2. ESSENTIALS OF GLYCOBIOLOGY LECTURE 33 GENETIC DISORDERS OF GLYCOSYLATION IN HUMANS Hud Freeze

3. OVERVIEW AND SUMMARY Many Human glycosylation disorders were discovered in the last 10 years, mostly in the N-linked pathway. Disorders are underdiagnosed. Alert physicians. Defects include monosaccharide activation, glycosyl transferases, transporters, biosynthetic glycosidases Golgi trafficking proteins. A few disorders can be treated with monosaccharides Misglycosylation causes some types of muscular dystrophy More glycosylation disorders will be found, phenotypes Will be broad and variable

4. GLYCOSYLATION DEFECTS IDENTIFIED Year 99 98 979695 94 80- 93 01 00 15 5 10 205 10 15 20 N-linked Disorders Muscular Dystrophy 02-04 Number of defects

5. -N-X-T/S On Protein Dol-P- P LLO TYPE I TYPE II CONGENITAL DISORDERS OF GLYCOSYLATION -N-X-T/S

6. CONGENITAL DISORDERS OF GLYCOSYLATION (CDG) TYPE I---Defects in synthesis or transfer of oligosaccharides from Dol-PP carrier to proteins in the lumen of the endoplasmic reticulum TYPE II--Defects in the processing of N-linked oligosaccharides OR in all other types of glycosylation TypesNumber of Patients Ia-IL-- >400 (Ia>300) IIa-IIe--~20 Ix or IIx-->50 CDG--often (not always) recognized by glycosylation-dependent alterations in serum transferrin isoelectric focusing pattern

7. Isoelectric focusing of transferrin 4 2 0 control CDG patients Sialic acid Galactose N-acetylglucosamine Mannose normal missing chains altered processing Sialic acids

8. 30-40 GENES REQUIRED TO MAKE AND ADD AN ASN-LINKED CHAIN

9. OVERVIEW OF SUGAR METABOLISM IN CELLS

10. Mannose supplements can treat CDG-Ib, Fru-6-P-->Man-6-P defect

12. anticoagulation (Marcumar R ) i. v. albumin substitution albumin (g/dl) fecal alpha1 AT (mg/g) AT III time in months BEFORE MANNOSEDURING MANNOSE

13. CDG defect sugar transporter Man GlcNAc -6-P -1-PGDP- Dol-P- Fru-6-P PMI PMM Therapy for CDG-Ib

14. Oral fucose supplements used to Treat CDG-IIc-- Defective in Golgi GDP-Fuc Transporter Patient is deficient in fucosylation What symptoms would you expect? Cause? What therapy?

15. FUCOSE THERAPY NORMALIZES NEUTROPHIL COUNTS OF ONE CDG-IIc PATIENT Serum Fucose (µM) Normal Range

17. CLINICAL FEATURES OF CDG PATIENTS Used with permission T. Marquardt. European J. Ped (2003)162 359-379.

18. Kindly provided by Dr. Thorsten Marquardt Dysmorphic features of a baby with CDG-Ia (PMM-deficiency) -6P GDP- Ia -6P-1P

19. DEFECT

20. -6P-1P GDP- -N-X-T/S Golgi Cytosol IIa IIb IIc UDP CMP UDP -N-X-T/S IId Dol-P- P LLO On Protein

21. -N-X-T/S T/S Possible Defect in Sialic Acid Synthesis or Transport? Defect is Shared by N-Linked and O-Linked Pathways Defects in Multiple Sialyl Transferases?

23. pmol/mg protein Endogenous acceptor pmol/mg protein 046810122 Exogenous acceptor (GAP) D V max 182 Lec2 0 50 100 150 200 Control CHO D E F Control V max 43 Control V max 306 HD V max 18 HD V max 92 0 50 150 250 300 200 100 0 10 20 30 40 50 CMP-Sia (uM) UDP-Gal (uM) CMP-Sia (uM) Nucleotide Sugar Transport is reduced Vmax is reduced, Km remains the same

25. H D Control F D Pre-bleach bleach 10min 30min 60 min Recovery ST-GFP Trafficking is reduced in HD and FD cells T/SST3Gal-I

26. ldlB=Cog1 ldlC=Cog2 ldlB and ldlC deficient cells are defective in glycosylation

27. Cog7 mutation decreases mRNA and protein Destabilizes COG complex, mislocalizing some COG subunits Decreases trafficking of glycosyltransferases and transporters Decreases enzyme/transporter stability and normal localization Decreases terminal modifications on glycans Mutation Function

28. SUMMARY Physicians must know many faces of glycosylation Defective Glycosylation causes disease Only a few of the potential types of CDG are known Simple therapy may help a few CDG patients CDG may be the cause of unexplained cases of developmental delay, coagulopathy, and hypoglycemia CDG presentations can be very mild and broad WHEN TO CONSIDER CDG? ”EVERY TIME YOU SUSPECT IT…. AND EVERY TIME YOU DON’T” -J. Jaeken

29. UDP-GlcNAc epimerase/kinase Defective in two human diseases Sialuria and Hereditary Inclusion Body Myopathy-II Sialuria--hypotonia, cerebellar ataxia, and mental retardation

31. Muscular Dystrophies Walker Warburg Syndrome--POMT1 mutations cause 30% MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a  1,2GlcNAc transferase specific for O-Man Fukuyma-type CMD--fukutin (9q31) Putative transferase in golgi (?) Fukutin Related protein--(19q13.3) Putative transferase (?) LARGE- cause of myd mouse, tandem glycosyltransfrases(?) Common Features: Affects  -dystroglycan glycosylation and not  -dystroglycan Hereditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia

32. O-Mannose: An Emerging Family of Glycans WWS-Mutated transferase

33. O-Mannose: An Emerging Family of Glycans MEB- Mutated transferase

34. MACULAR CORNEAL DYSTROPHY Autosomal recessive Progressive punctate corneal opacity, requires transplant Caused by mutation in GlcNAc-6-Sulfotransferase (CHST6) used for sulfation of keratan sulfate

36. http://www.burnham.org/presentations/hum an_glycosylation_disorders/index.htm