Congenital Disorders of Glycosylation:

Diagnostic steps

Dirk J. Lefeber (D.Lefeber@neuro.umcn.nl)

Nijmegen, The Netherlands

ERNDIM Meeting, Basel 2009

--

N 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

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

- -

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

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

= 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

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

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

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

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

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

Escape of the CDG-I vs CDG-II classification?

Type I

+

Type II

9 yr girl:cleft palate, dilated cardiomyopathy and

chronic hepatitis

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

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

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

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)

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

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

-

-

-

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

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

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

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

1 8cog32

5

67

cog8cog5

cog7cog6cog3

cog4

cog2cog1

X

Model of COG complex

COG complex is required for recycling of glycosyltransferases

Group 2: 14/21 patients with cutis laxa phenotype

group 2: ApoCIII-1

0

1

2

1

2

3

4

ATP6V0A2 and glycosylation?

6.4

6.24-5

pH gradient

ATP6V0A2

• <6 months of age: isolated

ApoCIII -1 profile

• Patients with normal ApoCIII

exist

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