Children’s Hospital of Philadelphia

Glycomics Profile Analysis by MALDI TOF/MS in Human CSF

Xueli Li, Ph.D., Children’s Hospital of

Philadelphia

Children’s Hospital of Philadelphia

Introduction

• Genetic defects in human glycome– 2-3% of the human genome encodes proteins for

glycosylation– 50% of proteins are glycosylated in human– 10 distinct glycoprotein biosynthesis pathways– >100 disease causing genes discovered– Many still remain unknown

Children’s Hospital of Philadelphia

Why CSF glycome?

• Glycoproteome and glycome in plasma are limited

• Unique glycosylation type and glycogene expression pattern in brain

• CSF glycome in human has not been well studied

• Does CSF glycome provide us more diagnostic value for CDG ?

Children’s Hospital of Philadelphia

Project

• CSF free glycome• CSF N-glycome• CSF O-glycome• Study cohort

– 166 CSF from patients with undiagnosed neurological disease (NIH/UDP)

– 10 CSF normal controls

Children’s Hospital of Philadelphia

Methods400 μl CSF400 μl CSF

Filter

Free glycanFree glycan

N-Glycan O-Glycan

PNGase FReduced β-elimination

Desalted & Purified by C18 column

Desalted & purified by acetic acid – methanol & resin

Permethylation Permethylation

MALDI-TOF MALDI-TOF

GlycoproteinGlycoprotein

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“Undiagnosed” Cases

Brother (MM, 11 year old)Encephalopathy/CP/Spastic ParaparesisCongenital HypotoniaGlobal Developmental DelayBilateral Hearing LossMultiple FracturesRecurrent PneumoniasLow immunoglobulin level, low IgAGeneralized AminoaciduriaCDG normal by CDT

Sister (IM, 6 year old)Profound Global Developmental DelayCongenital HypotoniaNeonatal SeizuresCerebral atrophy, small corpus callosumHistory of recurrent UTIsCDG normal by CDT

Children’s Hospital of Philadelphia

Sib’s Urine Free Glycan

1000 1500 2000 2500 3000

0

5000

10000

15000

20000

Inte

nsity

m/z

65

1.6

8

12

41

.30

88

5.9

2

12

75

.35

800 1600 2400 3200 4000

0

24000

48000

Inte

nsity

m/z

83

8.5

7

681.

578

85

.4

17

42

.86

Glu4 or Glu3Man1

Control patient

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Differential For Hex4Pompe Disease

acid alpha-1,4-glucosidase (GAA), 17q25.2-q25.3

CDG IIb

glucosidase I (GCS1), 2p13-p12

Polyglucose Diet

Glc(3)-Man(9)-GlcNAc(2)

X

α1,2

Glucosidase I

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Sib’s CSF Free Glycan

Control Patient

Children’s Hospital of Philadelphia

Abnormal CSF Free Glycans

m/z value Reference range

885.40 Hex4 (Glu4 or Glu3Man1) 43.21% H 1.37-8.59%

1538.697 Man6 GlcNAc1 or Glu3Man3GlcNAc1 0.41% H 0.0-0.13%

1742.86 Man7 GlcNAc1 or Glu3Man4GlcNAc1 17.07% H 0.0-0.0%

1946.96 Man8 GlcNAc1 or Glu3Man5GlcNAc1 0.32% H 0.0-0.0%

2151.06 Man9 GlcNAc1 or Glu3Man6GlcNAc1 0.14% H 0.0-0.0%

2355.06 Glu3Man7GlcNAc1 0.10% H 0.0-0.0%

Children’s Hospital of PhiladelphiaFreeze Nature Reviews Genetics advance online publication; published online 6 June 2006 | doi:10.1038/nrg1894

Children’s Hospital of Philadelphia

Sib’s Plasma N-glycan

999.0 1601.8 2204.6 2807.4 3410.2 4013.0

Mass (m/z)

1.1E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Reflector Spec #1 MC[BP = 2793.1, 11203]

12-17-2010-10BN09688

2792

.0989

2430

.9685

2822

.1096

1579

.6453

2778

.0840

2416

.9443

2966

.1733

2226

.9016

2600

.0334

2460

.9790

1981

.8041

3602

.4058

1783

.7192

3776

.4980

1053

.4089

2185

.8855

3047

.3540

2882

.1362

2738

.1023

2546

.9954

2069

.8403

2834

.0967

1118

.5491

1866

.7703

2359

.9280

3241

.2546

1620

.6633

2788

.0940

1553

.0438

1241

.4728

1000

.4803

1293

.5281

2302

.8977

1416

.5986

2506

.9702

2028

.8046

2686

.2212

1701

.6742

3415

.3765

1661

.6952

1925

.8187

1175

.5707

1823

.6553

1742

.6985

3648

.4072

3690

.3521

3002

.2876

3541

.3098

3833

.4614

3356

.3542

3140

.2556

3950

.5535

3283

.2417

3731

.4783

Glc3Man7

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Sib’s CSF N-Glycan Compare with Control

Control CSF N-Glycan Patient CSF N-Glycan

Wrong slides

3009

.22

2805

.08

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MOGS Seq• Affected male sibling

Exon 1 c.65C>A, p.A22E; c.329G>A, p.110R>H; Exon 2 c.370C>T, p.124Q>X

• Affected female siblingExon 1 c.65C>A, p.A22E; c.329G>A, p.110R>H Exon 2 c.370C>T, p.124Q>X

• Mother

Exon 2 c.370C>T, p.124Q>X

• FatherE

xon 1 c.65C>A, p.A22E; c.329G>A, p.110R>H;

Children’s Hospital of Philadelphia

GalNAc Type O-linked Protein Glycosylation

Core1

Core2

Biosynthesis of Core 1 and Core 2

Essentials of Glycobiology Second Edition

Children’s Hospital of Philadelphia

895.584

1391.894 1705.983

0

200

400

600

800

1000

1200In

ten

s. [a

.u.]

Monosialyl T antigen

Disialyl T

M-core 2

D-core 2

1344.874*

I.S.

m/z800 1200 1600

1256.790

Plasma O-glycan Profile

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CSF O-glycan Profiles

• Control and abnormal CSF O-linked glycan

Control CAD-Def

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CAD Deficiency

Children’s Hospital of Philadelphia

Conclusion

• CSF glycome is different and more complex than plasma glycome– Free glycome, N-glycome and O-glycome

• CSF glycome may detect CNS-specific defect in protein glycosylation

• CSF glycome provides additional information for the diagnosis of CDG

Children’s Hospital of Philadelphia

Acknowledgment

He Research Lab NIH/UDP Miao He, Ph.D. Mariska Davids, Ph.D.Mohd Raihan Megan Kane, Ph.D.

Lynne WolfeNeal Boerkoel, MD. Ph.D.Bill Gahl, MD. Ph.D.