Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type II
UNIT 17.14
Britt A. Johnson,1 Otto P. van Diggelen,2,3 Angela Dajnoki,1 and Olaf A. Bodamer1 1
Division of Clinical and Translational Genetics, Dr. John T. MacDonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, Florida 2 Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 3 Erasmus University Medical College, Rotterdam, The Netherlands
ABSTRACT Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder caused by a deficiency of iduronate 2-sulfatase (IDS). Progressive, intralysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate in almost all tissues leads to multi-organ involvement in affected males but to virtual absence of symptoms in heterozygote female carriers due to preferential inactivation of the mutant allele. Diagnosis of MPS II in males is based on IDS analysis in leukocytes, fibroblasts, plasma, or dried blood spots (DBS), whereas IDS activities may be within the normal range in heterozygote females. The advent of fluorometric and mass spectrometry methods for enzyme analysis in DBS has simplified the diagnostic approach for MPS II males. Molecular analysis of the IDS gene confirms the diagnosis of MPS II in males and is the only diagnostic test to confirm carrier status in females. This unit provides detailed analytical protocols for measurement of IDS activity in DBS and plasma using a fluorometric assay. C 2013 by John Wiley & Sons, Inc. Curr. Protoc. Hum. Genet. 79:17.14.1-17.14.9. Keywords: dried blood spot r alpha-iduronate-2-sulfatase r fluorometry r mucopolysaccharidosis Type II r MPS II r Hunter Syndrome r mucopolysaccharide r glycosaminoglycans
INTRODUCTION Mucopolysaccharidosis (MPS) type II (OMIM # 309900), also known as Hunter Syndrome, is a lysosomal storage disorder that results from a deficiency of iduronate 2-sulfatase (IDS) (Neufeld and Muenzer, 2001). Deficiency of IDS leads to the progressive intralysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate in nearly all tissues and organs (Wraith et al., 2008). The IDS enzyme catalyzes the removal of a sulfate group in the first enzymatic step in the breakdown of dermatan and heparan sulfate (Wraith et al., 2008). Alpha iduronidase, the deficient enzyme in MPS I, catalyzes the second step in the breakdown of these GAGs; consequently, patients with MPS I and MPS II have similar phenotypes. The analysis of urinary GAGs is used as a first-line screening for individuals suspected of MPS disorders. In patients with MPS II, the excretion of dermatan and heparan sulfate would be typically increased; however, some patients may be missed due to the mild nature of the disease and/or preanalytical errors, such as incorrect specimen handling. The diagnosis of MPS II in males is based on the identification of low IDS activity in leukocytes, fibroblasts, plasma, or (more recently) dried blood spots (Voznyi et al., 2001; Civallero et al., 2006). Since IDS enzyme activity can be normal in heterozygous females, Biochemical Genetics Current Protocols in Human Genetics 17.14.1-17.14.9, October 2013 Published online October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142905.hg1714s79 C 2013 John Wiley & Sons, Inc. Copyright
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confirmatory diagnosis of MPS II in affected females, to determine carrier screening and for prenatal diagnosis, should be performed using molecular analysis (Wang et al., 2011). Low IDS enzyme activity is typically present in Multiple Sulfatase Deficiency (MSD), so a second sulfatase activity needs to be measured to rule out MSD (Wang et al., 2011). In addition, for quality control purposes, a second lysosomal enzyme should always be tested in samples with low IDS activity to rule out sample mishandling as the cause of the low IDS activity. BASIC PROTOCOL
MPS II DRIED BLOOD SPOT ASSAY The dried blood spot (DBS) protocol is based on previously published methods (Voznyi et al., 2001; Civallero et al., 2006). This assay is a two-step enzyme reaction. In the first step, the IDS in the patient sample removes the sulfate group from the 4-methylumbelliferyl-iduronide 2-sulfate substrate. For the second reaction, purified lysosomal enzymes containing alpha-iduronidase are added to cleave the 4-methylumbelliferyl (4 MU) from the substrate. The free 4 MU generated is measured fluorometrically and is directly proportional to the amount of IDS enzyme in the patient sample.
Materials Filter card (Whatman multipart 903 neonatal screening filter paper) containing dried blood from patient suspected to have MPS II Filter card with normal and abnormal dried blood control samples 0.2% bovine serum albumin (BSA; see recipe) Substrate (4-methylumbelliferyl-iduronide-2-sulfate; Moscerdam Substrates #M2; http://moscerdam.com/) Stop buffer (see recipe) Pi/Ci buffer (see recipe) Purified lysosomal enzymes, reconstituted (supplied with substrate; see recipe) 4-methylumbelliferyl standard (4 MU; see recipe) Hand-held puncher for 3-mm punches or DBS puncher (automated system; Perkin Elmer) 96-well flat-bottom plate (Greiner Bio-One) NCS 96-well plate incubator (Perkin Elmer) with shaker Multichannel pipettor and tips Reagent reservoirs for use with multichannel pipettors (VWR) 96-well conical-bottom plate (Fisher Scientific BD Falcon) Silicone plate sealer (Pall) Aluminum foil White 96-well flat-bottom plate (Greiner Bio-One) 96-well plate fluorometer (e.g., Bio-Tek Synergy HT) Day 1: Dried blood spot samples 1. Check the filter card containing dried blood for an area where the blood has fully soaked the filter paper from front to back. 2. In that area, punch a single DBS (diameter 3 mm) from the filter card into the second well of a flat-bottom 96-well plate using a hand-held puncher or an automated DBS puncher. Continue punching one DBS sample per well (one well per sample). Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type II
3. Punch one normal control DBS, one abnormal control DBS, and an extra punch from one patient sample to be used as a blank.
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4. Add 100 μl of 0.2% BSA to each DBS. 5. Shake at 650 rpm for 30 min in a 96-well plate shaker at 37◦ C. Thaw substrate, protected from light, during this incubation step. 6. Using a multichannel pipettor, pipet 10 μl of eluted DBS into a 96-well conicalbottom plate in duplicate. 7. Add 200 μl of stop buffer to the two blank wells. 8. Using a multichannel pipettor, add 20 μl of substrate to all wells. 9. Seal plate well with a silicone plate sealer, protect from light by covering with aluminum foil, and shake at 650 rpm at 37◦ C for 24 hr in a 96-well plate incubator shaker.
Day 2: Dried blood spot samples 10. Remove the plate from the incubator and stop the iduronate-2-sulfatase enzyme reaction by adding 20 μl of Pi/Ci buffer to each well. 11. Add 10 μl of reconstituted purified lysosomal enzymes to each well for the secondstep enzyme reaction. 12. Seal plate well, protect from light, and shake at 650 rpm at 37◦ C for 24 hr in a 96-well plate incubator shaker.
Day 3: Dried blood spot samples 13. At 1 hr before the end of plate incubation, thaw stop buffer at 37◦ C in a water bath. Let the stop buffer thaw for the full hour to ensure that it is well dissolved. Also, take out 0.2% BSA and thaw at room temperature. 14. At 40 min before the end of plate incubation, punch a control DBS into a new 96-well flat-bottom plate to use for the standards. One punch is sufficient for both standards used in the assay.
15. Add 100 μl 0.2% BSA to new standard DBS and shake at 650 rpm for 30 min. 16. Prepare standards: a. Before using the 2-level standard below, the intraday precision, interday precision, and analytical measurement range of a full 5-point standard curve must be validated in your lab. The appropriate range for the 5-point standard curve will depend on the linear range of your plate fluorometer. For the Bio-Tek Synergy HT, we chose a 6-point standard curve consisting of 0, 0.01, 0.05, 0.1, 0.5, and 1 nmol. b. Label two 1.5-ml tubes as a 0-nmol standard (0 Std) and a 0.75-nmol standard (0.75 Std). c. Add 100 μl water to the 0 Std tube. d. Add 50 μl water to the 0.75 Std tube. e. Add 50 μl of 30 μM 4 MU to the 0.75 Std tube. f. Once the new standard DBS is finished incubating, add 20 μl of eluted DBS to each standard tube. It is imperative that the eluted DBS is added to the standard samples because the hemoglobin in the sample quenches the 4 MU signal by 33%. If eluted DBS is not added to the standards, the standards will be erroneously high and the activity of the samples containing eluted DBS will be falsely decreased.
g. Add 400 μl of thawed stop buffer to each standard tube. Vortex.
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17. Enter information into the 96-well plate fluorometer about the sample and sample location. 18. Once the sample plate has finished the second 24-hr incubation step, remove plate film and add 200 μl of stop buffer to the sample and control wells using a multichannel pipettor. Pipet up and down to mix. Do not add more stop buffer to the blank wells. 19. Centrifuge plate for 10 min at 3590 × g, 4◦ C, or room temperature. 20. Pipet 200 μl of 0 Std in duplicate into a white 96-well plate. 21. Pipet 200 μl of 0.75 Std in duplicate into the 96-well plate. Protect plate from light while the sample plate is still centrifuging. 22. Once the conical-bottom plate has finished centrifuging, using a multichannel pipettor, carefully transfer 200 μl of each well to the white 96-well plate containing the standards. When finished you should have duplicates for every sample, control, standard, and blank.
23. Read plate in plate fluorometer with an excitation of 365 nm and emission of 450 nm. 24. Calculate the enzyme activity by the following formula: RFUssample ∗ 0.75 nmol/ (RFUsstd 0.75 nmol – rfusstd 0 nmol ) ∗ 1/0.00032 ml ∗ 1/24 hr = sample activity nmol/ml/hr This formula assumes 3.2 μl of blood per 3-mm DBS punch. RFU, relative fluorescence unit. ALTERNATE PROTOCOL
MPS II PLASMA ASSAY The plasma protocol is based on a previously a published method (Voznyi et al., 2001). This assay is also a two-step enzyme reaction. In the first step, the IDS in the patient sample removes the sulfate group from the 4-MU-iduronide 2-sulfate. For the second reaction, purified lysosomal enzymes containing alpha-iduronidase are added to cleave the 4 MU from the substrate. The free 4 MU generated is measured fluorometrically and is directly proportional to the amount of IDS enzyme in the patient sample.
Additional Materials Plasma from patient suspected to have MPS II Plasma from normal and abnormal control samples Day 1: Plasma samples 1. Dilute plasma samples and controls 1:5 in water (7 μl of plasma +28 μl of water). For the blank, use 0.2% BSA (undiluted). 2. Using a multichannel pipettor, pipet 10 μl of diluted plasma into a 96-well conical bottom plate in duplicate. 3. Using a multichannel pipettor, add 20 μl of substrate to all wells.
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4. Seal plate well with a silicone plate sealer, protect from light by covering with aluminum foil, and shake at 650 rpm at 37◦ C for 4 hr in a 96-well plate incubator shaker. 5. Remove the plate from the incubator and stop the iduronate-2-sulfatase enzyme reaction by adding 20 μl of Pi/Ci buffer to each well. 6. Add 10 μl of reconstituted purified lysosomal enzymes to each well for the secondstep enzyme reaction. Current Protocols in Human Genetics
7. Seal plate well, protect from light, and shake at 650 rpm at 37◦ C for 24 hr in a 96-well plate incubator shaker.
Day 2: Plasma samples 8. At 1 hr before the end of plate incubation, thaw stop buffer at 37◦ C in a water bath. Let the stop buffer thaw for the full hour to ensure that it is well dissolved.
9. Prepare standards: a. Before using the 2-level standard below, intraday precision, interday precision, and the analytical measurement range of a full 5-point standard curve must be validated in your lab. b. Label two 1.5-ml tubes as a 0-nmol standard (0 Std) and a 0.75-nmol standard (0.75 Std). c. Add 120 μl water to the 0 Std tube. d. Add 70 μl water to the 0.75 Std tube. e. Add 50 μl of 30 μM 4 MU to the 0.75 Std tube. f. Add 400 μl of thawed stop buffer to each standard tube. Vortex. 10. Prepare the 96-well plate fluorometer with sample information and sample location. 11. Once the sample plate has finished the second 24-hr incubation step, remove plate film and add 200 μl of stop buffer using a multichannel pipettor to the sample, control, and blank wells. Pipet up and down to mix. 12. Pipet 200 μl of 0 Std in duplicate into a white 96-well plate. 13. Pipet 200 μl of 0.75 Std in duplicate into the 96-well plate. 14. Using a multichannel pipettor, carefully transfer 200 μl of each well to the white 96-well plate containing the standards. When finished, you should have duplicates for every sample, control, standard, and blank.
15. Read plate in plate fluorometer with an excitation of 365 nm and emission of 450 nm. 16. Calculate the enzyme activity by the following formula: RFUssample ∗ 0.75 nmol/ (RFUsstd 0.75 nmol – RFUsstd 0 nmol ) /0.01 ml activity nmol/ml/4 hr
∗
5 = sample
The 4 hr in the sample activity units is referring to the first enzyme reaction, not the second. Interpretation of results: For expected control and disease ranges, see Anticipated Results below.
REAGENTS AND SOLUTIONS Use HPLC-grade or deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2D; for suppliers, see SUPPLIERS APPENDIX.
Bovine serum albumin (BSA), 0.2% 1. Weigh 2.5 g BSA into a 100-ml beaker. 2. Add 50 ml water, making sure that the solid BSA does not stick to the sides of the beaker above the water level. continued
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3. Seal beaker with foil and leave overnight at 4◦ C (no stirring necessary). 4. Bring the 5% BSA solution to room temperature and adjust the pH to 10.0 with 6 M NaOH. 5. Heat BSA in a 50◦ C water bath for 4 hr without stirring. 6. Allow BSA to cool. 7. Adjust pH to 7.0 with 1 M HCl. 8. Centrifuge for 10 min at 3590 × g, 4◦ C or room temperature and collect supernatant. 9. Dilute the heat-inactivated 5% BSA solution 25-fold in 0.02% sodium azide in water. 10. Store the 0.2% BSA in 1-ml aliquots at −20◦ C.
4-methylumbelliferyl (4 MU) standard, 30 μM 1. First make a 30 mM concentrated stock solution: a. Add 52.85 mg of 4 MU (Sigma; MW = 176.17) to a 10-ml class A volumetric flask. b. Bring to 10 ml with 20 mM sodium phosphate buffer (see recipe below). c. Mix well and make sure everything is dissolved. 2. Take 1 ml of concentrated 4 MU stock and bring to 1000 ml with 20 mM sodium phosphate buffer (see recipe below) in a class A volumetric flask. Mix well. 3. Take 200 ml of solution made in step 2 and make 300-μl aliquots in 1.5-ml amber microcentrifuge tubes. 4. Store aliquots at −20◦ C. Use a freshly thawed tube of standard each time assay is run.
Pi/Ci buffer (McIlvains phosphate/citric acid buffer: 0.2 M Na2 HPO4 / 0.1 M citric acid buffer, pH 4.5 + 0.02% sodium azide) 1. Add the following to a 100 ml beaker: 2.84 g Na2 HPO4 (Sigma) 2.1 g citric acid monohydrate (Sigma) 20 mg sodium azide (Sigma). 2. Add 75 ml water. 3. pH to 4.5 with phosphoric acid. 4. Bring to 100 ml. 5. Aliquot in 15-ml conical tubes and store at −20◦ C. Keep one tube at room temperature.
Purified lysosomal enzymes, reconstituted 1. Reconstitute one vial of purified lysosomal enzymes from bovine testis (included with Moscerdam Substrates #M2) with 2.2 ml of water. 2. Store 500-μl aliquots at −80◦ C. Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type II
Sodium phosphate buffer, 20 mM Dissolve 2.84 g of Na2 HPO4 in 1000 ml of water.
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Stop buffer (0.5 M NaHCO3 /0.5 M Na2 CO3 buffer, pH 10.7 + 0.025% Triton-X 100) 1. Add the following to a 500 ml beaker: 26.5 g sodium carbonate (Na2 CO3 ; Sigma) 21 g sodium bicarbonate (NaHCO3 ; Sigma). 2. Add 450 ml water. 3. pH to 10.7 using 6 N NaOH. 4. Add 125 μl Triton-X 100 (Sigma). 5. Bring to 500 ml.
Substrate buffer (0.1 M sodium acetate/0.1 M acetic acid buffer pH 5.0, with 0.2% sodium azide and 10 mM lead acetate) 1. To a 100-ml beaker, add the following: 0.82 g sodium acetate (Sigma) 0.945 g lead acetate trihydrate (Sigma; MW 379) 571 μl glacial acetic acid (EMD). 2. Add 75 ml of 0.2% sodium azide in water. 3. pH to 5.0 with acetic acid or NaOH as needed. 4. Bring to 100 ml. 5. Store at room temperature.
Substrate (4-methylumbelliferyl-iduronide-2-sulfate) 1. Add 3 ml of substrate buffer (see recipe above) to vial containing 5 mg 4methylumbelliferyl-iduronide 2-sulfate (Moscerdam Substrates #M2), dissolve, and transfer to a 10-ml tube. 2. Rinse substrate vial with 3 ml of substrate buffer and add this to the 10-ml tube. 3. Add 2.33 ml of substrate buffer to the 10-ml tube for a total of 8.33 ml. 4. Make 500-μl aliquots of substrate and store immediately at −80◦ C.
COMMENTARY Background Information The main clinical features in MPS II include progressive facial coarsening, hepatosplenomegaly, short stature, dysostosis multiplex, joint contractures, obstructive airway disease, hearing loss, cardiomyopathy, and valvular heart disease (Wraith et al., 2008). Many but not all patients have central nervous system (CNS) involvement that leads to severe, progressive intellectual disability (Wraith et al., 2008). The clinical phenotype of MPS II follows a clinical continuum ranging from the more severe form with cognitive regression that is typically diagnosed between 18 and 36 months of life and resulting in death by the second decade of life to a milder form characterized with limited or no CNS involvement, an age of diagnosis around 4 to 8 years, and death
occurring as late as the sixth decade of life (Wraith et al., 2008; Wang et al., 2011). IDS is encoded by the IDS gene located on Xq28. The vast majority of MPS II patients are males. The estimated frequency of MPS II ranges from 1 in 76,000 to 1 in 320,000 live male births (Poorthuis et al., 1999; Nelson et al., 2003; Baehner et al., 2005). However, very few affected females have been reported (Tuschl et al., 2005). Affected females tend to have a severe phenotype due to skewed Xinactivation (Tuschl et al., 2005). Affected female patients have been reported with translocations or complex rearrangements involving the IDS gene or homozygous mutations in IDS (Tuschl et al., 2005). The majority of affected females reported thus far had a clinical phenotype comparable to the more severe forms seen in affected males (Tuschl et al., 2005).
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Table 17.14.1 MPS II Enzyme Expected Reference and Disease Ranges—Dried Blood Spots
Dried blood spots
N
Mean ± SD (nmol/ml/hr )
Range (nmol/ml/hr )
Control newborns
49
22.84 ± 5.66
13.51-44.18
Control adults
20
20.35 ± 4.11
13.45-27.52
MPS II patients
17
4.06 ± 1.15
1.60-5.95
Table 17.14.2 MPS II Enzyme Expected Reference and Disease Ranges—Plasma
Plasma
N
Range (nmol/ml/4 hr )
Controls
16
167-475
MPS II patients
19
0-8
Enzyme replacement therapy (ERT) using recombinant human IDS (Elaprase; Shire, Cambridge, MA) is available for the treatment for MPS II. ERT has been shown to reduce hepatosplenomegaly, increase joint motility, and normalize the urine GAG accumulation in the majority of patients (Wang et al., 2011). ERT may be able to slow disease progression when started early near the onset of disease. However, the recombinant enzyme does not cross the blood-brain barrier and has therefore no effect on brain function and intellectual abilities. Early diagnosis and initiation of treatment may be nevertheless beneficial for patients with MPS II, which has formed the basis of advocacy for newborn screening of lysosomal storage disorders.
Critical Parameters and Troubleshooting
Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type II
To save substrate costs, only one blank sample is used per plate. If patient samples being tested were collected at vastly different times, either each patient sample could also be tested with a blank reaction, or all positive samples could be measured a second time using their own blank. In our experience, using one blank per plate can give a false positive result rarely but does not give false negative results. It is suggested to analyze a second lysosomal enzyme, such as beta-galactosidase, in parallel in the same DBS filter card as internal quality control (QC) to correct for sample integrity and quality. In cases where both enzymes come back as low, a second specimen needs to be requested for retesting. For all positive specimens, a second sulfatase enzyme, such as arylsulfatase B, should be tested to rule out Multiple Sulfatase Deficiency.
Additional QC samples (high and low control) as well as blank samples (stop buffer added before the addition of substrate) need to be included with each batch (e.g., each 96well plate).
Anticipated Results IDS activity in DBS was tested in 49 control newborns, 20 control adults, and 17 male patients with MPS II (Table 17.14.1). IDS activity in plasma was tested previously in the laboratory (Table 17.14.2). These tables show the mean, standard deviation, and range of IDS activities for each group. The MPS II patients are clearly distinguishable from controls.
Time Considerations For dried blood spots, the protocol takes 2.5 business days including the two 24-hr overnight incubation steps. For plasma samples, the protocol takes 1.5 business days including the 4-hr and 24-hr overnight incubation steps. An analytical run on the fluorometer takes approximately 5 min for a full 96-well plate.
Acknowledgments O. van Diggelen serves as a scientific advisor to Moscerdam Substrates.
Literature Cited Baehner, F., Schmiedeskamp, C., Krummenauer, F., Miebach, E., Bajbouj, M., Whybra, C., Kohlschutter, A., Kampmann, C., and Beck, M. 2005. Cumulative incidence rates of the mucopolysaccharidoses in Germany. J. Inherit. Metab. Dis. 28:1011-1017. Civallero, G., Michelin, K., de Mari, J., Viapiana, M., Burin, M., Coelho, J.C., and Giugliani, R. 2006. Twelve different enzyme assays on
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dried-blood filter paper samples for detection of patients with selected inherited lysosomal storage diseases. Clin. Chim. Acta 372: 98-102.
Tuschl, K., Gal, A., Paschke, E., Kircher, S., and Bodamer, O.A. 2005. Mucopolysaccharidosis type II in females: Case report and review of literature. Pediatr. Neurol. 32:270-272.
Nelson, J., Crowhurst, J., Carey, B., and Greed, L. 2003. Incidence of the mucopolysaccharidoses in Western Australia. Am. J. Med. Genet. A 123A:310-313.
Voznyi, Y.V., Keulemans, J.L., and van Diggelen, O.P. 2001. A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease). J. Inherit. Metab. Dis. 24:675-680.
Neufeld, E.F. and Muenzer, J. 2001. The mucopolysaccharidoses. In The Metabolic and Molecular Basis of Inherited Disease, 8th ed. (C.R Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.) pp. 3421-3452. McGraw-Hill, New York.
Wang, R.Y., Bodamer, O.A., Watson, M.S., and Wilcox, W.R. 2011. Lysosomal storage diseases: Diagnostic confirmation and management of presymptomatic individuals. Genet. Med. 13:457-484.
Poorthuis, B.J., Wevers, R.A., Kleijer, W.J., Groener, J.E., de Jong, J.G., van Weely, S., Niezen-Koning, K.E., and van Diggelen, O.P. 1999. The frequency of lysosomal storage diseases in The Netherlands. Hum. Genet. 105: 151-156.
Wraith, J.E., Scarpa, M., Beck, M., Bodamer, O.A., De Meirleir, L., Guffon, N., Meldgaard Lund, A., Malm, G., Van der Ploeg, A.T., and Zeman, J. 2008. Mucopolysaccharidosis type II (Hunter syndrome): A clinical review and recommendations for treatment in the era of enzyme replacement therapy. Eur. J. Pediatr. 167:267-277.
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UNIT 17.14
Britt A. Johnson,1 Otto P. van Diggelen,2,3 Angela Dajnoki,1 and Olaf A. Bodamer1 1
Division of Clinical and Translational Genetics, Dr. John T. MacDonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, Florida 2 Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 3 Erasmus University Medical College, Rotterdam, The Netherlands
ABSTRACT Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder caused by a deficiency of iduronate 2-sulfatase (IDS). Progressive, intralysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate in almost all tissues leads to multi-organ involvement in affected males but to virtual absence of symptoms in heterozygote female carriers due to preferential inactivation of the mutant allele. Diagnosis of MPS II in males is based on IDS analysis in leukocytes, fibroblasts, plasma, or dried blood spots (DBS), whereas IDS activities may be within the normal range in heterozygote females. The advent of fluorometric and mass spectrometry methods for enzyme analysis in DBS has simplified the diagnostic approach for MPS II males. Molecular analysis of the IDS gene confirms the diagnosis of MPS II in males and is the only diagnostic test to confirm carrier status in females. This unit provides detailed analytical protocols for measurement of IDS activity in DBS and plasma using a fluorometric assay. C 2013 by John Wiley & Sons, Inc. Curr. Protoc. Hum. Genet. 79:17.14.1-17.14.9. Keywords: dried blood spot r alpha-iduronate-2-sulfatase r fluorometry r mucopolysaccharidosis Type II r MPS II r Hunter Syndrome r mucopolysaccharide r glycosaminoglycans
INTRODUCTION Mucopolysaccharidosis (MPS) type II (OMIM # 309900), also known as Hunter Syndrome, is a lysosomal storage disorder that results from a deficiency of iduronate 2-sulfatase (IDS) (Neufeld and Muenzer, 2001). Deficiency of IDS leads to the progressive intralysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate in nearly all tissues and organs (Wraith et al., 2008). The IDS enzyme catalyzes the removal of a sulfate group in the first enzymatic step in the breakdown of dermatan and heparan sulfate (Wraith et al., 2008). Alpha iduronidase, the deficient enzyme in MPS I, catalyzes the second step in the breakdown of these GAGs; consequently, patients with MPS I and MPS II have similar phenotypes. The analysis of urinary GAGs is used as a first-line screening for individuals suspected of MPS disorders. In patients with MPS II, the excretion of dermatan and heparan sulfate would be typically increased; however, some patients may be missed due to the mild nature of the disease and/or preanalytical errors, such as incorrect specimen handling. The diagnosis of MPS II in males is based on the identification of low IDS activity in leukocytes, fibroblasts, plasma, or (more recently) dried blood spots (Voznyi et al., 2001; Civallero et al., 2006). Since IDS enzyme activity can be normal in heterozygous females, Biochemical Genetics Current Protocols in Human Genetics 17.14.1-17.14.9, October 2013 Published online October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142905.hg1714s79 C 2013 John Wiley & Sons, Inc. Copyright
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confirmatory diagnosis of MPS II in affected females, to determine carrier screening and for prenatal diagnosis, should be performed using molecular analysis (Wang et al., 2011). Low IDS enzyme activity is typically present in Multiple Sulfatase Deficiency (MSD), so a second sulfatase activity needs to be measured to rule out MSD (Wang et al., 2011). In addition, for quality control purposes, a second lysosomal enzyme should always be tested in samples with low IDS activity to rule out sample mishandling as the cause of the low IDS activity. BASIC PROTOCOL
MPS II DRIED BLOOD SPOT ASSAY The dried blood spot (DBS) protocol is based on previously published methods (Voznyi et al., 2001; Civallero et al., 2006). This assay is a two-step enzyme reaction. In the first step, the IDS in the patient sample removes the sulfate group from the 4-methylumbelliferyl-iduronide 2-sulfate substrate. For the second reaction, purified lysosomal enzymes containing alpha-iduronidase are added to cleave the 4-methylumbelliferyl (4 MU) from the substrate. The free 4 MU generated is measured fluorometrically and is directly proportional to the amount of IDS enzyme in the patient sample.
Materials Filter card (Whatman multipart 903 neonatal screening filter paper) containing dried blood from patient suspected to have MPS II Filter card with normal and abnormal dried blood control samples 0.2% bovine serum albumin (BSA; see recipe) Substrate (4-methylumbelliferyl-iduronide-2-sulfate; Moscerdam Substrates #M2; http://moscerdam.com/) Stop buffer (see recipe) Pi/Ci buffer (see recipe) Purified lysosomal enzymes, reconstituted (supplied with substrate; see recipe) 4-methylumbelliferyl standard (4 MU; see recipe) Hand-held puncher for 3-mm punches or DBS puncher (automated system; Perkin Elmer) 96-well flat-bottom plate (Greiner Bio-One) NCS 96-well plate incubator (Perkin Elmer) with shaker Multichannel pipettor and tips Reagent reservoirs for use with multichannel pipettors (VWR) 96-well conical-bottom plate (Fisher Scientific BD Falcon) Silicone plate sealer (Pall) Aluminum foil White 96-well flat-bottom plate (Greiner Bio-One) 96-well plate fluorometer (e.g., Bio-Tek Synergy HT) Day 1: Dried blood spot samples 1. Check the filter card containing dried blood for an area where the blood has fully soaked the filter paper from front to back. 2. In that area, punch a single DBS (diameter 3 mm) from the filter card into the second well of a flat-bottom 96-well plate using a hand-held puncher or an automated DBS puncher. Continue punching one DBS sample per well (one well per sample). Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type II
3. Punch one normal control DBS, one abnormal control DBS, and an extra punch from one patient sample to be used as a blank.
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4. Add 100 μl of 0.2% BSA to each DBS. 5. Shake at 650 rpm for 30 min in a 96-well plate shaker at 37◦ C. Thaw substrate, protected from light, during this incubation step. 6. Using a multichannel pipettor, pipet 10 μl of eluted DBS into a 96-well conicalbottom plate in duplicate. 7. Add 200 μl of stop buffer to the two blank wells. 8. Using a multichannel pipettor, add 20 μl of substrate to all wells. 9. Seal plate well with a silicone plate sealer, protect from light by covering with aluminum foil, and shake at 650 rpm at 37◦ C for 24 hr in a 96-well plate incubator shaker.
Day 2: Dried blood spot samples 10. Remove the plate from the incubator and stop the iduronate-2-sulfatase enzyme reaction by adding 20 μl of Pi/Ci buffer to each well. 11. Add 10 μl of reconstituted purified lysosomal enzymes to each well for the secondstep enzyme reaction. 12. Seal plate well, protect from light, and shake at 650 rpm at 37◦ C for 24 hr in a 96-well plate incubator shaker.
Day 3: Dried blood spot samples 13. At 1 hr before the end of plate incubation, thaw stop buffer at 37◦ C in a water bath. Let the stop buffer thaw for the full hour to ensure that it is well dissolved. Also, take out 0.2% BSA and thaw at room temperature. 14. At 40 min before the end of plate incubation, punch a control DBS into a new 96-well flat-bottom plate to use for the standards. One punch is sufficient for both standards used in the assay.
15. Add 100 μl 0.2% BSA to new standard DBS and shake at 650 rpm for 30 min. 16. Prepare standards: a. Before using the 2-level standard below, the intraday precision, interday precision, and analytical measurement range of a full 5-point standard curve must be validated in your lab. The appropriate range for the 5-point standard curve will depend on the linear range of your plate fluorometer. For the Bio-Tek Synergy HT, we chose a 6-point standard curve consisting of 0, 0.01, 0.05, 0.1, 0.5, and 1 nmol. b. Label two 1.5-ml tubes as a 0-nmol standard (0 Std) and a 0.75-nmol standard (0.75 Std). c. Add 100 μl water to the 0 Std tube. d. Add 50 μl water to the 0.75 Std tube. e. Add 50 μl of 30 μM 4 MU to the 0.75 Std tube. f. Once the new standard DBS is finished incubating, add 20 μl of eluted DBS to each standard tube. It is imperative that the eluted DBS is added to the standard samples because the hemoglobin in the sample quenches the 4 MU signal by 33%. If eluted DBS is not added to the standards, the standards will be erroneously high and the activity of the samples containing eluted DBS will be falsely decreased.
g. Add 400 μl of thawed stop buffer to each standard tube. Vortex.
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17. Enter information into the 96-well plate fluorometer about the sample and sample location. 18. Once the sample plate has finished the second 24-hr incubation step, remove plate film and add 200 μl of stop buffer to the sample and control wells using a multichannel pipettor. Pipet up and down to mix. Do not add more stop buffer to the blank wells. 19. Centrifuge plate for 10 min at 3590 × g, 4◦ C, or room temperature. 20. Pipet 200 μl of 0 Std in duplicate into a white 96-well plate. 21. Pipet 200 μl of 0.75 Std in duplicate into the 96-well plate. Protect plate from light while the sample plate is still centrifuging. 22. Once the conical-bottom plate has finished centrifuging, using a multichannel pipettor, carefully transfer 200 μl of each well to the white 96-well plate containing the standards. When finished you should have duplicates for every sample, control, standard, and blank.
23. Read plate in plate fluorometer with an excitation of 365 nm and emission of 450 nm. 24. Calculate the enzyme activity by the following formula: RFUssample ∗ 0.75 nmol/ (RFUsstd 0.75 nmol – rfusstd 0 nmol ) ∗ 1/0.00032 ml ∗ 1/24 hr = sample activity nmol/ml/hr This formula assumes 3.2 μl of blood per 3-mm DBS punch. RFU, relative fluorescence unit. ALTERNATE PROTOCOL
MPS II PLASMA ASSAY The plasma protocol is based on a previously a published method (Voznyi et al., 2001). This assay is also a two-step enzyme reaction. In the first step, the IDS in the patient sample removes the sulfate group from the 4-MU-iduronide 2-sulfate. For the second reaction, purified lysosomal enzymes containing alpha-iduronidase are added to cleave the 4 MU from the substrate. The free 4 MU generated is measured fluorometrically and is directly proportional to the amount of IDS enzyme in the patient sample.
Additional Materials Plasma from patient suspected to have MPS II Plasma from normal and abnormal control samples Day 1: Plasma samples 1. Dilute plasma samples and controls 1:5 in water (7 μl of plasma +28 μl of water). For the blank, use 0.2% BSA (undiluted). 2. Using a multichannel pipettor, pipet 10 μl of diluted plasma into a 96-well conical bottom plate in duplicate. 3. Using a multichannel pipettor, add 20 μl of substrate to all wells.
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4. Seal plate well with a silicone plate sealer, protect from light by covering with aluminum foil, and shake at 650 rpm at 37◦ C for 4 hr in a 96-well plate incubator shaker. 5. Remove the plate from the incubator and stop the iduronate-2-sulfatase enzyme reaction by adding 20 μl of Pi/Ci buffer to each well. 6. Add 10 μl of reconstituted purified lysosomal enzymes to each well for the secondstep enzyme reaction. Current Protocols in Human Genetics
7. Seal plate well, protect from light, and shake at 650 rpm at 37◦ C for 24 hr in a 96-well plate incubator shaker.
Day 2: Plasma samples 8. At 1 hr before the end of plate incubation, thaw stop buffer at 37◦ C in a water bath. Let the stop buffer thaw for the full hour to ensure that it is well dissolved.
9. Prepare standards: a. Before using the 2-level standard below, intraday precision, interday precision, and the analytical measurement range of a full 5-point standard curve must be validated in your lab. b. Label two 1.5-ml tubes as a 0-nmol standard (0 Std) and a 0.75-nmol standard (0.75 Std). c. Add 120 μl water to the 0 Std tube. d. Add 70 μl water to the 0.75 Std tube. e. Add 50 μl of 30 μM 4 MU to the 0.75 Std tube. f. Add 400 μl of thawed stop buffer to each standard tube. Vortex. 10. Prepare the 96-well plate fluorometer with sample information and sample location. 11. Once the sample plate has finished the second 24-hr incubation step, remove plate film and add 200 μl of stop buffer using a multichannel pipettor to the sample, control, and blank wells. Pipet up and down to mix. 12. Pipet 200 μl of 0 Std in duplicate into a white 96-well plate. 13. Pipet 200 μl of 0.75 Std in duplicate into the 96-well plate. 14. Using a multichannel pipettor, carefully transfer 200 μl of each well to the white 96-well plate containing the standards. When finished, you should have duplicates for every sample, control, standard, and blank.
15. Read plate in plate fluorometer with an excitation of 365 nm and emission of 450 nm. 16. Calculate the enzyme activity by the following formula: RFUssample ∗ 0.75 nmol/ (RFUsstd 0.75 nmol – RFUsstd 0 nmol ) /0.01 ml activity nmol/ml/4 hr
∗
5 = sample
The 4 hr in the sample activity units is referring to the first enzyme reaction, not the second. Interpretation of results: For expected control and disease ranges, see Anticipated Results below.
REAGENTS AND SOLUTIONS Use HPLC-grade or deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2D; for suppliers, see SUPPLIERS APPENDIX.
Bovine serum albumin (BSA), 0.2% 1. Weigh 2.5 g BSA into a 100-ml beaker. 2. Add 50 ml water, making sure that the solid BSA does not stick to the sides of the beaker above the water level. continued
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3. Seal beaker with foil and leave overnight at 4◦ C (no stirring necessary). 4. Bring the 5% BSA solution to room temperature and adjust the pH to 10.0 with 6 M NaOH. 5. Heat BSA in a 50◦ C water bath for 4 hr without stirring. 6. Allow BSA to cool. 7. Adjust pH to 7.0 with 1 M HCl. 8. Centrifuge for 10 min at 3590 × g, 4◦ C or room temperature and collect supernatant. 9. Dilute the heat-inactivated 5% BSA solution 25-fold in 0.02% sodium azide in water. 10. Store the 0.2% BSA in 1-ml aliquots at −20◦ C.
4-methylumbelliferyl (4 MU) standard, 30 μM 1. First make a 30 mM concentrated stock solution: a. Add 52.85 mg of 4 MU (Sigma; MW = 176.17) to a 10-ml class A volumetric flask. b. Bring to 10 ml with 20 mM sodium phosphate buffer (see recipe below). c. Mix well and make sure everything is dissolved. 2. Take 1 ml of concentrated 4 MU stock and bring to 1000 ml with 20 mM sodium phosphate buffer (see recipe below) in a class A volumetric flask. Mix well. 3. Take 200 ml of solution made in step 2 and make 300-μl aliquots in 1.5-ml amber microcentrifuge tubes. 4. Store aliquots at −20◦ C. Use a freshly thawed tube of standard each time assay is run.
Pi/Ci buffer (McIlvains phosphate/citric acid buffer: 0.2 M Na2 HPO4 / 0.1 M citric acid buffer, pH 4.5 + 0.02% sodium azide) 1. Add the following to a 100 ml beaker: 2.84 g Na2 HPO4 (Sigma) 2.1 g citric acid monohydrate (Sigma) 20 mg sodium azide (Sigma). 2. Add 75 ml water. 3. pH to 4.5 with phosphoric acid. 4. Bring to 100 ml. 5. Aliquot in 15-ml conical tubes and store at −20◦ C. Keep one tube at room temperature.
Purified lysosomal enzymes, reconstituted 1. Reconstitute one vial of purified lysosomal enzymes from bovine testis (included with Moscerdam Substrates #M2) with 2.2 ml of water. 2. Store 500-μl aliquots at −80◦ C. Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type II
Sodium phosphate buffer, 20 mM Dissolve 2.84 g of Na2 HPO4 in 1000 ml of water.
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Stop buffer (0.5 M NaHCO3 /0.5 M Na2 CO3 buffer, pH 10.7 + 0.025% Triton-X 100) 1. Add the following to a 500 ml beaker: 26.5 g sodium carbonate (Na2 CO3 ; Sigma) 21 g sodium bicarbonate (NaHCO3 ; Sigma). 2. Add 450 ml water. 3. pH to 10.7 using 6 N NaOH. 4. Add 125 μl Triton-X 100 (Sigma). 5. Bring to 500 ml.
Substrate buffer (0.1 M sodium acetate/0.1 M acetic acid buffer pH 5.0, with 0.2% sodium azide and 10 mM lead acetate) 1. To a 100-ml beaker, add the following: 0.82 g sodium acetate (Sigma) 0.945 g lead acetate trihydrate (Sigma; MW 379) 571 μl glacial acetic acid (EMD). 2. Add 75 ml of 0.2% sodium azide in water. 3. pH to 5.0 with acetic acid or NaOH as needed. 4. Bring to 100 ml. 5. Store at room temperature.
Substrate (4-methylumbelliferyl-iduronide-2-sulfate) 1. Add 3 ml of substrate buffer (see recipe above) to vial containing 5 mg 4methylumbelliferyl-iduronide 2-sulfate (Moscerdam Substrates #M2), dissolve, and transfer to a 10-ml tube. 2. Rinse substrate vial with 3 ml of substrate buffer and add this to the 10-ml tube. 3. Add 2.33 ml of substrate buffer to the 10-ml tube for a total of 8.33 ml. 4. Make 500-μl aliquots of substrate and store immediately at −80◦ C.
COMMENTARY Background Information The main clinical features in MPS II include progressive facial coarsening, hepatosplenomegaly, short stature, dysostosis multiplex, joint contractures, obstructive airway disease, hearing loss, cardiomyopathy, and valvular heart disease (Wraith et al., 2008). Many but not all patients have central nervous system (CNS) involvement that leads to severe, progressive intellectual disability (Wraith et al., 2008). The clinical phenotype of MPS II follows a clinical continuum ranging from the more severe form with cognitive regression that is typically diagnosed between 18 and 36 months of life and resulting in death by the second decade of life to a milder form characterized with limited or no CNS involvement, an age of diagnosis around 4 to 8 years, and death
occurring as late as the sixth decade of life (Wraith et al., 2008; Wang et al., 2011). IDS is encoded by the IDS gene located on Xq28. The vast majority of MPS II patients are males. The estimated frequency of MPS II ranges from 1 in 76,000 to 1 in 320,000 live male births (Poorthuis et al., 1999; Nelson et al., 2003; Baehner et al., 2005). However, very few affected females have been reported (Tuschl et al., 2005). Affected females tend to have a severe phenotype due to skewed Xinactivation (Tuschl et al., 2005). Affected female patients have been reported with translocations or complex rearrangements involving the IDS gene or homozygous mutations in IDS (Tuschl et al., 2005). The majority of affected females reported thus far had a clinical phenotype comparable to the more severe forms seen in affected males (Tuschl et al., 2005).
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Table 17.14.1 MPS II Enzyme Expected Reference and Disease Ranges—Dried Blood Spots
Dried blood spots
N
Mean ± SD (nmol/ml/hr )
Range (nmol/ml/hr )
Control newborns
49
22.84 ± 5.66
13.51-44.18
Control adults
20
20.35 ± 4.11
13.45-27.52
MPS II patients
17
4.06 ± 1.15
1.60-5.95
Table 17.14.2 MPS II Enzyme Expected Reference and Disease Ranges—Plasma
Plasma
N
Range (nmol/ml/4 hr )
Controls
16
167-475
MPS II patients
19
0-8
Enzyme replacement therapy (ERT) using recombinant human IDS (Elaprase; Shire, Cambridge, MA) is available for the treatment for MPS II. ERT has been shown to reduce hepatosplenomegaly, increase joint motility, and normalize the urine GAG accumulation in the majority of patients (Wang et al., 2011). ERT may be able to slow disease progression when started early near the onset of disease. However, the recombinant enzyme does not cross the blood-brain barrier and has therefore no effect on brain function and intellectual abilities. Early diagnosis and initiation of treatment may be nevertheless beneficial for patients with MPS II, which has formed the basis of advocacy for newborn screening of lysosomal storage disorders.
Critical Parameters and Troubleshooting
Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type II
To save substrate costs, only one blank sample is used per plate. If patient samples being tested were collected at vastly different times, either each patient sample could also be tested with a blank reaction, or all positive samples could be measured a second time using their own blank. In our experience, using one blank per plate can give a false positive result rarely but does not give false negative results. It is suggested to analyze a second lysosomal enzyme, such as beta-galactosidase, in parallel in the same DBS filter card as internal quality control (QC) to correct for sample integrity and quality. In cases where both enzymes come back as low, a second specimen needs to be requested for retesting. For all positive specimens, a second sulfatase enzyme, such as arylsulfatase B, should be tested to rule out Multiple Sulfatase Deficiency.
Additional QC samples (high and low control) as well as blank samples (stop buffer added before the addition of substrate) need to be included with each batch (e.g., each 96well plate).
Anticipated Results IDS activity in DBS was tested in 49 control newborns, 20 control adults, and 17 male patients with MPS II (Table 17.14.1). IDS activity in plasma was tested previously in the laboratory (Table 17.14.2). These tables show the mean, standard deviation, and range of IDS activities for each group. The MPS II patients are clearly distinguishable from controls.
Time Considerations For dried blood spots, the protocol takes 2.5 business days including the two 24-hr overnight incubation steps. For plasma samples, the protocol takes 1.5 business days including the 4-hr and 24-hr overnight incubation steps. An analytical run on the fluorometer takes approximately 5 min for a full 96-well plate.
Acknowledgments O. van Diggelen serves as a scientific advisor to Moscerdam Substrates.
Literature Cited Baehner, F., Schmiedeskamp, C., Krummenauer, F., Miebach, E., Bajbouj, M., Whybra, C., Kohlschutter, A., Kampmann, C., and Beck, M. 2005. Cumulative incidence rates of the mucopolysaccharidoses in Germany. J. Inherit. Metab. Dis. 28:1011-1017. Civallero, G., Michelin, K., de Mari, J., Viapiana, M., Burin, M., Coelho, J.C., and Giugliani, R. 2006. Twelve different enzyme assays on
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dried-blood filter paper samples for detection of patients with selected inherited lysosomal storage diseases. Clin. Chim. Acta 372: 98-102.
Tuschl, K., Gal, A., Paschke, E., Kircher, S., and Bodamer, O.A. 2005. Mucopolysaccharidosis type II in females: Case report and review of literature. Pediatr. Neurol. 32:270-272.
Nelson, J., Crowhurst, J., Carey, B., and Greed, L. 2003. Incidence of the mucopolysaccharidoses in Western Australia. Am. J. Med. Genet. A 123A:310-313.
Voznyi, Y.V., Keulemans, J.L., and van Diggelen, O.P. 2001. A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease). J. Inherit. Metab. Dis. 24:675-680.
Neufeld, E.F. and Muenzer, J. 2001. The mucopolysaccharidoses. In The Metabolic and Molecular Basis of Inherited Disease, 8th ed. (C.R Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.) pp. 3421-3452. McGraw-Hill, New York.
Wang, R.Y., Bodamer, O.A., Watson, M.S., and Wilcox, W.R. 2011. Lysosomal storage diseases: Diagnostic confirmation and management of presymptomatic individuals. Genet. Med. 13:457-484.
Poorthuis, B.J., Wevers, R.A., Kleijer, W.J., Groener, J.E., de Jong, J.G., van Weely, S., Niezen-Koning, K.E., and van Diggelen, O.P. 1999. The frequency of lysosomal storage diseases in The Netherlands. Hum. Genet. 105: 151-156.
Wraith, J.E., Scarpa, M., Beck, M., Bodamer, O.A., De Meirleir, L., Guffon, N., Meldgaard Lund, A., Malm, G., Van der Ploeg, A.T., and Zeman, J. 2008. Mucopolysaccharidosis type II (Hunter syndrome): A clinical review and recommendations for treatment in the era of enzyme replacement therapy. Eur. J. Pediatr. 167:267-277.
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