Institute of Inherited Metabolic Disorders

de
rs
or
M
et
ab
ol
ic
D
is
Survey of diagnosis of lysosomal
storage disorders
In
st
itu
te
of
In
he
rit
ed
Milan Elleder
Institute of Inherited Metabolic Disorders
Charles University, 1st Faculty of Medicine and University Hospital
Prague
October 5, 2006
de
rs
ol
ic
D
is
or
prehistory – empirical part of the story
clinical reports by Tay (1881), Gaucher (1882) and Sachs (1896)
and by others
et
ab
modern history of the lysosomes
their discovery: C. de Duve et al.
(Biochem. J. 60, 604, 1955)
ed
M
Nobel Prize 1974
te
of
In
he
rit
modern history of the lysosomal storage
•H.G. Hers et al. (1963) Acid glucosidase deficiency in GSD II
•Austin et al. (1963) Arylsulphatase deficiency in MLD
In
st
itu
present state of the art (2006) – 48 defined entities
of different molecular basis (groups Ia,b and II)
e
as
de
rs
at
or
D
is
se
MPS
n=10
ic
co
u
erase
f
l
s
n
g
a
c -tr
D
A
N
e
inid
c -α
m
a
s
A
o
c
N
α-glu
:
A
o
hatase
p
l
C
u
s
e
t
-sulpha
6
c
A
N
Glc
GalNAc-6-sulphate sulphatase
GalNAc-4
-sulphate s
ulphatase
β-gluc
uronid
* hyaluro ase
* N-a nidase (hy
aluro
* asp cetylnic a
α-g
ar
cid)
ala
ty
cto
lg
sa m
lu
co
inid
sa
a se
m
in
id
as
e
ab
et
he
rit
ed
M
expanded by
storage
In
of
te
osa
glu
c
itu
NA
c-β
-
st
NA
c-β
In
2006
lysosome
se
min
B
i
d
a
α-ga
se A
lacto
sida
se A
α-neur
aminid
ase
m
in
id
a
-g
lu
co
sa
n=9
a
nid
i
sa m
ol
ida
se
acid lipa
se
β-glucosylceram
idase
ceramidase
idase *
m
a
lcer
se
y
a
s
o
t
n
lac
el i
y
β-ga
A
om
e
s
g
in
ata
f
l
sph
u
yl s
r
a
lipidoses
α
Id -L-id
he uro uron
ida
pa na
se
ra ten
2s
N
-s ulp
ul
fa hate
ta
su
se
lp
h
ase
lysosomal storage
disorders Ia
idase
s
o
n
n
β-Ma
ase
d
i
os
n
n
a
α-M
-glu
c os
ste
r
idase
thi
oe
β-galactos
α-1
,4
in
due to mutant
enzyme
protein
(n=30)
* α-Fucosidase
acid
l-p
ro
te
D
in
ep
eI
th
as
Ca
tid
ep
ylp
tid
GSD II
enzymopathies
p
pe
tr i
neuronal
ceroid
lipofuscinoses
Pa
lm
i to
y
glycoproteinoses
n=7
29 hydrolases
1 transferase
20. cathepsin D
or
mutated enzymes degrading lipids,
GPs, proteins, and glycogen
de
rs
I.A lysosomal enzymopathies caused by mutation of the
enzyme protein
mutated enzymes degrading GAGs
et
ab
ol
ic
D
is
21. α-L-iduronidase (DS, HS)
22.Iduronate-2-sulphate sulphatase(DS,HS)
23. heparan N-sulphatase (HS)
24. NAc-α-D-glucosaminidase (HS)
25. CoA:α-glucosaminid NAc-transferase (HS)
26. GlcNAc- 6-sulphate sulphatase (HS)
27. GalNAc-6-sulphate sulphatase (KS,C6S)
28. GalNAc-4-sulphate sulphatase (DS)
29. β-glucuronidase (DS,HS,C4S,C6S)
30. hyaluronidase (hyaluronic acid)
In
st
itu
te
of
In
he
rit
ed
M
1. ceramidase (N-Acyl-sfingoid-Cer)
2. β-glucosylceramidase (GlcCer)
3. β-galactosylceramidase (GalCer)
4. arylsulphatase A (SGalCer)
5. NAc-β-glucosaminidase A (GM2)
6. NAc-β-glucosaminidase B (GM2,GP)
7. β-galactosidase (GM1, OLS, GP, KS)
8. α-galactosidase A ( Gb3Cer)
9. sphingomyelinase (P-cholin-cer)
10. acid lipase (CE, TAG)
11. α-neuraminidase (GP, gangliosides?)
12. N-acetyl-α-galactosaminidase (GP,
blood gr. A: αGalNAc- [Fucα]- βgal-;)
13. acid α-1,4-glucosidase (glycogen)
14. α-Mannosidase (GP)
15. β-Mannosidase (GP)
16. α-Fucosidase (GP)
17. aspartylglucosaminidase (GP)
18 tripeptidylpeptidase I – TPP (prot.)
19. palmitoyl-protein thioesterase-PPT
30 lysosomal enzymes
29 hydrolases +1 transferase
30 proteins 30 genes 30 entities
memento – there is more than 40
lysosomal enzymes known !!
2006
de
rs
or
I.B deficient enzyme associated functions (n=8)
D
is
deficient posttranslational processing (n= 2)
ed
M
et
ab
ol
ic
•abnormal targeting of lys. enzymes extracellularly (deficient
synthesis of Mannose-6P label – mucolipidosis II/III
•deficient synthesis of active site in a group of sulphatases (special
enzyme in ER: FGE formylglycine generating enzyme or SUMF1
sulphatase modifying factor 1): cystein→ formylglycine
(PSD = MPS + sulphatidosis, ect)
In
he
rit
deficient protection=increased degradation (galactosialidosis)
(protection by cathepsine A) (n=1)
of
deficient lysosomal enzyme activators (n=5)
In
st
itu
te
•SAPs A-D (pSap deficiency): A (GALCase); B (ASA, αGalase);
C (GCase); D (ceramidase)
•hexosaminidase activator (alternat. Tay-Sachs)
de
rs
ab
ol
ic
D
is
or
total 38 enzymopathic entities to be diagnosed
prime importance: enzyme activity assay
he
rit
ed
M
et
mechanism of the deficient
activity should be specified
In
st
itu
te
of
In
DNA analysis is of
secondary importance
2006
D
is
or
de
rs
II. lysosomal disorders due to deficiency of
noncatalytic membrane components
(n = 10)
ab
ol
ic
A. transporters across the lysosomal membrane(n=2)
• cystinosis
• sialic acid storage disease
of
In
he
rit
ed
M
et
B. mutant lysosomal membrane proteins operating in the
membrane lipid trafficking and by so far unknown (albeit
essential) mechanisms (n<8)
NCL3, NCL5, NCL6, NCL 8 (neuronal ceroid lipofuscinoses)
proteins, ML IV, NPC1, NPC2 (Niemann-Pick disease type C),
LAMP 2 (Danon disease)
In
st
itu
te
total 10 entities to be diagnosed at the protein/metabolite levels
final diagnosis the DNA level
(gene coding the dysfunctional protein)
!! activities of all the lysosomal enzymes are in the normal range !!
de
rs
D
is
or
II. (n=10)
deficiencies of noncatalytic
lysosomal membrane
functions
ab
ol
ic
48
entities
et
I. A, B (n=38)
deficiencies
in breakdown
catalysis
itu
st
N
te
of
In
he
rit
ed
M
hall mark: „storage“- overfilling
and gradual lysosomal expansion by: • undegraded
enzyme substrates
• unremovable
enzyme products
• misshandeled
N
heterogenous
compounds
In
normal cell
storage cell
2006
B
A
A
A
B
B
A
A B
A
B
B
B
A B
A B
A B
B
de
rs
ultrastructure
of the stored material
he
rit
ed
M
et
B
Gb3Cer
OLS
st
itu
te
of
In
GM2
In
A
A
B
ol
A
B
A B
ab
A
B
cytology
or
A
B
A
D
is
A
ic
Lysosomal
enzymopathy
cytology - EM
SU
GlcCer
CE
low power EM: cell in advanced
stage of lysosomal storage
de
rs
or
D
is
loss of NPC1 protein function
(altered lipid strafficking)
mixture of lipids stored
ic
NCL6
loss of NCL 6 protein
(function unknown)
SCMAS storage
ISSD
loss of SA transporter
retention of sialic acid
ab
loss of mucolipin function
(function unknown)
mixture of lipids stored
NPC1
ol
ML IV
M
et
EM patterns in group II LSDs
he
rit
ed
basic cytological patterns
of lysosomal storage
EM patterns in uncleaved
substrate accumulation
foamy pattern (3D distension)
In
st
itu
te
of
In
striated pattern-GC (2D distension)
GSD II (glycogen)
Gaucher (GlcCer)
MPS (GAGs)
CESD (CE storage)
NCL6
Tay-Sachs (GM2)
de
rs
or
D
is
In
st
itu
te
of
In
he
rit
ed
M
et
ab
ol
ic
each of the molecular defects is a
specific biological entity informing
indirectly about the level of a critical
lysosomal function (e.g. degradative,
trafficking) in normal human
(eukaryotic) tissues
de
rs
or
D
is
ab
ol
ic
lysosomal storage disorders
the diagnostic approach
decisions at the
clinical level
(phenotype analysis)
decisions at the
biochemical/molecular
levels (48 entities !!)
ed
M
et
decisions at the tissue level
•storage cell analysis (biopsy)
•results of urine analysis
In
st
itu
te
of
In
he
rit
Intermediate auxiliary
direction indicator
„at a crossroad“
enzyme
deficiency
(and its
mechanism!)
deficient lysosomal
noncatalytic
membrane
protein
D
is
or
de
rs
specific storage pattern at the cell level:
uniform storage of SM liquid crystals
in a b.m. histiocyte
et
hepatosplenomegly
In
st
itu
te
of
In
he
rit
ed
M
Niemann-Pick type A
ab
ol
ic
recommendation:
ASM activity evaluation
foamy storage pattern
birefringence of SM liquid crystals
uniform staining
for phospholipids
de
rs
or
D
is
ic
ol
phase contrast
ab
autofluorescence (ceroid)
et
variable storage
of phospholipids
In isotropic state
(admixture of ceroid)
ed
M
bone marrow storage
pattern in NPC
Giemsa
Filipin test – NPC1,2 gene
In
st
itu
te
of
In
he
rit
cytology and
staining
birefringence (absent)
irregular accumulation of phospholipids (ferric hematoxylin)
et
ab
ol
ic
D
is
or
de
rs
storage pattern in NCL2 (TPP I deficiency)
activity of AcPhos - cryostat sections
NCL
In
st
itu
te
of
In
he
rit
ed
M
control
autofluorescent storage material
EM intralysosomal curvilinear bodies
he
rit
ed
M
et
ab
ol
ic
D
is
or
de
rs
Grays Anatomy, TB Johnston et al., eds.,1958
In
urine analysis = „chemical biopsy of the kidney“
In
st
itu
te
of
positive finding means presence of
lysosomal storage in the tubular and
glomerular cells; detached storage cells
should be the main source of the diagnostic
stored compound
D
is
or
de
rs
Lipidoses free of urinary findings
Gaucher
negative
Krabbe
negative
ed
M
et
ab
ol
ic
Lipidoses with positive findings in the urine
(currently not utilized for screening)
NPA/B
sphingomyelin/cholesterol
CESD/Wolman
cholesteryl esters
In
st
itu
te
of
In
he
rit
Lipidoses with positive findings in the urine
(recommended for screening)
Fabry (incl. SapB def.)
Gb3Cer
MLD (incl. SapB def.)
sulphatide
Farber
ceramide
GM1 gangliosidosis
βgal - OLS
GM2 gangliosidosis
GlcNAc – OLS
glycolipids in the urinary sediment
de
rs
kidney (frozen section)
D
is
or
storage in
glomerules
ab
ol
ic
storage in
medullary
tubules
et
Fabry disease
results of kidney
and urinary
analysis
Gb3Cer
N
N
C
C
C
H
N normal control; C carriers; H hemizygote
section of the kidney
u.sediment
In
st
itu
te
of
In
he
rit
ed
M
PAS +
Gb3Cer
EM – storage in tubular epithelium
birefringence of in situ
tubular cell storage
desqumated
storage cell
D
is
or
de
rs
Gb3Cer in urines of Fabry (FD) hemizygotes, female carriers
and controls.
600
ol
ic
nmol Gb3Cer/mmol creatinine
ab
500
M
et
400
3
5
7
9
300
200
100
In
1
he
rit
ed
cont rols
FD carriers
FD hemiz.
11
15
0
17
19
23
In
st
METHOD: Extraction of total urinary lipids –
reversed-phase chromatography
Ð
itu
25
Berná L. et al.,Anal Biochem, 269, 304-311 (1999)
cont rols
FD carriers
Ð
te
21
FD hemiz.
Lipid separation – HPTLC
orcinol detection
densitometrically (CAMAG II)
Ð
of
13
quantification
ic
D
is
or
de
rs
kidney tubules and urinary sediment in sulphatidosis
birefringence
rit
ed
M
et
ab
ol
Cresyl violet
In
st
itu
te
of
In
he
Urinary sediment
¨storage in kidney tubules
Urinary sediment
de
rs
of
In
he
rit
ed
M
et
ab
ol
ic
D
is
or
Sulfatides (SGalCer) in urines of pacients (P) with
sulfatidosis (C1,2=controls)
In
st
itu
te
A. Orcinol detection, B. Azur A detection (specific),
Berná L. et al.,Anal Biochem, 269, 304-311 (1999)
Stand.= sulfatide and GL standard
rit
ed
M
et
ab
ol
ic
CS DS2 HS
DS1 Hep
+
-/±
-/±
+
+
+ variable +
+
+
+ variable +
+
+ !!
+
++
+
+
+!! +!! ++
+!! +
+!! -
st
itu
te
of
In
he
KS
CONTROL MPS I
MPS II
MPS III
MPS IVA
+
MPS IV B -/±
MPS VI
MPS VII
-
D
is
or
de
rs
urine in mucopolysaccharidoses
(GAGs)
In
KS – keratan sulphate; DS – dermatansulphate;
CS – chondroitinsulphate; HS – heparan sulphate; Hep - heparin
∗
∗
∗
∗
∗
∗
In
st
itu
te
of
In
he
rit
ed
M
et
ab
ol
ic
D
is
or
de
rs
MPS I – Hurler disease (α −iduronidase deficiency)
3,5 =MPS I (excretion of dermatan sulphate/DS and
heparan sulphate/HS, see arrows), 4 = neonatal
control (traces of HS and DS1), 1,2,6 = controls
or
de
rs
Urine GAG ELFO in patients with MPS II, and
its comparison with MPS I a MPS III
ab
ol
ic
D
is
Urinary excretion of dermatan
sulphate (DS1,2) and heparan
sulphate (HS)
itu
te
of
In
he
rit
ed
M
et
MPS II
patients
KO = control
In
st
MPS II: iduronate-2-sulphate sulphatase deficiency, X – linked disorder
de
rs
or
MPS IVA
In
st
itu
te
of
In
he
rit
ed
M
et
ab
ol
ic
D
is
deficiency of
GalNAc-6-sulphate
sulfatase (excretion of
keratan sulfate,
chondroitin-6-sulphate)
2,6 = MPS IVA (see arrows)
4 = MPS I
1,3,5,7 = controls
de
rs
or
Urine in glycoproteinoses and related disorders
D
is
OLS (oligosaccharides) – low mol.w. glycoconjugates reflecting incomplete
degradation of glycoproteins
st
In
*
itu
te
of
In
he
rit
ed
M
et
ab
ol
ic
GM1 gangliosidosis…………….. βgal-OLS
α-mannosidosis…………………..αmann- OLS
β-mannosidosis ………………….βmann- OLS
α-Fucosidosis ………………….. αfuco - OLS
Sialidosis ……………………….. sialyl - OLS
Galactosialidosis ……………… gal- and sialyl – OLS
AGU ……………………………… aspartylglucosamine
(+ other glycoasparagines)
ISSD (SALLA dis.) ……………... free sialic acid
Schindler disease ……………. sialyl – OLS
GSD II ………………….………… occasionally (Glc)4 *
de
rs
st
itu
te
of
In
he
rit
ed
M
et
ab
ol
ic
D
is
or
Glycoproteinoses – HPTLC of urinary oligosacharides
In
P1, P2 = susp.sialidosis confirmed later by enzymology (sample applied in two concentrations), Sial=sialidosis(archived
pathologic control), GM1=GM1 gangliosidosis,
Fuc= fucosidosis, Sch=Schindler disease, NANA= N-acetylneuraminic acid (standard), Ko=control urine
de
rs
or
D
is
ic
ol
ab
et
M
AGU
itu
te
of
In
he
rit
ed
patient
In
st
ninhydrine detection – urine in AGU
de
rs
D
is
or
In
st
itu
te
ol
et
M
ed
rit
he
of
In
NPCs
NCLs
Danon dis.
ML IV
Cystinosis
ab
(negative or unsignificant findings)
ic
Urine in other LSDs
negative
negative (or SCMAS)
not studied
phospholipids
generalized AAU
de
rs
or
D
is
Clinical findings :
he
rit
ed
M
et
ab
ol
ic
dysmorphia; dysostosis
neurology; visceromegaly;
corneal clauding (absent in MPS II)
heart valvular disease;
storage vacuoles in lymphocytes incl.
Alder-Reily granules
In
Urine analysis:
In
st
itu
te
of
• GAGs
• OLS
• free silaic acid
• AGU
de
rs
ML
1%
In
te
st
itu
M PS
26%
of
In
he
rit
ed
M
et
ab
ol
ic
GP
2%
GSD II
3%
NCL
16%
D
is
or
LYSOSOMAL STORAGE DISORDERS IN THE CZECH and
SLOVAK REPUBLICS 1975-2005 (n=525)
LIPIDOSES
53%
ab
ol
Krabbe
8%
Gaucher
20%
ic
GM1
5%
other
2%
D
is
GM2
4%
or
de
rs
LIPIDOSES IN THE CZECH AND SLOVAK REPUBLICS
1975-2005 (n=279)
In
he
rit
ed
M
et
CESD
6%
itu
te
of
MLD
9%
In
st
NPA, B
10%
IIMD, 2005
Fabry
18%
NPC
18%
ic
ol
VII
1%
I
20%
M
et
ab
IV A
15%
III D
2%
VI
2%
IV B
1%
D
is
or
de
rs
MUCOPOLYSACCHARIDOSES 1975 - 2005 (n=103)
In
he
rit
ed
III C
4%
itu
te
of
III B
3%
In
st
III A
27%
IIMD, 2005
II
25%
Year
20
20
19
19
19
19
19
19
19
19
19
19
05
02
99
96
93
90
87
84
81
78
75
72
69
66
e
of
ed
rit
he
In
ic
ol
ab
et
M
40
35
30
25
20
15
10
5
0
19
19
st 1960
i1t9u
63
t
In
N u m b e r o f p a tie n ts
d ia g n o s e d in
p a rtic u la r y e a r
de
rs
or
D
is
Postnatal and prenatal diagnoses of
LSDs in our Institute (1960 - 2005)
de
rs
No. of live births (A), postnatal and prenatal diagnoses of LSDs (B)
in the Czech and Slovak Republics (1960 - 2003)
or
350000
D
is
A
300000
250000
ic
200000
ab
ol
150000
et
100000
M
50000
0
he
rit
B
25
In
20
15
of
10
Institute of Inherited Metabolic Disorders
Prague, March 2004
20
02
19
96
19
99
19
93
19
90
19
87
19
81
19
84
19
78
19
75
19
69
19
63
In19
s66t
0
19
72
itu
te
5
19
60
1: 6000
ed
30
ab
de
rs
or
ol
ic
D
is
The diagnostic procedures
decisions at the
clinical level
(phenotype analysis)
decisions at the
biochemical/molecular
levels (48 entities !!)
ed
M
et
decisions at the tissue level
storage cell analysis (biopsy)
results of urine analysis
In
st
itu
te
of
In
he
rit
Intermediate auxiliary
(directing) step
team work
enzyme
deficiency
(and its
mechanism!)
lys. noncatalytic
membrane
protein
dysfunction
de
rs
Selected syndromes suggestive of LSD
ab
ol
ic
D
is
or
systemic disorder in childhood mainly
dysmorphy + dysostosis + corneal clouding + valvular heart
disease + neurology
(MPS, GP, GM1, PSD)
rit
ed
M
et
adolescence - adulthood
progressive nephropathy + cardiomyopathy +
angiokeratomas + neuropathy (sensitive)
Fabry disease
te
of
In
he
early childhood
progressive neurol. disturbance + retinopathy (blindness)
neuronal ceroidlipofuscinoses
In
st
itu
childhood - adolescence
neurological disturbance + VSO + splenomegaly (even mild)
Niemann-Pick type C
de
rs
selected syndromes suggestive of LSD cont.
D
is
or
adulthood (adolescence)
et
ab
ol
ic
isolated splenomegaly + hypersplenism
splenomegaly + unexplained femoral head necrosis
Gaucher´s disease (glucocerebrosidase deficiency)
rit
ed
M
myoclonous epilepsy + cherry red spot
ML I (sialidase deficiency)
of
In
he
isolated hypertrophic CMP
cave! Fabry disease (αGal deficiency) (inter alia)
In
st
itu
te
isolated hepatomegaly with slightly altered LFTs
serum cholesterol increased in LDL (decreased in HDL)
risk of accelerated atherosclerosis
CESD (acid lipase deficiency)
de
rs
LSDs project into majority of clinical disciplins
or
(only significant involvement is included)
st
itu
te
of
In
he
rit
ed
M
et
ab
ol
ic
D
is
ophthalmology (NCL, GM1-2, NPA, Fabry, MPS, GP, ML IV)
neurology (GM1-2, NCL1,2, NCL3,5,6,8, NPCs, Krabbe, MLD, MPS, GP, GD)
psychiatry (adult neurolopidoses)
pneumology (Gaucher, NPA/B, NPC2)
cardiology (Fabry, MPS, GP, GSD II)
angiology (Fabry, CESD, MPS)
hepatology (CESD, NPC infantile, MPS, GSD II, GP)
nephrology (Fabry, nephrosialidosis)
dermatology (Fabry, Fuco-, βMannosidosis, PSD, Farber)
hematology (Gaucher, NPA/B, NPC, ect)
orthopedy/osteology (MPS, GP, GD, ML II/III)
stomatology (MPS, GP, ML II/III)
ORL (Fabry, MPS, GP)
In
GP glycoproteinoses, CESD acid lipase deficiency, PSD polysulphatase deficiency, GSD II Pompe disease
MPS mucopolysaccharidoses; GD Gaucher disease, Farber dis. = ceramidase deficiency
de
rs
or
D
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The end
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M
ACKNOWLEDGEMENT
clinical analysis (division A) – E. Hrubá, Jahnová, E. Košťálová, S. Štastná.
biochemical analyses (divisions A and B): J. Ledvinová, H. Poupětová, L.
Berná, O. Martinová, E. Pospíšilová, M. Hřebíček
DNA analysis (Div.B) : L. Dvořáková and her group
histology, EM, histochemistry: M. Elleder, H. Hůlková (div. B)