age-related macular degeneration and the y402h polymorphism in complement factor h
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Manchester Biomedical Research Centre. Age-related macular degeneration and the Y402H polymorphism in complement factor H. Tony Day. Simon Clark. Progression of AMD. Age-related maculopathy Age-related macular degeneration Atrophic Neovascular. - PowerPoint PPT PresentationTRANSCRIPT
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Age-related macular degeneration and the Y402H polymorphism in complement
factor H
Manchester Biomedical Research Centre
Simon ClarkTony Day
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Progression of AMD
Age-related maculopathy
Age-related macular degenerationAtrophic Neovascular
Soft drusen + pigmentary changes in RPE
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AMD is a condition that primarily affects RPE cells in the macula
Why particularly the macula?
• Most metabolically active
• Light damage
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Ageing changes within the RPE• Intracellular accumulation of lipofuscin• Autofluorescent material that accumulates in lysosomes• Contains lipid and protein• Formed from incompletely degraded photoreceptor outer segments and
autophagy• Induces free-radical formation
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Accumulation of extracellular material derived from RPE
RPE cells
Thickening of Bruch’s Membrane(basal linear deposits)
2 micron
Retinal pigment epithelium
Druse
Bruch’s membrane
Choroid
2 micron
Retinal pigment epithelium
Druse
Bruch’s membrane
Choroid
Extracellular material disrupts transport between RPE and choroid and thereby increases RPE stress
Drusen Diffuse thickening of Bruch’s membrane
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Ageing retina and progression to AMD
High metabolic activity, light, oxygen resulting in free radical formation and RPE dysfunction
Inflammation and accumulation of intracellular and extracellular debris
Death of retinal/RPE cellsAtrophy Choroidal neovascularisation
Critical point reached
Genetic susceptibility
Environmental factors
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What predisposes to AMD apart from ageing?
• Genetic risk factors: Major risk factors• Complement factor H (especially Y402H polymorphism)• ARMS2/HTRA1 locus (Chr 10q26) Intermediate risk factors• Complement factor 2/complement factor B locus • Complement factor 3 Minor risk factors• ABCA4• ApoE
Decreased riskCFHR1/CFHR3 deletion
• Environmental influences• Smoking ↑• Abdominal adiposity ↑• Anti-oxidant intake ↓• Omega-3 fatty acid intake ↓
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Complement cascade
Cellular damage, phagocytosis, inflammation
*
*
*
* Implicated in AMD
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Functions of Complement Factor H (CFH)There is tick over C3b is deposition on host surfaces which can amplify and lead to activation of alternative pathway unless suppressed
CFH is a soluble protein mainly produced by the liver that binds to polyanionic structures (such as glycosaminoglycans (GAGs)) on host surfaces and prevents C3b mediated complement activation
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Structure of CFH
71 20
402Y/H
864 191412
Facilitate decay of C3 convertase
Cofactor for factor I
GAG/polyanionCell surfaceC3b, C3dGAG/polyanion C3b
CRP
CFH consists of 20 complement control protein (CCP) modules Y402H polymorphism is a major genetic risk factor for AMD
402 residue contributes to GAG binding site (Prosser et al., J. Exp Med 2007)
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Glycosaminoglycans (GAGs)
Types of GAGs
Heparan sulfate (HS) proteoglycans
Heparin a highly sulfated form of HS made specifically by mast cells
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Immunolocalisation of CFH in human macula tissue (OX23/24 Abs)
RPE
Bruch’s membrane
Drusen
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Hypothesis and research question
• Hypothesis – differential binding of 402H and 402Y forms to GAGs explains the association of the polymorphism with AMD (still debate as to whether this is the functional change associated with this genetic locus)
• Question – Do the 402H and 402Y forms show differential binding to (normal) macular tissue
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Y
Full length CFH 402H
H6 8402H CCP6-8 (disease associated)
Y6 8 402Y CCP6-8
Methodology
H
Full length CFH 402Y
• 402H and 402Y CCP6-8 (recombinant) or CFH (full-length) were fluorescently-labelled (H - red) (Y - green) and applied in pairs to frozen sections of “normal” post mortem human macula tissue (age 46-90)
• Relative levels of binding of the 402H and 402Y forms were calculated after measuring fluorescence (and subtracting background fluorescence)
• In some experiments tissue sections treated with GAG degrading enzymes prior to application of fluorescently-labelled proteins
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Results402H and 402Y CCP6-8 and full-length CFH
flCFH
* P = 0.0005, ** P = 0.009
402H 402Y 402H 402Y
RPE Bruch’s membrane
200
Rel
ativ
e flu
ores
cenc
e (a
rbitr
ary
units
)
150
100
50
0
CCP6-8*
402H 402Y 402H 402Y
RPE Bruch’s membrane
80
Rel
ativ
e flu
ores
cenc
e (a
rbitr
ary
units
)
60
40
20
0
100flCFH
**
76 8 402H
76 8 402Y
76 8 402H76 8 402H
76 8 402Y76 8 402Y
YY
CFH 402HHH
CFH 402Y402H 402Y merge
Note similar pattern in choroidal vessels
Control experiments:Binding unaffected by genotype of tissueResults unaffected by exchanging fluorophores Unlabelled proteins effectively competed for binding
CC
P6-
8
402H 402Y mergeRPE
Bruch’s membrane
100 µm
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402H and 402Y (CCP6-8) binding sites are predominantly
heparan sulphate (HS) with a smaller amounts of dermatan sulphate (DS)
Relative binding of 402H and 402Y from previous slide
No evidence of interactions with hyaluronan or chondroitin sulfate
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Effects of heparinise and chondroitinase B digestion on binding of full length 402H and 402Y
and CFH antibody labelling
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Binding of CCP6-8 Y and H variants to selectively desulfated heparin
(Heparin is a highly sulfated form of HS)
0
Biotinylated protein/well (pmol)
0 20 40 60 80 100
Ab s
o rba
nce
(405
nm
)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
2ISre-N Ac6-O deS2-O deSN deS
FHCCP6-8(402Y)
2,6-O deS
0
Biotinylated protein/well (pmol)
0 20 40 60 80 100
Ab s
o rba
nce
(405
nm
)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
2ISre-N Ac6-O deS2-O deSN deS
FHCCP6-8(402Y)
2,6-O deS
Abs
orba
nce
(405
nm
)
0
0 20 40 60 80
Biotinylated protein/well (pmol)
0 20 40 60 80 100
0.2
0.4
0.6
0.82IS
6-O deS
2-O deS
N deS
N deSre-N Ac
FHCCP6-8(402H)
2,6-O deS
Abs
orba
nce
(405
nm
)
0
0 20 40 60 80
Biotinylated protein/well (pmol)
0 20 40 60 80 100
0.2
0.4
0.6
0.82IS
6-O deS
2-O deS
N deS
N deSre-N Ac
FHCCP6-8(402H)
2,6-O deS
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Conclusion
402Y form localises to Bruch’s membrane and thereby protects against complement activation on the membrane
402H form localises poorly to Bruch’s membrane and this may lead to complement over-activation, inflammation and eventually AMD
1. 402H form of CFH binds poorly to Bruch’s membrane and choroidal structures compared to 402Y form
2. CFH binds particularly to HS and the differing affinities of 402H and 402Y forms and dependent upon HS sulfation patterns
3. Suggests a possible disease mechanism…..
4. Supports the Y402H amino acid substitution being the genetic alteration that predisposes to AMD.