View
217
Download
0
Category
Preview:
Citation preview
1 / 58
Mucopolysaccharidoses
diagnostic approaches
George Ruijter
Center for Lysosomal and Metabolic Diseases
Erasmus University Medical Center
Rotterdam, The Netherlands
1/35
2 / 582/35
3 / 58
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
4 / 58
Degradation of glycosaminoglycans
(GAG; mucopolysaccharides)
nucleus
proteoglycansendocytosis
endoglycosidic
cleavage
proteolysis
lysosome
degradation to free
sugars and sulfate
endosome
4/35
5 / 58
Heparan sulfate degradation
5/35
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
6/35
7 / 58
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
8 / 58
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
9 / 58
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
9/35
10 / 58
1D/2D electrophoresis
Appl.
DS1
DS2
HS
CSKS
CS
?
normal
HS
MPS III
MPS IIIInormal
MPS I
CS
HS
DS
1-dimensional 2-dimensional
10/35
11 / 58
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
11/35
12 / 58
• 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
12/35
13 / 58
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
14 / 58
Chemistry of enzyme assays using synthetic
substrates
pH 10
Fluorogenic 4MU substrate Lipidated MS-MS substrate
IDUA IDUA
14/35
15 / 58
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
15/35
16 / 58
-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
16/35
17 / 58
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
17/35
18 / 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
19 / 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
20 / 58
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
20/35
21 / 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
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
21/35
22 / 58
Interpretation of quantitative GAG results
22/35
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
23 / 58
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
23/35
24 / 58
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
24/35
25 / 58
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
25/35
26 / 58
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%
26/35
Proficiency
27 / 58
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
27/35
28 / 58
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
28/35
29 / 58
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
29/35
30 / 58
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
30/35
31 / 58
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
31/35
32 / 58
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
32/35
33 / 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
3333/35
34 / 5834/35
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
Recommended