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Mucopolysaccharidoses

diagnostic approaches

George Ruijter

Center for Lysosomal and Metabolic Diseases

Erasmus University Medical Center

Rotterdam, The Netherlands

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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)

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Degradation of glycosaminoglycans

(GAG; mucopolysaccharides)

nucleus

proteoglycansendocytosis

endoglycosidic

cleavage

proteolysis

lysosome

degradation to free

sugars and sulfate

endosome

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Heparan sulfate degradation

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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 Chondroitinsulfate

DS Dermatansulfate

HS Heparansulfate

KS Keratansulfate

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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

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DMB test; normal controls vs. MPS

0

50

100

150

200

0 10 20 30 40 50

age (y)

GA

G (

mg

/mm

ol)

Controls

Ref values

MPS I

MPS II

MPS III

MPS IV

MPS VI

False positive results:

rheumatoid arthritis, heparin, glue from collection bags 8/35

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What deviation from normal is suspect?

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

Measured value – mean of age matched reference values

Standard deviation in age matched reference values

X =

X = no. of SD above average normal

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1D/2D electrophoresis

Appl.

DS1

DS2

HS

CSKS

CS

?

normal

HS

MPS III

MPS IIIInormal

MPS I

CS

HS

DS

1-dimensional 2-dimensional

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MPS I on ERT

0

5

10

15

20

25

30

35

40

45

7.8 8 8.2 8.4 8.6 8.8 9

Age (y)

GA

G (

mg

/mm

ol)

GAG reference values

0

20

40

60

80

0 5 10 15 20

Age (y)

GA

G (

mg

/mm

ol)

Controls

Ref values

Sensitivity/specificity of current quantitative

GAG analysis is not sufficient

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• ELISAe.g. Tomatsu et al (2005) J Inherit Metab Dis 28:743-57

• LC-MS-MS of 1-phenyl-3-methyl-pyrazolon (PMP)-derivatised

oligosaccharidese.g. Fuller et al (2004) Ped Res 56:733-738

• GAG degradation by bacterial GAG lyases followed by LC-

MS-MS of disaccharidese.g. Tomatsu et al (2010) Mol Gen Metab 99:124-131

• GAG degradation by methanolysis followed by LC-MS-MS of

disaccharidese.g. Auray-Blais et al (2011) Mol Gen Metab 102:49-56

Novel methods for GAG diagnostics in urine

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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 +

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Chemistry of enzyme assays using synthetic

substrates

pH 10

Fluorogenic 4MU substrate Lipidated MS-MS substrate

IDUA IDUA

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Improved method for 4MU assays in DBS

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

fluAdd substrate

Incub. 37o C

TCA prec.

flu

2.

1.

Add substrate

Incub. 37o C

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-110

-60

-10

40

90

AIDU -TCA AIDU +TCA

AF

U /p

un

ch

/17

h

-50

100

250

400

550

700

850

1000

1150

1300

AF

U /p

un

ch

/17

h

35 controls

9 patients

Hurler disease: α-L-iduronidase assay in DBS

(Raw fluorescence data AFU/ 3mm punch/ 17h

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Current newborn screening for LSDs

No MPS so far

2005 2006 2007 2008 2009 2010 2011 2012 2013

Taiwan NY Austria

Illinois?

NY state?

Missouri?

Pompe

Fabry

Pompe

Fabry

Krabbe

Fluorometry

LC-MS-MS LC-MS-MS?

Krabbe

Gaucher

Niemann-Pick

Pompe

Fabry

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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

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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

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Quantitative analysis of GAG in urine

% MPS pilot

Spot tests -

Turbidity tests 7

Hexuronic – carbazol (harmine) 4

• precipitation of GAG with CPC/CTAB

• spectrophotometry after reaction with carbazol

• low sensitivity for MPS IV

Spectrofotometry using dye

• Dimethylmethyleneblue (direct) 81

• Alcian blue (indirect) 8

DMB; 70

Alcian blue; 6

CPC; 5

Hexuronic-Carbazol;

3 Spot test; 0

DMB

Alcian blue

CPC

Hexuronic-Carbazol

Spot test

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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

21

0.31

14

6.05

0.56

9

1.09

0.35

32

3.14

0.31

10

1.66

0.22

13

3.95

0.48

12

GAG (mg/mmol)

Average

SD

CV

9.4

4.6

49

10.0

4.1

41

130

50

38

54.4

13.2

24

51.9

15.9

31

10.9

5.1

47

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Interpretation of quantitative GAG results

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0

10

20

30

40

50

60

70

80

90

100

MPS9; MPS IIIB

MPS10; Normal control

MPS11; MPS I

MPS12; MPS II

MPS13; MPS IIIA

MPS14; MPS VII

%

normal

increased

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Performance of quantitative methods

4 MPS samples: 3 x MPS III, 1 x MPS VII

GAG concentrations moderately increased

0

10

20

30

40

50

60

70

80

90

100

DMB Alcian Blue CPC

%

GAG normal

GAG increased

n=256 n=22 n=16

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Is quantitative analysis of total GAG sufficient?

MPS III: 4 samples in 2010-2011

Total sample reports: 301

N: 37

MPS III:

14

GAG : 264

GAG quantitative Analysis GAG

subfractions &

diagnosis

Normal:

37

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Qualitative analysis of GAG in urine

1D; 27

1D discontinuous; 25

2D; 11

TLC; 9

Other; 2

Multiple; 5

1D

1D discontinuous

2D

TLC

Other

Multiple

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Diagnostic proficiency summarised

Participants who submitted at least 10 out of 14 sample reports: 76

>90%; 15

70-90%; 36

50-70%; 18

<50%; 7

>90%

70-90%

50-70%

<50%

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Proficiency

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Reproducibility of diagnostic proficiency

0

10

20

30

40

50

60

70

80

90

100

Normal

control

MPS III MPS IV MPSVI

%

1 correct

2 correct

1 not correct

2 not correct

1 no diagnosis

2 no diagnosis

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Can MPS I, II and VI be distinguished by

GAG analysis?

Correct I / II

Partially correct I / II / VI

DS increased (%) 99

HS increased (%) 43

0

10

20

30

40

50

60

70

80

90

100

MPS I MPS II MPS VI

%

Correct

Partially correct

Not correct

No diagnosis

I / II

I / II / VI

96

77

VI

I / II / VI

91

25

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Diagnostic proficiency of MPS III

0

10

20

30

40

50

60

70

80

90

100

MPS IIIA MPS IIIA MPS IIIB

%

Correct

Not correct

No diagnosis

5 y

‘classic’

‘Normal’ (%) 5

HS increased (%) 94

11 y

mild

12

80

19 y

mild

23

66

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Overall performance of qualitative methods

n=27 n=25 n=11 n=9 n=5

0

10

20

30

40

50

60

70

80

90

100

1D 1D discont 2D TLC Multiple

%

(partially) correct

not correct

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Performance of qualitative methods for MPS IV

0

10

20

30

40

50

60

70

80

90

100

1D 1D discont 2D TLC Multiple

% (partially) correct

not correct

n=27 n=25 n=11 n=9 n=5

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Performance of qualitative methods for MPS III

n=27 n=25 n=11 n=9 n=5

0

10

20

30

40

50

60

70

80

90

100

1D 1D discont 2D TLC Multiple

% (partially) correct

not correct

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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

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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