Mucopolysaccharidoses diagnostic approaches

Transcription

1 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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3 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

4 Degradation of glycosaminoglycans (GAG; mucopolysaccharides) proteoglycans endocytosis proteolysis endosome endoglycosidic cleavage degradation to free sugars and sulfate nucleus lysosome 4/35 / 58

5 Heparan sulfate degradation 5/35 / 58

6 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

7 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

8 GAG (mg/mmol) DMB test; normal controls vs. MPS Controls Ref values MPS I MPS II MPS III MPS IV MPS VI 50 8/35 / age (y) False positive results: rheumatoid arthritis, heparin, glue from collection bags

9 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 II III IV VI /35 / 58

10 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

11 GAG (mg/mmol) GAG (mg/mmol) Sensitivity/specificity of current quantitative GAG analysis is not sufficient GAG reference values MPS I on ERT Controls Ref values Age (y) Age (y) 11/35 / 58

12 Novel methods for GAG diagnostics in urine ELISA e.g. Tomatsu et al (2005) J Inherit Metab Dis 28: LC-MS-MS of 1-phenyl-3-methyl-pyrazolon (PMP)-derivatised oligosaccharides e.g. Fuller et al (2004) Ped Res 56: GAG degradation by bacterial GAG lyases followed by LC- MS-MS of disaccharides e.g. Tomatsu et al (2010) Mol Gen Metab 99: /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

13 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

14 Chemistry of enzyme assays using synthetic substrates Fluorogenic 4MU substrate Lipidated MS-MS substrate IDUA IDUA ph 10 14/35 / 58

15 Improved method for 4MU assays in DBS 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

16 AFU /punch/17h AFU /punch/17h Hurler disease: α-l-iduronidase assay in DBS controls 9 patients AIDU -TCA AIDU +TCA -50 (Raw fluorescence data AFU/ 3mm punch/ 17h 16/35 / 58

17 Current newborn screening for LSDs No MPS so far Taiwan NY Austria Illinois? NY state? Missouri? Pompe Fabry Fluorometry Krabbe Pompe Fabry Krabbe Gaucher Niemann-Pick Pompe Fabry LC-MS-MS LC-MS-MS? 17/35 / 58

18 Mutation analysis The European Directory of DNA Diagnostic Laboratories (EDDNAL) Orphanet MPS I, II, III A/B/C/D, IV A/B, VI 18/35 / 58

19 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

20 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

21 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 Creatinine (mmol/l) Average SD CV GAG (mg/mmol) Average SD CV /35 / 58

22 % Interpretation of quantitative GAG results normal increased MPS9; MPS IIIB MPS10; Normal control MPS11; MPS I MPS12; MPS II MPS13; MPS IIIA MPS14; MPS VII 22/35 / 58

23 % Performance of quantitative methods 4 MPS samples: 3 x MPS III, 1 x MPS VII GAG concentrations moderately increased GAG normal GAG increased 0 DMB Alcian Blue CPC n=256 n=22 n=16 23/35 / 58

24 Is quantitative analysis of total GAG sufficient? MPS III: 4 samples in Total sample reports: 301 GAG quantitative Analysis GAG subfractions & diagnosis GAG : 264 N: 37 Normal: 37 MPS III: 14 24/35 / 58

25 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

26 Diagnostic proficiency summarised Participants who submitted at least 10 out of 14 sample reports: %; 18 <50%; 7 >90%; %; 36 Proficiency >90% 70-90% 50-70% <50% 26/35 / 58

27 % Reproducibility of diagnostic proficiency 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

28 Can MPS I, II and VI be distinguished by GAG analysis? % 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 (%) /35 / 58

29 % Diagnostic proficiency of MPS III MPS IIIA MPS IIIA MPS IIIB Correct Not correct No diagnosis 5 y classic 11 y mild 19 y mild Normal (%) 5 HS increased (%) /35 / 58

30 % Overall performance of qualitative methods (partially) correct not correct 0 1D 1D discont 2D TLC Multiple n=27 n=25 n=11 n=9 n=5 30/35 / 58

31 % Performance of qualitative methods for MPS IV (partially) correct not correct 0 1D 1D discont 2D TLC Multiple n=27 n=25 n=11 n=9 n=5 31/35 / 58

32 % Performance of qualitative methods for MPS III (partially) correct not correct 0 1D 1D discont 2D TLC Multiple n=27 n=25 n=11 n=9 n=5 32/35 / 58

33 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

34 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