Congenital Disorders of Glycosylation: Diagnostic steps

Transcription

Congenital Disorders of Glycosylation: Diagnostic steps
Congenital Disorders of Glycosylation:
Diagnostic steps
- -
N
ERNDIM Meeting, Basel 2009
Dirk J. Lefeber (D.Lefeber@neuro.umcn.nl)
Nijmegen, The Netherlands
O
Dynamic glycosylation pathway
Courtesy of Dr. T. Hennet (Zurich)
General principle of Congenital Disorders of Glycosylation
X
Congenital Disorders of Glycosylation:
Errors in the assembly line
Glycoprotein Function
• Protein stability, solubility
and structure
• Protection against proteases
• Cell – cell interactions
• Target – receptor interaction
• Localization of proteins
• Signal transduction
• Bacterial adhesion
•…
> 50 % of human proteins are glycosylated
> 1 % of our genes is involved in glycosylation
N-glycosylation: multisystemic
Liver (transferrin)
Brain (MAG, P0, neurexin)
Muscle (Dystroglycan,
agrin)
Glycoprotein
hormones
(LH/FSH/TSH)
Coagulation (factor VII, IX, ATIII)
N
G
M
-
-
-
N
G
N
G
G
M
M
M
N
M
M
Asn
Asn
Clinical aspects of Congenital
Disorders of Glycosylation
Classical picture of CDG:
•
hypotonia/epilepsy/cerebellar atrophy, inverted nipples,
fat pads, strabismus, feeding problems, ataxia,
hypogonadism, mental retardation
Control
Patient
When to perform transferrin isofocusing?
• All patients with a suspicion of a metabolic disorder
• Reason:
• CDG: wide spectrum, mild isolated to severe
multisystem
• CDG-Ib/h, fructosemia
• CDG-Ix with isolated myopathy, optic nerve
atrophy, DCM, ichthyosis
• New phenotypes in CDG-II: adducted
•
thumbs/microcephaly; cutis laxa; complex
vertebral malformations
CDG-II with liver pathology as main feature
Ib
Ia
Fru-6-P
Diagnostic approach
Man-1-P
Man-6-P
UDP UDP -
M
If
M
Ij
Ik
Ie
P
P
M
P
P
M
G
M
M
M
Id
M M
MM
M
M
M
IL
M M
MM M
M
M
M
Ig
IL
P
M M M
M M M
M
M
M
Ic
G
M M M
M M M
M
M
M
G
G
G
M M M
M M M
M
M
M
Endoplasmatic Reticulum
G
G
G
M M M
M M M
M
M
OTase M
G
G
G
M M M
M M M
IIb
M
M
Ih
Im
G
G
M M
M M
M
M
M
M
M
M M M
M M M
M
M
M
M
M
M M M
M
M
M
M
M
M
M
M
M
M M
M
M
M
UDP- G
P
M
M
M
Cytoplasm
M
M
M
M
M
M
M
UDP
G
M
Ii
N-glycosylation
GDP
GDP - M
CMP-
GDP
UDP
IIf
UDP
IIc
Median Golgi
Trans Golgi
Cis Golgi
M
M
M
M
M
M
M
IId
M
M
M
M
IIa
M
M
M
M
G
= mannose
= glucose
= galactose
= sialic acid
M
= fucose
= COG complex
M
M
M
IIe/IIg/IIh
= dolichol-PP
= dolichol
= GlcNAc
M
M
M
M
M
M
M
CDG patients with different profiles
Type II; Golgi defects
Type I; ER defects
-
4
2
-
4
3
2
N
N
G
G
M
M
M
0
Asn
control patient
86 CDG-I; solved
8 CDG-Ix; unsolved
1
0
Asn
-
-N
N
G
G
G
M
M
M
M
M
M
Asn
Asn
patients
17 CDG-II; solved
29 CDG-IIx; unsolved
Nomenclature changes in CDG
• 1999: CDGS-I to VI to CDG-I(a-o) and CDG-II(a-h)
• 2009: from CDG-I//II to PMM2-CDG
PMM2-CDG
MGAT2-CDG
ALG6-CDG
B4GALT1-CDG
PMI-CDG
POMT1-CDG
CDG-I
CDG-II
Walker Warburg Syndrome (WWS)
Keep CDG-I/II with gene name??
CDG-I(ALG3)
Stage 1: Secondary causes & Type I/II determination
Type I:
1. Galactosemia
2. Fructosemia
3. Alcohol abuse
1
2
2
4
2
0
Type II:
4. Haemolytic Uremic Syndrome (HUS)
4
5. Severe liver disease
0
6. Young age (<1-2 months)
7. Transferrin protein polymorphism
4
5
6
Transferrin protein polymorphism
Lanes 1 - 4: No neuraminidase treatment
Lanes 5 - 8: Neuraminidase treatment
-
+
1
2
3
4
5
6
7
8
0
1
2 3
4 5 -sialotransferrin
1/5: normal
2/6: The frequently occurring C1/C3 variant
3/7: protein polymorphism shifting towards anode (= B variants)
4/8: protein polymorphism shifting towards cathode ( D variants)
Type I/II classification
• In some cases, assignment of type I or type II is difficult
• SDS-PAGE of transferrin might help
SDS-PAGE
TIEF
4
2
0
1a
1b
1c
C
2
LC-MS of lane 2
Escape of the CDG-I vs CDG-II classification?
9 yr girl:
cleft palate, dilated cardiomyopathy and
chronic hepatitis
Type I
+
Type II
Stage 2: CDG-I diagnostic work-up
Mevalonaat
Short LLO:
normal
Dehydrodolichol-PP
Ib
Ia
dolichol
M-1-P
GDP
GDP - M
UDP UDP M
Ij
Ik
P
Ie
G
P
M
P
P
P
M
G
M
M
M
M
M
M
M M
M
M
M
Id
IL
M M
M M
M
M
M
Ig
M M
M M M
M
M
M
IL
UDP
M M M
M M M
M
M
M
Ic
G
M M M
M M M
M
M
M
G
GG
M M
M M
M
Ih
M
M
M
M
P
Endoplasmatic Reticulum
G
G
G
M M
M M
M
M
M
M
G
G
G
M M M
M M M
M
M
OTase M
G
G
M M
M M
M
M
M
M M
M M
M
M
M
M
M
M
M
M
M M M
M
M
M
M
M
M
M
M
M
M
Cytoplasm
M
M
M
M
M
M
M
UDP- G
If
M
M
Ii
Fr-6-P
M-6-P
= dolichol
= GlcNAc
M
G
= mannose
= glucose
Lipid-linked Oligosaccharide (LLO) analysis
16
Stage 2: CDG-II diagnostic work-up
-
-
-
M
M
M
N-Glycan structural
analysis
M
M
M
control
patient
UDP
UDP
CMP-
GDP
Golgi
- -
M
Exit
M
M
M
M
M
M
M
M
M
M
M
M
MGAT2
CDG-IIa
M
M
M
M
M
M
M
M
M
10
0
1500
1782
1706
2000
2500
m/z
2936
2968
2607
2637
2690
2762
2794
2824
2593
2392
2432
2441
60
2158
70
2187
2239
2245
3000
3500
3982
3779
3809
3864
3634
3687
3417
3327
3213
3243
3197
3056
3605
2968
2824
2852
2762
2780
2607
2517
2433
2419
2145
2230
2246
2288
1970
40
2043
1956
1838
1766
1693
50
2013
2065
100
1984
CDG-IIa
1951
1796
1937
20
1609
10
1580
30
20
1579
Relative Abundance
2794
Control
100
90
80
70
60
0
90
80
50
40
30
4000
Glycan types
•
•
N-glycosylation: amide (NH2) binding with Asparagine (ASN)
O-glycosylation: hydroxy (OH) binding with Serine (Ser) or Threonine (Thr)
N-glycan
Neu5Ac
6Gal1
4GlcNAc1
Man1
Man1
Neu5Ac
6Gal1
4GlcNAc1
4GlcNAc1
Man1
O-glycan
Neu5Ac2
3Gal1
3GalNAc1
4GlcNAc1
More options in the Golgi
UDP
Cytoplasm
UDP
CMP-
GDP
Golgi
- -
M
Exit
M
CMP-
Exit
- -
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
N
UDP
CMP
CMP
-
UDP
UDP
UDP
UDP
O
Isoelectric focusing of serum apolipoproteinC-III
Core 1 mucin type O-glycan in position Thr-94
-
pH 3.5
Apo CIII - 2
-
Apo CIII - 1
-
Apo CIII - 0
pH 5.0
Wopereis et al. Clin Chem 2003;49(11):1839-45.
Profile types of ApoCIII in CDG type II patients
Transferrin
ApoCIII
ApoCIII-0
profile
ApoCIII-1
profile
4
3
2
2
1
1
0
0
control
group 1
46 CDG-II: 15 N glycosylation
10 N+O glycosylation group 1
21 N+O glycosylation group 2
group 2
Wopereis, Glycobiology 2005
Options for a combined N+O glycosylation defect
2
6 steps
UDP
Cytoplasm
UDP
CMP-
Golgi
- -
GDP
1
M
Exit
CMP-
Exit
- -
M
3
M
M
M
M
M
M
M
M
M
N
UDP
CMP
CMP
M
M
M
M
M
M
M
M
M
M
M
-
UDP
1
UDP
UDP
UDP
O
Option 4: Trafficking in the secretory pathway
a. Indirect
COG
Golgi defects
Conserved Oligomeric Golgi (COG)
complex
• Transport between Golgi vesicles
Cutis laxa
• ATPase defect influencing Golgi pH
Group 1: COG defect in 5/10 patients
COG7:
• Microcephaly, adducted thumbs, growth retardation,
VSD, episodes of hyperthermia, early fatal
4
3
2
1
2
1
0
0
group 1: ApoCIII-0
Morava J Hum Genet 2007
26
Model of COG complex
cog2
2
cog3
cog3
cog1
cog8
1
8
cog4
X
cog7
7
6
cog6
cog5
5
COG complex is required for recycling of glycosyltransferases
Group 2: 14/21 patients with cutis laxa phenotype
4
2
3
1
2
1
0
group 2: ApoCIII-1
ATP6V0A2 and glycosylation?
pH gradient
6.2
6.4
ATP6V0A2
•
•
<6 months of age: isolated
ApoCIII -1 profile
Patients with normal ApoCIII
exist
4-5
Step by step diagnostic approach for CDG
Stage 1:
Interpretation of transferrin isofocusing gel
Confirmation of generalized glycoprotein abnormality
Exclude transferrin protein polymorphism
Exclude secondary causes of N-glycan biosynthesis
abnormalities
Discriminate between CDG-I and CDG-II
•
•
•
•
•
Stage 2; CDG-I:
PMM/PMI measurement; LLO analysis in fibroblasts
Genetic tools & clinical information
Stage 2; CDG II:
Check O-glycan abnormalities and N-glycan structure
Genetic tools & clinical information
•
•
•
•

Similar documents