Congenital Disorders of Glycosylation: Diagnostic steps - ERNDIM

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N
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Congenital Disorders of Glycosylation:
Diagnostic steps
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

Muscle (Dystroglycan,
agrin)
Glycoprotein
hormones
(LH/FSH/TSH)
N-glycosylation: multisystemic
Liver (transferrin)
Coagulation (factor VII, IX, ATIII)
Brain (MAG, P0, neurexin)
N
- -
G G
M
M
N
M
- -
N
G G
M
M
Asn Asn
N
M

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

M
M M
M
M
M
M
M
M
Ii
Fru-6-P Man-6-P Man-1-P
CMP-
M
M
M
M
M M
M
M
M
M
M M
M
M
IIf
Ib
Ik
M
M M
M M
M
Id
Trans Golgi
M
M M
= dolichol-PP
= dolichol
= GlcNAc
UDP UDP-
IL
M M
M M
M M
M
GDP - M
P
M
M
M
M
M M
M
M
M
GDP
M
P
M
M
M
M
M
P P
M
M M
M M
M
M
G
M M M
M M M
M M
M
Cytoplasm
G
G
M M M
M M M
M M
M
G
G
G
M M M
M M M
M M
M
UDP GDP
UDP
IId
Ia
Ij
Ie
IIc
UDP
G
Median Golgi Cis Golgi
M M M M
M
M
M = mannose
= galactose
= fucose
G
= glucose
= sialic acid
= COG complex
If
Ig IL Ic Ih
IIe/IIg/IIh
Diagnostic approach
N-glycosylation
G
IIa
P
UDP- G
G
G
G
M
P
M
M
M M M
M M
OTase M
Im
IIb
Endoplasmatic Reticulum
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

4
2
0
control
CDG patients with different profiles
Type I; ER defects
patient
N
- -
G G
M
M
N
M
86 CDG-I; solved
8 CDG-Ix; unsolved
Asn Asn
Type II; Golgi defects
patients
4
3
2
1
0
- -
N
G G G
M
M
17 CDG-II; solved
29 CDG-IIx; unsolved
N
M M M
M
Asn Asn

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
Type II:
4. Haemolytic Uremic Syndrome (HUS)
5. Severe liver disease
1 2
6. Young age (

Transferrin protein polymorphism
Lanes 1 - 4: No neuraminidase treatment
Lanes 5 - 8: Neuraminidase treatment
1/5: normal
2/6: The frequently occurring C 1/C 3 variant
3/7: protein polymorphism shifting towards anode (= B variants)
4/8: protein polymorphism shifting towards cathode ( D variants)
-
1
2
3
4
5
6
7
8
0 1 2 3 4 5 -sialotransferrin
+

Type I/II classification
• In some cases, assignment of type I or type II is difficult
• SDS-PAGE of transferrin might help
4
2
0
TIEF
1a 1b 1c C 2
LC-MS of lane 2
SDS-PAGE

Escape of the CDG-I vs CDG-II classification?
9 yr girl:
cleft palate, dilated cardiomyopathy and
chronic hepatitis
Type I
+
Type II

Short LLO:
normal
M
M M
M
M
M
M
M
M
Ii
M
M
M
M
M
M
M
M M
M
M
Ik
M
M M
M M
M
Id
Mevalonaat
Dehydrodolichol-PP
dolichol
UDP
IL
UDP -
M
M
M
M
Ij
M M
M
P
M
M
M
Stage 2: CDG-I diagnostic work-up
M-1-P M-6-P
GDP - M
Ie
M
M M
M
M
M
P
M
M
M
Ia
GDP
P P
M
M M
M M
M
M
G
M M M
M M M
M M
M
Ig IL Ic Ih
G
G
M M M
M M M
M M
M
Cytoplasm
Fr-6-P
G
G
G
M M M
M M M
M M
M
UDP- G UDP
Lipid-linked Oligosaccharide (LLO) analysis
If
Ib
G
G
P
G
G
G
M
P
M
M
M M M
M M
OTase M
Endoplasmatic Reticulum
G
G
M M M
M M M
M M
M
= dolichol
= GlcNAc
M M M
M M M
M M
M
M
G
M M
M M M
M M
M
= mannose
= glucose
16

Stage 2: CDG-II diagnostic work-up
Exit
- -
M
M
M
control patient
-
M M
M
-
CMP-
M M
M
-
M
UDP
M
M
M M
M
Golgi
M M
M
MGAT2
CDG-IIa
N-Glycan structural
analysis
UDP
M M
M M
M
GDP
M M
M M
M

Control
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
2794
3605
1970
2433
2780
2968
2419
2043
2824
2762
1838
2607
2852
1956
1766
2246
2517
1580
3243
1693
3213
2145
2288
2230
3779
3056
3634
3687
3327
3197
3417
3809
3864
3982
CDG-IIa
1500 2000 2500 3000 3500 4000
0
10
20
30
40
50
60
70
80
90
100
1984
2158
2607
1609
1706
1782
1951
2187
2013
2794
2968
2065
1579
2239
2245
1937
1796
2762
2392
2432
2936
2593
2637
2441
2690
2824

Glycan types
• N-glycosylation: amide (NH2) binding with Asparagine (ASN)
• O-glycosylation: hydroxy (OH) binding with Serine (Ser) or Threonine (Thr)
Neu5Ac 6Gal1 4GlcNAc1
Neu5Ac
6Gal1
4GlcNAc1
Man1
Man1
Man1
4GlcNAc1
O-glycan
N-glycan
Neu5Ac2 3Gal1 3GalNAc1
4GlcNAc1

Exit
Exit
More options in the Golgi
-
M M
M
-
CMP-
- -
M M
M
CMP-
UDP
CMP
CMP
Cytoplasm
M M M M
M
M
-
Golgi
UDP
UDP
UDP
UDP
M M
M M
M
GDP
UDP
UDP
M M
M M
M
N
O

Isoelectric focusing of serum apolipoproteinC-III
Core 1 mucin type O-glycan in position Thr-94
pH 3.5
pH 5.0
Apo CIII - 2
Apo CIII - 1
Apo CIII - 0
Wopereis et al. Clin Chem 2003;49(11):1839-45.
-
-
-

Profile types of ApoCIII in CDG type II patients
Transferrin
ApoCIII
4
3
2
1
0
control
2
1
0
46 CDG-II: 15 N glycosylation
ApoCIII-0
profile
ApoCIII-1
profile
group 1 group 2
10 N+O glycosylation group 1
21 N+O glycosylation group 2 Wopereis, Glycobiology 2005

Exit
Exit
Options for a combined N+O glycosylation defect
2
-
M M
M
-
CMP-
6 steps
- -
M M
M
CMP-
UDP
3
CMP
CMP
Cytoplasm
M M M M
M
M
-
Golgi
UDP
1
1
UDP
UDP
UDP
M M
M M
M
GDP
UDP
UDP
M M
M M
M
N
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

COG7:
4
3
2
1
0
Group 1: COG defect in 5/10 patients
• Microcephaly, adducted thumbs, growth retardation,
VSD, episodes of hyperthermia, early fatal
2
1
0
group 1: ApoCIII-0
Morava J Hum Genet 2007
26

Model of COG complex
X
cog2 2
cog7 7
cog3
cog1 1 cog8 8 cog6 6
cog4
cog5 5
COG complex is required for recycling of glycosyltransferases

Group 2: 14/21 patients with cutis laxa phenotype
4
3
2
1
2
1
0
group 2: ApoCIII-1

ATP6V0A2 and glycosylation?
•

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