1' How do you define a glycosylation disorder

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1. How do you define a glycosylation
disorder?
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GLYCOSYLATION DISORDERS IDENTIFIED
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30
30
25
25
20
20
O-Mannose
Number of defects
15
15
10
10
O-Fucose
5
5
1980
03
99
01
02
04
05
06
07
Year
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Stephanie Grunewald. (2007) Early Human Develop
83 825-830
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Why were these disorders missed for so long?

• Physician awareness of glycosylation was
  low--still is!
• Symptoms are broad, diverse and variable that
  most specialists could not specify typical
  symptoms.
• When should you think CDG? Every time you
  suspect it and every time you dont!

What breakthroughs made the difference?

• Availability of a simple blood test--transferrin
• Increased awareness of glycosylation
• Possibility to treat a few of the disorders
• Sequencing of the human genome
• Availability of models to show pathology

Describe the methods used today to identify a
glycosylation disorder.
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Isoelectric focusing of transferrin
Sialic acids
NOTE Not all defects show transferrin differences
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SLOWEST STEP
What are the advantages and disadvantages of
isolating mutants in cultured cell lines compared
to deriving cell lines from mutant animals or
humans afflicted with glycosylation disorders?
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From Chapter 8--most of these genes first were
identified in yeast
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Whats an ldlD?
How was it found?
How might you use ldlD cells, in the presence and
absence of galactose and N-acetylgalactosamine,
to test the role(s) of glycans in
biological processes?
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ApoC-III in Normal and CDG-II patient
Control Patient
Some CDG mutations effect several pathways
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Refined view of mammalian COGs
ldlBCog1 ldlCCog2
ldlB and ldlC deficient cells are defective in
glycosylation
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Mutation Function
Cog7 splice site mutation decreases mRNA and
protein
Destabilizes COG complex, mis-localizing some COG
subunits
X
X
Decreases trafficking of glycosyltransferases and
transporters
X
X
Decreases enzyme/transporter stability and normal
localization
Decreases terminal modifications on sugar chain
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SEVERAL DEFECTS ARE DUE TO A MUTATIONS IN
COGs, AND IN OTHER PROTEINS IN TRAFFICKING
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Autosomal recessive cutis laxa Wrikly skin. Large
fontanelle, delayed closure, connective tissue
weakness, growth developmental delay,
neurological anbormalities, seizures and mental
deterioration in later life. Skin phenotype
becomes milder with age Abnormal N-and O-glycans
in serum. Homozygosity (10K and 250K) mapping 15
consanguineous families. Lod Score of 3.2 region
on 12q24. Micro satellite mapping gave 5.7Mb.
ATP6V0A2 in endosomes and TGN
Mechanism Alters intragolgi pH
Affects trafficking of Golgi proteins
Impaired glycosylation and cutis laxa caused by
mutations in the vesicular H-ATPase subunit
ATP6V0A2. Kornak, et al Nat Genet. 2008
Jan40(1)32-4.
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SOME PATIENTS HAVE HEPATIC FIBROSIS AND
PROTEIN- LOSING ENTEROPATHY
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BEFORE MANNOSE
DURING MANNOSE
AT III
albumin (g/dl)
anticoagulation (Marcumar R )
i. v. albumin substitution
fecal alpha1 AT (mg/g)
time in months
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Therapy for CDG-Ib
CDG defect sugar transporter Man GlcNAc
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PMI haploinsufficiency(heterozygous PMI knockout
mice)
Protein leakage in vivo in wild type and PMI/-
mice with or without Mannose intervention
Mannose (10 in drinking water)

• How long mannose

3-day-average (!)
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Tracking glycosylation defects is easier when
only one protein is affected
FromTamao Endo, in Beyond Glycogenes , Aug 20,
2003
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GLYCOSYLATION-BASED CONGENITAL MUSCULAR
DYSTROPHIES
POMT1 and POMT2
POMTGnT1
FCMD
FKRP
LARGE
Congenital Muscular Dystrophy 1D
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CONGENITAL MUSCULAR DYSTROPHIES

• Type II lissencephaly (type II LIS) (CMD) defects
  in ?-DG glycosylation
• Walker-Warburg syndrome, Fukuyama cerebral and
  muscular dystrophy, or muscle-eye-brain disease.
• Fetal presentation often elects pregnancy
  termination.
• 47 fetuses from 41 unrelated families. Sequenced
  ?-DG glycosylation genes and found 22 families 30
  different mutations
• RESULTS
• POMT1 32
• POMGNT1 15 and POMT2 7
• No mutations in FKRP, FCMD, and LARGE. No
  definitive molecular diagnosis in half the cases.
• CONCLUSIONS
• Type II LIS POMT1 and POMT2 are severest
  clinical picture and POMGNT1 for milder disease.
• FKRP, FCMD, and LARGE, not seen in the fetal
  CMD.
• Bouchet C, et al, Hum Mutat. 2007
  Oct28(10)1020-7.

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The phenotypic spectrum of glyco-deficient
patients with mutations in these genes is
broader than previously thought. Many patients
had no mutations, suggesting additional,
undefined genes are involved in dystroglycan
glycosylation.
Godfrey et al,
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Sialyl-Tn expression is a prominent feature of
many carcinomas. What explains the high frequency
of this expression despite the fact that
the enzyme responsible for it's synthesis is not
always upregulated? This is related to the
underlying mechanism of other diseases too.
Tn
S/T
Sialyl-Tn
S/T
S/T
S/T
How does this relate to CMD?
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Which disease has a really cool therapy??
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BEFORE AND AFTER HEPARIN THERAPY
Although heparin is primarily used as an
anticoagulant, its use has been proposed in
connection with several other diseases. How can
one drug have relevance to so many different
mechanisms?
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How would you assess the genetic and
environmental contributions to a "glycosylation
disorder?
Provide examples of gain-of-function mutations.
Consider mutations that create protein
glycosylation sites as well as those that change
the expression of glycosylation genes.
Youre a doctor and a young and very sick patient
arrives in your clinic. Fortunately, you were
trained in Glycobiology and suspect this young
one might have a glycosylation disorder What
would you do to confirm your suspicion?
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