Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type I
UNIT 17.17
Britt A. Johnson,1 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
Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha iduronidase (IDUA). Progressive storage of dermatan and heparan sulfate throughout the body lead to a multiorgan presentation including short stature, dysostosis multiplex, corneal clouding, hearing loss, coarse facies, hepatosplenomegaly, and intellectual disability. Diagnosis of MPS I is based on IDUA enzyme analysis in leukocytes or dried blood spots (DBS) followed by molecular confirmation of the IDUA gene mutations in individuals with low enzyme activity. The advent of mass spectrometry methods for enzyme analysis in DBS has enabled high-throughput screening for MPS I in symptomatic individuals and newborn infants. The following unit provides the detailed analytical protocol for measurement of IDUA activity in DBS using C 2015 by John Wiley & Sons, Inc. tandem mass spectrometry. Keywords: dried blood spot r alpha-iduronidase r tandem mass spectrometry r MPS I r Mucopolysaccharidosis type I r Hurler Syndrome r Scheie Syndrome
How to cite this article: Johnson, B.A., Dajnoki, A., and Bodamer, O.A. 2015. Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type I. Curr. Protoc. Hum. Genet. 84:17.17.1-17.17.8. doi: 10.1002/0471142905.hg1717s84
Mucopolysaccharidosis type I (MPS I, OMIM #607014, 607015, 607016) is a progressive lysosomal storage disorder that results from a defect in alpha-L-iduronidase activity (Bach et al., 1972). The mucopolysaccharides, dermatan sulfate and heparan sulfate, accumulate in patients with MPS I (Neufeld and Muenzer, 2001), resulting in multiorgan dysfunction that includes hepatosplenomegaly, dysostosis multiplex, short stature, coarse facial features, corneal clouding, joint contractures, umbilical hernias, failure to thrive, intellectual disability, and commonly developmental delay (Beck et al., 2014). Classically, patients were characterized as having Hurler Syndrome (most severe), HurlerScheie Syndrome, or Scheie Syndrome (least severe) (Neufeld and Muenzer, 2001). Given the same biochemical defect in all of these patients with overlapping phenotypes, patients are now characterized as having severe or attenuated MPS I (Vijay and Wraith, 2005). MPS I is autosomal recessive and is caused by mutations in the IDUA gene located on chromosome 4p16.3 (Scott et al., 1992a,b). Enzyme replacement therapy (ERT) is available for patients with MPS I (Wraith, 2005). Although urine glycosaminoglycan analysis (UNIT 7.12; Zhang et al., 2013) may be helpful as a screening tool for MPS I, the diagnosis of MPS I has traditionally been based on the identification of low IDUA enzyme activity in leukocytes or fibroblasts (Minami et al., 1980; Oussoren et al., 2013). More recently, the analysis of IDUA activity in dried blood spots has proven effective for the diagnosis of MPS I (Chamoles et al., 2001; Wang et al., 2005; Blanchard et al., 2008; Duffey et al., 2010; Muller et al., 2011; Campos et al., Current Protocols in Human Genetics 17.17.1-17.17.8, January 2015 Published online January 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142905.hg1717s84 C 2015 John Wiley & Sons, Inc. Copyright
BASIC PROTOCOL
Biochemical Genetics
17.17.1 Supplement 84
2013). The use of dried blood spots and tandem mass spectrometry for IDUA analysis has increased test throughput capacity, making large-scale screening studies possible (Blanchard et al., 2008; Lin et al., 2013; Scott et al., 2013). Molecular analysis of the IDUA gene, however, is needed for the definitive diagnosis for MPS I. This protocol details the methodology needed to perform IDUA enzyme analysis for the diagnosis of MPS I using DBS. Briefly, DBS are eluted by incubation with extraction buffer for one hour. Next, an assay cocktail containing D-saccharic acid 1,4-lactone (Sigma), IDUA assay buffer, internal standard [7-hydroxycoumarin-4-acetic acid-(1 ,3 N-boc-diaminopropane)-amide; CDC, Atlanta], and IDUA substrate [7-(1-iduronic acid)oxycoumarin-4-acetic acid-(1 ,4 -N-boc-diaminobutane)-amide; CDC, Atlanta] is added and incubated with the extracted DBS for 20 hr. D-Saccharic acid 1,4-lactone is a glucuronidase inhibitor that is added to the assay cocktail to prevent β-glucuronidase in the patient samples from acting on the substrate. The alpha-L-iduronidase in the sample hydrolyzes the substrate to yield iduronic acid and 7-hydroxycoumarin-4-acetic acid(1 ,4 -N-boc-diaminobutane)-amide. On day 2, the reaction is stopped and the samples are purified using liquid:liquid extraction. Next, the samples are further purified using solid-phase extraction (SPE). The samples are then dried, reconstituted, resuspended in mobile phase buffer, and analyzed on a triple quad tandem mass spectrometer. The minimal amount of sample needed (a single 3mm punch), as well as the portability of DBS make this protocol ideal for high-throughput, large population screening for MPS I.
Materials Filter card (Whatman multipart 903 neonatal screening filter paper) containing dried blood Extraction buffer (see recipe) IDUA assay cocktail (see recipe) HPLC-grade water (J.T. Baker) Ethyl acetate (Merck) Methanol (Merck) Nitrogen Silica gel (Sigma, 230-400 mesh) Acetonitrile (Fisher Scientific) Formic acid 96-well deep-well plate (VWR) Hand-held puncher for 3-mm punches or DBS puncher (automated system; Perkin Elmer) Multichannel pipets Silicone plate sealer (Pall) iEMS Incubator/Shaker for 96 well plates (Thermo scientific) Sonicator 96-well round-bottom solvent resistant plate (Corning) Centrifuge (Beckmann Coulter) Vortex mixer, optional MiniVap (Perkin Elmer) or custom-made system 96-well 0.45-μm polypropylene filter plate (Pall) 96-well plate vacuum manifold (Porvair Sciences) Tandem mass spectrometer (eg, API 4000) and autosampler 96-well plate conical-bottom solvent-resistant plate (NUNC) Diagnosing Mucopolysaccharidosis Type I
NOTE: A multichannel pipet is used when multiple samples are analyzed.
17.17.2 Supplement 84
Current Protocols in Human Genetics
Day 1 1. For high-throughput testing, prepare a 96-well deep-well plate map for your samples. The plate should include 3 to 4 filter only (no blood) spots for background subtraction, controls, samples, and a blank well to check for carryover. 2. Before punching each sample, turn the filter card over and check to make sure that the blood has soaked all the way through. If the sample has not soaked all the way through, it should be rejected. 3. In that area, punch a single dried blood spot (DBS; diameter 3 mm) from the filter card into the well of a 96-well plate using a hand-held puncher or an automated DBS puncher. 4. Continue punching the samples. For the filter only spots, add a 3-mm spot from blank filter paper. 5. Extract samples as follows: a. Add 70 μl extraction buffer into each well and seal the plate with a silicone plate-sealer. b. Transfer the plate into a 37°C incubator that contains a shaker. c. Incubate for 1 hr while shaking at 750 rpm. Take the IDUA assay cocktail out of the −20°C freezer to thaw at room temperature. d. Thirty min into the incubation, sonicate the assay cocktail for 3 min to make sure everything is dissolved. e. Pipet 15 μl of the IDUA assay cocktail into each well of a new round-bottom 96-well plate. However, for the blank well (carryover check), add HPLC-grade water instead of assay cocktail. For enzyme testing, it is recommended to test a second enzyme to ensure the quality of the sample. The leftover extracted sample can be used for this purpose.
f. Once the 1 hr incubation is complete, centrifuge the plate for 1 min at 3000 × g, room temperature. g. Add 10 μl of the extracted DBS to the corresponding well in the new plate. Add the extract to the side of the well so that once all extracts are added, the reactions can be started at the same time by tapping the plate. h. Seal the plate and centrifuge for 1 min at 3000 × g, room temperature. i. Incubate for 20 hr at 37°C with shaking at 400 rpm.
Day 2 6. Remove the plate from the incubator and centrifuge for 1 min at 3000 × g, room temperature. 7. Stop the enzyme reaction by adding 100 μl of 1:1 ethyl acetate:methanol. Mix the samples by aspirating and dispensing using a pipet or alternatively, a plate vortex mixer. 8. Centrifuge the plate for 1 min at 3000 × g, room temperature. 9. Transfer the samples into a 96-well deep-well plate for the liquid-liquid extraction. If multiple enzymes are being tested for each sample, they can be combined at this step. 10. Add 400 μl of ethyl acetate into each well, followed by 400 μl of HPLC-grade water. Mix well. 11. Centrifuge for 5 min at 3500 × g, room temperature, to separate the phases.
Biochemical Genetics
17.17.3 Current Protocols in Human Genetics
Supplement 84
12. Transfer 300 μl of the upper phase into a new 96-well deep-well plate and dry the samples under nitrogen at ambient temperature using either a minivap or an equivalent custom-made system. This may take up to 15 min. 13. While the samples are drying, prepare the filter plate for solid-phase extraction (SPE) as follows: a. Fill the wells of the round-bottom plate with silica gel. Make sure that the gel is filled to the top in all wells that are used. b. Remove the excess amount of silica gel. c. Place a 96-well 0.45-μm polypropylene filter plate upside down on the roundbottom plate so that the wells are aligned. d. Flip them over, while firmly holding the plate together. e. Tap on the bottom of the round-bottom plate a few times so that all silica gel is transferred into the filter plate wells. f. Place the filter plate onto a deep-well plate and place these into a 96-well plate vacuum manifold. g. Prewash the silica with 250 μl 19:1 ethyl acetate:methanol using the vacuum manifold. The first wash goes in a waste plate. Change the deep-well plate for a fresh one to collect the samples. 14. Reconstitute the dried samples in 100 μl of 19:1 ethyl acetate:methanol, cover the plate, and shake for 5 min at ambient temperature. 15. Pipet the samples onto the silica gel. Turn the vacuum on and let the samples completely absorb into the silica gel. 16. Add 3 × 400 μl of 19:1 ethyl acetate:methanol to the wells for the SPE extraction. 17. Once all 3 SPE eluates have been collected, dry the samples under nitrogen at ambient temperature using either a minivap or a custom-made system. This may take up to 30 min. Samples can be covered and stored at −20°C for up to 2 days before proceeding.
18. Reconstitute the samples in 100 μl of mobile phase (e.g., 80:20 acetonitrile:water, 0.2% formic acid) or in 200 μl if more than one enzyme is assayed and SPE is performed. Transfer the samples into a new 96-well conical-bottom solvent-resistant plate. 19. Inject 10 μl of the reconstituted sample with a flow rate of 250 μl/min.
Perform tandem mass spectrometry 20. Perform tandem mass spectrometry. The following settings were used in the authors’ laboratory, using an API4000 as noted above:
Diagnosing Mucopolysaccharidosis Type I
Positive ion mode Curtain gas pressure: 20 Torr Collision cell pressure: 10 Torr Ion spray voltage: 5000 V Source temperature: 200°C Gas 1: 30 psi Gas 2: 35 psi Declustering potential: 30 V Entrance potential: 10 V Collision energy: 15 V Collision exit potential: 14 V
17.17.4 Supplement 84
Current Protocols in Human Genetics
XIC of +MRM (12 pairs): 377.331/277.100 Da ID: IDUA-IS from Sample 59 (H 14-2483) of 20140501_H14-2461-2535.wiff (Turbo Spray) Max 7620.0 cps
0.25
7500 7000 6500 6000 5500
Intensity (cps)
5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Time (min) XIC of +MRM (12 pairs): 377.331/277.100 Da ID: IDUA-IS from Sample 60 (H 14-2484) of 20140501_H14-2461-2535.wiff (Turbo Spray)
8365 8000
0.32
Max 8365.0 cps
7500 7000 6500 6000
Intensity (cps)
5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Time (min)
Figure 17.17.1 MPS I patients show reduced enzyme activity compared to controls. An example chromatogram is shown from a control individual (top) and a confirmed MPS I patient (bottom). The internal standard MRM transition (m/z 377>277) is shown in blue and the product MRM transition (m/z 391>291) is shown in red.
21. Use multiple-reaction monitoring (MRM) mode for the analysis of the IDUA IS and product with the following transitions (precursor ion m/z >product ion m/z): 377>277 (IS) and 391>291 (product; see Fig. 17.17.1). These settings are not only analyte specific, but also instrument specific, thus they need to be optimized to give the highest positive sensitivity for the transitions.
Biochemical Genetics
17.17.5 Current Protocols in Human Genetics
Supplement 84
REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2D; for suppliers, see SUPPLIERS APPENDIX.
Extraction buffer 20 mM sodium-phosphate monobasic (Sigma) solution Adjust pH to 7.1 using NaOH Store up to 6 months at ambient temperature IDUA assay buffer 0.112 M sodium formate (Sigma) 0.158 M formic acid (EMD) HPLC-grade water (J.T. Baker) Adjust pH to 3.6 using formic acid or NaOH, if necessary Store up to 6 months at 2° to 8°C IDUA inhibitor solution 3 mM D-saccharic acid 1,4-lactone (Sigma) HPLC-grade water (J.T. Baker) Divide into 2-ml aliquots Store up to 6 months at −20°C Stable for up to 3 freeze/thaw cycles.
IDUA assay cocktail Allow all reagents to reach room temperature Add the following solutions 17.5 ml IDUA assay buffer (see recipe) 0.5 ml IDUA inhibitor (see recipe) to a vial containing 0.21 μmol internal standard [7-hydroxycoumarin-4-acetic acid-(1 ,3 -N-boc-diaminopropane)-amide; CDC, Atlanta] 30 μmol IDUA substrate [7-(1-iduronic acid)-oxycoumarin-4-acetic acid-(1 ,4 -N-boc-diaminobutane)-amide; CDC, Atlanta] Vortex after addition of each solution to vial Store up to 6 month at −20°C The final IDUA assay cocktail contains 1.7 mM substrate, 11.7 μM internal standard, and 83 μM D-saccharic acid 1,4-lactone. Internal standard and IDUA substrate are provided free of charge through the Centers for Disease Control and Prevention (CDC; http://www.cdc.org)
COMMENTARY Background Information
Diagnosing Mucopolysaccharidosis Type I
MPS I is a pan ethnic disorder with original prevalence estimates of 1/100,000 individuals. However, pilot newborn screening studies have suggested that the incidence may be much higher (Lin et al., 2013; Scott et al., 2013). Enzyme-replacement therapy (ERT) with laronidase is available for patients with MPS I. The recombinant enzyme is given intravenously at weekly intervals (Wraith, 2005). Studies have shown that ERT improves
cardiac function (Harada et al., 2011, 2014), respiratory function (Wraith, 2005), and range of motion (Tylki-Szymanska et al., 2010), and is most effective at halting or slowing disease progression if started before the onset of irreversible damage (Muenzer, 2014). The advent of MS/MS and its widespread use for neonatal screening triggered the development of a MS/MS based method for analysis of different lysosomal enzyme activities (Li et al., 2004; Gelb et al., 2006). This approach
17.17.6 Supplement 84
Current Protocols in Human Genetics
Table 17.17.1 IDUA Activities in Normal Controls and Molecularly Confirmed MPS I Patients (O. Bodamer, unpub. observ.)
n
Mean ± SD (μmol/hr/liter)
CI - 95% (μmol/hr/liter)
Normal samples screened
4968
8.60 ± 5.3
8.45-8.75
Confirmed IDUA patients
12
UNIT 17.17
Britt A. Johnson,1 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
Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha iduronidase (IDUA). Progressive storage of dermatan and heparan sulfate throughout the body lead to a multiorgan presentation including short stature, dysostosis multiplex, corneal clouding, hearing loss, coarse facies, hepatosplenomegaly, and intellectual disability. Diagnosis of MPS I is based on IDUA enzyme analysis in leukocytes or dried blood spots (DBS) followed by molecular confirmation of the IDUA gene mutations in individuals with low enzyme activity. The advent of mass spectrometry methods for enzyme analysis in DBS has enabled high-throughput screening for MPS I in symptomatic individuals and newborn infants. The following unit provides the detailed analytical protocol for measurement of IDUA activity in DBS using C 2015 by John Wiley & Sons, Inc. tandem mass spectrometry. Keywords: dried blood spot r alpha-iduronidase r tandem mass spectrometry r MPS I r Mucopolysaccharidosis type I r Hurler Syndrome r Scheie Syndrome
How to cite this article: Johnson, B.A., Dajnoki, A., and Bodamer, O.A. 2015. Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type I. Curr. Protoc. Hum. Genet. 84:17.17.1-17.17.8. doi: 10.1002/0471142905.hg1717s84
Mucopolysaccharidosis type I (MPS I, OMIM #607014, 607015, 607016) is a progressive lysosomal storage disorder that results from a defect in alpha-L-iduronidase activity (Bach et al., 1972). The mucopolysaccharides, dermatan sulfate and heparan sulfate, accumulate in patients with MPS I (Neufeld and Muenzer, 2001), resulting in multiorgan dysfunction that includes hepatosplenomegaly, dysostosis multiplex, short stature, coarse facial features, corneal clouding, joint contractures, umbilical hernias, failure to thrive, intellectual disability, and commonly developmental delay (Beck et al., 2014). Classically, patients were characterized as having Hurler Syndrome (most severe), HurlerScheie Syndrome, or Scheie Syndrome (least severe) (Neufeld and Muenzer, 2001). Given the same biochemical defect in all of these patients with overlapping phenotypes, patients are now characterized as having severe or attenuated MPS I (Vijay and Wraith, 2005). MPS I is autosomal recessive and is caused by mutations in the IDUA gene located on chromosome 4p16.3 (Scott et al., 1992a,b). Enzyme replacement therapy (ERT) is available for patients with MPS I (Wraith, 2005). Although urine glycosaminoglycan analysis (UNIT 7.12; Zhang et al., 2013) may be helpful as a screening tool for MPS I, the diagnosis of MPS I has traditionally been based on the identification of low IDUA enzyme activity in leukocytes or fibroblasts (Minami et al., 1980; Oussoren et al., 2013). More recently, the analysis of IDUA activity in dried blood spots has proven effective for the diagnosis of MPS I (Chamoles et al., 2001; Wang et al., 2005; Blanchard et al., 2008; Duffey et al., 2010; Muller et al., 2011; Campos et al., Current Protocols in Human Genetics 17.17.1-17.17.8, January 2015 Published online January 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142905.hg1717s84 C 2015 John Wiley & Sons, Inc. Copyright
BASIC PROTOCOL
Biochemical Genetics
17.17.1 Supplement 84
2013). The use of dried blood spots and tandem mass spectrometry for IDUA analysis has increased test throughput capacity, making large-scale screening studies possible (Blanchard et al., 2008; Lin et al., 2013; Scott et al., 2013). Molecular analysis of the IDUA gene, however, is needed for the definitive diagnosis for MPS I. This protocol details the methodology needed to perform IDUA enzyme analysis for the diagnosis of MPS I using DBS. Briefly, DBS are eluted by incubation with extraction buffer for one hour. Next, an assay cocktail containing D-saccharic acid 1,4-lactone (Sigma), IDUA assay buffer, internal standard [7-hydroxycoumarin-4-acetic acid-(1 ,3 N-boc-diaminopropane)-amide; CDC, Atlanta], and IDUA substrate [7-(1-iduronic acid)oxycoumarin-4-acetic acid-(1 ,4 -N-boc-diaminobutane)-amide; CDC, Atlanta] is added and incubated with the extracted DBS for 20 hr. D-Saccharic acid 1,4-lactone is a glucuronidase inhibitor that is added to the assay cocktail to prevent β-glucuronidase in the patient samples from acting on the substrate. The alpha-L-iduronidase in the sample hydrolyzes the substrate to yield iduronic acid and 7-hydroxycoumarin-4-acetic acid(1 ,4 -N-boc-diaminobutane)-amide. On day 2, the reaction is stopped and the samples are purified using liquid:liquid extraction. Next, the samples are further purified using solid-phase extraction (SPE). The samples are then dried, reconstituted, resuspended in mobile phase buffer, and analyzed on a triple quad tandem mass spectrometer. The minimal amount of sample needed (a single 3mm punch), as well as the portability of DBS make this protocol ideal for high-throughput, large population screening for MPS I.
Materials Filter card (Whatman multipart 903 neonatal screening filter paper) containing dried blood Extraction buffer (see recipe) IDUA assay cocktail (see recipe) HPLC-grade water (J.T. Baker) Ethyl acetate (Merck) Methanol (Merck) Nitrogen Silica gel (Sigma, 230-400 mesh) Acetonitrile (Fisher Scientific) Formic acid 96-well deep-well plate (VWR) Hand-held puncher for 3-mm punches or DBS puncher (automated system; Perkin Elmer) Multichannel pipets Silicone plate sealer (Pall) iEMS Incubator/Shaker for 96 well plates (Thermo scientific) Sonicator 96-well round-bottom solvent resistant plate (Corning) Centrifuge (Beckmann Coulter) Vortex mixer, optional MiniVap (Perkin Elmer) or custom-made system 96-well 0.45-μm polypropylene filter plate (Pall) 96-well plate vacuum manifold (Porvair Sciences) Tandem mass spectrometer (eg, API 4000) and autosampler 96-well plate conical-bottom solvent-resistant plate (NUNC) Diagnosing Mucopolysaccharidosis Type I
NOTE: A multichannel pipet is used when multiple samples are analyzed.
17.17.2 Supplement 84
Current Protocols in Human Genetics
Day 1 1. For high-throughput testing, prepare a 96-well deep-well plate map for your samples. The plate should include 3 to 4 filter only (no blood) spots for background subtraction, controls, samples, and a blank well to check for carryover. 2. Before punching each sample, turn the filter card over and check to make sure that the blood has soaked all the way through. If the sample has not soaked all the way through, it should be rejected. 3. In that area, punch a single dried blood spot (DBS; diameter 3 mm) from the filter card into the well of a 96-well plate using a hand-held puncher or an automated DBS puncher. 4. Continue punching the samples. For the filter only spots, add a 3-mm spot from blank filter paper. 5. Extract samples as follows: a. Add 70 μl extraction buffer into each well and seal the plate with a silicone plate-sealer. b. Transfer the plate into a 37°C incubator that contains a shaker. c. Incubate for 1 hr while shaking at 750 rpm. Take the IDUA assay cocktail out of the −20°C freezer to thaw at room temperature. d. Thirty min into the incubation, sonicate the assay cocktail for 3 min to make sure everything is dissolved. e. Pipet 15 μl of the IDUA assay cocktail into each well of a new round-bottom 96-well plate. However, for the blank well (carryover check), add HPLC-grade water instead of assay cocktail. For enzyme testing, it is recommended to test a second enzyme to ensure the quality of the sample. The leftover extracted sample can be used for this purpose.
f. Once the 1 hr incubation is complete, centrifuge the plate for 1 min at 3000 × g, room temperature. g. Add 10 μl of the extracted DBS to the corresponding well in the new plate. Add the extract to the side of the well so that once all extracts are added, the reactions can be started at the same time by tapping the plate. h. Seal the plate and centrifuge for 1 min at 3000 × g, room temperature. i. Incubate for 20 hr at 37°C with shaking at 400 rpm.
Day 2 6. Remove the plate from the incubator and centrifuge for 1 min at 3000 × g, room temperature. 7. Stop the enzyme reaction by adding 100 μl of 1:1 ethyl acetate:methanol. Mix the samples by aspirating and dispensing using a pipet or alternatively, a plate vortex mixer. 8. Centrifuge the plate for 1 min at 3000 × g, room temperature. 9. Transfer the samples into a 96-well deep-well plate for the liquid-liquid extraction. If multiple enzymes are being tested for each sample, they can be combined at this step. 10. Add 400 μl of ethyl acetate into each well, followed by 400 μl of HPLC-grade water. Mix well. 11. Centrifuge for 5 min at 3500 × g, room temperature, to separate the phases.
Biochemical Genetics
17.17.3 Current Protocols in Human Genetics
Supplement 84
12. Transfer 300 μl of the upper phase into a new 96-well deep-well plate and dry the samples under nitrogen at ambient temperature using either a minivap or an equivalent custom-made system. This may take up to 15 min. 13. While the samples are drying, prepare the filter plate for solid-phase extraction (SPE) as follows: a. Fill the wells of the round-bottom plate with silica gel. Make sure that the gel is filled to the top in all wells that are used. b. Remove the excess amount of silica gel. c. Place a 96-well 0.45-μm polypropylene filter plate upside down on the roundbottom plate so that the wells are aligned. d. Flip them over, while firmly holding the plate together. e. Tap on the bottom of the round-bottom plate a few times so that all silica gel is transferred into the filter plate wells. f. Place the filter plate onto a deep-well plate and place these into a 96-well plate vacuum manifold. g. Prewash the silica with 250 μl 19:1 ethyl acetate:methanol using the vacuum manifold. The first wash goes in a waste plate. Change the deep-well plate for a fresh one to collect the samples. 14. Reconstitute the dried samples in 100 μl of 19:1 ethyl acetate:methanol, cover the plate, and shake for 5 min at ambient temperature. 15. Pipet the samples onto the silica gel. Turn the vacuum on and let the samples completely absorb into the silica gel. 16. Add 3 × 400 μl of 19:1 ethyl acetate:methanol to the wells for the SPE extraction. 17. Once all 3 SPE eluates have been collected, dry the samples under nitrogen at ambient temperature using either a minivap or a custom-made system. This may take up to 30 min. Samples can be covered and stored at −20°C for up to 2 days before proceeding.
18. Reconstitute the samples in 100 μl of mobile phase (e.g., 80:20 acetonitrile:water, 0.2% formic acid) or in 200 μl if more than one enzyme is assayed and SPE is performed. Transfer the samples into a new 96-well conical-bottom solvent-resistant plate. 19. Inject 10 μl of the reconstituted sample with a flow rate of 250 μl/min.
Perform tandem mass spectrometry 20. Perform tandem mass spectrometry. The following settings were used in the authors’ laboratory, using an API4000 as noted above:
Diagnosing Mucopolysaccharidosis Type I
Positive ion mode Curtain gas pressure: 20 Torr Collision cell pressure: 10 Torr Ion spray voltage: 5000 V Source temperature: 200°C Gas 1: 30 psi Gas 2: 35 psi Declustering potential: 30 V Entrance potential: 10 V Collision energy: 15 V Collision exit potential: 14 V
17.17.4 Supplement 84
Current Protocols in Human Genetics
XIC of +MRM (12 pairs): 377.331/277.100 Da ID: IDUA-IS from Sample 59 (H 14-2483) of 20140501_H14-2461-2535.wiff (Turbo Spray) Max 7620.0 cps
0.25
7500 7000 6500 6000 5500
Intensity (cps)
5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Time (min) XIC of +MRM (12 pairs): 377.331/277.100 Da ID: IDUA-IS from Sample 60 (H 14-2484) of 20140501_H14-2461-2535.wiff (Turbo Spray)
8365 8000
0.32
Max 8365.0 cps
7500 7000 6500 6000
Intensity (cps)
5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Time (min)
Figure 17.17.1 MPS I patients show reduced enzyme activity compared to controls. An example chromatogram is shown from a control individual (top) and a confirmed MPS I patient (bottom). The internal standard MRM transition (m/z 377>277) is shown in blue and the product MRM transition (m/z 391>291) is shown in red.
21. Use multiple-reaction monitoring (MRM) mode for the analysis of the IDUA IS and product with the following transitions (precursor ion m/z >product ion m/z): 377>277 (IS) and 391>291 (product; see Fig. 17.17.1). These settings are not only analyte specific, but also instrument specific, thus they need to be optimized to give the highest positive sensitivity for the transitions.
Biochemical Genetics
17.17.5 Current Protocols in Human Genetics
Supplement 84
REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2D; for suppliers, see SUPPLIERS APPENDIX.
Extraction buffer 20 mM sodium-phosphate monobasic (Sigma) solution Adjust pH to 7.1 using NaOH Store up to 6 months at ambient temperature IDUA assay buffer 0.112 M sodium formate (Sigma) 0.158 M formic acid (EMD) HPLC-grade water (J.T. Baker) Adjust pH to 3.6 using formic acid or NaOH, if necessary Store up to 6 months at 2° to 8°C IDUA inhibitor solution 3 mM D-saccharic acid 1,4-lactone (Sigma) HPLC-grade water (J.T. Baker) Divide into 2-ml aliquots Store up to 6 months at −20°C Stable for up to 3 freeze/thaw cycles.
IDUA assay cocktail Allow all reagents to reach room temperature Add the following solutions 17.5 ml IDUA assay buffer (see recipe) 0.5 ml IDUA inhibitor (see recipe) to a vial containing 0.21 μmol internal standard [7-hydroxycoumarin-4-acetic acid-(1 ,3 -N-boc-diaminopropane)-amide; CDC, Atlanta] 30 μmol IDUA substrate [7-(1-iduronic acid)-oxycoumarin-4-acetic acid-(1 ,4 -N-boc-diaminobutane)-amide; CDC, Atlanta] Vortex after addition of each solution to vial Store up to 6 month at −20°C The final IDUA assay cocktail contains 1.7 mM substrate, 11.7 μM internal standard, and 83 μM D-saccharic acid 1,4-lactone. Internal standard and IDUA substrate are provided free of charge through the Centers for Disease Control and Prevention (CDC; http://www.cdc.org)
COMMENTARY Background Information
Diagnosing Mucopolysaccharidosis Type I
MPS I is a pan ethnic disorder with original prevalence estimates of 1/100,000 individuals. However, pilot newborn screening studies have suggested that the incidence may be much higher (Lin et al., 2013; Scott et al., 2013). Enzyme-replacement therapy (ERT) with laronidase is available for patients with MPS I. The recombinant enzyme is given intravenously at weekly intervals (Wraith, 2005). Studies have shown that ERT improves
cardiac function (Harada et al., 2011, 2014), respiratory function (Wraith, 2005), and range of motion (Tylki-Szymanska et al., 2010), and is most effective at halting or slowing disease progression if started before the onset of irreversible damage (Muenzer, 2014). The advent of MS/MS and its widespread use for neonatal screening triggered the development of a MS/MS based method for analysis of different lysosomal enzyme activities (Li et al., 2004; Gelb et al., 2006). This approach
17.17.6 Supplement 84
Current Protocols in Human Genetics
Table 17.17.1 IDUA Activities in Normal Controls and Molecularly Confirmed MPS I Patients (O. Bodamer, unpub. observ.)
n
Mean ± SD (μmol/hr/liter)
CI - 95% (μmol/hr/liter)
Normal samples screened
4968
8.60 ± 5.3
8.45-8.75
Confirmed IDUA patients
12
