NPSR Gene & Panic Disorder NPSR A/T (Asn 107 Ile) variant (rs324981) Timothy M. Hanna, MS.
Congenital Disorders of Glycosylation: Diagnostic steps · Coagulation (factor VII, IX, ATIII)...
Transcript of Congenital Disorders of Glycosylation: Diagnostic steps · Coagulation (factor VII, IX, ATIII)...
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Congenital Disorders of Glycosylation:
Diagnostic steps
Dirk J. Lefeber ([email protected])
Nijmegen, The Netherlands
ERNDIM Meeting, Basel 2009
--
N O
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Dynamic glycosylation pathway
Courtesy of Dr. T. Hennet (Zurich)
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General principle of Congenital Disorders of Glycosylation
X
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Congenital Disorders of Glycosylation:
Errors in the assembly line
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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
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N-glycosylation: multisystemic
Brain (MAG, P0, neurexin)Liver (transferrin)
Coagulation (factor VII, IX, ATIII)
Glycoprotein
hormones
(LH/FSH/TSH)
Muscle (Dystroglycan,
agrin)
Asn Asn
MM
M
G G
NN
- -
MM
M
G G
NN
- -
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Classical picture of CDG:• hypotonia/epilepsy/cerebellar atrophy, inverted nipples,
fat pads, strabismus, feeding problems, ataxia,
hypogonadism, mental retardation
Clinical aspects of Congenital
Disorders of Glycosylation
Control Patient
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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
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= mannose
= glucose
= dolichol
= GlcNAc
= galactose
= sialic acid
= fucoseM
G = COG complex
P P
P
GDP
Fru-6-P Man-1-PMan-6-P
GDP
Median Golgi Cis GolgiTrans Golgi
M
M
M M
MM
MMM
M MM
M
P
UDP
OTase
M
P
-
M
M M
M
M
MM
M
M
M M
M M
M
MM
M
M
M
MM
M
M
M
M
MM M
M
M
MM
M
GDP
Endoplasmatic Reticulum
M M
MM
M
CMP-
MMM
M
MM
MM
MM
M MM
M
M
MM
M
M
M
M M
M
M
M
MM
M
M
M M
M
G G
M
M
MM
M
M
M
M
M
G
G
MM
M
MM
M
M
M
M
G
G
G
G
M
M
M
M
MM
M
M
M
G
G
P
G
UDP- GUDP-UDP
Cytoplasm
M
M
M
M
MM
M
M
M
M M
M
M
M
M
M
M
M
G
G
G
UDP UDP
MM
M
M
MM
MM
M
M
M
IIa
IIb
IIc
IId
IIf
IIe/IIg/IIh
Diagnostic approachN-glycosylation
IaIb
IcId
If
Ig Ih
Ii IjIk
IL
Ie
IL
Im
= dolichol-PP
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CDG patients with different profiles
control
Type I; ER defects
0
2
4
MM
M
G G
NN
Asn Asn
- -
patient
Type II; Golgi defects
4
3
2
1
0
MM
M M M
G G G
NN
Asn Asn
M
- -
patients
86 CDG-I; solved8 CDG-Ix; unsolved
17 CDG-II; solved29 CDG-IIx; unsolved
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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
Keep CDG-I/II with gene name?? CDG-I(ALG3)
CDG-II
CDG-I
Walker Warburg Syndrome (WWS)
PMM2-CDG
MGAT2-CDG
ALG6-CDG
B4GALT1-CDG
PMI-CDG
POMT1-CDG
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1. Galactosemia
2. Fructosemia
3. Alcohol abuse
4. Haemolytic Uremic Syndrome (HUS)
5. Severe liver disease
6. Young age (<1-2 months)
7. Transferrin protein polymorphism
Stage 1: Secondary causes & Type I/II determination
4
0
4
0
2
Type I:
Type II:
1 22
45 6
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Transferrin protein polymorphism
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)
+-
0 1 2 3 4 5 -sialotransferrin
Lanes 1 - 4: No neuraminidase treatment
Lanes 5 - 8: Neuraminidase treatment1
2
3
4
5
6
7
8
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Type I/II classification
1a 1b 1c C 2
• In some cases, assignment of type I or type II is difficult
• SDS-PAGE of transferrin might help
4
0
2
SDS-PAGETIEF
LC-MS of lane 2
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Escape of the CDG-I vs CDG-II classification?
Type I
+
Type II
9 yr girl:cleft palate, dilated cardiomyopathy and
chronic hepatitis
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Short LLO:
normal
Lipid-linked Oligosaccharide (LLO) analysis
16
= mannose= glucose
= dolichol= GlcNAc
M
G
Endoplasmatic ReticulumP P
P
GDP
Fr-6-PM-1-P M-6-P
GDP
M
M
P
UDP- UDP
OTase
M
P
-
M
M M
M
M
MM
M
M
M M
M M
M
MM
M
M
M
MM
M
M
M
M
MM M
M
M
M
MM
MM
MM
M MM
M
M
MM
M
M
M
M M
M
M
M
MM
M
M
M M
M
G G
M
M
MM
M
M
M
M
MG
G
MM
M
MM
M
M
M
M
G
G
G
G
M
M
M
M
MM
M
M
MG
G
P
G
GUDP -UDP
Cytoplasm
M
M
M
M
MM
M
M
M
M M
M
M
M
M
M
M
MG
G
G
M
MM
MM
M
M
M
IcId
If
Ig Ih
Ii IjIk
IL
Ie
IL
IbIa
Stage 2: CDG-I diagnostic work-upMevalonaat
Dehydrodolichol-PP
dolichol
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Stage 2: CDG-II diagnostic work-up
M M
MM
MMM
M MM
M
M M
MM
MMMM
GDPUDPUDP
MM
M
CMP-
Exit
Golgi--
M
M
M
control patient
M
M
M
N-Glycan structural
analysis
-- -
MGAT2
CDG-IIa
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Control
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
2794
36
05
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
28
24
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Glycan types
Neu5Ac2 3GalNAc13Gal1
4GlcNAc14GlcNAc1Man1
Man1
Man14GlcNAc16Gal1
6Gal1
Neu5Ac
Neu5Ac 4GlcNAc1
N-glycan
O-glycan
• N-glycosylation: amide (NH2) binding with Asparagine (ASN)
• O-glycosylation: hydroxy (OH) binding with Serine (Ser) or Threonine (Thr)
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CytoplasmGDPUDPUDPCMP-
CMP-
Exit
UDP
UDP
UDP
UDP
CMP
CMP
O
UDP
-- -
M M
MM
MMM
M MM
M
M M
MM
MMMM
MM
M NExit
Golgi--
More options in the Golgi
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Isoelectric focusing of serum apolipoproteinC-IIICore 1 mucin type O-glycan in position Thr-94
Wopereis et al. Clin Chem 2003;49(11):1839-45.
Apo CIII - 0
Apo CIII - 1
Apo CIII - 2
pH 5.0
pH 3.5
-
-
-
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Profile types of ApoCIII in CDG type II patients
0
1
2
ApoCIII
group 1 group 2
ApoCIII-0
profileApoCIII-1
profile
control
0
1
2
3
4
Transferrin
46 CDG-II: 15 N glycosylation
10 N+O glycosylation group 1
21 N+O glycosylation group 2Wopereis, Glycobiology 2005
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CytoplasmGDPUDPUDPCMP-
CMP-
Exit
UDP
UDP
UDP
UDP
CMP
CMP
O
UDP
-- -
M M
MM
MMM
M MM
M
M M
MM
MMMM
MM
M NExit
Golgi--
6 steps
Options for a combined N+O glycosylation defect
1
1
2
3
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Option 4: Trafficking in the secretory pathway
a. Indirect
COG
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Golgi defects
Conserved Oligomeric Golgi (COG) complex• Transport between Golgi vesicles
Cutis laxa• ATPase defect influencing Golgi pH
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26
Group 1: COG defect in 5/10 patients
• Microcephaly, adducted thumbs, growth retardation,
VSD, episodes of hyperthermia, early fatal
Morava J Hum Genet 2007
group 1: ApoCIII-0
COG7:
0
1
2
0
1
2
3
4
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1 8cog32
5
67
cog8cog5
cog7cog6cog3
cog4
cog2cog1
X
Model of COG complex
COG complex is required for recycling of glycosyltransferases
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Group 2: 14/21 patients with cutis laxa phenotype
group 2: ApoCIII-1
0
1
2
1
2
3
4
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ATP6V0A2 and glycosylation?
6.4
6.24-5
pH gradient
ATP6V0A2
• <6 months of age: isolated
ApoCIII -1 profile
• Patients with normal ApoCIII
exist
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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
Step by step diagnostic approach for CDG
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