening for other treatable conditions, including lysosomal storage diseases. Limitations to expansion include labor and equipment costs. We describe a cost-effective new platform that reduces the time to result reporting and can perform multiplexing assays requiring different platforms. Immunoassays and enzyme activity assays currently used in newborn screening have been translated to a disposable microchip programmed to dispense, transport, mix, wash, and incubate individual microdroplets from specimens, including dried blood spot extracts, and reagents all under software control. The specimen and reagents consumed are approximately 1% of those required by equivalent bench assays. In addition to immunologic and enzymatic assays, DNA amplification, amplicon detection, and sequencing have been demonstrated using the same microchips and control equipment. Recently, the multiplexing of 4 different enzyme activities has also been demonstrated with negligible cross-contamination. We review assays relevant to newborn screening.
- 43Millington, D. S.; Bali, D. M. Misinformation regarding tandem mass spectrometric vs fluorometric assays to screen newborns for LSDs. Mol. Genet. Metab. Rep. 2017, 11, 72– 73, DOI: 10.1016/j.ymgmr.2017.04.009Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnsFentro%253D&md5=d68e577d456a648e7bcb0b3fba99f3b7Misinformation regarding tandem mass spectrometric vs fluorometric assays to screen newborns for LSDsMillington, David S.; Bali, Deeksha M.Molecular Genetics and Metabolism Reports (2017), 11 (), 72-73CODEN: MGMRDI; ISSN:2214-4269. (Elsevier B.V.)There is no expanded citation for this reference.
- 44Millington, D.; Norton, S.; Singh, R.; Sista, R.; Srinivasan, V.; Pamula, V. Digital microfluidics comes of age: high-throughput screening to bedside diagnostic testing for genetic disorders in newborns. Expert Rev. Mol. Diagn. 2018, 18, 701– 712, DOI: 10.1080/14737159.2018.1495076Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlahtLjE&md5=23778480e146d2a3266def7e268653b1Digital microfluidics comes of age: high-throughput screening to bedside diagnostic testing for genetic disorders in newbornsMillington, David; Norton, Scott; Singh, Raj; Sista, Rama; Srinivasan, Vijay; Pamula, VamseeExpert Review of Molecular Diagnostics (2018), 18 (8), 701-712CODEN: ERMDCW; ISSN:1473-7159. (Taylor & Francis Ltd.)A review. Introduction: Digital microfluidics (DMF) is an emerging technol. with the appropriate metrics for application to newborn and high-risk screening for inherited metabolic disease and other conditions that benefit from early treatment. Areas covered: This review traces the development of electrowetting-based DMF technol. toward the fulfillment of its promise to provide an inexpensive platform to conduct enzymic assays and targeted biomarker assays at the bedside. The high-throughput DMF platform, referred to as SEEKER, was recently authorized by the United States Food and Drug Administration to screen newborns for four lysosomal storage disorders (LSDs) and is deployed in newborn screening programs in the United States. The development of reagents and methods for LSD screening and results from screening centers are reviewed. Preliminary results from a more compact DMF device, to perform disease-specific test panels from small vols. of blood, are also reviewed. Literature for this review was sourced using principal author and subject searches in PubMed. Expert commentary: Newborn screening is a vital and highly successful public health program. DMF technol. adds value to the current testing platforms that will benefit apparently healthy newborns with underlying genetic disorders and infants at-risk for conditions that present with symptoms in the newborn period.
- 45Cecioni, S.; Vocadlo, D. J. Carbohydrate bis-acetal-based substrates as tunable fluorescence-quenched probes for monitoring exo-glycosidase activity. J. Am. Chem. Soc. 2017, 139, 8392– 8395, DOI: 10.1021/jacs.7b01948Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVWitL%252FJ&md5=67e3b0771281b90a2c51dbe8237d7487Carbohydrate bis-acetal-based substrates as tunable fluorescence-quenched probes for monitoring exo-glycosidase activityCecioni, Samy; Vocadlo, David J.Journal of the American Chemical Society (2017), 139 (25), 8392-8395CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Tunable Forster resonance energy transfer (FRET)-quenched substrates are useful for monitoring the activity of various enzymes within their relevant physiol. environments. Development of FRET-quenched substrates for exo-glycosidases, however, has been hindered by their constrained pocket-shaped active sites. Here we report the design of a new class of substrate that overcomes this problem. These Bis-Acetal-Based Substrates (BABS) bear a hemiacetal aglycon leaving group that tethers fluorochromes in close proximity, also positioning them distant from the active site pocket. Following cleavage of the glycosidic bond, the liberated hemiacetal spontaneously breaks down, leading to sepn. of the fluorophore and quencher. We detail the synthesis and characterization of GlcNAc-BABS, revealing a striking 99.9% quenching efficiency. These substrates are efficiently turned over by the human exo-glycosidase O-GlcNAcase (OGA). We find the hemiacetal leaving group rapidly breaks down, enabling quant. monitoring of OGA activity. We expect this strategy to be broadly useful for the development of substrate probes for monitoring exo-glycosidases, as well as a range of other enzymes having constrained pocket-shaped active sites.
- 46Geyer, K.; Seeberger, P. H. Optimization of glycosylation reactions in a microreactor. Helv. Chim. Acta 2007, 90, 395– 403, DOI: 10.1002/hlca.200790046Google ScholarThere is no corresponding record for this reference.
- 47Scott, H. S.; Anson, D. S.; Orsborn, A. M.; Nelson, P. V.; Clements, P. R.; Morris, C. P.; Hopwood, J. J. Human alpha-L-iduronidase: cDNA isolation and expression. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 9695– 9699, DOI: 10.1073/pnas.88.21.9695Google ScholarThere is no corresponding record for this reference.
- 48Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem., Int. Ed. 2001, 40, 2004– 2021, DOI: 10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXksVOis78%253D&md5=61c28b416c42c87821bc8a5bc2fdec36Click chemistry: diverse chemical function from a few good reactionsKolb, Hartmuth C.; Finn, M. G.; Sharpless, K. BarryAngewandte Chemie, International Edition (2001), 40 (11), 2004-2021CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)Review with > 88 refs. Examn. of nature's favorite mols. reveals a striking preference for making carbon-heteroatom bonds over carbon-carbon bonds - surely no surprise given that carbon dioxide is nature's starting material and that most reactions are performed in water. Nucleic acids, proteins, and polysaccharides are condensation polymers of small subunits stitched together by carbon-heteroatom bonds. Even the 35 or so building blocks from which these crucial mols. are made each contain, at most, six contiguous C-C bonds, except for the three arom. amino acids. Taking a cue from nature's approach, the development of a set of powerful, highly reliable, and selective reactions for the rapid synthesis of useful new compds. and combinatorial libraries through heteroatom links (C-X-C), an approach called "click chem." is addressed. Click chem. is at once defined, enabled, and constrained by a handful of nearly perfect "spring-loaded" reactions. The stringent criteria for a process to earn click chem. status are described along with examples of the mol. frameworks that are easily made using this spartan, but powerful, synthetic strategy.
- 49Kolb, H. C.; Sharpless, K. B. The growing impact of click chemistry on drug discovery. Drug Discovery Today 2003, 8, 1128– 1137, DOI: 10.1016/S1359-6446(03)02933-7Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXpvVWkuro%253D&md5=f0042f3a8f153d1c1192533d90699d19The growing impact of click chemistry on drug discoveryKolb, Hartmuth C.; Sharpless, K. BarryDrug Discovery Today (2003), 8 (24), 1128-1137CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Science Ltd.)A review. Click chem. is a modular approach that uses only the most practical and reliable chem. transformations. Its applications are increasingly found in all aspects of drug discovery, ranging from lead finding through combinatorial chem. and target-templated in situ chem., to proteomics and DNA research, using bioconjugation reactions. The copper-(I)-catalyzed 1,2,3-triazole formation from azides and terminal acetylenes is a particularly powerful linking reaction, due to its high degree of dependability, complete specificity, and the bio-compatibility of the reactants. The triazole products are more than just passive linkers; they readily assoc. with biol. targets, through hydrogen bonding and dipole interactions. Click chem., a modular approach based on highly reliable chem. transformations, is being applied in all aspects of drug discovery, ranging from lead finding to proteomics and DNA research.
- 50Moses, J. E.; Moorhouse, A. D. The growing applications of click chemistry. Chem. Soc. Rev. 2007, 36, 1249– 1262, DOI: 10.1039/B613014NGoogle Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXnsVChu7w%253D&md5=3351a2d4815715d0746e45e8bfae5bb2The growing applications of Click chemistryMoses, John E.; Moorhouse, Adam D.Chemical Society Reviews (2007), 36 (8), 1249-1262CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Click chem. is a modular synthetic approach towards the assembly of new mol. entities. This powerful strategy relies mainly upon the construction of carbon-heteroatom bonds using "spring-loaded" reactants. Its growing no. of applications are found in nearly all areas of modern chem. from drug discovery to materials science. The copper(I)-catalyzed 1,2,3-triazole forming reaction between azides and terminal alkynes has become the gold std. of Click chem. due to its reliability, specificity and biocompatibility.
- 51McKay, C. S.; Finn, M. G. Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem. Biol. 2014, 21, 1075– 1101, DOI: 10.1016/j.chembiol.2014.09.002Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFyrur7N&md5=64a6e5ea8a232b97fce61d1268093fe4Click Chemistry in Complex Mixtures: Bioorthogonal BioconjugationMcKay, Craig S.; Finn, M. G.Chemistry & Biology (Oxford, United Kingdom) (2014), 21 (9), 1075-1101CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)A review. The selective chem. modification of biol. mols. drives a good portion of modern drug development and fundamental biol. research. While a few early examples of reactions that engage amine and thiol groups on proteins helped establish the value of such processes, the development of reactions that avoid most biol. mols. so as to achieve selectivity in desired bond-forming events has revolutionized the field. We provide an update on recent developments in bioorthogonal chem. that highlights key advances in reaction rates, biocompatibility, and applications. While not exhaustive, we hope this summary allows the reader to appreciate the rich continuing development of good chem. that operates in the biol. setting.
- 52Peracha, H.; Sawamoto, K.; Averill, L.; Kecskemethy, H.; Theroux, M.; Thacker, M.; Nagao, K.; Pizarro, C.; Mackenzie, W.; Kobayashi, H.; Yamaguchi, S.; Suzuki, Y.; Orii, K.; Orii, T.; Fukao, T.; Tomatsu, S. Molecular genetics and metabolism, special edition: diagnosis, diagnosis and prognosis of Mucopolysaccharidosis IVA. Mol. Genet. Metab. 2018, 125, 18– 37, DOI: 10.1016/j.ymgme.2018.05.004Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpslOrtL4%253D&md5=070925e02db747d32705ecf7bdd06081Molecular genetics and metabolism, special edition: Diagnosis, diagnosis and prognosis of Mucopolysaccharidosis IVAPeracha, Hira; Sawamoto, Kazuki; Averill, Lauren; Kecskemethy, Heidi; Theroux, Mary; Thacker, Mihir; Nagao, Kyoko; Pizarro, Christian; MacKenzie, William; Kobayashi, Hironori; Yamaguchi, Seiji; Suzuki, Yasuyuki; Orii, Kenji; Orii, Tadao; Fukao, Toshiyuki; Tomatsu, ShunjiMolecular Genetics and Metabolism (2018), 125 (1-2), 18-37CODEN: MGMEFF; ISSN:1096-7192. (Elsevier B.V.)A review. Mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) is an autosomal recessive disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. Deficiency of this enzyme leads to the accumulation of specific glycosaminoglycans (GAGs), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), which are mainly synthesized in the cartilage. Therefore, the substrates are stored primarily in the cartilage and its extracellular matrix (ECM), leading to a direct impact on bone development and successive systemic skeletal spondylepiphyseal dysplasia. The skeletal-related symptoms for MPS IVA include short stature with short neck and trunk, odontoid hypoplasia, spinal cord compression, tracheal obstruction, obstructive airway, pectus carinatum, restrictive lung, kyphoscoliosis, platyspondyly, coxa valga, genu valgum, waddling gait, and laxity of joints. The degree of imbalance of growth in bone and other organs and tissues largely contributes to unique skeletal dysplasia and clin. severity. Diagnosis of MPS IVA needs clin., radiog., and lab. testing to make a complete conclusion. To diagnose MPS IVA, total urinary GAG anal. which has been used is problematic since the values overlap with those in age-matched controls. Currently, urinary and blood KS and C6S, the enzyme activity of GALNS, and GALNS mol. anal. are used for diagnosis and prognosis of clin. phenotype in MPS IVA. MPS IVA can be diagnosed with unique characters although this disorder relates closely to other disorders in some characteristics. In this review article, we comprehensively describe clin., radiog., biochem., and mol. diagnosis and clin. assessment tests for MPS IVA. We also compare MPS IVA to other closely related disorders to differentiate MPS IVA. Overall, imbalance of growth in MPS IVA patients underlies unique skeletal manifestations leading to a crit. indicator for diagnosis.
- 53Khan, S.; Alméciga-Díaz, C. J.; Sawamoto, K.; Mackenzie, W. G.; Theroux, M. C.; Pizarro, C.; Mason, R. W.; Orii, T.; Tomatsu, S. Mucopolysaccharidosis IVA and glycosaminoglycans. Mol. Genet. Metab. 2017, 120, 78– 95, DOI: 10.1016/j.ymgme.2016.11.007Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVyisbfP&md5=7eb2530048576d15dd5030fe48f18cf9Mucopolysaccharidosis IVA and glycosaminoglycansKhan, Shaukat; Almeciga-Diaz, Carlos J.; Sawamoto, Kazuki; Mackenzie, William G.; Theroux, Mary C.; Pizarro, Christian; Mason, Robert W.; Orii, Tadao; Tomatsu, ShunjiMolecular Genetics and Metabolism (2017), 120 (1-2), 78-95CODEN: MGMEFF; ISSN:1096-7192. (Elsevier B.V.)Mucopolysaccharidosis IVA (MPS IVA; Morquio A: OMIM 253000) is a lysosomal storage disease with an autosomal recessive trait caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. Deficiency of this enzyme leads to accumulation of specific glycosaminoglycans (GAGs): chondroitin-6-sulfate (C6S) and keratan sulfate (KS). C6S and KS are mainly produced in the cartilage. Therefore, the undegraded substrates are stored primarily in cartilage and in its extracellular matrix (ECM), leading to a direct impact on cartilage and bone development, and successive systemic skeletal dysplasia. Chondrogenesis, the earliest phase of skeletal formation, is maintained by cellular interactions with the ECM, growth and differentiation factors, signaling pathways, and transcription factors in a temporal-spatial manner. In patients with MPS IVA, the cartilage is disrupted at birth as a consequence of abnormal chondrogenesis and/or endochondral ossification. The unique skeletal features are distinguished by a disproportional short stature, odontoid hypoplasia, spinal cord compression, tracheal obstruction, pectus carinatum, kyphoscoliosis, platyspondyly, coxa valga, genu valgum, waddling gait, and laxity of joints. In spite of many descriptions of these unique clin. features, delay of diagnosis still happens. The pathogenesis and treatment of systemic skeletal dysplasia in MPS IVA remains an unmet challenge. In this review article, we comprehensively describe historical aspect, property of GAGs, diagnosis, screening, pathogenesis, and current and future therapies of MPS IVA.
- 54Kumar, A. B.; Spacil, Z.; Ghomashchi, F.; Masi, S.; Sumida, T.; Ito, M.; Turecek, F.; Scott, C. R.; Gelb, M. H. Fluorimetric assays for N-acetylgalactosamine-6-sulfatase and arylsulfatase B based on the natural substrates for confirmation of mucopolysaccharidoses types IVA and VI. Clin. Chim. Acta 2015, 451, 125– 128, DOI: 10.1016/j.cca.2015.08.010Google ScholarThere is no corresponding record for this reference.
- 55Ullal, A. J.; Millington, D. S.; Bali, D. S. Development of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spots. Mol. Genet. Metab. Rep. 2014, 1, 461– 464, DOI: 10.1016/j.ymgmr.2014.10.004Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmtVamu7o%253D&md5=1cd9f842afddc7cb04ffe232f6f0bfbcDevelopment of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spotsUllal, Anirudh J.; Millington, David S.; Bali, Deeksha S.Molecular Genetics and Metabolism Reports (2014), 1 (), 461-464CODEN: MGMRDI; ISSN:2214-4269. (Elsevier B.V.)Mucopolysaccharidosis type IVA or Morquio type-A disease is a hereditary lysosomal storage disorder caused by deficient activity of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The disease is caused by lysosomal accumulation of unprocessed glycosaminoglycans (GAGs) that manifests with severe to mild skeletal and cardiopulmonary abnormalities. We have developed a modified microtiter plate-based enzyme activity assay using dried blood spots and a fluorescent substrate for measuring specific GALNS activity to identify patients with MPS IVA.
- 56Camelier, M. V.; Burin, M. G.; De Mari, J.; Vieira, T. A.; Marasca, G.; Giugliani, R. Practical and reliable enzyme test for the detection of Mucopolysaccharidosis IVA (Morquio Syndrome type A) in dried blood samples. Clin. Chim. Acta 2011, 412, 1805– 1808, DOI: 10.1016/j.cca.2011.06.001Google ScholarThere is no corresponding record for this reference.
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Abstract
Figure 1
Figure 1. (a) General concept of LC-MS/MS-based glycosidase assays. Glycosylated substrates are cleaved by the enzyme yielding the product (P) as analyte for subsequent analysis. An isotope-labeled internal standard (IS; here shown as deuterated product, P*) is required and used for quantification. (b) Divergent building block assembly of substrates using a single optimized glycosylation reaction and click chemistry to access a library of click substrates (CS). (c) Corresponding internal standards (IS) can easily be prepared using a single deuterated linker in combination with other building blocks.
Figure 2
Figure 2. Cleavage of α-l-iduronates by IDUA.
Figure 3
Figure 3. (a) Synthesis of nonlabeled and isotope-labeled azide-modified linkers 5a and 5b, respectively, and subsequent conjugation to modifier compounds (M) to obtain a library of click markers (CM). (b) Glycosylation of propargyl alcohol using iduronyl donor 6 and click assembly with CM1–CM5 to afford click substrates CS1–CS5. (c) Analytical sets of click substrates (CS) and corresponding internal standards (IS) for the development of LC-MS/MS-based α-l-iduronidase assays.
Figure 4
Figure 4. (a) Enzyme assays using recombinant human α-l-iduronidase (IDUA) and sets 1–5 of click substrate (CS1–CS5) and corresponding internal standard (IS1–IS5) (calculated specific activities are shown in μmol per min and mg enzyme). (b) Analysis of dried blood spots (DBS) using CDC control cards (QCL, QCM, QCH = quality control low, medium, high). (c) Analysis of DBS of confirmed MPS I patients (n = 9, anonymized) and random newborns (n = 88, anonymized). [****p < 0.0001.]
Figure 5
Figure 5. (a) Simultaneous analysis of three different samples using a single UHPLC-MS/MS run. (b) Chromatographic separation of CS/IS sets 3, 4, and 5 (including the products P3–P5 of the corresponding enzyme assays) using a 5 min gradient. (c) Triplex assay of three combined DBS samples using CS/IS sets 3, 4, and 5 (affected, n = 8; random, n = 23; ****p < 0.0001). (d) Analyzed DBS samples (random) in triplex vs singleplex assays (n = 23). [S = substrate, P* = IS, P = product.]
Figure 6
Figure 6. (a) Deficiency of GALNS leads to accumulation of chondroitin-6-sulfate and keratan sulfate causing MPS IVa. (b) Click assembly of GALNS substrates and internal standards using clickable galactose derivatives and already available linker–modifier building blocks. (c) Synthesis of GALNS sets 6–8 of click substrates (CS6–CS8) and corresponding internal standards (IS6–IS8) using click markers CM3a–CM5a and isotope-labeled CM3b–CM5b, respectively. (d) Analysis of dried blood spots (DBS) using CDC control cards. (e) Analysis of DBS of confirmed affected patients (n = 9, anonymized) and random newborns (n = 116, anonymized). [****p < 0.0001.]
References
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- 1Futerman, A. H.; van Meer, G. The cell biology of lysosomal storage disorders. Nat. Rev. Mol. Cell Biol. 2004, 5, 554– 565, DOI: 10.1038/nrm1423Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlt1Ortb8%253D&md5=929c8b53bf57c04520cdd01d7b8974edThe cell biology of lysosomal storage disordersFuterman, Anthony H.; van Meer, GerritNature Reviews Molecular Cell Biology (2004), 5 (7), 554-565CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Lysosomal storage disorders, of which more than 40 are known, are caused by the defective activity of lysosomal proteins, which results in the intra-lysosomal accumulation of undegraded metabolites. Despite years of study of the genetic and mol. bases of lysosomal storage disorders, little is known about the events that lead from this intra-lysosomal accumulation to pathol. Here, we summarize the biochem. of lysosomal storage disorders. We then discuss downstream cellular pathways that are potentially affected in these disorders and that might help us to delineate their pathol. mechanisms.
- 2Vellodi, A. Lysosomal storage disorders. Br. J. Haematol. 2005, 128, 413– 431, DOI: 10.1111/j.1365-2141.2004.05293.xGoogle Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXit1Wmur8%253D&md5=665737194c681c93b01e9a0555074c69Lysosomal storage disordersVellodi, AshokBritish Journal of Haematology (2005), 128 (4), 413-431CODEN: BJHEAL; ISSN:0007-1048. (Blackwell Publishing Ltd.)A review. Although the first description of a lysosomal storage disorder was that of Tay-Sachs disease in 1881, the lysosome was not discovered until 1955, by Christian De Duve. The first demonstration by Hers in 1963 of a link between an enzyme deficiency and a storage disorder (Pompe's disease) paved the way for a series of seminal discoveries about the intracellular biol. of these enzymes and their substrates, culminating in the successful treatment of Gaucher's disease with β-glucosidase in the early 1990s. It is now recognized that these disorders are not simply a consequence of pure storage, but result from perturbation of complex cell signalling mechanisms. These in turn give rise to secondary structural and biochem. changes, which have important implications for therapy. Significant challenges remain, particularly the treatment of central nervous system disease. It is hoped that recent advances in our understanding of lysosomal biol. will enable successful therapies to be developed.
- 3Wang, R. Y.; Bodamer, O. A.; Watson, M. S.; Wilcox, W. R. Lysosomal storage diseases: diagnostic confirmation and management of presymptomatic individuals. Genet. Med. 2011, 13, 457– 484, DOI: 10.1097/GIM.0b013e318211a7e1Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3Mnjt1ygsw%253D%253D&md5=e80dbf16cb613bf7fe369f956fbaf031Lysosomal storage diseases: diagnostic confirmation and management of presymptomatic individualsWang Raymond Y; Bodamer Olaf A; Watson Michael S; Wilcox William RGenetics in medicine : official journal of the American College of Medical Genetics (2011), 13 (5), 457-84 ISSN:.PURPOSE: To develop educational guidelines for the diagnostic confirmation and management of individuals identified by newborn screening, family-based testing after proband identification, or carrier testing in at-risk populations, and subsequent prenatal or postnatal testing of those who are presymptomatic for a lysosomal storage disease. METHODS: Review of English language literature and discussions in a consensus development panel comprised an international group of experts in the clinical and laboratory diagnosis, treatment and management, newborn screening, and genetic aspects of lysosomal storage diseases. RESULTS: Although clinical trial and longitudinal data were used when available, the evidence in the literature is limited and consequently the recommendations must be considered as expert opinion. Guidelines were developed for Fabry, Gaucher, and Niemann-Pick A/B diseases, glycogen storage type II (Pompe disease), globoid cell leukodystrophy (Krabbe disease), metachromatic leukodystrophy, and mucopolysaccharidoses types I, II, and VI. CONCLUSION: These guidelines serve as an educational resource for confirmatory testing and subsequent clinical management of presymptomatic individuals suspected to have a lysosomal storage disease; they also help to define a research agenda for longitudinal studies such as the American College of Medical Genetics/National Institutes of Health Newborn Screening Translational Research Network.
- 4Meikle, P. J.; Hopwood, J. J.; Clague, A. E.; Carey, W. F. Prevalence of lysosomal storage disorders. JAMA 1999, 281, 249– 254, DOI: 10.1001/jama.281.3.249Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK1M7hsFSguw%253D%253D&md5=26f325562b3ff5e300adf6e5660c0a04Prevalence of lysosomal storage disordersMeikle P J; Hopwood J J; Clague A E; Carey W FJAMA (1999), 281 (3), 249-54 ISSN:0098-7484.CONTEXT: Lysosomal storage disorders represent a group of at least 41 genetically distinct, biochemically related, inherited diseases. Individually, these disorders are considered rare, although high prevalence values have been reported in some populations. These disorders are devastating for individuals and their families and result in considerable use of resources from health care systems; however, the magnitude of the problem is not well defined. To date, no comprehensive study has been performed on the prevalence of these disorders as a group. OBJECTIVE: To determine the prevalence of lysosomal storage disorders individually and as a group in the Australian population. DESIGN: Retrospective case studies. SETTING: Australia, from January 1, 1980, through December 31, 1996. MAIN OUTCOME MEASURE: Enzymatic diagnosis of a lysosomal storage disorder. RESULTS: Twenty-seven different lysosomal storage disorders were diagnosed in 545 individuals. The prevalence ranged from 1 per 57000 live births for Gaucher disease to 1 per 4.2 million live births for sialidosis. Eighteen of 27 disorders had more than 10 diagnosed cases. As a group of disorders, the combined prevalence was 1 per 7700 live births. There was no significant increase in the rate of either clinical diagnoses or prenatal diagnoses of lysosomal storage disorders during the study period. CONCLUSIONS: Individually, lysosomal storage disorders are rare genetic diseases. However, as a group, they are relatively common and represent an important health problem in Australia.
- 5Pastores, G. M. Therapeutic approaches for lysosomal storage diseases. Ther. Adv. Endocrinol. Metab. 2010, 1, 177– 188, DOI: 10.1177/2042018810384429Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVKisLzK&md5=6ddf6c6f96172aee21e75b79c7d5febaTherapeutic approaches for lysosomal storage diseasesPastores, Gregory M.Therapeutic Advances in Endocrinology and Metabolism (2010), 1 (4), 177-188CODEN: TAEMBU; ISSN:2042-0188. (Sage Publications Ltd.)A review. The lysosomal storage disorders (LSDs) comprise a heterogeneous group of inborn errors of metab. characterized by tissue substrate deposits, most often caused by a deficiency of the enzyme normally responsible for catabolism of various byproducts of cellular turnover. Individual entities are typified by involvement of multiple body organs, in a pattern reflecting the sites of substrate storage. It is increasingly recognized that one or more processes, such as aberrant inflammation, dysregulation of apoptosis and/or defects of autophagy, may mediate organ dysfunction or failure. Several therapeutic options for various LSDs have been introduced, including hematopoietic stem cell transplantation, enzyme replacement therapy and substrate redn. therapy. Addnl. strategies are being explored, including the use of pharmacol. chaperones and gene therapy. Most LSDs include a variant characterized by primary central nervous system (CNS) involvement. At present, therapy of the CNS manifestations remains a major challenge because of the inability to deliver therapeutic agents across the intact blood-brain barrier. With improved understanding of underlying disease mechanisms, addnl. therapeutic options may be developed, complemented by various strategies to deliver the therapeutic agent(s) to recalcitrant sites of pathol. such as brain, bones and lungs.
- 6Desnick, R. J.; Schuchman, E. H. Enzyme replacement and enhancement therapies: lessons from lysosomal disorders. Nat. Rev. Genet. 2002, 3, 954– 966, DOI: 10.1038/nrg963Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XptFCjtL8%253D&md5=9fde9e7a61c0f781b9e6a3d0b76e0bb1Enzyme replacement and enhancement therapies: lessons from lysosomal disordersDesnick, Robert J.; Schuchman, Edward H.Nature Reviews Genetics (2002), 3 (12), 954-966CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A review. The past decade has witnessed remarkable advances in our ability to treat inherited metabolic disorders, esp. the lysosomal storage diseases, a group of more than 40 disorders, each of which is caused by the deficiency of a lysosomal enzyme or protein. During the past few years, both enzyme replacement and enhancement therapies have been developed to treat these disorders. This review discusses the successes and shortcomings of these therapeutic strategies, and the contributions that they have made to treating lysosomal storage diseases.
- 7Coutinho, M. F.; Santos, J. I.; Alves, S. Less is more: substrate reduction therapy for lysosomal storage disorders. Int. J. Mol. Sci. 2016, 17, 1065, DOI: 10.3390/ijms17071065Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVGntLjI&md5=c36d29878ee3883ad97972199e079220Less is more: substrate reduction therapy for lysosomal storage disordersCoutinho, Maria Francisca; Santos, Juliana Ines; Alves, SandraInternational Journal of Molecular Sciences (2016), 17 (7), 1065/1-1065/22CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)Lysosomal storage diseases (LSDs) are a group of rare, life-threatening genetic disorders, usually caused by a dysfunction in one of the many enzymes responsible for intralysosomal digestion. Even though no cure is available for any LSD, a few treatment strategies do exist. Traditionally, efforts have been mainly targeting the functional loss of the enzyme, by injection of a recombinant formulation, in a process called enzyme replacement therapy (ERT), with no impact on neuropathol. This ineffectiveness, together with its high cost and lifelong dependence is amongst the main reasons why addnl. therapeutic approaches are being (and have to be) investigated: chaperone therapy; gene enhancement; gene therapy; and, alternatively, substrate redn. therapy (SRT), whose aim is to prevent storage not by correcting the original enzymic defect but, instead, by decreasing the levels of biosynthesis of the accumulating substrate(s). Here we review the concept of substrate redn., highlighting the major breakthroughs in the field and discussing the future of SRT, not only as a monotherapy but also, esp., as complementary approach for LSDs.
- 8Biffi, A. Hematopoietic stem cell gene therapy for storage disease: current and new indications. Mol. Ther. 2017, 25, 1155– 1162, DOI: 10.1016/j.ymthe.2017.03.025Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXntFelt7c%253D&md5=eb2da5f3983ae056908ad9d132769efeHematopoietic Stem Cell Gene Therapy for Storage Disease: Current and New IndicationsBiffi, AlessandraMolecular Therapy (2017), 25 (5), 1155-1162CODEN: MTOHCK; ISSN:1525-0024. (Cell Press)Lysosomal storage disorders (LSDs) are a broad class of monogenic diseases with an overall incidence of 1:7,000 newborns, due to the defective activity of one or more lysosomal hydrolases or related proteins resulting in storage of un-degraded substrates in the lysosomes. The over 40 different known LSDs share a life-threatening nature, but they are present with extremely variable clin. manifestations, detd. by the characteristics and tissue distribution of the material accumulating due to the lysosomal dysfunction. The majority of LSDs lack a curative treatment. This is particularly true for LSDs severely affecting the CNS. Based on current preclin. and clin. evidences, among other treatment modalities, hematopoietic stem cell gene therapy could potentially result in robust therapeutic benefit for LSD patients, with particular indication for those characterized by severe brain damage. Optimization of current approaches and technol., as well as implementation of clin. trials for novel indications, and prolonged and more extensive follow-up of the already treated patients will allow translating this promise into new medicinal products.
- 9Shihabuddin, L. S.; Cheng, S. H. Neural stem cell transplantation as a therapeutic approach for treating lysosomal storage diseases. Neurotherapeutics 2011, 8, 659– 667, DOI: 10.1007/s13311-011-0067-8Google ScholarThere is no corresponding record for this reference.
- 10Gelb, M. H.; Scott, C. R.; Turecek, F. Newborn screening for lysosomal storage diseases. Clin. Chem. 2015, 61, 335– 346, DOI: 10.1373/clinchem.2014.225771Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXislGnsrg%253D&md5=7e4232da6432b577779e8d18de6ed7ffNewborn screening for lysosomal storage diseasesGelb, Michael H.; Scott, C. Ronald; Turecek, FrantisekClinical Chemistry (Washington, DC, United States) (2015), 61 (2), 335-346CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)A review. Background: There is worldwide interest in newborn screening for lysosomal storage diseases because of the development of treatment options that give better results when carried out early in life. Screens with high differentiation between affected and nonaffected individuals are crit. because of the large no. of potential false positives. Content: This review summarizes 3 screening methods: (a) direct assay of enzymic activities using tandem mass spectrometry or fluorometry, (b) immunocapture-based measurement of lysosomal enzyme abundance, and (c) measurement of biomarkers. Assay performance is compared on the basis of small-scale studies as well as on large-scale pilot studies of mass spectrometric and fluorometric screens. Summary: Tandem mass spectrometry and fluorometry techniques for direct assay of lysosomal enzymic activity in dried blood spots have emerged as the most studied approaches. Comparative mass spectrometry vs fluorometry studies show that the former better differentiates between nonaffected vs affected individuals. This in turn leads to a manageable no. of screen positives that can be further evaluated with second-tier methods.
- 11Chamoles, N. A.; Blanco, M.; Gaggioli, D. Diagnosis of α-l-iduronidase deficiency in dried blood spots on filter paper: the possibility of newborn diagnosis. Clin. Chem. 2001, 47, 780– 781Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXis1Kis7s%253D&md5=c986557f2b6616968e2af7ad9eb379c4Diagnosis of α-L-iduronidase deficiency in dried blood spots on filter paper: the possibility of newborn diagnosisChamoles, Nestor A.; Blanco, Mariana; Gaggioli, DanielaClinical Chemistry (Washington, DC, United States) (2001), 47 (4), 780-781CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Mucopolysaccharidosis type I (MPS I), produced by deficiency of a-L-iduronidase. In the last few years treatment of MPS I became possible by bone marrow transplantation (2), enzyme replacement The filter paper need not be removed during the therapy (3), and gene transfer or gene modification. The effectiveness of these therapies, particularly for MPS tube was run for each sample of the assay. The presymptomatic detection of MPS I can be achieved only by newborn screening. In this case, a simple technique suitable for dried blood spots on filter paper (DBFP) is needed. The present methodol. is easier, faster, and less expensive than the leukocyte assay. A drop of blood obtained through heel prick is sufficient to perform the assay in duplicate plus one blank. Sample transportation is safe. Minimal activity loss occurs during storage at room temp. up to 20 days. The iduronidase activity test in DBFP samples appears to be a reasonable approach for the initial diagnosis of MPS I.
- 12Chamoles, N. A.; Blanco, M.; Gaggioli, D.; Casentini, C. Gaucher and Niemann–Pick diseases—enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards. Clin. Chim. Acta 2002, 317, 191– 197, DOI: 10.1016/S0009-8981(01)00798-7Google ScholarThere is no corresponding record for this reference.
- 13Chamoles, N. A.; Blanco, M. B.; Gaggioli, D.; Casentini, C. Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paper. Clin. Chem. 2001, 47, 2098– 2102Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXovVOmsrs%253D&md5=16560c7cd5ce59dad07cc45fd9976d03Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paperChamoles, Nestor A.; Blanco, Mariana B.; Gaggioli, Daniela; Casentini, CarinaClinical Chemistry (Washington, DC, United States) (2001), 47 (12), 2098-2102CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Background: Clin. differentiation among mucopolysaccharidosis, oligosaccharidosis, and mucolipidosis II and III is difficult. We describe methods for the assay of 8 lysosomal enzymes in dried blood spots on filter paper that allow screening for 12 lysosomal storage diseases that present with a Hurler-like phenotype. Methods: To test tubes contg. 3-mm blood spots, we added elution liq. and fluorescent or radioactive substrate soln. After incubation at 37°, the reaction was terminated by the addn. of a stop buffer. The amt. of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. Sample stability was studied during storage for 21 days and during shipment of samples. We measured enzyme activities in 85 healthy controls (35 newborn, 50 adult), 57 patients suffering from 11 lysosomal storage diseases, and 46 obligate carriers. Results: Intra- and interassay CVs were <9% and <15%, resp. Mean activity losses during transportation or storage for up to 21 days at 4° were ≤27%. Enzyme activities in all patients were outside the ranges of values seen for carriers and controls. Conclusions: The described methodol. distinguishes between patients and controls with samples that are sufficiently stable to be mailed to the testing lab.
- 14Ceci, R.; Francesco, P.; Mucci, J.; Cancelarich, L.; Fossati, C.; Rozenfeld, P. Reliability of enzyme assays in dried blood spots for diagnosis of 4 lysosomal storage disorders. Adv. Biol. Chem. 2011, 1, 58– 64, DOI: 10.4236/abc.2011.13008Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XltFKht7k%253D&md5=6805941b51f8b416bb27817b0aac4cc9Reliability of enzyme assays in dried blood spots for diagnosis of 4 lysosomal storage disordersCeci, Romina; de Francesco, Pablo N.; Mucci, Juan M.; Cancelarich, Lorena N.; Fossati, Carlos A.; Rozenfeld, Paula A.Advances in Biological Chemistry (2011), 1 (3), 58-64CODEN: ABCDAS; ISSN:2162-2183. (Scientific Research Publishing, Inc.)Lysosomal storage disorders (LSD) are inherited diseases caused, in the majority of them, by the deficiency of lysosomal enzymic activities. We aimed to analyze the usefulness of DBS samples for diagnosis of 4 LSDs, with the availability of a large quantity of patient samples. Blood samples from previously diagnosed patients with Fabry, Gaucher, Hunter, and Maroteaux-Lamy syndromes and normal control individuals, were collected and dispensed in filter paper, and used for enzymic activity detn. Diagnosis of hemi/homo-zygous patients with Fabry, Hunter and Maroteaux-Lamy diseases using DBS samples showed ideal parameters of 100% sensitivity and specificity. DBS assay for Gaucher disease would need a posterior confirmatory step. Leukocyte measurement is the only reliable way to diagnose Gaucher disease. For Hunter, Fabry and Maroteaux-Lamy disorders discrimination between patients and controls seems adequate by DBS.
- 15Garg, U.; Dasouki, M. Expanded newborn screening of inherited metabolic disorders by tandem mass spectrometry: Clinical and laboratory aspects. Clin. Biochem. 2006, 39, 315– 332, DOI: 10.1016/j.clinbiochem.2005.12.009Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjvVGisLg%253D&md5=aa3ec45e117e16efe534286ccead4db7Expanded newborn screening of inherited metabolic disorders by tandem mass spectrometry: clinical and laboratory aspectsGarg, Uttam; Dasouki, MajedClinical Biochemistry (2006), 39 (4), 315-332CODEN: CLBIAS; ISSN:0009-9120. (Elsevier)A review. Newborn screening started in the 1960 s for the purpose of identifying phenylketonuric patients to begin early intervention and to prevent mental retardation in these patients. Soon thereafter, screening programs expanded to include addnl. genetic disorders added individually one at a time. In the 1980 s, tandem mass spectrometry (MS/MS) was introduced in clin. labs., and in the 1990 s, the technique was used for newborn screening. Unlike measuring one analyte at a time, MS/MS allows measurement of > 40 analytes, in a few minutes with the use of a single assay. Currently, MS/MS is being used for the identification of several amino acid, org. acid and fatty acid disorders. Several states in the United States and many other countries are using MS/MS in newborn screening. However, there is a significant disparity among different newborn screening programs for disorders being screened by MS/MS and many other challenges are faced by the expanded newborn screening. It is anticipated that in the future the use of MS/MS in newborn screening will expand both at the analyte and geog. levels. Clinicians and lab. scientists should become familiar with MS/MS, disorders being screened in their patients' population and the future of this emerging technol.
- 16Blanchard, S.; Sadilek, M.; Scott, C. R.; Turecek, F.; Gelb, M. H. Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis I. Clin. Chem. 2008, 54, 2067– 2070, DOI: 10.1373/clinchem.2008.115410Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjtFeitg%253D%253D&md5=10a78214da663c38b4339aa0be6c91d1Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis IBlanchard, Sophie; Sadilek, Martin; Scott, C. Ronald; Turecek, Frantisek; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2008), 54 (12), 2067-2070CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)BACKGROUND: Treatments now available for mucopolysaccharidosis I require early detection for optimum therapy. Therefore, we have developed an assay appropriate for newborn screening of the activity of the relevant enzyme, α-L-iduronidase. METHODS: We synthesized a new α-L-iduronidase substrate that can be used to assay the enzyme by use of tandem mass spectrometry together with an internal std. or by fluorometry. The assay uses a dried blood spot on a newborn screening card as the enzyme source. The assay protocol uses a simple liq.-liq. extn. step before mass spectrometry. We optimized enzyme reaction conditions and procedures for the assay, including the concn. of substrate, the reaction pH, the incubation time, and mass spectrometer operation. We also assessed inter- and intraassay imprecision. RESULTS: When the assay was tested on dried blood spots, the α-L-iduronidase activity measured for 5 patients with mucopolysaccharidosis I was well below the interval found for 10 randomly chosen newborns. Inter- and intraassay imprecision were <10%. The synthesis of the α-L-iduronidase substrate is practical for use on a scale needed to support newborn screening demands. CONCLUSIONS: This newly developed tandem mass spectrometry assay has the potential to be adopted for newborn screening of mucopolysaccharidosis I. This assay has advantages over a previously reported assay also developed in this lab. and has the potential to be performed in a multiplex fashion to measure several lysosomal enzymes relevant to treatable lysosomal storage diseases.
- 17Chennamaneni, N. K.; Kumar, A. B.; Barcenas, M.; Spacil, Z.; Scott, C. R.; Turecek, F.; Gelb, M. H. Improved reagents for newborn screening of mucopolysaccharidosis types I, II, and VI by tandem mass spectrometry. Anal. Chem. 2014, 86, 4508– 4514, DOI: 10.1021/ac5004135Google ScholarThere is no corresponding record for this reference.
- 18Duffey, T. A.; Khaliq, T.; Scott, C. R.; Turecek, F.; Gelb, M. H. Design and synthesis of substrates for newborn screening of Maroteaux-Lamy and Morquio A syndromes. Bioorg. Med. Chem. Lett. 2010, 20, 5994– 5996, DOI: 10.1016/j.bmcl.2010.08.080Google ScholarThere is no corresponding record for this reference.
- 19Gelb, M. H.; Turecek, F.; Scott, C. R.; Chamoles, N. A. Direct multiplex assay of enzymes in dried blood spots by tandem mass spectrometry for the newborn screening of lysosomal storage disorders. J. Inherited Metab. Dis. 2006, 29, 397– 404, DOI: 10.1007/s10545-006-0265-4Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlsV2ltbc%253D&md5=891bb8799620949e228a8088a137c6c1Direct multiplex assay of enzymes in dried blood spots by tandem mass spectrometry for the newborn screening of lysosomal storage disordersGelb, Michael H.; Turecek, Frantisek; Scott, C. Ron; Chamoles, Nestor A.Journal of Inherited Metabolic Disease (2006), 29 (2/3), 397-404CODEN: JIMDDP; ISSN:0141-8955. (Springer)A review. Tandem mass spectrometry is currently used in newborn screening programs to quantify the level of amino acids and acylcarnitines in dried blood spots for detection of metabolites assocd. with treatable diseases. We have developed assays for lysosomal enzymes in rehydrated dried blood spots in which a set of substrates is added and the set of corresponding enzymic products are quantified using tandem mass spectrometry with the aid of mass-differentiated internal stds. We have developed a multiplex assay of the set of enzymes that, when deficient, cause the lysosomal storage disorders Fabry, Gaucher, Hurler, Krabbe, Niemann-Pick A/B and Pompe diseases. These diseases were selected because treatments are now available or expected to emerge shortly. The discovery that acarbose is a selective inhibitor of maltase glucoamylase allows the Pompe disease enzyme, acid α-glucosidase, to be selectively assayed in white blood cells and dried blood spots. When tested with dried blood spots from 40 unaffected individuals and 10-12 individuals with the lysosomal storage disorder, the tandem mass spectrometry assay led to the correct identification of the affected individuals with 100% sensitivity. Many of the reagents needed for the new assays are com. available, and those that are not are being prepd. under Good Manufg. Procedures for approval by the FDA. Our newborn screening assay for Krabbe disease is currently being put in place at the Wadsworth Center in New York State for the anal. of ∼1000 dried blood spots per day. Summary We have developed tandem mass spectrometry for the direct assay of lysosomal enzymes in rehydrated dried blood spots that can be implemented for newborn screening of lysosomal storage disorders. Several enzymes can be analyzed by a single method (multiplex anal.) and in a high-throughput manner appropriate for newborn screening labs.
- 20Khaliq, T.; Sadilek, M.; Scott, C. R.; Turecek, F.; Gelb, M. H. Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis IVA. Clin. Chem. 2011, 57, 128– 131, DOI: 10.1373/clinchem.2010.149880Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXitFSksro%253D&md5=a44d9eb0fda8bb9f807704fd35212f69Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis IVAKhaliq, Tanvir; Sadilek, Martin; Scott, C. Ronald; Turecek, Frantisek; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2011), 57 (1), 128-131CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Treatments are being developed for an increasing no. of mucopolysaccharidoses, and early diagnosis is expected to be necessary to maximize the benefits of therapy. Therefore, we developed an assay for N-acetylgalactosamine-6-sulfate sulfatase (GALNS), the enzyme deficient in mucopolysaccharidosis IVA (Morquio A syndrome), that is applicable for clin. diagnosis. A novel substrate for GALNS was synthesized for a new enzyme activity assay that is based on tandem mass spectrometry and uses dried blood spots (DBSs) as the enzyme source. We optimized the assay conditions, including the substrate concn., reaction pH, lead formate concn., incubation time, punch size of the DBS, and mass spectrometer conditions. We also assessed inter- and intraassay variation. The assay uses either solid-phase or liq.-phase extn. before anal. by mass spectrometry. An evaluation of blood spots from 90 randomly chosen healthy newborns and 9 patients with Morquio A syndrome showed a well-defined interval between their resp. enzyme activities. Inter- and intraassay imprecision was <10%. This tandem mass spectrometry assay requires a minimal no. of sample-prepn. steps, thus making it easy to implement. The assay has the potential to be adopted for early diagnosis of Morquio A syndrome. We believe this assay could be performed in a multiplex fashion with assays for other lysosomal enzymes.
- 21Li, Y.; Brockmann, K.; Turecek, F.; Scott, C. R.; Gelb, M. H. Tandem mass spectrometry for the direct assay of enzymes in dried blood spots: application to newborn screening for Krabbe disease. Clin. Chem. 2004, 50, 638– 640, DOI: 10.1373/clinchem.2003.028381Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhvVChs7g%253D&md5=7dcd950e60f9765fdcbb3357a038c90eTandem mass spectrometry for the direct assay of enzymes in dried blood spots: Application to newborn screening for Krabbe diseaseLi, Yijun; Brockmann, Knut; Turecek, Frantisek; Scott, C. Ronald; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2004), 50 (3), 638-640CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)A study was conducted to investigate whether MS/MS can be used to directly assay enzymes in dried blood spots, in this case, galactocerebroside β-galactosidase (GALC) for the detection of Krabbe disease. Results suggest that an assay for GALC in dried blood spots from newborns can be implemented and evaluated for the early detection of Krabbe disease. Measurement of GALC activity with a series of substrates that differed in the lengths of their fatty acyl groups revealed that C8:0 ceramide gives fivefold higher activity than substrates contg. ceramides with the longer chain fatty acids found in natural ceramides, a factor that was important in the ability to detect GALC in dried blood spots. The use of β-Gal-C8-Cer also has an advantage in that it generates the unnatural product C8-Cer, thus avoiding interference from the natural ceramides present in biol. specimens.
- 22Li, Y.; Scott, C. R.; Chamoles, N. A.; Ghavami, A.; Pinto, B. M.; Turecek, F.; Gelb, M. H. Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin. Chem. 2004, 50, 1785– 1796, DOI: 10.1373/clinchem.2004.035907Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXotVylt7s%253D&md5=f6fb0eb04e31d75bc440b1223e82c589Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screeningLi, Yijun; Scott, C. Ronald; Chamoles, Nestor A.; Ghavami, Ahmad; Pinto, B. Mario; Turecek, Frantisek; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2004), 50 (10), 1785-1796CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Background: Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards contg. dried blood spots as the enzyme source. Methods: We used a cassette of substrates and internal stds. to directly quantify the enzymic activities, and tandem mass spectrometry for enzymic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liq.-liq. extn. followed by solid-phase extn. with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid α-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed. Results: We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann-Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the min. activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5-9 heterozygous carriers were approx. one-half those measured with 15-32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals. Conclusion: For all five diseases, the affected individuals were detected. The assay can be readily automated, and the anticipated reagent and supply costs are well within the budget limits of newborn-screening centers.
- 23Wang, D.; Eadala, B.; Sadilek, M.; Chamoles, N. A.; Turecek, F.; Scott, C. R.; Gelb, M. H. Tandem mass spectrometric analysis of dried blood spots for screening of mucopolysaccharidosis I in newborns. Clin. Chem. 2005, 51, 898– 900, DOI: 10.1373/clinchem.2004.047167Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjvVKjsrk%253D&md5=49a454b7ad48bf47fa6af79ee30cb5cdTandem mass spectrometric analysis of dried blood spots for screening of mucopolysaccharidosis I in newbornsWang, Ding; Eadala, Bhramara; Sadilek, Martin; Chamoles, Nestor A.; Turecek, Frantisek; Scott, C. Ronald; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2005), 51 (5), 898-900CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)An electrospray ionization tandem mass spectrometry (ESI-MS/MS) assay that directly measures the reaction velocity of α-L-iduronidase (IDUA) in rehydrated dried blood spots (DBS) for the newborn screening of mucopolysaccharidosis type I (MPS-I) is described. The assay can be combined with ESI-MS/MS assays of Niemann-Pick type A/B, Krabbe, Gaucher, Pompe, and Fabry diseases for the simultaneous anal. of six lysosomal storage diseases. The assay is compatible with microtiter plate and multichannel pipetting techniques. Each IDUA assay requires only 13.3 μg of substrate, which can be readily prepd. from com. available heparin and 0.1 μg of internal std.
- 24Wolfe, B. J.; Ghomashchi, F.; Kim, T.; Abam, C. A.; Sadilek, M.; Jack, R.; Thompson, J. N.; Scott, C. R.; Gelb, M. H.; Turecek, F. New substrates and enzyme assays for the detection of mucopolysaccharidosis III (Sanfilippo Syndrome) types A, B, C, and D by tandem mass spectrometry. Bioconjugate Chem. 2012, 23, 557– 564, DOI: 10.1021/bc200609xGoogle Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtVWiur8%253D&md5=21afdbb74116b48f6531ecaed0094d8fNew Substrates and Enzyme Assays for the Detection of Mucopolysaccharidosis III (Sanfilippo Syndrome) Types A, B, C, and D by Tandem Mass SpectrometryWolfe, Brian J.; Ghomashchi, Farideh; Kim, Tim; Abam, Cynthia A.; Sadilek, Martin; Jack, Rhona; Thompson, Jerry N.; Scott, C. Ronald; Gelb, Michael H.; Turecek, FrantisekBioconjugate Chemistry (2012), 23 (3), 557-564CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)The clin. phenotype of Sanfilippo Syndrome is caused by one of four enzyme deficiencies that are assocd. with a defect in mucopolysaccharide metab. The four subtypes (A, B, C, and D) are each caused by an enzyme deficiency involved in the degrdn. of heparan sulfate. A highly efficient synthesis of the substrates and internal stds. required for the enzymic assay of each of the four enzymes was developed. The synthesis of the substrates involves chem. modification of a common intermediate. The substrates and internal stds. allow the measurement of the enzymes relevant to heparan N-sulfatase (type A); N-acetyl-α-glucosaminidase (type B); acetyl-CoA:α-glucosamide N-acetyltransferase (type C); and N-acetylglucosamine 6-sulfatase (type D). The internal stds. are similar to the substrates and allow for the accurate quantification of the enzyme assays using tandem mass spectrometry. The synthetic substrates incorporate a coumarin moiety and can also be used in fluorometric enzyme assays. It was confirmed that all four substrates can detect the appropriate Sanfilippo Syndrome in fibroblast lysates, and the measured enzyme activities are distinctly lower by a factor of 10 when compared to fibroblast lysates from unaffected persons.
- 25Spáčil, Z.; Elliott, S.; Reeber, S. L.; Gelb, M. H.; Scott, C. R.; Tureček, F. Comparative triplex tandem mass spectrometry assays of lysosomal enzyme activities in dried blood spots using fast liquid chromatography: application to newborn screening of Pompe, Fabry, and Hurler diseases. Anal. Chem. 2011, 83, 4822– 4828, DOI: 10.1021/ac200417uGoogle ScholarThere is no corresponding record for this reference.
- 26Spacil, Z.; Tatipaka, H.; Barcenas, M.; Scott, C. R.; Turecek, F.; Gelb, M. H. High-throughput assay of 9 lysosomal enzymes for newborn screening. Clin. Chem. 2013, 59, 502– 511, DOI: 10.1373/clinchem.2012.189936Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjvFOhu7g%253D&md5=9f66f6221142df65473ea4350333aa5fHigh-throughput assay of 9 lysosomal enzymes for newborn screeningSpacil, Zdenek; Tatipaka, Haribabu; Barcenas, Mariana; Scott, C. Ronald; Turecek, Frantisek; Gelb, Michael H.Clinical Chemistry (Washington, DC, United States) (2013), 59 (3), 502-511CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymic assays that uses HPLC-tandem mass spectrometry (MS/MS). The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal stds. and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre-HPLC-MS/MS sample prepn. required only 4 liq. transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to sepn. and column equilibration. Product-specific and internal std.-specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode. Anal. of blood spots from 58 random newborns and lysosomal storage disease-affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex anal. (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening labs. HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the no. of sep. incubations necessary.
- 27la Marca, G.; Casetta, B.; Malvagia, S.; Guerrini, R.; Zammarchi, E. New strategy for the screening of lysosomal storage disorders: the use of the online trapping-and-cleanup liquid chromatography/mass spectrometry. Anal. Chem. 2009, 81, 6113– 6121, DOI: 10.1021/ac900504sGoogle ScholarThere is no corresponding record for this reference.
- 28Kasper, D. C.; Herman, J.; De Jesus, V. R.; Mechtler, T. P.; Metz, T. F.; Shushan, B. The application of multiplexed, multi-dimensional ultra-high-performance liquid chromatography/tandem mass spectrometry to the high-throughput screening of lysosomal storage disorders in newborn dried bloodspots. Rapid Commun. Mass Spectrom. 2010, 24, 986– 994, DOI: 10.1002/rcm.4496Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjt1Gmtbw%253D&md5=2163c3dfc20bd7bd9c46d73f2bd7f5a3The application of multiplexed, multi-dimensional ultra-high-performance liquid chromatography/tandem mass spectrometry to the high-throughput screening of lysosomal storage disorders in newborn dried bloodspotsKasper, David C.; Herman, Joseph; De Jesus, Victor R.; Mechtler, Thomas P.; Metz, Thomas F.; Shushan, BoriRapid Communications in Mass Spectrometry (2010), 24 (7), 986-994CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)Lysosomal storage disorders are just beginning to be routinely screened using enzyme activity assays involving dried blood spots and tandem mass spectrometry (MS/MS). This paper discusses some of the anal. challenges assocd. with published assays including complex sample prepn. and potential interference from excess residual substrate. Solns. to these challenges are presented in the form of online two-dimensional chromatog. to eliminate off-line liq.-liq. extn. (LLE) and solid-phase extn. (SPE), the use of ultra-HPLC (UHPLC) to sep. excess substrate from all other analytes and multiplexed sample introduction for higher throughput required of a population screening assay. High sensitivity, specificity and throughput were demonstrated using this novel method. Copyright © 2010 John Wiley & Sons, Ltd.
- 29Mechtler, T. P.; Stary, S.; Metz, T. F.; De Jesus, V. R.; Greber-Platzer, S.; Pollak, A.; Herkner, K. R.; Streubel, B.; Kasper, D. C. Neonatal screening for lysosomal storage disorders: feasibility and incidence from a nationwide study in Austria. Lancet 2012, 379, 335– 341, DOI: 10.1016/S0140-6736(11)61266-XGoogle ScholarThere is no corresponding record for this reference.
- 30Metz, T. F.; Mechtler, T. P.; Orsini, J. J.; Martin, M.; Shushan, B.; Herman, J. L.; Ratschmann, R.; Item, C. B.; Streubel, B.; Herkner, K. R.; Kasper, D. C. Simplified newborn screening protocol for lysosomal storage disorders. Clin. Chem. 2011, 57, 1286– 1294, DOI: 10.1373/clinchem.2011.164640Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFOjtbrJ&md5=af79f7ce24b6387ea71c9320b7069596Simplified newborn screening protocol for lysosomal storage disordersMetz, Thomas F.; Mechtler, Thomas P.; Orsini, Joseph J.; Martin, Monica; Shushan, Bori; Herman, Joseph L.; Ratschmann, Rene; Item, Chike B.; Streubel, Berthold; Herkner, Kurt R.; Kasper, David C.Clinical Chemistry (Washington, DC, United States) (2011), 57 (9), 1286-1294CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Interest in lysosomal storage disorders, a collection of more than 40 inherited metabolic disorders, has increased because of new therapy options such as enzyme replacement, stem cell transplantation, and substrate redn. therapy. We developed a high-throughput protocol that simplifies anal. challenges such as complex sample prepn. and potential interference from excess residual substrate assocd. with previously reported assays. After overnight incubation (16-20 h) of dried blood spots with a cassette of substrates and deuterated internal stds., we used a TLX-2 system to quantify 6 lysosomal enzyme activities for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and mucopolysaccharidosis I disease. This multiplexed, multidimensional ultra-HPLC-tandem mass spectrometry assay included Cyclone P Turbo Flow and Hypersil Gold C8 columns. The method did not require offline sample prepn. such as liq.-liq. and solid-phase extn., or hazardous reagents such as Et acetate. Obviating the offline sample prepn. steps led to substantial savings in anal. time (approx. 70%) and reagent costs (approx. 50%). In a pilot study, lysosomal enzyme activities of 8586 newborns were measured, including 51 pos. controls, and the results demonstrated 100% diagnostic sensitivity and high specificity. The results for Krabbe disease were validated with parallel measurements by the New York State Screening Lab. Turboflow online sample cleanup and the use of an addnl. anal. column enabled the implementation of lysosomal storage disorder testing in a nationwide screening program while keeping the total anal. time to <2 min per sample.
- 31Gucciardi, A.; Legnini, E.; Di Gangi, I. M.; Corbetta, C.; Tomanin, R.; Scarpa, M.; Giordano, G. A column-switching HPLC-MS/MS method for mucopolysaccharidosis type I analysis in a multiplex assay for the simultaneous newborn screening of six lysosomal storage disorders. Biomed. Chromatogr. 2014, 28, 1131– 1139, DOI: 10.1002/bmc.3133Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXps1yqug%253D%253D&md5=b04eba1bdfc8c819db44dac8be1e6ca9A column-switching HPLC-MS/MS method for mucopolysaccharidosis type I analysis in a multiplex assay for the simultaneous newborn screening of six lysosomal storage disordersGucciardi, Antonina; Legnini, Elisa; Di Gangi, Iole Maria; Corbetta, Carlo; Tomanin, Rosella; Scarpa, Maurizio; Giordano, GiuseppeBiomedical Chromatography (2014), 28 (8), 1131-1139CODEN: BICHE2; ISSN:0269-3879. (John Wiley & Sons Ltd.)Lysosomal storage disorders comprise a group of rare genetic diseases in which a deficit of specific hydrolases leads to the storage of undegraded substrates in lysosomes. Impaired enzyme activities can be assessed by MS/MS quantification of the reaction products obtained after incubation with specific substrates. In this study, a column-switching HPLC-MS/MS method for multiplex screening in dried blood spot of the lysosomal enzymes activities was developed. Mucopolysaccharidosis type I, Fabry, Gaucher, Krabbe, Niemann-Pick A/B and Pompe diseases were simultaneously assayed. Dried blood spots were incubated with substrates and internal stds.; thereafter, supernatants were collected with minor manipulations. Samples were injected, trapped into an online perfusion column and, by a six-port valve, switched online through the C18 anal. column to perform sepn. of metabolites followed by MS/MS anal. A total of 1136 de-identified newborn screening samples were analyzed to det. refs. for enzymes activity values. As pos. controls, we analyzed dried blood spots from three patients with Pompe, one with Fabry, one with Krabbe disease and two with MPS I, and in all cases the enzyme activities were below the cutoff values measured for newborns, except for an MPS I patient after successful hematopoietic stem cell transplantation. Copyright © 2014 John Wiley & Sons, Ltd.
- 32He, W.; Voznyi, Y. V.; Boer, A. M.; Kleijer, W. J.; van Diggelen, O. P. A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type D (MPS IIID). J. Inherited Metab. Dis. 1993, 16, 935– 941, DOI: 10.1007/BF00711508Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXis1Chsbo%253D&md5=4180310d828c6e95f2526cf4431c115fA fluorometric enzyme assay for the diagnosis of Sanfilippo disease type D (MPS IIID)He, Wang; Voznyi, Ya. V.; Boer, A. M.; Kleijer, W. J.; van Diggelen, O. P.Journal of Inherited Metabolic Disease (1993), 16 (6), 935-41CODEN: JIMDDP; ISSN:0141-8955.4-Methylumbelliferyl-α-N-acetylglucosamine 6-sulfate was synthesized and shown to be a substrate for the lysosomal N-acetylglucosamine-6-sulfate sulfatase (GlcNAc-6S sulfatase). Fibroblasts and leukocytes from 3 different Sanfilippo D patients showed <1% of mean normal GlcNAc-6S sulfatase activity. The enzymic liberation of the fluorochrome from 4-methylumbelliferyl-α-N-acetylglucosamine 6-sulfate requires the sequential action of the GlcNAc-6S sulfatase and α-N-acetylglucosaminidase. A normal level of α-N-acetylglucosaminidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl-α-N-acetylglucosaminide formed by the GlcNAc-6S sulfatase. A second incubation in the presence of excess α-N-acetylglucosaminidase is needed to avoid underestimation of the GlcNAc-6S sulfatase activity.
- 33Karpova, E. A.; Voznyi, Y. V.; Keulemans, J. L. M.; Hoogeveen, A. T.; Winchester, B.; Tsvetkova, I. V.; van Diggelen, O. P. A fluorimetric enzyme assay for the diagnosis of sanfilippo disease type A (MPS IIIA). J. Inherited Metab. Dis. 1996, 19, 278– 285, DOI: 10.1007/BF01799255Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XksFentLg%253D&md5=5700f006001507b1011dee9b4e1afcd6A fluorometric enzyme assay for the diagnosis of Sanfilippo disease type A (MPS IIIA)Karpova, E. A.; Voznyi, Ya. V.; Keulemans, J. L. M.; Hoogeveen, A. T.; Winchester, B.; Tsvetkova, I. V.; Van Diggelen, O. P.Journal of Inherited Metabolic Disease (1996), 19 (3), 278-285CODEN: JIMDDP; ISSN:0141-8955. (Kluwer)4-Methylumbelliferyl-α-D-N-sulfoglucosaminide (MU-α-GlcNS) was synthesized and shown to be a substrate for the lysosomal heparin sulfamidase. Sanfilippo A patients' fibroblasts and lymphocytes showed 0-3% of mean normal heparin sulfamidase activity; in total leukocytes from patients sulfamidase activity was clearly deficient. In fibroblasts from obligate heterozygotes for Sanfilippo A, the sulfamidase activity was reduced in 9 out of 10 cases. Heparin sulfamidase desulfates MU-αGlcNS to MU-αGlcNH2 and further hydrolysis during a second incubation is required to liberate 4-methylumbelliferone, which can be measured. Yeast α-glucosidase, which has low but sufficient α-glucosaminidase activity, was used to hydrolyze the reaction intermediate MU-αGlcNH2 to release 4-methylumbelliferone and free glucosamine.
- 34Voznyi, Y. V.; Karpova, E. A.; Dudukina, T. V.; Tsvetkova, I. V.; Boer, A. M.; Janse, H. C.; van Diggelen, O. P. A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease C (MPS III C). J. Inherited Metab. Dis. 1993, 16, 465– 472, DOI: 10.1007/BF00710299Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXjtFyjtbY%253D&md5=f72815d38131e1d593ff14573b5a2948A fluorometric enzyme assay for the diagnosis of Sanfilippo disease C (MPS III C)Voznyi, Ya. V.; Karpova, E. A.; Dudukina, T. V.; Tsvetkova, I. V.; Boer, A. M.; Janse, H. C.; van Diggelen, O. P.Journal of Inherited Metabolic Disease (1993), 16 (2), 465-72CODEN: JIMDDP; ISSN:0141-8955.Both the α- and β-anomers of 4-methylumbelliferyl-D-glucosaminide were synthesized and shown to be substrates for the lysosomal acetyl-CoA:glucosaminide N-acetyltransferase. Using the β-anomer, fibroblasts and leukocytes from 11 different Sanfilippo C patients showed < 1% of mean normal N-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymic liberation of the fluorochrome from 4-methylumbelliferyl-β-D-glucosaminide requires the sequential action of the N-acetyltransferase and β-hexosaminidase. Normal β-hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl-β-D-N-acetylglucosaminide formed by the N-acetyltransferase. In cell exts. with a β-hexosaminidase deficiency, however, a second incubation in the presence of excess β-hexosaminidase is needed to avoid underestimation of the N-acetyltransferase activity.
- 35Voznyi, Y. V.; Keulemans, J. L. M.; van Diggelen, O. P. A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease). J. Inherited Metab. Dis. 2001, 24, 675– 680, DOI: 10.1023/A:1012763026526Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XltVSisA%253D%253D&md5=a06782de1b11dafe842ccc5d9b61c9e9A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease)Voznyi, Ya. V.; Keulemans, J. L. M.; van Diggelen, O. P.Journal of Inherited Metabolic Disease (2001), 24 (6), 675-680CODEN: JIMDDP; ISSN:0141-8955. (Kluwer Academic Publishers)4-Methylumbelliferyl-α-iduronate 2-sulfate was synthesized and shown to be a specific substrate for the lysosomal iduronate-2-sulfate sulfatase (IDS). Fibroblasts (n = 17), leukocytes (n = 3) and plasmas (n = 9) from different MPS II patients showed < 5% of mean normal IDS activity. The enzymic liberation of the fluorochrome from 4-methylumbelliferyl-α-iduronate 2-sulfate requires the sequential action of IDS and α-iduronidase. A normal level of α-iduronidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl-α-iduronide formed by IDS. A second incubation step in the presence of excess purified α-iduronidase is needed to avoid underestimation of the IDS activity.
- 36van Diggelen, O. P.; Voznyi, Y. V.; Keulemans, J. L. M.; Schoonderwoerd, K.; Ledvinova, J.; Mengel, E.; Zschiesche, M.; Santer, R.; Harzer, K. A new fluorimetric enzyme assay for the diagnosis of Niemann–Pick A/B, with specificity of natural sphingomyelinase substrate. J. Inherited Metab. Dis. 2005, 28, 733– 741, DOI: 10.1007/s10545-005-0105-yGoogle Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2MvotFSgug%253D%253D&md5=b8625aa93b719824525ec8739e9af75cA new fluorimetric enzyme assay for the diagnosis of Niemann-Pick A/B, with specificity of natural sphingomyelinase substratevan Diggelen O P; Voznyi Ya V; Keulemans J L M; Schoonderwoerd K; Ledvinova J; Mengel E; Zschiesche M; Santer R; Harzer KJournal of inherited metabolic disease (2005), 28 (5), 733-41 ISSN:0141-8955.6-Hexadecanoylamino-4-methylumbelliferylphosphorylcholine (HMUPC) was shown to be a specific substrate for the determination of acid (lysosomal) sphingomyelinase (ASM; gene SMPD1). Fibroblasts (n = 27) and leukocytes (n = 8) from both the A and B types of Niemann-Pick disease showed < 6% and < 10% of mean normal ASM activity, respectively. Niemann-Pick A or B patients bearing the Q292K mutation had apparently normal ASM activity with our new artificial substrate. These patients with false-normal sphingomyelinase activity, however, could readily be detected by determining the extent of inhibition of enzymatic hydrolysis of the artificial substrate HMU-PC by an unlabelled natural substrate, in particular lysosphingomyelin. This approach is generally applicable. Our novel assay for ASM combines the ease of a rapid and robust enzyme assay using a fluorogenic substrate with the specificity of an ASM assay using a natural substrate. Such assays are obviously more convenient to the diagnostic laboratory, since radiolabelled substrates are not required.
- 37van Diggelen, O. P.; Zhao, H.; Kleijer, W. J.; Janse, H. C.; Poorthuis, B. J. H. M.; van Pelt, J.; Kamerling, J. P.; Galjaard, H. A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A). Clin. Chim. Acta 1990, 187, 131– 139, DOI: 10.1016/0009-8981(90)90339-TGoogle ScholarThere is no corresponding record for this reference.
- 38Gasparotto, N.; Tomanin, R.; Frigo, A. C.; Niizawa, G.; Pasquini, E.; Blanco, M.; Donati, M. A.; Keutzer, J.; Zacchello, F.; Scarpa, M. Rapid diagnostic testing procedures for lysosomal storage disorders: alpha-glucosidase and beta-galactosidase assays on dried blood spots. Clin. Chim. Acta 2009, 402, 38– 41, DOI: 10.1016/j.cca.2008.12.006Google ScholarThere is no corresponding record for this reference.
- 39Chien, Y.-H.; Chiang, S.-C.; Zhang, X. K.; Keutzer, J.; Lee, N.-C.; Huang, A.-C.; Chen, C.-A.; Wu, M.-H.; Huang, P.-H.; Tsai, F.-J.; Chen, Y.-T.; Hwu, W.-L. Early detection of Pompe disease by newborn screening is feasible: results from the Taiwan screening program. Pediatrics 2008, 122, e39– 45, DOI: 10.1542/peds.2007-2222Google ScholarThere is no corresponding record for this reference.
- 40Sista, R.; Eckhardt, A. E.; Wang, T.; Sellos-Moura, M.; Pamula, V. K. Rapid, single-step assay for Hunter syndrome in dried blood spots using digital microfluidics. Clin. Chim. Acta 2011, 412, 1895– 1897, DOI: 10.1016/j.cca.2011.06.015Google ScholarThere is no corresponding record for this reference.
- 41Sista, R. S.; Eckhardt, A. E.; Wang, T.; Graham, C.; Rouse, J. L.; Norton, S. M.; Srinivasan, V.; Pollack, M. G.; Tolun, A. A.; Bali, D.; Millington, D. S.; Pamula, V. K. Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns. Clin. Chem. 2011, 57, 1444– 1451, DOI: 10.1373/clinchem.2011.163139Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlSht7vE&md5=525b2fc48d2157574e36fa05034d3d1dDigital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newbornsSista, Ramakrishna S.; Eckhardt, Allen E.; Wang, Tong; Graham, Carrie; Rouse, Jeremy L.; Norton, Scott M.; Srinivasan, Vijay; Pollack, Michael G.; Tolun, Adviye A.; Bali, Deeksha; Millington, David S.; Pamula, Vamsee K.Clinical Chemistry (Washington, DC, United States) (2011), 57 (10), 1444-1451CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymic anal. of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using std. fluorometric methods. We performed bench-based, fluorometric enzymic anal. on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochem. Genetics Lab. using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymic assays at Advanced Liq. Logic Inc. using ext. from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h. Assay results were generally comparable, although mean enzymic activity for GAA using microfluidics was approx. 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concn. Clear sepn. was obsd. between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics. A digital microfluidic platform compared favorably with a clin. ref. lab. to perform enzymic anal. in DBSs for Pompe and Fabry disorders. This platform presents a new technol. for a newborn screening lab. to screen LSDs by fully automating all the liq.-handling operations in an inexpensive system, providing rapid results.
- 42Millington, D. S.; Sista, R.; Eckhardt, A.; Rouse, J.; Bali, D.; Goldberg, R.; Cotten, M.; Buckley, R.; Pamula, V. Digital Microfluidics: A Future technology in the newborn screening laboratory?. Semin. Perinatol 2010, 34, 163– 169, DOI: 10.1053/j.semperi.2009.12.008Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3c7osVGmug%253D%253D&md5=7b2cf37b11a1d466399d362f40eaecb2Digital microfluidics: a future technology in the newborn screening laboratory?Millington David S; Sista Ramakrishna; Eckhardt Allen; Rouse Jeremy; Bali Deeksha; Goldberg Ronald; Cotten Michael; Buckley Rebecca; Pamula VamseeSeminars in perinatology (2010), 34 (2), 163-9 ISSN:.Expansion of newborn screening for inherited metabolic disorders using tandem mass spectrometry has generated interest in screening for other treatable conditions, including lysosomal storage diseases. Limitations to expansion include labor and equipment costs. We describe a cost-effective new platform that reduces the time to result reporting and can perform multiplexing assays requiring different platforms. Immunoassays and enzyme activity assays currently used in newborn screening have been translated to a disposable microchip programmed to dispense, transport, mix, wash, and incubate individual microdroplets from specimens, including dried blood spot extracts, and reagents all under software control. The specimen and reagents consumed are approximately 1% of those required by equivalent bench assays. In addition to immunologic and enzymatic assays, DNA amplification, amplicon detection, and sequencing have been demonstrated using the same microchips and control equipment. Recently, the multiplexing of 4 different enzyme activities has also been demonstrated with negligible cross-contamination. We review assays relevant to newborn screening.
- 43Millington, D. S.; Bali, D. M. Misinformation regarding tandem mass spectrometric vs fluorometric assays to screen newborns for LSDs. Mol. Genet. Metab. Rep. 2017, 11, 72– 73, DOI: 10.1016/j.ymgmr.2017.04.009Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnsFentro%253D&md5=d68e577d456a648e7bcb0b3fba99f3b7Misinformation regarding tandem mass spectrometric vs fluorometric assays to screen newborns for LSDsMillington, David S.; Bali, Deeksha M.Molecular Genetics and Metabolism Reports (2017), 11 (), 72-73CODEN: MGMRDI; ISSN:2214-4269. (Elsevier B.V.)There is no expanded citation for this reference.
- 44Millington, D.; Norton, S.; Singh, R.; Sista, R.; Srinivasan, V.; Pamula, V. Digital microfluidics comes of age: high-throughput screening to bedside diagnostic testing for genetic disorders in newborns. Expert Rev. Mol. Diagn. 2018, 18, 701– 712, DOI: 10.1080/14737159.2018.1495076Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlahtLjE&md5=23778480e146d2a3266def7e268653b1Digital microfluidics comes of age: high-throughput screening to bedside diagnostic testing for genetic disorders in newbornsMillington, David; Norton, Scott; Singh, Raj; Sista, Rama; Srinivasan, Vijay; Pamula, VamseeExpert Review of Molecular Diagnostics (2018), 18 (8), 701-712CODEN: ERMDCW; ISSN:1473-7159. (Taylor & Francis Ltd.)A review. Introduction: Digital microfluidics (DMF) is an emerging technol. with the appropriate metrics for application to newborn and high-risk screening for inherited metabolic disease and other conditions that benefit from early treatment. Areas covered: This review traces the development of electrowetting-based DMF technol. toward the fulfillment of its promise to provide an inexpensive platform to conduct enzymic assays and targeted biomarker assays at the bedside. The high-throughput DMF platform, referred to as SEEKER, was recently authorized by the United States Food and Drug Administration to screen newborns for four lysosomal storage disorders (LSDs) and is deployed in newborn screening programs in the United States. The development of reagents and methods for LSD screening and results from screening centers are reviewed. Preliminary results from a more compact DMF device, to perform disease-specific test panels from small vols. of blood, are also reviewed. Literature for this review was sourced using principal author and subject searches in PubMed. Expert commentary: Newborn screening is a vital and highly successful public health program. DMF technol. adds value to the current testing platforms that will benefit apparently healthy newborns with underlying genetic disorders and infants at-risk for conditions that present with symptoms in the newborn period.
- 45Cecioni, S.; Vocadlo, D. J. Carbohydrate bis-acetal-based substrates as tunable fluorescence-quenched probes for monitoring exo-glycosidase activity. J. Am. Chem. Soc. 2017, 139, 8392– 8395, DOI: 10.1021/jacs.7b01948Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVWitL%252FJ&md5=67e3b0771281b90a2c51dbe8237d7487Carbohydrate bis-acetal-based substrates as tunable fluorescence-quenched probes for monitoring exo-glycosidase activityCecioni, Samy; Vocadlo, David J.Journal of the American Chemical Society (2017), 139 (25), 8392-8395CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Tunable Forster resonance energy transfer (FRET)-quenched substrates are useful for monitoring the activity of various enzymes within their relevant physiol. environments. Development of FRET-quenched substrates for exo-glycosidases, however, has been hindered by their constrained pocket-shaped active sites. Here we report the design of a new class of substrate that overcomes this problem. These Bis-Acetal-Based Substrates (BABS) bear a hemiacetal aglycon leaving group that tethers fluorochromes in close proximity, also positioning them distant from the active site pocket. Following cleavage of the glycosidic bond, the liberated hemiacetal spontaneously breaks down, leading to sepn. of the fluorophore and quencher. We detail the synthesis and characterization of GlcNAc-BABS, revealing a striking 99.9% quenching efficiency. These substrates are efficiently turned over by the human exo-glycosidase O-GlcNAcase (OGA). We find the hemiacetal leaving group rapidly breaks down, enabling quant. monitoring of OGA activity. We expect this strategy to be broadly useful for the development of substrate probes for monitoring exo-glycosidases, as well as a range of other enzymes having constrained pocket-shaped active sites.
- 46Geyer, K.; Seeberger, P. H. Optimization of glycosylation reactions in a microreactor. Helv. Chim. Acta 2007, 90, 395– 403, DOI: 10.1002/hlca.200790046Google ScholarThere is no corresponding record for this reference.
- 47Scott, H. S.; Anson, D. S.; Orsborn, A. M.; Nelson, P. V.; Clements, P. R.; Morris, C. P.; Hopwood, J. J. Human alpha-L-iduronidase: cDNA isolation and expression. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 9695– 9699, DOI: 10.1073/pnas.88.21.9695Google ScholarThere is no corresponding record for this reference.
- 48Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem., Int. Ed. 2001, 40, 2004– 2021, DOI: 10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXksVOis78%253D&md5=61c28b416c42c87821bc8a5bc2fdec36Click chemistry: diverse chemical function from a few good reactionsKolb, Hartmuth C.; Finn, M. G.; Sharpless, K. BarryAngewandte Chemie, International Edition (2001), 40 (11), 2004-2021CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)Review with > 88 refs. Examn. of nature's favorite mols. reveals a striking preference for making carbon-heteroatom bonds over carbon-carbon bonds - surely no surprise given that carbon dioxide is nature's starting material and that most reactions are performed in water. Nucleic acids, proteins, and polysaccharides are condensation polymers of small subunits stitched together by carbon-heteroatom bonds. Even the 35 or so building blocks from which these crucial mols. are made each contain, at most, six contiguous C-C bonds, except for the three arom. amino acids. Taking a cue from nature's approach, the development of a set of powerful, highly reliable, and selective reactions for the rapid synthesis of useful new compds. and combinatorial libraries through heteroatom links (C-X-C), an approach called "click chem." is addressed. Click chem. is at once defined, enabled, and constrained by a handful of nearly perfect "spring-loaded" reactions. The stringent criteria for a process to earn click chem. status are described along with examples of the mol. frameworks that are easily made using this spartan, but powerful, synthetic strategy.
- 49Kolb, H. C.; Sharpless, K. B. The growing impact of click chemistry on drug discovery. Drug Discovery Today 2003, 8, 1128– 1137, DOI: 10.1016/S1359-6446(03)02933-7Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXpvVWkuro%253D&md5=f0042f3a8f153d1c1192533d90699d19The growing impact of click chemistry on drug discoveryKolb, Hartmuth C.; Sharpless, K. BarryDrug Discovery Today (2003), 8 (24), 1128-1137CODEN: DDTOFS; ISSN:1359-6446. (Elsevier Science Ltd.)A review. Click chem. is a modular approach that uses only the most practical and reliable chem. transformations. Its applications are increasingly found in all aspects of drug discovery, ranging from lead finding through combinatorial chem. and target-templated in situ chem., to proteomics and DNA research, using bioconjugation reactions. The copper-(I)-catalyzed 1,2,3-triazole formation from azides and terminal acetylenes is a particularly powerful linking reaction, due to its high degree of dependability, complete specificity, and the bio-compatibility of the reactants. The triazole products are more than just passive linkers; they readily assoc. with biol. targets, through hydrogen bonding and dipole interactions. Click chem., a modular approach based on highly reliable chem. transformations, is being applied in all aspects of drug discovery, ranging from lead finding to proteomics and DNA research.
- 50Moses, J. E.; Moorhouse, A. D. The growing applications of click chemistry. Chem. Soc. Rev. 2007, 36, 1249– 1262, DOI: 10.1039/B613014NGoogle Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXnsVChu7w%253D&md5=3351a2d4815715d0746e45e8bfae5bb2The growing applications of Click chemistryMoses, John E.; Moorhouse, Adam D.Chemical Society Reviews (2007), 36 (8), 1249-1262CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Click chem. is a modular synthetic approach towards the assembly of new mol. entities. This powerful strategy relies mainly upon the construction of carbon-heteroatom bonds using "spring-loaded" reactants. Its growing no. of applications are found in nearly all areas of modern chem. from drug discovery to materials science. The copper(I)-catalyzed 1,2,3-triazole forming reaction between azides and terminal alkynes has become the gold std. of Click chem. due to its reliability, specificity and biocompatibility.
- 51McKay, C. S.; Finn, M. G. Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem. Biol. 2014, 21, 1075– 1101, DOI: 10.1016/j.chembiol.2014.09.002Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFyrur7N&md5=64a6e5ea8a232b97fce61d1268093fe4Click Chemistry in Complex Mixtures: Bioorthogonal BioconjugationMcKay, Craig S.; Finn, M. G.Chemistry & Biology (Oxford, United Kingdom) (2014), 21 (9), 1075-1101CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)A review. The selective chem. modification of biol. mols. drives a good portion of modern drug development and fundamental biol. research. While a few early examples of reactions that engage amine and thiol groups on proteins helped establish the value of such processes, the development of reactions that avoid most biol. mols. so as to achieve selectivity in desired bond-forming events has revolutionized the field. We provide an update on recent developments in bioorthogonal chem. that highlights key advances in reaction rates, biocompatibility, and applications. While not exhaustive, we hope this summary allows the reader to appreciate the rich continuing development of good chem. that operates in the biol. setting.
- 52Peracha, H.; Sawamoto, K.; Averill, L.; Kecskemethy, H.; Theroux, M.; Thacker, M.; Nagao, K.; Pizarro, C.; Mackenzie, W.; Kobayashi, H.; Yamaguchi, S.; Suzuki, Y.; Orii, K.; Orii, T.; Fukao, T.; Tomatsu, S. Molecular genetics and metabolism, special edition: diagnosis, diagnosis and prognosis of Mucopolysaccharidosis IVA. Mol. Genet. Metab. 2018, 125, 18– 37, DOI: 10.1016/j.ymgme.2018.05.004Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpslOrtL4%253D&md5=070925e02db747d32705ecf7bdd06081Molecular genetics and metabolism, special edition: Diagnosis, diagnosis and prognosis of Mucopolysaccharidosis IVAPeracha, Hira; Sawamoto, Kazuki; Averill, Lauren; Kecskemethy, Heidi; Theroux, Mary; Thacker, Mihir; Nagao, Kyoko; Pizarro, Christian; MacKenzie, William; Kobayashi, Hironori; Yamaguchi, Seiji; Suzuki, Yasuyuki; Orii, Kenji; Orii, Tadao; Fukao, Toshiyuki; Tomatsu, ShunjiMolecular Genetics and Metabolism (2018), 125 (1-2), 18-37CODEN: MGMEFF; ISSN:1096-7192. (Elsevier B.V.)A review. Mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) is an autosomal recessive disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. Deficiency of this enzyme leads to the accumulation of specific glycosaminoglycans (GAGs), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), which are mainly synthesized in the cartilage. Therefore, the substrates are stored primarily in the cartilage and its extracellular matrix (ECM), leading to a direct impact on bone development and successive systemic skeletal spondylepiphyseal dysplasia. The skeletal-related symptoms for MPS IVA include short stature with short neck and trunk, odontoid hypoplasia, spinal cord compression, tracheal obstruction, obstructive airway, pectus carinatum, restrictive lung, kyphoscoliosis, platyspondyly, coxa valga, genu valgum, waddling gait, and laxity of joints. The degree of imbalance of growth in bone and other organs and tissues largely contributes to unique skeletal dysplasia and clin. severity. Diagnosis of MPS IVA needs clin., radiog., and lab. testing to make a complete conclusion. To diagnose MPS IVA, total urinary GAG anal. which has been used is problematic since the values overlap with those in age-matched controls. Currently, urinary and blood KS and C6S, the enzyme activity of GALNS, and GALNS mol. anal. are used for diagnosis and prognosis of clin. phenotype in MPS IVA. MPS IVA can be diagnosed with unique characters although this disorder relates closely to other disorders in some characteristics. In this review article, we comprehensively describe clin., radiog., biochem., and mol. diagnosis and clin. assessment tests for MPS IVA. We also compare MPS IVA to other closely related disorders to differentiate MPS IVA. Overall, imbalance of growth in MPS IVA patients underlies unique skeletal manifestations leading to a crit. indicator for diagnosis.
- 53Khan, S.; Alméciga-Díaz, C. J.; Sawamoto, K.; Mackenzie, W. G.; Theroux, M. C.; Pizarro, C.; Mason, R. W.; Orii, T.; Tomatsu, S. Mucopolysaccharidosis IVA and glycosaminoglycans. Mol. Genet. Metab. 2017, 120, 78– 95, DOI: 10.1016/j.ymgme.2016.11.007Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVyisbfP&md5=7eb2530048576d15dd5030fe48f18cf9Mucopolysaccharidosis IVA and glycosaminoglycansKhan, Shaukat; Almeciga-Diaz, Carlos J.; Sawamoto, Kazuki; Mackenzie, William G.; Theroux, Mary C.; Pizarro, Christian; Mason, Robert W.; Orii, Tadao; Tomatsu, ShunjiMolecular Genetics and Metabolism (2017), 120 (1-2), 78-95CODEN: MGMEFF; ISSN:1096-7192. (Elsevier B.V.)Mucopolysaccharidosis IVA (MPS IVA; Morquio A: OMIM 253000) is a lysosomal storage disease with an autosomal recessive trait caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. Deficiency of this enzyme leads to accumulation of specific glycosaminoglycans (GAGs): chondroitin-6-sulfate (C6S) and keratan sulfate (KS). C6S and KS are mainly produced in the cartilage. Therefore, the undegraded substrates are stored primarily in cartilage and in its extracellular matrix (ECM), leading to a direct impact on cartilage and bone development, and successive systemic skeletal dysplasia. Chondrogenesis, the earliest phase of skeletal formation, is maintained by cellular interactions with the ECM, growth and differentiation factors, signaling pathways, and transcription factors in a temporal-spatial manner. In patients with MPS IVA, the cartilage is disrupted at birth as a consequence of abnormal chondrogenesis and/or endochondral ossification. The unique skeletal features are distinguished by a disproportional short stature, odontoid hypoplasia, spinal cord compression, tracheal obstruction, pectus carinatum, kyphoscoliosis, platyspondyly, coxa valga, genu valgum, waddling gait, and laxity of joints. In spite of many descriptions of these unique clin. features, delay of diagnosis still happens. The pathogenesis and treatment of systemic skeletal dysplasia in MPS IVA remains an unmet challenge. In this review article, we comprehensively describe historical aspect, property of GAGs, diagnosis, screening, pathogenesis, and current and future therapies of MPS IVA.
- 54Kumar, A. B.; Spacil, Z.; Ghomashchi, F.; Masi, S.; Sumida, T.; Ito, M.; Turecek, F.; Scott, C. R.; Gelb, M. H. Fluorimetric assays for N-acetylgalactosamine-6-sulfatase and arylsulfatase B based on the natural substrates for confirmation of mucopolysaccharidoses types IVA and VI. Clin. Chim. Acta 2015, 451, 125– 128, DOI: 10.1016/j.cca.2015.08.010Google ScholarThere is no corresponding record for this reference.
- 55Ullal, A. J.; Millington, D. S.; Bali, D. S. Development of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spots. Mol. Genet. Metab. Rep. 2014, 1, 461– 464, DOI: 10.1016/j.ymgmr.2014.10.004Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmtVamu7o%253D&md5=1cd9f842afddc7cb04ffe232f6f0bfbcDevelopment of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spotsUllal, Anirudh J.; Millington, David S.; Bali, Deeksha S.Molecular Genetics and Metabolism Reports (2014), 1 (), 461-464CODEN: MGMRDI; ISSN:2214-4269. (Elsevier B.V.)Mucopolysaccharidosis type IVA or Morquio type-A disease is a hereditary lysosomal storage disorder caused by deficient activity of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The disease is caused by lysosomal accumulation of unprocessed glycosaminoglycans (GAGs) that manifests with severe to mild skeletal and cardiopulmonary abnormalities. We have developed a modified microtiter plate-based enzyme activity assay using dried blood spots and a fluorescent substrate for measuring specific GALNS activity to identify patients with MPS IVA.
- 56Camelier, M. V.; Burin, M. G.; De Mari, J.; Vieira, T. A.; Marasca, G.; Giugliani, R. Practical and reliable enzyme test for the detection of Mucopolysaccharidosis IVA (Morquio Syndrome type A) in dried blood samples. Clin. Chim. Acta 2011, 412, 1805– 1808, DOI: 10.1016/j.cca.2011.06.001Google ScholarThere is no corresponding record for this reference.
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