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Glycomics Profile Analysis by MALDI TOF/MS in Human CSF

Glycomics Profile Analysis by MALDI TOF/MS in Human CSF. Xueli Li, Ph.D., 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

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Glycomics Profile Analysis by MALDI TOF/MS in Human CSF

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  1. Glycomics Profile Analysis by MALDI TOF/MS in Human CSF Xueli Li, Ph.D., Children’s Hospital of Philadelphia

  2. 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

  3. 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 ?

  4. 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

  5. Methods 400 μl CSF Filter Glycoprotein Free glycan O-Glycan N-Glycan Reduced β-elimination PNGase F Desalted & purified by acetic acid – methanol & resin Desalted & Purified by C18 column Permethylation Permethylation MALDI-TOF MALDI-TOF

  6. “Undiagnosed” Cases • Brother (MM, 11 year old) • Encephalopathy/CP/Spastic Paraparesis • Congenital Hypotonia • Global Developmental Delay • Bilateral Hearing Loss • Multiple Fractures • Recurrent Pneumonias • Low immunoglobulin level, low IgA • Generalized Aminoaciduria • CDG normal by CDT • Sister (IM, 6 year old) • Profound Global Developmental Delay • Congenital Hypotonia • Neonatal Seizures • Cerebral atrophy, small corpus callosum • History of recurrent UTIs • CDG normal by CDT

  7. Sib’s Urine Free Glycan 838.57 885.4 Glu4 or Glu3Man1 Control patient 651.68 1275.35 1241.30 681.57 885.92 1742.86

  8. Differential For Hex4 α1,2 Glucosidase I • Pompe Disease • acid alpha-1,4-glucosidase (GAA), 17q25.2-q25.3 • CDG IIb • glucosidase I (GCS1), 2p13-p12 • Polyglucose Diet X Glc(3)-Man(9)-GlcNAc(2)

  9. Sib’s CSF Free Glycan Control Patient

  10. Abnormal CSF Free Glycans

  11. Freeze Nature Reviews Genetics advance online publication; published online 6 June 2006 | doi:10.1038/nrg1894

  12. Sib’s Plasma N-glycan Glc3Man7

  13. Sib’s CSF N-Glycan Compare with Control Control CSF N-Glycan Patient CSF N-Glycan Wrong slides 3009.22 2805.08

  14. 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 sibling Exon 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 • Father Exon 1 c.65C>A, p.A22E; c.329G>A, p.110R>H;

  15. GalNAc Type O-linked Protein Glycosylation Core1 Biosynthesis of Core 1 and Core 2 Core2 Essentials of Glycobiology Second Edition

  16. Plasma O-glycan Profile Monosialyl T antigen 1200 895.584 1000 800 M-core 2 Intens. [a.u.] Disialyl T 600 D-core 2 400 1256.790 I.S. 200 * 1391.894 1705.983 1344.874 0 800 1200 1600 m/z

  17. CSF O-glycan Profiles Control CAD-Def Control and abnormal CSF O-linked glycan

  18. CAD Deficiency

  19. 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

  20. Acknowledgment He Research Lab NIH/UDP Miao He, Ph.D. Mariska Davids, Ph.D. Mohd Raihan Megan Kane, Ph.D. Lynne Wolfe Neal Boerkoel, MD. Ph.D. Bill Gahl, MD. Ph.D.

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