Leading Article. Enzyme Assays on Dried Blood Filter Paper Samples for Specific Detection of Selected Inherited Lysosomal Storage Diseases

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1 103 Leading Article Enzyme Assays on Dried Blood Filter Paper Samples for Specific Detection of Selected Inherited Lysosomal Storage Diseases GABRIEL CIVALLERO 1, MAIRA BURIN 1,2, JUREMA DE MARI 1, MARLI VIAPIANA 1, KRISTIANE MICHELIN 1,2, JANICE C COELHO 1,2, AND ROBERTO GIUGLIANI 1,2,3 1 Medical Genetics Service, Hospital de Clínicas de Porto Alegre; 2 Postgraduate Program of Biochemistry; 3 Postgraduate Program of Genetics and Molecular Biology, UFRGS. Porto Alegre, RS, Brazil. Inherited lysosomal storage diseases (LSDs) are severe conditions with a broad spectrum of manifestations that may include skeletal abnormalities, organ dysfunction, neuronal involvement, and tissue accumulation of specific substances, including glycosaminoglycans, carbohydrate chains of glycosylated proteins, or membrane lipids [1]. Diagnosis of these diseases is based on specific enzymatic assays performed on plasma, leukocytes, and fibroblasts. Recently, dried blood filter paper (DBFP) samples have been used as a novel way to conduct these assays [2 8]. In addition, enzyme replacement therapy is approved or is being developed for Gaucher s, Anderson Fabry (AFD), Pompe (PD), and Niemann Pick diseases and for mucopolysaccharidosis types I (MPS I), II, and VI. Early identification of affected individuals seems to be critical for the success of the therapy in most disease cases; therefore, the neonatal screening of LSDs using DBFP samples and different analytical methodologies have been proposed [9 14]. Rapid Enzyme Assays for the Biochemical Diagnosis of Several LSDs In this article our findings on the detection of patients with several inherited LSDs using specific enzyme assays on DBFP samples are reported. DBFP samples were obtained from healthy individuals (adults and newborns), obligate heterozygotes, and patients with confirmed LSDs. Fluorometric analysis methods that had already been validated [3 5] were implemented for the evaluation of: Arylsulfatase B. Chitotriosidase. α-galactosidase (α-gal). β-galactosidase (α-gal). β-glucuronidase. Total hexosaminidases (HEXT). In addition, the methods from a number of different enzymes were adapted to evaluate [10,15 20]: α-glucosidase (GAA). β-glucosidase (GBA). Hexosaminidase A (HEXA). α-iduronidase. Iduronate-2-sulfatase (IDS). α-n-acetylglucosaminidase (NAGLU). Acid sphingomyelinase (ASM). Samples were derived from healthy individuals, obligate heterozygotes, and patients with confirmed LSDs. In all the assays, individual 3-mm diameter disks containing the dried-blood samples (approximately 3.6 µl of whole blood) were incubated at 37 C with

2 104 Gabriel Civallero, Maira Burin, Jurema de Mari, et al. Table 1. Lysosomal enzyme activities in dried-blood filter paper samples [8]. Enzyme n Activities (nmol/ml/h) p value* Cut-off range (mean±sd) value** α-n-acetylglucosaminidase MPS III-B patients (a) 1 ND b vs. c: Normal controls (adults) (b) (2.42±1.24) Normal controls (newborns) (c) (1.40±0.49) Arylsulfatase B MPS VI patients (a) (2.01±0.57) a vs. b: < MPS VI heterozygotes (d) (5.95±1.32) b vs. c: Normal controls (adults) (b) (11.56±4.19) a vs. d: <0.001 Normal controls (newborns) (c) (17.34±8.92) b vs. d: <0.001 Chitotriosidase GD patients (a1) (576.90±353.21) a1 vs. b: < GD patients (on therapy) (a2) (161.35±121.88) b vs. c: Normal controls (adults) (b) 56 ND (6.44±8.11) a1 vs. a2: <0.001 Normal controls (newborns) (c) 26 ND 95.6 (12.61±20.18) α-glucosidase*** Pompe disease patients (a) (1.68±1.10) a vs. b: < Normal controls (adults) (b) (27.30±6.97) b vs. c: <0.001 Normal controls (newborns) (c) (37.29±15.14) α-galactosidase AFD patients (a) 12 ND 0.79 (0.42±0.25) a vs. b: < AFD heterozygotes (d) (1.63±0.80) b vs. c: Normal controls (adults) (b) (8.11±3.48) b vs. d: <0.001 Normal controls (newborns) (c) (10.26±4.06) a vs. d: β-galactosidase G M1 gangliosidosis patients (a) (1.99±0.74) a vs. b: < G M1 gangliosidosis heterozygotes (d) (26.19±9.76) b vs. c: Normal controls (adults) (b) (69.18±24.24) b vs. d: <0.001 Normal controls (newborns) (c) (90.19±27.24) a vs. d: β-glucosidase GD patients (a) (0.8±0.31) a vs. b: < GD heterozygotes (d) (1.71±0.02) b vs. c: Normal controls (adults) (b) (4.92±2.73) b vs. d: <0.001 Normal controls (newborns) (c) (4.93±1.54) a vs. d: <0.001 β-glucuronidase MPS VII patients (a) b vs. c: Normal controls (adults) (b) (8.97±4.39) Normal controls (newborns) (c) (9.80±8.91) Total hexosaminidases TSD patient b vs. c: G M2 gangliosidosis B1 variant patient Normal controls (adults) (b) (71.19±28.95) Normal controls (newborns) (c) (93.85±53.22) Hexosaminidase A TSD patient b vs. c: < G M2 gangliosidosis B1variant patient b vs. d: TSD heterozygotes (d) (24.27±2.02) Normal controls (adults) (b) (53.29±17.02) Normal controls (newborns) (c) (87.77±41.21) α-iduronidase MPS I patient (a) 9 ND 0.23 (0.06±0.09) a vs. b: < Normal controls (adults) (b) (3.82±2.15) b vs. c: <0.001 Normal controls (newborns) (c) (7.62±3.63) MPS II patient (a) (7.22±1.79) a vs. b: <0.001

3 Enzyme Assays on Dried Blood Filter Paper Samples 105 Table 1. continued Enzyme n Activities (nmol/ml/h) p value* Cut-off range (mean±sd) value** Iduronate-2-sulfatase Normal controls (adults) (b) (16.71±2.58) b vs. c: < Normal controls (newborns) (c) (21.26±5.40) Acid spingomyelinase Niemann Pick patient (a) (0.61±0.18) a vs. b: <0.001 Niemann-Pick heterozygotes (d) b vs. c: Normal controls (adults) (b) (8.23±5.22) Normal controls (newborns) (c) (5.08±2.46) *Correspond to an asymptotic significance (2-tailed) from the Mann-Whitney U test. (a), (b), (c), and (d) correspond to patient, normal adult, normal newborn, and heterozygote groups. **Obtained from ROC curve value of patient and adult control groups (99% confidence. 100 % of selectivity and 100% of specificity). ***Activity nmol/ml/20h. Activity nmol/ml/24h. AFD: Anderson Fabry disease; GD: Gaucher s disease; MPS: Mucopolysaccharidosis type; ND: no activity detected; SD: standard deviation; TSD: Tay Sachs disease. appropriate artificial substrates and dilution buffers. Samples were centrifuged and then tested using fluorometric analysis for activity (nanomoles of substrate hydrolyzed/h/ml of blood) [8]. Results Table 1 shows specific lysosomal enzyme activities measured using the DBFP samples. It is interesting to note that all the patients had proven specific enzyme deficiencies and were significantly differentiated from the control group in all cases (p<0.001), which concurs with the findings of previous reports [3 5,6]. Patients with B1 and B0 variants of G M2 gangliosidosis were correctly discriminated using the HEXA test. In order to confirm this finding, further patients with these variants need to be investigated. DBFP samples from patients with Sandhoff disease were not available to test for HEXA and HEXT and this is a potential area for future study. Individuals with NAGLU and IDS deficiencies were identified in our assays; the first reports of fluorometric enzyme assays on DBFP samples for these enzymes. The range of activity in healthy newborns and adults showed variable overlap. Heterozygote ranges partially overlapped with healthy controls for different assays, except for the GLA test, making it unsuitable for carrier detection. Cut-off values were estimated for the patients and corresponding adult controls (Table 1). These values overlapped with those of the heterozygote groups, and with the IDS control group. We consider as a tentative cutoff value for affected newborns (not available in this study) the mean minus 1.96 standard deviation (SD) (mean plus 1.96 SD for CHI assay) of the newborn control groups. Unfortunately, we have not yet been able to assay samples from affected newborns to completely validate this methodology. The assays show that the diagnosis methods used on the DBFP samples evaluated are valid for the detection of patients with selected LSDs. Their incorporation as regular laboratory procedures should be considered to improve screening programs. As a likely consequence of this, the number of pathological cases detected will increase, as has been proven in our laboratory and therefore the actual incidence of LSDs in the population can be better appreciated, especially in large countries with many areas that are difficult to access, or where the conditions for collection and mailing of regular samples are limited. Use of Acarbose for Diagnosis of PD The diagnosis of PD is mainly based on an assay of acid GAA activity in fibroblasts [21]. The procedure involves the collection of skin samples and a subsequent 2 4 week period of cell culture growth, which increases the cost of the test and delays the diagnosis. Plasma, leukocyte, and DBFP samples are not usually used for the enzyme assay due to presence of other unrelated less acidic GAA isoenzymes (especially maltase glucoamylase) that

4 106 Gabriel Civallero, Maira Burin, Jurema de Mari, et al. interfere in the detection technique. To solve this, Li et al. used acarbose for selective inhibition of GAA and were therefore able to detect patients with PD, using combined enzyme and tandem mass spectrometry assays [10]. Based on their findings, we tested a fluorometric enzyme assay using 8 mmol/l acarbose and DBFP samples for the diagnosis of PD. As a result, patients with infantile and adult forms of PD were discriminated from healthy controls (Table 1). The difference between the enzyme activities of patients with infantile and adult forms of PD was not significant and the ranges overlapped. Our results agree with many previous reports, and support the proposed selective action of acarbose, inhibiting less acid GAA than other isoenzymes present in DBFP samples [6,10,22 24]. Neonatal Screening of LSD Recently, neonatal screening of LSDs was proposed [9 14,25]. Diagnostic methods in any neonatal program should meet with the usual requirements, such as: Screening of pathologies with a relatively high frequency. Availability of treatment. Early start of the treatment. Availability of equipment. Assay simplicity. Low cost. Quick results. The whole group of LSDs show a reasonably high incidence while individually the diseases are relatively rare. However, the figures can record change as neonatal screening is implemented, as suggested by a pioneer study on AFD in Italy using a newborn screening approach, which showed an unexpectedly high incidence of the disease [9]. Multiplex enzyme assays, and analysis of substrate derivatives and LSD markers using tandem mass spectrometry have been proposed for the simultaneous screening of several LSDs [10,14,25]. Nevertheless, the identification of non-treatable diseases is an ethical problem that should be considered. Alternatively, the proposed individual enzyme assays [9, 11 13] meet the screening requirements in terms of equipment availability and low cost, with the advantage that programs could select for individual diseases meant to be screened. Therefore, these methods should be considered for the screening of selected diseases with relatively increased regional incidence and a positive outcome after the early introduction of available therapy. Acknowledgements We wish to thank Dr Nestor Chamoles laboratory and Dr Philip Lee for the supply of samples from affected Pompe disease patients. We are grateful to Juarez Huve, Regis Guidobono, and Marylin Tsao for their technical assistance and express our gratitude to CNPq, CAPES, FAPERGS, NORD, PROPESQ/UFRGS, FIPE/HCPA for their partial support of this work. Table 1 is reproduced from Clinica Chimica Acta 372 Civallero G, Michelin K, de Mari J et al. Twelve different enzyme assays on dried-blood filter paper samples to detection of patients with selected inherited lysosomal storage diseases. p98 102, 2006 with permission from Elsevier. Adress for correspondence: Medical Genetics Service. Hospital de Clinicas de Porto Alegre. Rua Ramiro Barcelos 2350 ( ), RS, Brazil. address: gecivallero@yahoo.com References 1. Scriver CR, Beaudet AL, Sly WS et al. The metabolic and molecular basis of inherited disease, 8th ed. New York, NY: McGraw Hill Umapathysivam K, Hopwood J, Meikle P. Determination of acid α-glucosidase activity in blood spots as a diagnostic test for Pompe disease. Clin Chem 2001;47: Chamoles NA, Blanco MB, Gaggioli D et al. Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paper. Clin Chem 2001;47: Chamoles NA, Blanco MB, Gaggioli D. Fabry disease: enzymatic diagnosis in dried blood spots on filter paper. Clin Chim Acta 2001;308: Chamoles NA, Blanco MB, Gaggioli D et al. Gaucher and Niemann-Pick diseases enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newbornscreening cards. Clin Chim Acta 2002;317: Kallwass H, Carr S, Chamoles N et al. Diagnosis of Pompe disease by enzyme assay in dried-blood filter paper spots. J Inherit Metab Dis 2005;28(Suppl 1): Hein L, Meikle P, Dean C et al. Development of an assay for the detection of mucopolysaccharidosis type VI patients using dried blood-spots. Clin Chim Acta 2005;353:67 74.

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