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Letters to the Editor |
2 Lysosomal Diseases Research Unit Department of Genetic Medicine, Children Youth and Women’s Health Service, North Adelaide, South Australia, Australia
3 Department of Paediatrics, University of Adelaide, Adelaide, South Australia, Australia
4 Department of Chemical Pathology, Universiti Sains Malaysia, Health Campus, Malaysia
5 Present address: Baker IDI Heart and Diabetes Institute, Melbourne, Victoria 3004, Australia
aAddress correspondence to this author at: Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, Victoria 3004, Australia;, PO Box 6492, St Kilda Road Central, Melbourne, Victoria 8008, Australia, Fax (613) 8532 1100, E-mail peter.meikle{at}baker.edu.au
To the Editor:
Metachromatic leukodystrophy (MLD),1 an autosomal recessive neurodegenerative disease resulting from a deficiency of arylsulfatase A (ASA), results in the lysosomal accumulation of sulfatide in several peripheral organs, notably the central nervous system. The central nervous system exhibits progressive demyelination, leading to impaired neurological function with a fatal outcome (1). Biochemical diagnosis of MLD is achieved by enzymatic analysis of ASA activity in peripheral blood leukocytes and cultured fibroblasts. However, diagnosis based on enzyme activity alone is complicated by the high frequency of ASA pseudo-deficiency (ASA-PD). ASA enzyme activities for individuals with ASA-PD are 5%–15% of normal. ASA-PD individuals are clinically normal, but compound heterozygotes (with 1 ASA-PD and 1 MLD allele) can have impaired capacity for sulfatide degradation and exhibit neuropsychiatric symptoms(2). Approximately 1%–2% of the European population is homozygous for ASA-PD(1). With the carrier frequency of MLD at 1:152, this frequency will lead to a prevalence of compound heterozygotes as high as 1:2300. Diagnostic laboratories are often required to perform additional complex sulfatide assays or molecular analysis to obtain a definitive diagnosis.
We have developed and evaluated 2 immune-based assays that enable the differentiation of MLD individuals from individuals with ASA-PD and unaffected controls. These assays use an immune-quantification assay to quantify the amount of ASA protein and an immune-capture activity assay to determine enzyme activity. Both assays are performed on 3-mm dried blood spots (DBS) collected on filter paper.
Affinity-purified sheep anti-ASA polyclonal antibody was produced and Eu3+-labeled as previously described (3). Recombinant human ASA protein was expressed in a CHO-K1 expression system from a full-length ASA cDNA clone in HT 14/CP 8 pBluescript® vector. Calibrators and quality control materials were prepared by diluting recombinant human ASA in working buffer (0.1 mol/L sodium acetate/acetic acid, 0.1% heat-treated BSA, pH 5.0) or Delfia® assay buffer (Perkin-Elmer Life Sciences) to measure ASA activity and protein, respectively.
All DBS were stored at –20 °C before analysis. The institute’s ethics committee approved use of the patient samples. ASA protein from DBS was assayed using a single 3-mm disk as described previously (3). A calibration curve (2.0–1000 pg/well) was included in each assay. ASA activity in DBS was determined as described previously(4), using two 3-mm disks per assay, 0.1 mol/L sodium acetate/acetic acid, 0.1% heat-treated BSA, pH 5.0, as elution buffer; 20 mmol/L sodium acetate/acetic acid, pH 5.0, as wash buffer; and 5 mmol/L 4-methylumbelliferyl sulfate in 0.2 mol/L sodium acetate/acetic acid, pH 5.0, 0.1% BSA (100 µL/well) as substrate. A calibration curve using recombinant human ASA (2.0 –800 pmol/h/well) was included in each assay.
The ASA protein and activity assays gave linear responses over the biological range (R2 > 0.999). The detection limits were 0.8 pg/well and 1.5 pmol/h/well. The intra- and interassay CVs were 9% and 10%, respectively, for the protein assay and 6% and 13% for the activity assay. The median amount of ASA protein in whole blood from unaffected, ASA-PD, and heterozygote individuals was 31.9 µg/L (range 21.0–46.3 µg/L), 13.0 µg/L (range 8.3–17.0 µg/L), and 12.5 µg/L (range 12.0–13.0 µg/L), respectively; no detectable ASA protein was observed in MLD individuals (Fig. 1A
). The median ASA activity in unaffected and heterozygote individuals was 4.16 µmol/min/L (range = 1.48–7.72 µmol/min/L) and 2.62 µmol/min/L (range = 2.44–2.80 µmol/min/L); no detectable activity was observed in ASA-PD or MLD individuals (Fig. 1B
).
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ASA activity did not enable differentiation of ASA-PD individuals from MLD patients; however, the ASA protein quantification assay enabled clear differentiation between controls and ASA-PD and MLD individuals. To further investigate why ASA-PD showed no ASA activity, we analyzed DBS from an unaffected individual. This DBS was stored at room temperature for up to 30 days, and during this period ASA activity decreased such that only 39% of activity remained after 30 days; in contrast, no decrease was observed in the ASA protein. Measured ASA protein and activity in heterozygotes were both in the lower end of the reference interval.
The ability to use DBS to measure ASA protein and activity will simplify procedures for collection, handling, and storage of patient samples. The ease of transporting DBS, combined with the diagnostic sensitivity and specificity of these assays, provide a powerful approach to the diagnosis of MLD. After further investigation and validation, these assays may be used for newborn screening for MLD and other lysosomal storage disorders. Importantly, the inability to differentiate ASA-PD from ASA by the activity assay alone suggests that ASA protein determination from DBS may be the most appropriate screening approach for newborns, as has recently been proposed in a report of a multiplex immune-quantification assay (5).
Acknowledgments
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: This work was supported by the National Health and Medical Research Council (Australia).
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
Footnotes
1 Nonstandard abbreviations: MLD, metachromatic leukodystrophy; ASA, arylsulfatase A; ASA-PD, ASA pseudo-deficiency; DBS, dried blood spots. 
References
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