melting curves for the typing of two variants ... - Clinical Chemistry

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melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nat Genet 1995;11:328–30. 2. Bliss JM, Ford D, Swerdlow ...
Clinical Chemistry 49, No. 3, 2003

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Fig. 1. Melting profiles of representative samples of the three detected MC1R genotypes at position 160 (Arg3 Trp) and 163 (Arg3 Gln).

melting curves for the typing of two variants (Arg160Trp and Arg163Gln) are shown in Fig. 1 as examples. LightCycler results were compared with the results achieved by PCR–restriction fragment length polymorphism typing (11, 14, 15 ) and DNA sequencing (PrismTM 310 Genetic Analyzer; Applied Biosystems Applera) and correlated perfectly. In conclusion, the established LightCycler-based assays offer a fast, accurate, and reproducible method for detecting point mutations in MC1R by melting curve analysis. Thus, they can be valuable tools for the screening of volunteers or patients when evaluating cutaneous UV sensitivity. References 1. Valverde P, Healy E, Jackson I, Rees JL, Thody AJ. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nat Genet 1995;11:328 –30. 2. Bliss JM, Ford D, Swerdlow AJ, Armstrong BK, Cristofolini M, Elwood JM, et al. Risk of cutaneous melanoma associated with pigmentation characteristics and freckling: systematic overview of 10 case-control studies. Int J Cancer 1995;62:367–76. 3. Kricker A, Armstrong BK, English DR, Heenan PJ. Pigmentary and cutaneous risk factors for non-melanocytic skin cancer: a case-control study. Int J Cancer 1991;48:650 – 62. 4. Box NF, Wyeth JR, O’Gorman LE, Martin NG, Sturm RA. Characterisation of melanocyte stimulating hormone receptor variant alleles in twins with red hair. Hum Mol Genet 1997;6:1891–7. 5. Smith R, Healy E, Siddiqui S, Flanagan N, Steijlen PM, Rosdahl I, et al. Melanocortin 1 receptor variants in an Irish population. J Invest Dermatol 1998;111:119 –22. 6. Flanagan N, Healy E, Ray A, Philips S, Todd C, Jackson IJ, et al. Pleiotropic effects of the melanocortin 1 receptor (MC1R) gene on human pigmentation. Hum Mol Genet 2000;9:2531–7. 7. Palmer JS, Duffy DL, Box NF, Aitken JF, O’Gorman LE, Green AC, et al. Melanocortin-1 receptor polymorphisms and risk of melanoma: is the association explained solely by pigmentation phenotype? Am J Hum Genet 2000;66:176 – 86. 8. Valverde P, Healy E, Sikkink S, Haldane F, Thody AJ, Carothers A, et al. The Asp84Glu variant of the melanocortin 1 receptor (MC1R) is associated with melanoma. Hum Mol Genet 1996;5:1663– 6. 9. Healy E, Todd C, Jackson IJ, Birch-Machin M, Rees JL. Skin type, melanoma, and melanocortin 1 receptor variants. J Invest Dermatol 1999;112:512–3. 10. Rees JL, Healy E. Melanocortin receptors, red hair, and skin cancer. J Investig Dermatol Symp Proc 1997;2:94 – 8. 11. Koppula SV, Robbins LS, Lu DS, Baack E, White CR, Swanson NA, et al. Identification of common polymorphisms in the coding sequence of the human MSH receptor (MC1R) with possible biological effects. Hum Mutat 1997;9:30 – 6. 12. Fra¨ ndberg PA, Doufexis M, Kapas S, Chhajlani V. Human pigmentation phenotype: a point mutation generates nonfunctional MSH receptor. Biochem Biophys Res Commun 1998;245:490 –2. 13. Schio¨ th HB, Phillips SR, Rudzish R, Birch-Machin MA, Wikberg JES, Rees JL. Loss of function mutations of the human melanocortin 1 receptor are

common and are associated with red hair. Biochem Biophys Res Commun 1999;260:488 –91. 14. Chhajlani V, Wikberg JE. Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. FEBS Lett 1992;309:417– 20. 15. Ichii-Jones F, Lear JT, Heagerty AH, Smith AG, Hutchinson PE, Osborne J, et al. Susceptibility to melanoma: influence of skin type and polymorphism in the melanocyte stimulating hormone receptor gene. J Invest Dermatol 1998;111:218 –2.

Rapid and Simple Assay for the Determination of Tripeptidyl Peptidase and Palmitoyl Protein Thioesterase Activities in Dried Blood Spots, Zoltan Lukacs,1 Pirkko Santavuori,2 Angelika Keil,1 Robert Steinfeld,1 and Alfried Kohlschu¨ tter1* (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, Stenba¨ ckinkatu 11, 00290 Helsinki, Finland; * author for correspondence: fax 49-40-42803-5137, e-mail [email protected]) 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

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Technical Briefs

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 ␤-galactosidase, we observed that activities on filterpaper 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-␤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 ␮L chloroform–methanol containing 50 mL/L Triton X-100. Subsequently, the solvent was evaporated, and 400 ␮L of phosphate– citrate buffer (0.4 mol/L; pH 4.0) and 40 ␮L of ␤-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 ␮L of substrate buffer (0.64 mmol/L) and 40 ␮L 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 ␮L of substrate buffer and 40 ␮L 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. 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.

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Table 1. PPT1 and TPP1 activities in dried blood samples from patients with different forms of neuronal ceroid lipofuscinosis and from healthy controls. Patient

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Controls (n ⫽ 70)

Diagnosis

PPT1 activity, nmol/spot

TPP1 activity, nmol/spot

CLN1 CLN1 CLN1 CLN1 CLN1 CLN1 CLN2 CLN2 CLN2 CLN2 CLN2 CLN2 carrier CLN2 carrier CLN3 CLN3

0.02 0 0.02 0.02 0.03 0.01 1.13 0.8 0.49 0.41 0.88 0.42 0.46 0.54 0.49

0.22 0.34 0.39 0.27 0.31 0.3 0 0 0 0 0 0.05 0.04 0.21 0.11

0.4–1.52

0.1–0.67

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 1. Goebel HH, Mole SE, Lake BD, eds. The neuronal ceroid lipofuscinoses (Batten disease). Amsterdam: IOS Press, 1999:197 pp. 2. Van Diggelen OP, Keulemans JL, Winchester B, Hofman IL, Vanhanen SL, Santavuori P, et al. A rapid fluorogenic palmitoyl-protein thioesterase assay: pre- and postnatal diagnosis of INCL. Mol Genet Metab 1999;66:240 – 4. 3. Ezaki J, Takeda-Ezaki M, Oda K, Kominami E. Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis. Biochem Biophys Res Commun 2000;268:904 – 8. 4. Chamoles NA, Blanco MB, Gaggioli D, Casentini C. Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paper. Clin Chem 2001; 47:2098 –102. 5. Vanhanen SL, Raininko R, Autti T, Santavuori P. MRI evaluation of the brain in infantile neuronal ceroid-lipofuscinosis. Part 2. MRI findings in 21 patients. J Child Neurol 1995;10:444 –50. 6. Salonen T, Jarvela I, Peltonen L, Jalanko A. Detection of eight novel palmitoyl protein thioesterase (PPT) mutations underlying infantile neuronal ceroid lipofuscinosis (INCL; CLN1). Hum Mutat 2000;15:273–9. 7. Steinfeld R, Heim P, von Gregory H, Meyer K, Ullrich K, Goebel HH, et al. Late infantile neuronal ceroid lipofuscinosis: quantitative description of the clinical course in patients with CLN2 mutations. Am J Med Genet 2002;112:347–54. 8. Kohlschu¨ tter A, Laabs R, Albani M. Juvenile neuronal ceroid lipofuscinosis: quantitative description of its clinical variability. Acta Paediatr Scand 1988; 77:867–72.

Evaluation of a Turbidimetric Denka Seiken C-Reactive Protein Assay for Cardiovascular Risk Estimation and Conventional Inflammation Diagnosis, Thomas Christian Vukovich,* Stefan Mustafa, Helmut Rumpold, and Oswald Wagner (Institute of Medical and Chemical Laboratory Diagnostics, University Hospital of Vienna, AKH Leitstelle 5H, Waehringerguertel 18, A-1090 Vienna, Austria; * author for correspondence: fax 43-1-40400-5389, e-mail [email protected]) Measurement of C-reactive protein (CRP) is used for conventional inflammation diagnosis (1 ) and diagnosis of low-grade inflammation for risk estimation of cardiovascular events (2, 3 ). Because diagnostic measurement ranges for those two indications differ by two orders of magnitude, different methods or different applications of one method must be used at present to cover both diagnostic measurement ranges (4, 5 ). The aim of this study was to evaluate the analytical performance of the Denka Seiken turbidimetric CRP assay compared with the Dade Behring nephelometric assay across a concentration range of 0.2–300 mg/L. For this evaluation, leftover material was used, which is in concordance with the European Law for Medical and Diagnostic Products. For precision and linearity studies, we prepared serum pools from blood samples with previously measured CRP (BN II nephelometer; Dade Behring). The low and high pools were prepared by combining samples with CRP ⬍1 and 200 –300 mg/L, respectively. The high pool was diluted with the low pool to the following final percentages of high pool: 100%, 33%, 11%, 3.7%, 1.2%, 0.41%, 0.14%, and 0%. The dilutions were aliquoted and stored at ⫺20 °C for a maximum period of 4 weeks until use. Both CRP methods were used according the manufacturers’ instructions. The turbidimetric wide-range CRP assay provided by Denka Seiken [CRP-latex (II)X2 assay, calibrated against reference preparation CRM 470] was performed on a Hitachi 911 (Roche), and the nephelometric assay provided by Dade Behring was performed on a BN II nephelometer (Dade Behring). To examine the precision of the Denka Seiken method compared with the established method, aliquots of serum pool dilutions were measured in duplicate on 10 different days (Table 1). CVs were ⱕ6.8% for the Denka Seiken method and ⱕ4.4% for the Dade Behring method. For all Table 1. Summary of precision and linearity data. Denka Seiken (Hitachi 911)

Pool

Mean, mg/L

1 2 3 4 5 6 7 8

254 84.7 29.7 10.1 3.62 1.57 0.81 0.38

CV, Target, Deviation, % mg/L %

1.3 1.1 84.2 1.6 28.3 2.1 9.78 2.8 3.5 2.0 1.42 3.7 0.74 6.8

0.6 5 3 3 11 9

Dade Behring (BN II) Mean, mg/L

243 82.9 28.9 8.61 3.15 1.27 0.68 0.32

CV, Target, Deviation, % mg/L %

2.1 2.5 80.2 2.3 27 2.9 9.28 2.5 3.3 3.1 1.31 2.6 0.66 4.4

3 7 ⫺7 ⫺5 ⫺3 3