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Systemic Complement Activation in Age-Related Macular
Degeneration
Hendrik P. N. Scholl
1.
, Peter Charbel Issa
1.
, Maja Walier
2
, Stefanie Janzer
1
, Beatrix Pollok-Kopp
3
,
Florian Bo
¨
rncke
3
, Lars G. Fritsche
4
, Ngaihang V. Chong
5
, Rolf Fimmers
2
, Thomas Wienker
2
, Frank G.
Holz
1
, Bernhard H. F. Weber
4
, Martin Oppermann
3
*
1 Department of Ophthalmology, University of Bonn, Bonn, Germany, 2 Institute of Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany,
3 Department of Cellular and Molecular Immunology, University of Go
¨
ttingen, Go
¨
ttingen, Germany, 4 Institute of Human Genetics, University of Regensburg, Regensburg,
Germany, 5 Oxford Eye Hospital, University of Oxford, Oxford, United Kingdom
Abstract
Dysregulation of the alternative pathway (AP) of complement cascade has been implicated in the pathogenesis of age-
related macular degeneration (AMD), the leading cause of blindness in the elderly. To further test the hypothesis that
defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were
determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation
products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified
in patients (n = 112) and controls (n = 67). Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH),
factor B-C2 (BF-C2) and complement C3 (C3) genes which were previously found to be associated with AMD. All activation
products, especially markers of chronic complement activation Ba and C3d (p,0.001), were significantly elevated in AMD
patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic
regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers
Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study
population. In both the controls’ and AMD patients’ group, the protein markers of complement activation were correlated
with CFH haplotypes. This study is the first to show systemic complement activation in AMD patients. This suggests that
AMD is a systemic disease with local disease manifestation at the ageing macula. Furthermore, the data provide evidence
for an association of systemic activation of the alternative complement pathway with genetic variants of CFH that were
previously linked to AMD susceptibility.
Citation: Scholl HPN, Issa PC, Walier M, Janzer S, Pollok-Kopp B, et al. (2008) Systemic Complement Activation in Age-Related Macular Degeneration. PLoS
ONE 3(7): e2593. doi:10.1371/journal.pone.0002593
Editor: Amanda Ewart Toland, Ohio State University Medical Center, United States of America
Received March 12, 2008; Accepted May 31, 2008; Publi shed July 2, 2008
Copyright: ß 2008 Scholl et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Pro Retina Foundation Pro-Re/Seed/Issa.1; European Commission (EU FP6), Integrated Project ‘‘EVIGENORET’’ (LSHG-CT-2005-512036); German
Research Foundation (DFG) Heisenberg Fellowship SCHO 734/2-1, HO 1926/1-3. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: mopperm@gwdg.de
. These authors contributed equally to this work.
Introduction
Age-related macular degeneration (AMD) is the leading cause of
blindness in all populations of European origin [1]. The
pathogenesis of AMD is not well understood with both genetic
and environmental factors known to influence susceptibility to this
disease [2]. A hallmark of early disease are drusen, lipoproteinac-
eous deposits which accumulate in the space between the retinal
pigment epithelium (RPE) and Bruch’s membrane. Late AMD is
broadly classified into two clinical forms, a dry form with
geographic atrophy (GA), characterized by loss of RPE and outer
neurosensory retinal cells, and a wet form with choroidal
neovascularization (CNV). An estimated 1.75 million US Amer-
icans suffer from late AMD and another 7.3 million have signs of
early AMD putting them at substantial risk for vision loss from this
devastating disease [1].
Studies on the molecular composition of drusen have implicated
inflammation, and particularly local activation of the alternative
pathway (AP) of the complement cascade in the retina, in the
pathogenesis of AMD [3]. Furthermore, strong evidence for a role of
complement in this disease derives from an independent line of
research which showed that variants in the complement factor H
(CFH) gene are significantly associated with an increased risk for
AMD in Caucasian populations [4–7]. These genetic studies were
recently extended by the observation that polymorphisms in other
complement genes, notably those coding for factor B-complement
component C2 (BF-C2) and complement C3 (C3), are also associated
with AMD [8–11]. Multiple haplotypes in the CFH and BF genes
appear to modulate the AMD disease risk and both disease-
predisposing and protective gene variants were identified [7,8,12].
Activation of the AP of complement on cellular surfaces results
from the failure to downregulate the spontaneous low-level
activation of C3. Factor H, the major inhibitor of the AP of
complement activation in the fluid-phase, binds to host cells and
inhibits complement activation by its ability to interfere with the
formation and activity of the alternative C3 convertase, C3bBb. It
PLoS ONE | www.plosone.org 1 July 2008 | Volume 3 | Issue 7 | e2593
accelerates the decay of this convertase and acts as a cofactor for
the factor I-mediated proteolytic inactivation of C3b into iC3b
and C3dg [13]. In the absence of factor H, C3b binds factor B,
allowing its cleavage by the serine protease factor D to yield the
fragments Ba and Bb. Eventually, this results in the formation of
the alternative C5 convertase and assembly of terminal comple-
ment components into the C5b-9 membrane-attack complex
(Fig. 1). Factor H is among the most abundant complement
proteins in serum, synthesized predominantly in the liver, but to a
lesser extent also locally in the eye by RPE cells [14]. Within the
superfamily of functionally and structurally related cofactors for
Factor I-mediated C3b degradation (factor H, CR1, CR2, MCP)
and for the acceleration of the decay of the C3 convertases (factor
H, CR1, DAF), factor H is the main regulator which acts as a
soluble protein. This possibly explains systemic consequences
which could result from polymorphic variation of this protein.
Subtle differences in plasma concentrations or functional
activities of negative (factors H and I) or positive (factor D)
complement regulatory proteins, as well as differences in the
substrates factor B and C3, could have a significant impact on the
magnitude of local complement activation in response to a given
stimulus. Consequently, low-level activation of the AP of
complement may result in the local release of pro-inflammatory
and angiogenic mediators as well as tissue damage in the retina
which eventually may lead to manifest disease. Based on the
hypothesis that defective control of complement activation leads to
the release of complement cleavage products which are detectable
in the circulation, we performed a comprehensive investigation of
AP of complement protein plasma concentrations in a cohort of
AMD patients and controls. The findings were correlated with
polymorphisms in the CFH, BF-C2, and C3 genes.
Results
The study population included 112 AMD patients and 67 control
subjects of similar age, gender and smoking habits which showed no
signs of macular disease (Table 1). Plasma concentrations of
complement proteins in the study population are shown in Table 2.
All complement activation products, and most prominently markers
of chronic complement activation C3d and Ba (p,0.001), were
significantly elevated in AMD patients as compared to controls. The
small C3a and C5a anaphylatoxins, which are rapidly eliminated
from blood plasma, and SC5b-9, which is generated downstream of
C3 and factor B activation, were also detectable at higher levels,
although these differences were less pronounced. Complement
activation cannot be attributed to different plasma concentrations of
factor H, nor of C3 or C4, which were found to be very similar in
both study groups. Factor D plasma levels were significantly
(p,0.001) higher in the patients’ group.
An analysis of phenotypic subgroups revealed that only the C3d
concentration in plasma was differently distributed (ANOVA,
uncorrected p,0.001), the CNV subgroup exhibiting lowest C3d
levels compared to patients with geographic atrophy and patients
with early AMD. The degree to which this finding can be
generalized is limited due to the small number of patients which
were studied within the different phenotypic subgroups.
To determine whether AP of complement activation in AMD
patients is related to complement gene polymorphisms previously
associated with the disease, all probands were genotyped for six
SNPs in the CFH gene, five in BF-C2, and one polymorphism in
C3 (Fig. 1 and Table S1). In accordance with previous reports [9–
12], four markers in CFH (I62V, Y402H, A473A, IVS 15), two
markers in BF-C2 (IVS 10, R32Q) and R102G in C3 were
Figure 1. The Alternative Pathway of Complement: Polymorphic Variants and Complement Proteins under Study. Complement gene
SNPs (boxed with dotted lines) and protein plasma concentrations (boxed with solid lines) were determined in all AMD patients and controls. C3, C4
and factor B are substrates (open rectangles), factor H and factor D are regulators (open ellipses), Ba, C3d and SC5b-9 are markers of chronic
activation (filled rectangles), and C3a and C5a are markers of acute activation (filled ellipses) of the alternative complement pathway.
doi:10.1371/journal.pone.0002593.g001
Complement Activation in AMD
PLoS ONE | www.plosone.org 2 July 2008 | Volume 3 | Issue 7 | e2593
significantly associated with AMD. Haplotype analysis of the SNPs
in CFH revealed one risk haplotype, GCGGGC, and two
protective haplotypes, ATGAAC and GTGAAC (Table S2). For
the definition of the CFH risk haplotype, the Y402H polymor-
phism was sufficient. The risk haplotype was observed in 57% of
cases but only 36% of controls, whereas the protective haplotypes
were observed in 12% and 8% of cases and in 23% and 18% of
controls, respectively. The distribution of haplotypes was different
between cases and controls for CFH (p,0.001), but not for BF-C2
(p = 0.14).
Logistic regression analysis yielded significance for six variables
including three protein markers and three genetic markers. A
subsequent stepwise logistic regression analysis including exclu-
sively genetic markers revealed the three genetic markers A473A
at CFH, IVS 10 at BF-C2, and R102G at C3 to be the best genetic
predictors of risk for AMD with a high accuracy for discrimination
and an area under the curve (AUC) value of 0.726 (Fig. 2). A
stepwise logistic regression analysis including only protein markers
resulted in a model with the three markers Ba, C3d and factor D.
The corresponding ROC curve showed an even better discrim-
inatory accuracy (AUC = 0.816; p = 0.05). When both genetic and
protein markers were included into the stepwise regression
procedure, the resulting model revealed only a marginally better
fit (AUC = 0.850).
To detect a direct link between genetic variants at CFH and
protein markers of AP of complement activation, plasma levels
were compared between carriers of the CFH risk haplotype and
carriers of the protective haplotypes, both within the controls’ and
the AMD patients’ group (Table 3). Within both subject groups
(with the exception of C5a in the patients’ group), carriers of the
CFH risk haplotype showed consistently higher complement
activation parameters than those carrying the protective haplo-
types. A correlation between genetic variants in BF-C2 or C3 and
complement protein levels was not observed.
Discussion
A low-key but persistent inflammatory process which involves
activation of the complement system has been proposed to underlie
the sight-threatening manifestations in AMD. In support of this
hypothesis, various inflammatory mediators including complement
proteins, their activation products and regulators have been
identified in retinal deposits of AMD patients. In particular,
complement components C3 and C5, the membrane attack complex
C5b-9 and factor H, the main regulator of alternative pathway
activation, are constituents of drusen in AMD patients [16]. Further
evidence for a role of complement in this disease derives from two
recent studies which showed that laser-induced CNV, an accelerated
Table 1. Clinical and Demographic Characteristics of the
Study Population.
Variable Controls AMD patients
(N = 67) (N = 112)
Disease status - no. (%) *
Early AMD (Drusen) - 9 (8%)
Choroidal neovascularization - 78 (70%)
Geographic atrophy - 25 (22%)
Gender - no. (%) **
Male 37 (55%) 41 (37%)
Female 30 (45%) 71 (63%)
Mean age - years (SD; rang e)*** 70.1 (6.0; 60–86) 75.6 (6.6; 59–94)
Smoking history – no. (%)
Never smoked 35 (52%) 59 (53%)
Ex-smoker 26 (39%) 43 (38%)
Current smoker 6 (9%) 10 (9%)
*
AMD patients were categorized into mutually exclusive groups: All subjects
classified as ‘‘early AMD’’ (n = 9) had extensive intermediate and/or large
drusen in at least one eye and all qualified as category III of the Age-Related
Eye Disease Study group. Patients with GA in both eyes or GA in one eye in the
absence of CNV in the fell ow eye were classified as ‘‘GA’’ (n = 25). Nineteen of
these had subfoveal GA in at least one eye; the remaining six patients
exclusively showed extrafoveal GA. If a CNV due to AMD was present in at least
one eye, patients were classified as ‘‘CNV’’ (n = 78). Subjects with changes such
as many hard drusen and/or pigmentary alterations in both eyes were
excluded as these changes may refer to normal aging processes not necessarily
linked to AMD.
** Difference between cases and controls, p = 0.02;
***
p,0.001.
doi:10.1371/journal.pone.0002593.t001
Table 2. Plasma Concentrations of Complement Proteins in AMD Patients and Controls.
Complement protein * Units
Controls AMD patients
p**
Median 5
th
,95
th
Ptcl. Median 5
th
,95
th
Ptcl.
C3 [mg/ml] 1.18 0.85–1.48 1.12 0.89–1.53 0.85
C4 [mg/ml] 0.24 0.15–0.34 0.23 0.15–0.38 1.0
Factor B [mg/ml] 642 378–1354 803 497–1489 0.02
Factor H [mg/ml] 515 365–711 546 396–758 0.21
Factor D [mg/ml] 0.95 0.50–1.65 1.26 0.69–2.30 ,0.001
C3a [ng/ml] 14.3 10.6–21.2 15.5 11.2–24.1 0.03
C5a [ng/ml] 1.67 0.66–2.32 1.85 0.78–2.66 0.04
Ba [mg/ml] 1.09 0.60–1.71 1.33 0.90–2.09 ,0.001
C3d [mg/ml] 46.9 32.2–68.5 55.2 35.7–94.1 ,0.001
SC5b-9 [units] 159 90–710 188 107–777 0.01
*
Complement proteins in this table are arranged in groups of substrates (C3, C4, factor B), regulators (factor H, factor D), markers of acute (C3a, C5a) and chronic
activation (Ba, C3d, SC5b-9) of the alternative pathway of complement (Fig. 1).
**
Wilcoxon rank-sum test; corrected for multiple testing by Bonferroni-Holm procedure.
doi:10.1371/journal.pone.0002593.t002
Complement Activation in AMD
PLoS ONE | www.plosone.org 3 July 2008 | Volume 3 | Issue 7 | e2593
model of neovascular AMD in mice, requires intact C3 and cellular
C3aR/C5aR receptors [17,18]. Thus, uncontrolled activation of the
AP of complement within the central retina may play a major role in
the pathophysiology of AMD through its ability to promote local
tissue damage and angiogenesis [17].
In the present study we show that several parameters which
reflect systemic complement activation are significantly elevated in
the circulation of AMD patients as compared to controls. We
exercised great caution to avoid in vitro activation of complement.
As illustrated in a recent study on complement activation in AMD
patients, erroneously elevated concentrations of complement
fragments are detected, when heparin- rather than EDTA-
anticoagulated blood was used for analysis [19]. Although we
concur with the overall conclusions from this particular study, the
presented data are largely artifactual and result from ex vivo
complement activation in the heparinized blood tubes [40,41]. As
previous studies pointed to a role of complement activation in
AMD we focused our analysis on the quantification of the major
breakdown products of C3 and factor B, i.e. C3a, C3d, and Ba.
Among these, Ba and C3d are particularly well suited as markers
of chronic AP of complement activation at low levels, since their
respective half-lives in-vivo are in the order of several hours rather
than minutes [20]. Since complement turnover in vivo is further
determined by biosynthetic rates of the precursor proteins as well
as by concentrations of complement control proteins, we
determined plasma levels of the corresponding precursor proteins
C3 and factor B, and the main positive and negative regulators of
the AP of complement, factors D and H. C5a and SC5b-9 were
quantified as markers of terminal pathway of complement
activation which are generated downstream of C3 and factor B.
The simultaneous quantification of the main cleavage products,
substrates and of control proteins of the AP of complement is a
unique feature of this study and allowed to precisely document the
state of complement activation in all patients.
Our notion of enhanced systemic complement activation in
AMD is mainly based on the finding that in patients all
complement activation products determined in this study were
elevated as compared to controls. This difference was most
strikingly observed with regard to Ba and C3d, two sensitive
markers of chronic AP of complement activation in-vivo. In
contrast, the complement proteins C3, C4 and factor H did not
significantly differ between the two groups. We also observed
elevated levels of factors B and D in AMD patients and this could
possibly be due to an acute phase response-mediated upregulation
of factor B or by polymorphic variation in the FD gene which may
affect factor D plasma levels. Upregulation of these two positive
regulators may further contribute to enhanced AP of complement
activation, however, subtle changes in factors B and D alone
cannot explain the enhanced turnover of complement substrates
[21]. While Ba and C3d concentrations in AMD patients were
only modestly elevated by a factor of 1.2 to 1.3 compared to
controls, C3d levels in the plasma of patients with rheumatoid
arthritis are also increased to a similar degree [22]. Since local C3d
concentrations in synovial fluids from these patients are much
higher than in plasma [23], a similar gradient between sites of local
complement activation and blood plasma may also exist in AMD.
Our results further demonstrate that a combination of
complement activation markers can be used to most reliably
discriminate AMD patients from controls in our study population.
The discriminatory ability of these complement proteins
(AUC = 0.816) appears superior or at least similar to the
discriminatory ability of genetic markers of complement genes
Figure 2. Receiver Operating Characteristic (ROC) Curves for
the Discriminative Capability of Genetic and Protein markers
of the Complement System. Receiver operating characteristic curves
for genetic markers (dotted line; A473A of CFH, IVS 10 of BF-C2 and
R102G of C3) and complement protein markers (solid line; Ba, C3d, and
factor D) are shown. AUC = Area under ROC curve.
doi:10.1371/journal.pone.0002593.g002
Table 3. Plasma Concentrations (mean6S.E.M.) of Complement Proteins in Carriers of Risk-Conferring and Protective Haplotypes
of the CFH Gene.
Complement
protein Units
Controls AMD patients
All Risk haplotype
Protective
haplotypes All Risk haplotype
Protective
haplotypes
(n = 67) (n = 17) (n = 23) (n = 112) (n = 67) (n = 16)
Ba [mg/ml] 1.1160.04 1.2160.25 1.0460.08 1.4160.04 1.4660.05 1.2760.11
C3d [mg/ml] 48.161.40 50.963.71 45.462.34 59.161.80 60.662.11 52.764.88
C3a [ng/ml] 14.760.38 15.360.70 14.060.62 16.460.37 16.660.53 15.360.89
C5a [ng/ml] 1.5560.06 1.6460.12 1.4660.11 1.7760.06 1.7260.07 1.8660.16
Factor D [mg/ml] 0.9860.04 1.0460.05 0.8560.07 1.3560.05 1.3860.06 1.2660.12
doi:10.1371/journal.pone.0002593.t003
Complement Activation in AMD
PLoS ONE | www.plosone.org 4 July 2008 | Volume 3 | Issue 7 | e2593
for the prediction of AMD as determined in this and other
similarly designed studies [24]. Because the protein markers are
closer to the presumed mechanisms of AMD pathogenesis, this
finding appears plausible. Prospective studies will be required to
determine whether plasma concentrations of complement proteins
could be useful as markers of AMD at less advanced stages, either
alone or in combination with genetic markers. Since this study was
limited to genes and proteins of the alternative pathway of
complement, a general conclusion on the superiority of protein
markers compared to genetic markers is not possible. Such a
conclusion may be derived from an even more comprehensive
study including additional genetic and protein markers. Since the
LOC387715/HTRA1/ARMS2 locus has been shown to be of
similar significance as CFH [25], polymorphisms within ARMS2
would have to be considered. Recently, the discriminative
accuracy of testing polymorphisms in CFH and BF-C2 together
with ARMS2 for the prediction of AMD was found to be 80% [24],
which is similar to the score we found for testing protein markers
of the alternative pathway of complement.
Our study has linked elevated plasma concentrations of AP of
complement activation products in AMD patients to polymorphic
variations in the CFH gene which codes for the main regulator of the
AP of complement activation in the fluid phase and on cell surfaces
[13]. We observed within both study groups that individuals who
carry the AMD-associated CFH risk haplotype had higher plasma
concentrations of complement activation products, and conversely
that protective CFH haplotypes were associated with lower levels of
activation products (p = 0.05, MANOVA).
In particular, the association between complement activation
and the CFH risk haplotype which includes Y402H, a non-
synonymous SNP in CFH which leads to a tyrosine to histidine
exchange at position 402, is biologically plausible. Several recent
studies have shown that the His402 variant binds less well to
heparin, C reactive protein and RPE cell surfaces [14,26,27].
While the consequences of defective factor H function are most
likely not restricted to the eye, but result in inappropriate
complement control at other cell surfaces throughout the body,
the retina appears to be especially sensitive towards the effects of
complement activation.
Our data suggest that AMD indeed is a systemic disease with
local disease manifestation at the ageing macula. Such local
manifestation of systemic pathophysiology is not without prece-
dence. An example are mutations in the PRPF3 gene causing a
tissue-specific phenotype of autosomal dominant retinitis pigmen-
tosa although PRPF3 is an element of the ubiquitously expressed
RNA splicing machinery [28]. In addition, the macula is more
easily damaged than the retinal periphery, e.g. because it exhibits
decreased thickness and integrity of the elastic layer of Bruch’s
membrane [29]. The hypothesis of AMD being a systemic disease
certainly raises the possibility of other organ manifestations that
have so far not been detected. In support of this hypothesis,
patients with membranoproliferative glomerulonephritis type II
(MPGN II) and systemic complement activation develop retinal
deposits at early ages which resemble drusen in AMD patients
[30]. Since MPGN II patients benefit from substitution therapy
with intact complement control proteins, local or systemic
administration of AP of complement inhibitors may be considered
as a future therapeutic option in AMD.
Materials and Methods
Cases and Controls
112 patients with a clinical diagnosis of AMD and 67 control
subjects were included in the study. Absence of AMD, early and
late AMD was defined according to the criteria of the ARM
Epidemiological Study Group [31]. Exclusion criteria included age
below 55 years; any evidence of retinal disease (in the control
group) with the exception of AMD (in the AMD group)
ascertained by history, clinical examination, digital fundus
photography and grading of fundus images (see below); any
systemic disease known to affect the complement system (e.g.
rheumatoid arthritis) ascertained by a standardized case report
form (CRF) derived from the multicenter FAM-Study [32] and
abnormal renal clearance (ascertained by creatinine and cystatin C
values). In the recruitment of the control subjects particular
attention was paid to match for smoking habits (ascertained by the
CRF), since smoking has been shown to be by far the most
significant environmental risk factor for AMD. Moreover, earlier
studies have suggested an effect of smoking on factor H blood
levels [13]. As a result, the control group was of similar age and
gender, and exhibited identical smoking habits; minor differences
in age and gender of the two groups were regarded irrelevant for
complement protein concentrations [41]. All subjects were of
Caucasian descent and were recruited within the same time period
from the Department of Ophthalmology, University of Bonn
between January and October 2006. Control subjects did not have
any signs of AMD, especially no early changes such as many small
drusen, intermediate or large drusen. Informed consent was
obtained from all subjects. The research protocol was in keeping
with the provisions of the Declaration of Helsinki, and approval
was obtained from the institutional ethics committee. Digital
fundus photographs were obtained from all participants. In
patients with CNV, optical coherence tomography and fluorescein
angiography were performed. Fundus autofluorescence imaging
was performed in patients with GA. All fundus images were
evaluated separately by two independent readers (HPNS and PCI);
digital fundus images were graded according to the classification
system of the International ARM Epidemiological Study Group
[31,33,34]. Clinical characteristics and demographic data of the
study populations are provided in Table 1.
Blood samples
Venous blood was collected from all subjects into tubes
containing dipotassium EDTA at a final concentration of 8 mM.
The plasma was separated from the blood cells by centrifugation
(20 min/10006 g) within 3 hours after venipuncture and frozen in
aliquots at 280uC until analysis. One subject with elevated
complement levels due to mild chronic renal failure was excluded
from the analysis of factor D and Ba since the catabolism of these
two complement proteins is determined by the glomerular
filtration rate [21]. All other subjects had normal creatinine and
cystatin C values; both subject groups were not significantly
different for these variables.
Analysis of comp lement proteins
Assays for the quantitation of complement components factor B,
Ba, C3a, C3d, C5a, SC5b-9, factor D and factor H have been
developed previously [35–37]. All complement activation assays
were based on monoclonal antibodies with specificities for
activation-induced neoepitopes present on the different comple-
ment split products which are absent from the respective native
precursor proteins. In the case of the ‘C3d’-assay, a capture mAb
(I3/15) was used which reacts with a common neoepitope on C3b,
iC3b, and C3dg, in combination with a polyclonal rabbit anti-C3d
as the detecting antibody [35]. Plasma concentrations of C3 and
C4 were determined by rate nephelometry. All patients and
control samples were handled identically and analyzed simulta-
neously in order to ensure stable assay conditions. As reported
Complement Activation in AMD
PLoS ONE | www.plosone.org 5 July 2008 | Volume 3 | Issue 7 | e2593
before [36], the inter-assay coefficient of variation of ELISA
procedures was below 10%. The intra-individual stability of
complement plasma levels was assessed in a subset of AMD
patients (n = 14). From these patients, a second ETDA plasma
sample was obtained 12 months after the first venipuncture.
Within this period of time, Ba, C3d and factor D values varied by
less than 15% compared to the initial values.
Genotyping
Genomic DNA was extracted from peripheral blood leukocytes
following established protocols. Genotyping was done by TaqMan
SNP Genotyping or by direct sequencing of SNPs. TaqMan Pre-
Designed SNP Genotyping Assays (Applied Biosystems, Foster
City, U.S.A.) were performed according to the manufacturer’s
instructions and were analyzed with a 7900HT Fast Real-Time
PCR System (Applied Biosystems). Direct sequencing was
performed with the Big Dye Terminator Cycle Sequencing Kit
Version 1.1 (Applied Biosystems) according to the manufacturer’s
instructions. Reactions were analyzed with an ABI Prism Model
3130xl Sequencer (Applied Biosystems). Individual genotypes that
were ambiguous or missing were reanalyzed resulting in a call-rate
of 100% for all SNPs tested. SNP-IDs for three complement gene
loci CFH, BF-C2, and C3 are indicated in Fig. 1.
Statistical Analysis
Based on data of assay variability of the key proteins of chronic
AP of complement activation (C3d, Ba, SC5b-9) derived from our
laboratory [35,36], the study was designed to detect a difference of
2/3 of a standard deviation between cases and controls with a
power of at least 90%, with a two sided test at a level of a = 0.05/
3. The level had been chosen to account for the fact that three
biochemical markers had to be compared. The recruitment was
planned to be imbalanced with a case control ratio of 2:1 resulting
in 120 cases and 60 controls to be included.
Allele and genotype frequencies were determined. All markers
were in Hardy-Weinberg equilibrium (all p.0.20). All allele
frequencies were within the ranges reported in the Entrez SNP
database (www.ncbi.nih.gov) and in previous publications [4–12].
To test for genetic association, genotype frequencies were
compared between cases and controls by Armitage’s trend test.
A retrospective power analysis based on the empirical values for
allele frequencies and relative risks found in our data was
performed [38]. Results are reported in Table S3. Haplotypes
for the markers in CFH and BF-C2 were estimated using
FAMHAP [39]. The difference in distribution of haplotypes was
tested by likelihood ratio test.
Stepwise logistic regression analysis was used to explore models
to predict the risk for AMD depending on genetic markers and
complement protein markers and to gain more insight into the
relevance of these risk parameters in relation to one another.
Results were visualized by receiver operating characteristic (ROC)
curves for the scores resulting from the logistic regression. ROC
curves were compared using the method proposed by DeLong et
al [15]. Multivariate analysis of variance (MANOVA) was applied
to the joint distribution of complement activation markers to verify
the observation of simultaneously increased values depending on
CFH haplotypes and disease status. Data were handled in SAS
(SAS software package for Windows, version 9.1.; SAS Institute
Inc., Cary, NC, USA; http://www.sas.com).
Supporting Information
Table S1 Genotypes
Found at: doi:10.1371/journal.pone.0002593.s001 (0.09 MB
PDF)
Table S2 Haplotypes
Found at: doi:10.1371/journal.pone.0002593.s002 (0.08 MB
PDF)
Table S3 Retrospective Power Analysis
Found at: doi:10.1371/journal.pone.0002593.s003 (0.17 MB
PDF)
Author Contributions
Concei ved and designed the experiments: MO HS NC TW BW.
Performed the experiments: LF MO HS PI SJ BP FB. Analyzed the data:
RF MW TW. Wrote the paper: LF RF MO HS PI MW SJ BP FB NC TW
FH BW.
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