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Technical Briefs |
1 Department of Pediatrics, Metabolic Laboratory, University of Hamburg, Martinistrasse 52, 20246 Hamburg, Germany
2 Department of Child Neurology, Hospital for Children and Adolescents, University of Helsinki, Stenbäckinkatu 11, 00290 Helsinki, Finland
aauthor for correspondence: fax 49-40-42803-5137, e-mail kohlschuetter{at}uke.uni-hamburg.de
Neuronal ceroid lipofuscinoses constitute a group of at least eight inherited, progressive encephalopathies that are characterized by lipofuscin-like inclusions in various tissues and that have recently been classified as CLN1 to CLN8 according to their genetic defects (1). A form with mostly infantile manifestation (CLN1) and the classical late infantile form (CLN2) are caused by deficiencies of the lysosomal enzymes palmitoyl protein thioesterase 1 (PPT1) and tripeptidyl peptidase 1 (TPP1), respectively. The basic defects underlying the other forms are still ill-defined or unknown. To date, more than 30 mutations have been reported in the PPT1 and TPP1 genes, rendering molecular genetic analysis impractical as a primary means of diagnosis. Electron microscopy of characteristic cellular inclusions remains an important diagnostic method, but it is also tedious and not readily available.
Enzymatic assays based on fluorescent substrates have recently been reported for PPT1 (2) and TPP1(3). These assays can also be used with isolated leukocytes. However, because few laboratories are experienced in diagnosing these rare disorders, blood samples must be sent to specialized centers by expensive express mail services to avoid loss of enzymatic activity. In our experience, samples for this kind of study frequently arrive in poor condition. As an alternative to the use of leukocytes, methods for determining several lysosomal enzymes from dried blood spots (DBS) have recently been described (4). DBS are also used routinely for enzyme measurements within newborn-screening programs for the detection of biotinidase and galactose-uridyltransferase deficiencies in neonates. DBS require only minute amounts of blood, are easy to handle, and can be sent to the laboratory at ambient temperature by regular mail with little risk of a decrease in enzyme activity. In preliminary experiments with other lysosomal enzymes, such as hexosaminidase and ß-galactosidase, we observed that activities on filter-paper cards were stable over a period of several weeks when stored dry at room temperature. We have therefore developed and optimized enzymatic assays for PPT1 and TPP1 in dried blood.
DBS were obtained from 6 patients with CLN1, 5 patients with CLN2, and 2 patients with the juvenile form CLN3, as well as from 70 control individuals. The parents of the patients were informed about the aims of the study, and all consented to the use of dried blood specimens from their children. All patients showed a typical picture and clinical course of the disease. The patients with the infantile form of this disease (CLN1) were severely visually impaired, mentally retarded, and could not speak until the age of 2. All had lost the ability to crawl or walk. Their magnetic resonance imaging findings showed characteristic hypointense thalami in T2-weighted images (5), and DNA tests revealed homozygosity for the major Finnish mutation (6). The patients with the classical late infantile form (CLN2) had severe epilepsy starting around the third year of life, loss of psychomotor functioning, characteristic curvilinear cell inclusions, and mutations in the CLN2 gene (7). The patients with the juvenile form (CLN3) started to lose their vision because of retinopathy at 
6 years of age. They had conspicuous lymphocyte vacuoles and a progressive downhill course, which is typical of the disease (8).
We purchased 4-methylumbelliferyl-6-thiopalmitoyl-ß-glucoside (MUTG) from Moscerdam Substrates. The substrate for TPP (Ala-Ala-Phe-7-amido-4-methylcoumarin) was obtained from Bachem. All other chemicals were purchased from Sigma-Aldrich.
For the PPT1 assay, MUTG (3.4 mmol/L) was dissolved in 150 µL chloroform–methanol containing 50 mL/L Triton X-100. Subsequently, the solvent was evaporated, and 400 µL of phosphate–citrate buffer (0.4 mol/L; pH 4.0) and 40 µL of ß-glucosidase (100 U/mL) were added. Blood spots (3 mm) were punched on a 1296-031 Delfia puncher (PerkinElmer) into microtiter plates, and each spot was eluted with 20 µL of substrate buffer (0.64 mmol/L) and 40 µL of phosphate–citrate buffer to allow quantitative elution of proteins. Plates were shaken for 45 min at room temperature and then placed in an incubator at 37 °C for 45 h. The filter-paper spots were not removed after incubation. All assays were performed in duplicate. A calibration curve for 4-methylumbelliferone over the range 250–5000 nmol was recorded for each plate. For the TPP assay, the substrate (30 mmol/L) was dissolved in dimethyl sulfoxide and mixed with pepstatin A (8.34 mmol/L) and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (24 mmol/L) in acetate buffer at pH 4.0. DBS were eluted with a solution of 20 µL of substrate buffer and 40 µL of NaCl (9 g/L). All other steps were carried out as described above. A calibration curve for 4-methylcoumarin over the range 285-1472 nmol was prepared for each plate. Two blanks without added dried blood were run for each batch. After incubation, the reaction was stopped with a carbonate–glycine buffer (pH 9.7). To accommodate fluorescence quenching of hemoglobin, blood spots were subsequently added to the blanks and hemoglobin was eluted for 15 min. Plates were measured on a Fluoroscan II instrument (Labsystems) with excitation at 355 nm and emission at 460 nm. All results were corrected for the blank, and the released amounts of 4-methylumbelliferone or 4-methylcoumarin were calculated from the respective calibration curves. Enzyme activities are given in nmol of hydrolyzed substrate per DBS.
Storage of cards at room temperature did not diminish enzyme activities over a period of 2 weeks. In addition, heating of dried blood cards to 50 °C for 3 h, which inactivates enzymes in liquid EDTA-blood samples, did not affect TPP1 or PPT1 activities in DBS (data not shown). This indicates an improved stability of DBS compared with EDTA blood because neither degradation of enzymes by proteases nor changes in the folding of the protein could occur in the dried state. Intraassay variations were 8% for PPT1 and 12% for TPP1, respectively. Blanks displayed a fluorescence of 20 to 50 counts, whereas samples from healthy controls showed a fluorescence of 300 to 500 counts for TPP1 and 1000–3000 counts for PPT1. The resulting enzyme activities for homozygous patients, heterozygous carriers, and healthy controls are shown in Table 1
. PPT1 activities in healthy controls (n = 70) ranged from 0.4 to 1.52 nmol/spot with a mean value of 0.82 nmol/spot. Samples from patients with CLN1 displayed activities that were <3% of the mean for the controls, i.e., values that were 10-fold lower than the lowest control value. TPP1 activities in healthy controls (n = 70) were 0.1–0.67 nmol/spot with a mean value of 0.27 nmol/spot. The assay did not show any activity for patients with CLN2. Two heterozygous carriers (the parents of one patient) had TPP1 activities of 0.04 and 0.05 nmol/spot, which were 50% below the lowest control value. In both assays, normal activities were found in samples from patients with CLN3, as would be expected. To date, no false-positive results have been detected with our method.
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In conclusion, this new method allows the rapid and accurate determination of PPT1 and TPP1 activities from dried blood samples and a clear differentiation between healthy individuals, patients with neuronal ceroid lipofuscinoses (CLN1 and CLN2), and heterozygous carriers. Additionally, because the enzyme activities remain stable over several days, mailing to specialized centers is easier and less expensive. Furthermore, the assay requires only a few drops of blood in contrast to the 2–5 mL of EDTA blood needed for leukocyte assays. We consider the dried blood tests for PPT1 and TPP1 a very useful approach to the diagnosis of CLN1 and CLN2. However, the diagnosis should be confirmed by DNA tests, electron microscopy, and enzyme measurements in skin fibroblasts if only very low or no enzyme activities are detectable.
References
The following articles in journals at HighWire Press have cited this article:
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  L. B. Augestad and W. D. Flanders Occurrence of and Mortality From Childhood Neuronal Ceroid Lipofuscinoses in Norway J Child Neurol, November 1, 2006; 21(11): 917 - 922. [Abstract] [PDF]
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 Y. Tian, I. Sohar, J. W. Taylor, and P. Lobel Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates J. Biol. Chem., March 10, 2006; 281(10): 6559 - 6572. [Abstract] [Full Text] [PDF]
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 K. Tsiakas, R. Steinfeld, S. Storch, J. Ezaki, Z. Lukacs, E. Kominami, A. Kohlschutter, K. Ullrich, and T. Braulke Mutation of the glycosylated asparagine residue 286 in human CLN2 protein results in loss of enzymatic activity Glycobiology, April 1, 2004; 14(4): 1C - 5C. [Abstract] [Full Text] [PDF]
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