Mucopolysaccharidoses diagnostic approaches George Ruijter Center for Lysosomal and Metabolic Diseases Erasmus University Medical Center Rotterdam, The Netherlands 1/35 / 58
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Mucopolysaccharidoses MPS I Hurler -L-iduronidase MPS II Hunter iduronate sulfatase MPS III A Sanfilippo heparan N-sulfatase (sulfamidase) B -NAc-glucosaminidase C AcCoA: -glucosaminide Ac trf. D GlcNAc 6-sulfatase E? glucuronate sulfatase MPS IV A Morquio Galactose 6-sulfatase B -galactosidase MPS VI Maroteaux-Lamy GalNAc 4-sulfatase (arylsulfatase B) MPS VII Sly -glucuronidase MPS IX? Natowicz hyaluronidase (endo-hexosaminidase) 3/35 / 58
Degradation of glycosaminoglycans (GAG; mucopolysaccharides) proteoglycans endocytosis proteolysis endosome endoglycosidic cleavage degradation to free sugars and sulfate nucleus lysosome 4/35 / 58
Heparan sulfate degradation 5/35 / 58
GAG in urine Type GAG storage Normal - MPS I DS + HS MPS II DS + HS MPS III HS MPS IV KS + C6S MPS VI DS MPS VII CS + DS + HS CS DS HS KS Chondroitinsulfate Dermatansulfate Heparansulfate Keratansulfate 6/35
Diagnostic tests for mucopolysaccharidoses Screening in urine Quantitative analysis of total GAG Qualitative analysis: separation of GAG subfractions Enzyme tests in leukocytes/fibroblasts to establish subtype Activity assays of 1 to 4 enzymes Mutation analysis 7/35 / 58
GAG (mg/mmol) DMB test; normal controls vs. MPS 200 150 100 Controls Ref values MPS I MPS II MPS III MPS IV MPS VI 50 8/35 / 58 0 0 10 20 30 40 50 age (y) False positive results: rheumatoid arthritis, heparin, glue from collection bags
What deviation from normal is suspect? X = Measured value mean of age matched reference values Standard deviation in age matched reference values X = no. of SD above average normal MPS X n median range I 17 3.0 50 22 II 19 2.9 31 18 III 14 3.6 66 39 IV 7 1.0 16 19 VI 18 4.5 30 14 9/35 / 58
1D/2D electrophoresis 1-dimensional 2-dimensional KS CS DS2? CS CS DS HS HS normal MPS I DS1 Appl. HS normal III MPS I MPS III 10/35 / 58
GAG (mg/mmol) GAG (mg/mmol) Sensitivity/specificity of current quantitative GAG analysis is not sufficient GAG reference values MPS I on ERT 80 60 40 20 0 Controls Ref values 0 5 10 15 20 Age (y) 45 40 35 30 25 20 15 10 5 0 7.8 8 8.2 8.4 8.6 8.8 9 Age (y) 11/35 / 58
Novel methods for GAG diagnostics in urine ELISA e.g. Tomatsu et al (2005) J Inherit Metab Dis 28:743-57 LC-MS-MS of 1-phenyl-3-methyl-pyrazolon (PMP)-derivatised oligosaccharides e.g. Fuller et al (2004) Ped Res 56:733-738 GAG degradation by bacterial GAG lyases followed by LC- MS-MS of disaccharides e.g. Tomatsu et al (2010) Mol Gen Metab 99:124-131 12/35 / 58 GAG degradation by methanolysis followed by LC-MS-MS of disaccharides e.g. Auray-Blais et al (2011) Mol Gen Metab 102:49-56
Enzyme assays for MPS 4MU MPS I -L-iduronidase + MPS II iduronate sulfatase + (2 step) MPS III A sulfamidase + (2 step) B -NAc-glucosaminidase + C AcCoA: -glucosaminide Ac trf. + (2 step) D GlcNAc 6-sulfatase + (2 step) E? glucuronate sulfatase + (2 step) MPS IV A GalNAc 6-sulfatase + (2 step) B -galactosidase + MPS VI arylsulfatase B (-) pncs MPS VII -glucuronidase + 13/35 / 58
Chemistry of enzyme assays using synthetic substrates Fluorogenic 4MU substrate Lipidated MS-MS substrate IDUA IDUA ph 10 14/35 / 58
Improved method for 4MU assays in DBS 1. 2. Add substrate Incub. 37 o C Add substrate Incub. 37 o C TCA prec. flu flu 1. Traditional direct fluorescence determination Drawback quenching by hemoglobin 2. TCA precipitation hemoglobin after enzyme incubatio Quenching decreased, FLU about 8-fold increased More sensitive and reliable assay 15/35 / 58
AFU /punch/17h AFU /punch/17h Hurler disease: α-l-iduronidase assay in DBS 90 1300 1150 1000 40 850-10 700 550 35 controls 9 patients 400-60 250 100-110 AIDU -TCA AIDU +TCA -50 (Raw fluorescence data AFU/ 3mm punch/ 17h 16/35 / 58
Current newborn screening for LSDs No MPS so far Taiwan NY Austria Illinois? NY state? Missouri? 2005 2006 2007 2008 2009 2010 2011 2012 2013 Pompe Fabry Fluorometry Krabbe Pompe Fabry Krabbe Gaucher Niemann-Pick Pompe Fabry LC-MS-MS LC-MS-MS? 17/35 / 58
Mutation analysis The European Directory of DNA Diagnostic Laboratories (EDDNAL) www.eddnal.com Orphanet www.orpha.net MPS I, II, III A/B/C/D, IV A/B, VI 18/35 / 58
ERNDIM MPS pilot scheme 2010: 8 samples 88 participants 2011: 6 samples 89 participants Determine creatinine and GAG concentration Qualify GAG level according to age-matched reference values (i.e normal or increased) Analyse GAG subfractions and qualify (i.e. normal or increased CS, HS, DS and KS) Give most likely diagnosis 19/35 / 58
Quantitative analysis of GAG in urine Hexuronic-Carbazol; % MPS pilot 3 Spot tests Spot test; 0 - CPC; 5 Turbidity Alcian blue; tests 6 7 DMB Hexuronic carbazol (harmine) 4 precipitation of GAG with CPC/CTAB Alcian blue spectrophotometry after reaction with carbazol CPC low sensitivity for MPS IV Hexuronic-Carbazol Spot test Spectrofotometry using dye Dimethylmethyleneblue (direct) 81 Alcian blue (indirect) 8 DMB; 70 20/35 / 58
Quantitative GAG results 2011 Sample ID MPS9 MPS10 MPS11 MPS12 MPS13 MPS14 Diagnosis Age of patient MPS III 19 y Normal 5 y MPS I 2 y 4 m MPS II 5 y MPS III 5 y MPS VII 19 y No. of reports 78 78 78 79 79 79 Creatinine (mmol/l) Average SD CV GAG (mg/mmol) Average SD CV 21 0.31 14 9.4 4.6 49 6.05 0.56 9 10.0 4.1 41 1.09 0.35 32 130 50 38 3.14 0.31 10 54.4 13.2 24 1.66 0.22 13 51.9 15.9 31 3.95 0.48 12 10.9 5.1 47 21/35 / 58
% Interpretation of quantitative GAG results 100 90 80 70 60 50 40 normal increased 30 20 10 0 MPS9; MPS IIIB MPS10; Normal control MPS11; MPS I MPS12; MPS II MPS13; MPS IIIA MPS14; MPS VII 22/35 / 58
% Performance of quantitative methods 4 MPS samples: 3 x MPS III, 1 x MPS VII GAG concentrations moderately increased 100 90 80 70 60 50 40 30 20 10 GAG normal GAG increased 0 DMB Alcian Blue CPC n=256 n=22 n=16 23/35 / 58
Is quantitative analysis of total GAG sufficient? MPS III: 4 samples in 2010-2011 Total sample reports: 301 GAG quantitative Analysis GAG subfractions & diagnosis GAG : 264 N: 37 Normal: 37 MPS III: 14 24/35 / 58
Qualitative analysis of GAG in urine Other; 2 Multiple; 5 TLC; 9 1D; 27 2D; 11 1D 1D discontinuous 2D TLC Other Multiple 1D discontinuous; 25 25/35 / 58
Diagnostic proficiency summarised Participants who submitted at least 10 out of 14 sample reports: 76 50-70%; 18 <50%; 7 >90%; 15 70-90%; 36 Proficiency >90% 70-90% 50-70% <50% 26/35 / 58
% Reproducibility of diagnostic proficiency 100 90 80 70 60 50 40 30 20 10 0 Normal control MPS III MPS IV MPSVI 1 correct 2 correct 1 not correct 2 not correct 1 no diagnosis 2 no diagnosis 27/35 / 58
Can MPS I, II and VI be distinguished by GAG analysis? % 100 90 80 70 60 50 40 30 20 10 0 MPS I MPS II MPS VI Correct Partially correct Not correct No diagnosis Correct Partially correct I / II I / II / VI I / II I / II / VI VI I / II / VI DS increased (%) 99 HS increased (%) 43 96 77 91 25 28/35 / 58
% Diagnostic proficiency of MPS III 100 90 80 70 60 50 40 30 20 10 0 MPS IIIA MPS IIIA MPS IIIB Correct Not correct No diagnosis 5 y classic 11 y mild 19 y mild Normal (%) 5 HS increased (%) 94 12 80 23 66 29/35 / 58
% Overall performance of qualitative methods 100 90 80 70 60 50 40 30 20 10 (partially) correct not correct 0 1D 1D discont 2D TLC Multiple n=27 n=25 n=11 n=9 n=5 30/35 / 58
% Performance of qualitative methods for MPS IV 100 90 80 70 60 50 40 30 20 10 (partially) correct not correct 0 1D 1D discont 2D TLC Multiple n=27 n=25 n=11 n=9 n=5 31/35 / 58
% Performance of qualitative methods for MPS III 100 90 80 70 60 50 40 30 20 10 (partially) correct not correct 0 1D 1D discont 2D TLC Multiple n=27 n=25 n=11 n=9 n=5 32/35 / 58
Summary/conclusions MPS pilot scheme started in 2010; ~90 participants Quantitative GAG analysis: 83 % DMB DMB, Alcian Blue perform better than CPC for mildly elevated GAG Analysis GAG sub fractions: 66% 1-dimensional electrophoresis Difficult to distinguish MPS I, II and VI on the basis of GAG analysis Use of multiple methods for analysis of GAG sub fractions improves diagnostic proficiency Sensitivity/specificity of current GAG analysis not sufficient: novel methods required 33/35 / 58
Thank you! Erasmus MC: Jan Huijmans, Rolanda van den Berg, Eric van der Meijden SKML: Cas Weykamp, Irene de Graaf All participants of the ERNDIM MPS pilot study 34 / 58 34/35