Elevated membrane attack complex in human choroid with high risk complement factor H genotypes.

Abstract

Data from human genetics, histopathology, and animal models reveal a major role for the complement system in the development of age-related macular degeneration (AMD). Genetic variations in the complement factor H (CFH) gene are associated with an elevated risk of AMD. In this study we sought to determine whether eyes from donors with a high-risk genotype (homozygosity for the histidine allele at codon 402) exhibit altered levels of membrane attack complex (MAC) in the choroid, compared to eyes with a low risk genotype (homozygosity for tyrosine). Proteins were extracted from the RPE/choroid of 18 donors (10 low risk and 8 high risk) and levels of MAC were assessed using an ELISA assay. Eyes from donors homozygous for the histidine allele showed 69% higher levels of MAC than those homozygous for the tyrosine allele (p < 0.05), independent of whether the eyes showed signs of early AMD. Our results provide evidence that high-risk CFH genotypes may affect AMD risk by increased deposition of MAC around the aging choriocapillaris.

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Letter to the Editor
Elevated membrane attack complex in human choroid with high risk complement
factor H genotypesq
Robert F. Mullins*, Aaron D. Dewald, Luan M. Streb, Kai Wang, Markus H. Kuehn, Edwin M. Stone
University of Iowa, 4135E MERF, 375 Newton Rd, Iowa City, IA 52242, United States
a r t i c l e i n f o
Article history:
Received 10 June 2011
Accepted in revised form 20 June 2011
Available online 26 June 2011
Keywords:
macular degeneration
complement
choroid
choriocapillaris
a b s t r a c t
Data from human genetics, histopathology, and animal models reveal a major role for the complement
system in the development of age-related macular degeneration (AMD). Genetic variations in the
complement factor H (CFH) gene are associated with an elevated risk of AMD. In this study we sought to
determine whether eyes from donors with a high-risk genotype (homozygosity for the histidine allele at
codon 402) exhibit altered levels of membrane attack complex (MAC) in the choroid, compared to eyes
with a low risk genotype (homozygosity for tyrosine). Proteins were extracted from the RPE/choroid of
18 donors (10 low risk and 8 high risk) and levels of MAC were assessed using an ELISA assay. Eyes from
donors homozygous for the histidine allele showed 69% higher levels of MAC than those homozygous for
the tyrosine allele (p < 0.05), independent of whether the eyes showed signs of early AMD. Our results
provide evidence that high-risk CFH genotypes may affect AMD risk by increased deposition of MAC
around the aging choriocapillaris.
? 2011 Elsevier Ltd. All rights reserved.
Therelationshipbetweenvariationsingenesencodingmembers
of the complement cascade and age-related macular degeneration
(AMD) is well established. AMD-associated variations include
polymorphisms in C3 (Maller et al., 2007; Yates et al., 2007), the
C2/CFB locus (Gold et al., 2006), and, most notably, complement
factor H (CFH) (Edwards et al., 2005; Hageman et al., 2005; Haines
et al., 2005; Klein et al., 2005).
In view of the role of CFH as an inhibitor of the alternative
pathway of complement activation, it seems likely that some poly-
morphisms affecting protein sequence would result in loss of func-
tion, and thus increased complement activation (Anderson et al.,
2010). We and others have described the deposition of terminal
complement complexes in the eyes of donors with AMD (Abrera-
Abeleda et al., 2006; Hageman et al., 2005; Seth et al., 2008; Skeie
et al., 2010). Complement complexes deposited on and around the
choriocapillaris may place stress on the choroidal endothelium and
could be related to the choriocapillaris loss observed in early AMD
that is associated with abundance and size of drusen (Mullins et al.,
2011). While the assumption that CFH polymorphisms alter the
inhibitory function of CFH appears reasonable, there is little empir-
ical evidence to date that showalteredcomplementinhibitioninthe
human choroid. While C-reactive protein levels have been found
elevated in donor eyes homozygous for the high-risk allele (Johnson
etal.,2006)andtheabundanceofsystemiccomplementcomponents
have been shown to vary with AMD affection status and genotype
(see for example (Hecker and Edwards, 2011; Sivaprasad et al.,
2007)), the levels of the membrane attack complex (MAC) have not
been quantified in eyes with high and low risk CFH genotypes.
In the current study we sought to evaluate the impact of the
Y402H CFH polymorphism on choroidal deposition of the MAC in
aged human eyes. Other components of complement activation are
deposited in aging Bruch’s membrane/choroid, and some of these
have effects on RPE and/or choroidal behavior (Nozaki et al., 2006;
Skeie et al., 2010). MAC formation is likelythe most crucial outcome
of complement activation as it results from uninhibited C5 con-
vertase activity and failure of vitronectin or CD59 to impair C9
polymerization, and has the potential to injure the RPE and/or
choriocapillaris.
Human donor eyes were obtained from the Iowa Lions Eye Bank
(Iowa City, IA). Eyes were obtained within 8 h of death, an interval
in which protein content is largely unchanged (Ethen et al., 2006).
All experiments were performed in accordance with the Declara-
tion of Helsinki and with consent of the donors’ families. Eyes were
dissected and a 6 mm-diameter trephine punch was collected from
qSupported in part by NEI EY017451 (RFM), NEI 016822 (EMS), Foundation
Fighting Blindness (RFM), the Macula Vision Research Foundation (RFM), the
Hansjoerg E.J.W. Kolder Professorship for Best Disease Research (RFM) and the
Howard Hughes Medical Institute (EMS). RM is co-inventor on patents related to
complement and AMD.
* Corresponding author.
E-mail address: robert-mullins@uiowa.edu (R.F. Mullins).
Contents lists available at ScienceDirect
Experimental Eye Research
journal homepage: www.elsevier.com/locate/yexer
0014-4835/$ e see front matter ? 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2011.06.015
Experimental Eye Research 93 (2011) 565e567

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the inferotemporal, juxtamacular RPE-choroid complex. Punches of
RPE and choroid were snap frozen in liquid nitrogen and stored
at ?80?C until processed for protein. For immunohistochemistry,
a 6 mm macular punch was collected, fixed, and embedded as
described previously (Mullins et al., 2011).
Genotyping was performed on either whole blood obtained at
time of enucleation or from a fragment of extraocular muscle using
established techniques (Qiagen DNeasy kit, Valencia CA). Geno-
typing was performed on subjects using TaqMan pre-designed SNP
genotyping assays (SNP rs1061170, Applied Biosystems) in a high-
throughput micro-fluidic system (Fluidigm, San Francisco, CA).
Samples were selected for ELISA analysis on the basis of CFH
genotype. Protein from donors homozygous for the Y402H high-
risk allele (“HH”, n ¼ 8) and from donors homozygous for the low
Fig. 1. MAC associated with CFH genotype in the human RPE-choroid. A, B Graphs showing normalized levels of MAC in human RPE-choroid. Levels of MAC were significantly
elevated in donor eyes homozygous for the high-risk (HH) allele compared to the low risk (YY) allele (A). The presence of early AMD was associated with a trend toward increased
levels of MAC but this did not reach statistical significance. Asterisks indicate mean value. CeF, Distribution of the membrane attack complex (green fluorescence) in donor maculae.
In most cases, MAC is localized to domains surrounding the choriocapillaris in both HH (C, D) and YY (E, F) homozygotes. Drusen (arrows) are often immunoreactive. Yelloweorange
fluorescence indicates RPE lipofuscin and blue fluorescence is due to the nuclear counterstain diamidino-phenol-indole. CC, choriocapillaris. Scale bar ¼ 50 mm. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)
R.F. Mullins et al. / Experimental Eye Research 93 (2011) 565e567
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risk allele (“YY”, n ¼ 10) were processed for MAC quantification.
Punches of RPE-choroid were raised in 60 mL of PBS with 1% Triton
X-100 with protease inhibitors (Roche Complete Mini tablets,
Indianapolis, IN) and homogenized using disposable pestles.
Following centrifugation on a benchtop microcentrifuge, protein
concentrations of supernatants were determined using a commer-
cial protein assay kit (BioRad DC Protein Assay, Hercules, CA). Thirty
micrograms of total proteinwere then loaded in each well of a MAC
ELISA kit (Quidel MicroVue SC5b-9 Plus Kit) in triplicate, according
to the manufacturer’s instructions. The concentration of MAC was
determined in each sample based on absorbance and by linear
interpolation using standards in the kit. For three samples (two YY
and one HH) in which the loaded amount of protein was slightly
lower than 30 mg, the final protein content was adjusted accord-
ingly. MAC levels were assessed both as a fraction of total protein
and as mass extracted from each punch. To determine fraction of
MAC to total protein, the MAC concentration was divided by the
total protein content. To determine the mass of MAC extracted from
each punch, the MAC concentration was multiplied by the volume
of diluent used to extract the protein.
The MAC levels were assessed between the two genotypes. We
observed an approximately 69% increase in MAC content in the
choroids of donors homozygous for the histidine allele. Statistical
analysis was performed using a permutation test with 10,000
permutations (Streitherg and Röhmel, 1986). Results showed
a marginally statistically significant elevation in MAC in HH as
compared to YY homozygotes (p ¼ 0.039) (Fig. 1A).
In addition to CFH genotype, premortem clinical information
was available for most of the donors. None of the eyes in this study
manifested late atrophic or exudative AMD, whereas 6 had early
AMD (as defined previously (Mullins et al., 2011)), 9 were unaf-
fected controls, and no data were available for 3. When data were
analyzed on the basis of AMD affection status for the 15 eyes for
which these data were available, increased MAC content was
observed on average (35% higher than control levels; Fig. 1B). In
contrast to CFH genotype, however, this trend was not significant in
our data set (p ¼ 0.97). Interestingly, although the sample size is
small, the samples from donors with AMD showed a bimodal
distribution, with both the highest and lowest values in the group
being present in the RPE-choroid from AMD samples. Further study
will be necessary to confirm and explore the clinical significance of
this observation.
In summary, we observed that donor eyes homozygous for high
and low risk CFH variants showed different levels of choroidal
membrane attack complex, with histidine homozygotes possessing
over 60% elevated MAC compared to tyrosine homozygotes.There are
limitations to our study. Our approach using human donor eyes does
not discriminate between circulating and localized MAC. However, in
light of the distribution of MAC in the human maculadinwhich most
of the protein appears matrix associated rather than in vascular
lumensdit is likely that the circulating component makes a relatively
small contribution (Fig. 1CeF). This may be in contrast to abundant
plasma proteins like C-reactive protein. Overall, this study lends
support to the notion that the Y402H polymorphism in CFH is asso-
ciated with decreased complement cascade inhibition in the choroid.
Strategies to attenuate complement complex formation in AMD are
now in trials (recently reviewed, (Yehoshua et al., 2011)). Our results
suggest that these modalities may relieve the vascular and RPE loss
observedinAMD,especiallyinpatientswith high-riskCFHgenotypes.
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