Age-related macular degeneration and the Y402H polymorphism in complement

factor H

Manchester Biomedical Research Centre

Simon ClarkTony Day

Progression of AMD

Age-related maculopathy

Age-related macular degenerationAtrophic Neovascular

Soft drusen + pigmentary changes in RPE

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• 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

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

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

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 ↓

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

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)

Glycosaminoglycans (GAGs)

Types of GAGs

Heparan sulfate (HS) proteoglycans

Heparin a highly sulfated form of HS made specifically by mast cells

Immunolocalisation of CFH in human macula tissue (OX23/24 Abs)

RPE

Bruch’s membrane

Drusen

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

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

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

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)

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

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.