Transcript Age-related macular degeneration and the Y402H polymorphism in

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
Soft drusen + pigmentary changes in RPE
Age-related macular degeneration
Atrophic
Neovascular
AMD is a condition that primarily
affects RPE cells in the macula
Why particularly the macula?
• Most metabolically active
• Light damage
Ageing changes within the RPE
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•
•
•
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
Accumulation of extracellular material derived from RPE
Drusen
Diffuse thickening of Bruch’s membrane
2 micron
Retinal pigment epithelium
Druse
RPE cells
Bruch’s membrane
Choroid
Thickening of Bruch’s
Membrane
(basal linear deposits)
Extracellular material disrupts transport between RPE and choroid and thereby
increases RPE stress
Ageing retina and progression
to AMD
High metabolic activity, light, oxygen resulting in free radical
formation and RPE dysfunction
Genetic
susceptibility
Inflammation and accumulation of
intracellular and extracellular debris
Environmental
factors
Critical point reached
Death of retinal/RPE cells
Atrophy
Choroidal neovascularisation
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 risk
CFHR1/CFHR3 deletion
• Environmental influences
•
•
•
•
Smoking ↑
Abdominal adiposity ↑
Anti-oxidant intake ↓
Omega-3 fatty acid intake ↓
Complement cascade
*
*
*
*
Cellular damage, phagocytosis, inflammation
Implicated in AMD
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
Structure of CFH
CFH consists of 20 complement control protein (CCP) modules
Y402H polymorphism is a major genetic risk factor for AMD
Facilitate decay of
C3 convertase
Cofactor for factor I
1
4
CRP
C3b
GAG/polyanion
6 7 8
GAG/polyanion
Cell surface
C3b, C3d
12
14
19 20
402Y/H
402 residue contributes to GAG
binding site (Prosser et al., J. Exp
Med 2007)
Glycosaminoglycans (GAGs)
Heparan sulfate (HS) proteoglycans
Heparin a highly sulfated form of HS
made specifically by mast cells
Types of GAGs
Immunolocalisation of CFH in human
macula tissue (OX23/24 Abs)
RPE
Drusen
Bruch’s
membrane
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
Methodology
6
H 8
402H CCP6-8 (disease associated)
6
Y 8
402Y CCP6-8
H
Full length CFH 402H
Y
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
Results
402H and 402Y CCP6-8 and full-length CFH
merge
402Y
RPE
CCP6-8
100 µm
Bruch’s
membrane
Relative fluorescence (arbitrary units)
402H
200
CCP6-8
6
7 8
402H
6
7 8
402Y
*
150
100
50
402H
402Y
402H
402Y
0
Bruch’s
membrane
RPE
Note similar pattern in choroidal vessels
402Y
merge
flCFH
Relative fluorescence (arbitrary units)
402H
H
CFH 402H
Y
CFH 402Y
flCFH
100
**
80
60
40
20
402H
RPE
Control experiments:
Binding unaffected by genotype of tissue
Results unaffected by exchanging fluorophores
Unlabelled proteins effectively competed for binding
402Y
402H
402Y
0
Bruch’s
membrane
* P = 0.0005, ** P = 0.009
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
Effects of heparinise and chondroitinase B
digestion on binding of full length 402H and 402Y
and CFH antibody labelling
Binding of CCP6-8 Y and H variants to
selectively desulfated heparin
(Heparin is a highly sulfated form of HS)
Conclusion
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…..
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
4. Supports the Y402H amino acid substitution being the genetic
alteration that predisposes to AMD.