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Technical Briefs |
1 Lysosomal Diseases Research Unit, Department of Genetic Medicine, Children, Youth and Women’s Health Service, North Adelaide, Australia; 2 Department of Pediatrics, University of Adelaide, Adelaide, Australia;
aaddress correspondence to this author at: Lysosomal Diseases Research Unit, Department of Genetic Medicine, Children, Youth and Women’s Health Service, 72 King William Road, North Adelaide, SA 5006, Australia; fax 61-8-8161-7100, e-mail maria.fuller{at}adelaide.edu.au
Gaucher disease is an inborn error of glycosphingolipid metabolism resulting from a deficiency of the lysosomal enzyme ß-glucosidase (1)(2). Acid ß-glucosidase is responsible for the cleavage of the ß-glucosidic bond of its primary substrate glucosylceramide, an intermediate in the catabolism of globoside and gangliosides. Gaucher disease is the most prevalent lysosomal storage disorder with an estimated frequency of 1 in 57 000 births in Australia(3). The disease has been broadly classified into 3 clinical subtypes, but a broad spectrum of phenotypes exists within each group(1). Type 1, the most common, is a chronic nonneuronopathic form of the disease associated with various degrees of anemia, thrombocytopenia, hepatosplenomegaly, and bone disease. Clinical onset may occur at any age, but usually occurs after childhood and in some instances does not manifest until later adulthood. Type 2 is an acute neuronopathic form of the disease, characterized by early onset, severe central nervous system impairment, and death, usually by the second year of life. Type 3 is a subacute neuronopathic form, usually with a more chronic course and later onset than type 2.
More than 187 mutations in the ß-glucosidase gene have been associated with Gaucher disease (4), although there are rare instances of mutations in the prosaposin gene producing a Gaucher phenotype(5). Defined correlations between genotype and phenotype are not overly apparent, and there is broad phenotypic expression among all genotypes. A notable exception is the association of homozygosity for the L444P mutation with neuronopathic disease; similarly, the presence of the N370S allele precludes neuronopathic disease(6)(7).
Patients with the nonneuronopathic form of the disease are treated with enzyme replacement therapy (8)(9), which may also slow the progression of neuronopathic disease(10). Early and accurate diagnosis for Gaucher disease, as well as the prediction of disease severity, is paramount for the efficacy of current and proposed treatment strategies. To address this need, we evaluated measurements of acid ß-glucosidase activity and protein as markers for the diagnosis of Gaucher disease and for the prediction of neurologic involvement.
A monoclonal antibody (1D6.9.9) was produced (11) against recombinant human ß-glucosidase (rhß-gluc; Genzyme Corporation). An anti-rhß-gluc polyclonal antibody was produced in sheep and affinity-purified as described for 
-glucosidase(12). The purified polyclonal antibody was labeled with Eu3+(13). Calibrators and quality-control material were prepared by diluting rhß-gluc in working buffer [0.1 mol/L citric acid, 0.2 mol/L Na2HPO4 (pH 5.5), 10 g/L bovine serum albumin, 2.5 g/L taurocholate, and 2.5 g/L Triton-X-100] or DELFIA® assay buffer (Perkin-Elmer Life Sciences) to measure ß-glucosidase activity and protein, respectively.
ß-Glucosidase protein in cell extracts or dried filter-paper blood spots was determined by either 1- or 2-step immunoquantification assays, respectively. Briefly, microtiter plates were coated with monoclonal antibody 1D6.9.9 at 5 mg/L in 0.1 mol/L NaHCO3 (100 µL/well; incubation for 16 h at 4 °C). Wells were washed twice with DELFIA wash buffer (Wallac), and then samples or calibrators diluted in 100 µL of DELFIA assay buffer (Wallac) containing 200 µg/L Eu3+-labeled sheep anti-rhß-gluc polyclonal antibody were added to each well. Plates were shaken (10 min), incubated (16 h at 4 °C), and washed 6 times, and then DELFIA enhancement solution (Wallac; 200 µL) was added to each well. After shaking (10 min), the fluorescence was measured on a DELFIA 1234 Research Fluorometer. For the 2-step assay in which the capture and detection steps are performed separately, 2 dried blood spots were placed in each coated well with 200 µL of DELFIA assay buffer. The plates were shaken (1 h at room temperature), incubated overnight (4 °C), shaken again (1 h at room temperature), and then washed twice. The Eu3+-labeled sheep anti-rhß-gluc polyclonal antibody was added, and the plates were processed as described for the 1-step assay. The concentrations of ß-glucosidase in both the fibroblast extracts and the blood spots were calculated from calibration curves by use of Multicalc data analysis software (Wallac).
To determine ß-glucosidase activity, microtiter plates were coated with 1D6.9.9 and washed as described above. Samples were diluted in 100 µL of working buffer and added to the coated wells. A calibration curve (0–0.5 ng/well) was included with each assay. The activity of rhß-gluc was determined with the fluorogenic substrate 4-methylumbelliferyl ß-D-glucoside (9) before each assay, and the calibration curve was expressed as activity per well. The plates were shaken (1 h at room temperature) and then incubated (4 °C overnight). Plates were shaken (1 h at room temperature) and washed 6 times, and 7.5 mmol/L 4-methylumbelliferyl ß-D-glucoside in working buffer was added (100 µL/well). The plates were shaken (5 min at room temperature) and then incubated (4 h at 37 °C). The enzyme reaction was stopped by addition of 100 µL of glycine buffer (200 mmol/L glycine, 158 mmol/L sodium bicarbonate, 146 mmol/L sodium hydroxide, pH 10.7) to each well. The fluorescence was measured on a Wallac Victor2 1420 multilabel counter, and the ß-glucosidase activity was calculated from the calibration curve by use of Multicalc data analysis software.
The proportion of ß-glucosidase protein captured by the 1D6.9.9-coated plates was 56% as measured by immunoquantification of the uncaptured ß-glucosidase protein. The specific activity of the captured protein was 3.0 µmol · min–1 · mg–1, which was 45% of the value for the free enzyme (6.7 µmol · min–1 · mg–1). The immunoquantification assays for protein and activity were optimized to give a linear response over the biological range (see Figs. 1 and 2 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue11). The ß-glucosidase protein assay showed 9% inhibition from dried blood spots, and fibroblast extracts produced 4% and 2% inhibition in the protein and activity assays, respectively. The detection limits and CVs for each assay are shown in Table 1
. Blood spots from controls (n = 20) and patients with Gaucher disease types 1 (n = 8) and 3 (n = 1) were stored at –20 °C before assay.
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Measurement of ß-glucosidase protein did not differentiate the Gaucher patients from the controls, but the ß-glucosidase activities of all of the Gaucher blood spots were below the detection limit of the assay and were clearly differentiated from the control group (see Fig. 3 in the online Data Supplement).
Skin fibroblasts from controls and patients with Gaucher disease types 1, 2, and 3 were cultured as described previously (14), and cell extracts were prepared by sonication for 20 s in 20 mmol/L Tris-HCl–0.25 mol/L NaCl (pH 7.2) containing 1 mL/L NP40. Total cell protein was determined by the method of Lowry et al.(15). Extracts were assayed for ß-glucosidase protein and activity (Fig. 1
). The measurement of either ß-glucosidase protein or activity enabled differentiation of all 15 Gaucher patients from the 7 controls. The measurement of ß-glucosidase protein concentrations also allowed differentiation of the type 1 fibroblast extracts from the Gaucher type 2 and 3 extracts (Fig. 1A
). All Gaucher fibroblast extracts were clearly separated from controls by ß-glucosidase activity, and there was some separation of the 3 subtypes (Fig. 1B
). The results of specific activity measurements (Fig. 1C
) indicated that most fibroblast extracts from the patients with neuronopathic disease had a low amount of residual protein with a higher specific activity compared with the majority of cell extracts from individuals with nonneuronopathic disease, which had higher amounts of residual ß-glucosidase protein with a reduced specific activity. This enables a relatively clear grouping of the nonneuronopathic from the neuronopathic patients despite the ranges of ß-glucosidase activities and protein concentrations in patients with the same genotype.
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Early diagnosis of Gaucher disease may enable early treatment of patients before the onset of irreversible pathology, which will be particularly important for those patients who will go on to develop neuronopathic disease. Although enzyme replacement therapy is unlikely to provide an effective therapy for all neuronopathic patients, early diagnosis could allow for other potentially effective interventions, such as bone marrow transplantation, earlier in the disease process. A practical way to achieve early diagnosis is through a newborn screening program, as has been suggested previously (16)(17). The measurement of ß-glucosidase activity in blood spots could be used for the diagnosis of Gaucher disease, and with the expansion of sample numbers may have applicability in newborn screening programs. One of the challenges for the effective management and treatment of Gaucher disease is accurate prediction of clinical phenotype. The determination of residual ß-glucosidase activity and protein in cultured skin fibroblasts may be useful for the prediction of phenotype in patients with Gaucher disease.
Acknowledgments
We thank M.L. Harkin for assistance with cell culture. This work was supported by Genzyme (United States) and in part by the National Health and Medical Research Council (NH&MRC), Australia.
References
-glucosidase protein: evaluation as a screening marker for Pompe disease and other lysosomal storage disorders. Clin Chem 2000;46:1318-1325.The following articles in journals at HighWire Press have cited this article:
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  E. Parkinson-Lawrence, M. Fuller, J. J. Hopwood, P. J. Meikle, and D. A. Brooks Immunochemistry of Lysosomal Storage Disorders Clin. Chem., September 1, 2006; 52(9): 1660 - 1668. [Abstract] [Full Text] [PDF]
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and * represent statistical outliers and extreme statistical outliers, respectively. (C), ß-gluc activity and protein in fibroblast extracts from Gaucher patients by genotype. The nonneuronopathic (symbols inside the dot-dashed ellipse) and neuronopathic (symbols inside the dashed ellipse) fibroblast extracts are grouped: N370S/nd (
; n = 2), N370S/N370S (
; n = 2), N370S/84GG (
; n = 2), S196P/S196P (
; n = 1), L444P/L444P (
; n = 2), L444P/nd (+; n = 3), del155bp/R257Q (x; n = 1), D409H/nd (•; n = 1). Results are expressed per mg of total cell protein. nd, not determined.