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com
BJO Online First, published on May 10, 2011 as 10.1136/bjo.2010.193680
1
Eye Clinic, St. James’s
University Hospital, Leeds, UK
2
Section of Ophthalmology and
Neuroscience, Leeds Institute of
Molecular Medicine, University
of Leeds, Leeds, UK
3
Division of Biostatistics, Leeds
Institute of Genetics, Health and
Therapeutics, University of
Leeds, Leeds, UK
Correspondence to
Martin McKibbin, Eye Clinic, St
James’s University Hospital,
Leeds LS9 7TF, UK; martin.
mckibbin@leedsth.nhs.uk
A poster including some of
these data has been presented
at the ARVO meeting, May
2010.
Accepted 13 April 2011
McKibbin M, Ali M,Article
Copyright Bansal S, et al. Br J Ophthalmol
author
Clinical science
CFH, VEGF and HTRA1 promoter genotype may
influence the response to intravitreal ranibizumab
therapy for neovascular age-related
macular degeneration
Martin McKibbin,1,2 Manir Ali,2 Shveta Bansal,1,2 Paul D Baxter,3 Kumi West,1
Grange Williams,2 Frances Cassidy,1 Chris F Inglehearn2
ABSTRACT
Aims To investigate an association between genotype for
three single nucleotide polymorphisms strongly
associated with the development of age-related macular
degeneration (AMD) and the early response to treatment
with intravitreal ranibizumab for neovascular AMD.
Methods Best corrected visual acuity letter score was
recorded at baseline and each subsequent visit. Age,
sex, smoking history, lesion type and the number of
injections were also recorded. Genotypes were obtained
for rs11200638 in HTRA1, rs1061170 in CFH and
rs1413711 in VEGF. Data were analysed with treatment
response at month 6 as both a binary (>5 letter
improvement vs #5 letter gain) and a linear trait.
Results This initial study cohort consisted of 104
Caucasian neovascular AMD patients treated with
intravitreal ranibizumab. Trends towards a more
favourable outcome were seen with the higher AMD
risk genotypes in CFH and VEGF in both the linear and
binary models and in HTRA1 in the linear model alone.
For CFH, mean letter score change after 6 months
was +1.6, +5.9 and +7.2 letters for the TT, TC and
CC genotypes and a >5 letter gain was seen in
34.6%, 56.6% and 56%, respectively. For VEGF, mean
letter score change after 6 months was +1.3, +5.8 and
+7.4 letters for the TT, TC and CC genotypes and a >5
letter gain was seen in 40%, 55.8% and 51.9%,
respectively. For HTRA1, mean letter score change was
+2.2, +7.5 and +2.9 letters for the GG, GA and AA
genotypes.
Conclusions This study reports preliminary evidence
suggesting that the higher AMD risk genotypes in CFH,
VEGF and HTRA1 may influence the short-term response
to treatment with ranibizumab for neovascular AMD.
INTRODUCTION
Age-related macular degeneration (AMD) is the
most common cause of visual impairment in the
Western world, accounting for over 50% of certifi-
able sight impairment in the UK alone.1 Epidemi-
ological and association studies have shown that
both genetic and environmental factors play
a major role in the development of AMD.2 Associ-
ation studies have implicated variants in a number
of genes, with the largest effects observed with
alleles of the complement factor H (CFH), HTRA1 and
Apolipoprotein E genes.3e6 Association with AMD
has also been reported for polymorphisms in the
VEGF gene.7 8
(or their (2011). doi:10.1136/bjo.2010.193680
employer) 2011. Produced
by BMJ Publishing Group Ltd under licence.
Given the greater understanding of the genetic
changes underlying AMD, we hypothesised that
intravitreal ranibizumab (Lucentis, Novartis Phar-
maceuticals, Camberley, UK) therapy may be more
effective in patients with certain combinations of
AMD susceptibility alleles than with others. In this
study, we therefore compared genotypes for CFH,
HTRA1 and VEGF, and other potential variables,
with visual acuity response after 6 months of
intravitreal ranibizumab therapy for neovascular
AMD.
METHODS
This study was approved by the Leeds (East)
Research Ethics Committee and registered as
a clinical trial with the Medicines and Healthcare
Regulatory Authority. Participants were recruited
from the intravitreal injection clinics at St James’s
University Hospital, Leeds. Inclusion and exclusion
criteria are detailed in box 1. Written informed
consent was taken prior to enrolment.
Baseline stereoscopic colour fundus, fluorescein
and indocyanine green (when available) angiogram
images were reviewed and graded by a single
observer (MM) to identify lesion characteristics.9
The baseline choroidal neovascularisation pheno-
type was classified as being classic only (100%
classic), predominantly classic (50e99%), mini-
mally classic (1e49%) and occult only or retinal
angiomatous proliferation (RAP). (Given the
uncertainty surrounding response to treatment,
data from eyes with RAP lesions were also analysed
as a subgroup and compared with eyes with other
baseline lesion characteristics.) Information
regarding sex, smoking history and the number of
injections before the month 6 visit was also
recorded.
At baseline and subsequent visits, best corrected
visual acuity letter score was measured using an
Early Treatment Diabetic Retinopathy Study
(ETDRS) chart and recorded for both eyes. Optical
coherence tomography (OCT) examination was
performed using either Stratus OCT (Carl Zeiss
Meditec, Dublin, California, USA) or Spectralis
OCT (Heidelberg Engineering, Heidelberg,
Germany). Intravitreal ranibizumab therapy was
administered in accordance with the European
Medicines Agency marketing authorisation and
standard UK practice, with three consecutive
monthly injections in a loading phase and further
injections as required in a maintenance phase.
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Clinical science
Box 1 Main inclusion and exclusion criteria
Inclusion criteria
< Age 65 years or more
< Choroidal neovascularisation (CNV) secondary to age-related
macular degeneration and involving the foveal centre
< CNV occupying at least 50% of the total lesion area
< Greatest linear diameter #5400 mm
< No subfoveal atrophy or fibrosis
< Recent disease progression
< Minimum of 6 months follow-up after first intravitreal
ranibizumab injection
Exclusion criteria
< Other ocular disease that may influence the treatment
outcome
< CNV secondary to pathological myopia, inflammatory disease,
angioid streaks or trauma
< Peripapillary CNV extending to the foveal centre or polypoidal
choroidal vasculopathy
< Cataract surgery in the study eye any time from 3 months
before until 6 months after the initiation of intravitreal
ranibizumab therapy
< Failure to complete 6 months of intravitreal ranibizumab
therapy
Retreatment in the maintenance phase was prompted by stan-
dard criteria, namely a decrease in best corrected visual acuity
of $5 letters and/or persistent or recurrent subretinal fluid,
intraretinal cysts or thickening on OCT or new subretinal
haemorrhage on clinical examination.
DNA was extracted from peripheral blood lymphocytes and
genotyped for single nucleotide polymorphisms (SNPs) in the
CFH (Y402H, rs1061170), VEGF (+674, rs1413711) and HTRA1
(
512, rs11200638) genes. Amplimers of 220, 246 and 348 bp in
length were generated by the PCR using the PCRx Enhancer
System (Invitrogen, Paisley, UK). Briefly, a 20 ml reaction volume
contained 100 mM dNTPs, 1.5 mM MgSO4, 1 3 PCRx Amplifi-
cation buffer, 0.15 units Taq DNA polymerase, 100 ng genomic
DNA and 20 pmol of each of the F and R primers. The HTRA1
PCR also used 23 Enhancer solution. For the CFH poly-
morphism, the primers used were CFH-F (dtgg tcc tta gga aaa
tgt ta) and CFH-R (dgaa cat gct agg att tca gag tag tg); for the
VEGF polymorphism, the primers consisted of VEGF-F (dtgg cta
cag gcc tcc aag ta) and VEGF-R (dtca ccc att ccc atg aca c) and,
for the HTRA1 polymorphism, the primers were HTRA1-F (dtag
gct ctc tgc gaa tac gg) and HTRA1-R (dggg gaa agt tcc tgc aaa
tc). The reactions were performed in a DNA thermal cycler for
2 min at 958C followed by 40 cycles of 948C denaturation for
30 s, an annealing temperature of 588C for the CFH primers,
568C for the VEGF primers and 56.58C for the HTRA1 primers
for 45 s and an extension step of 728C for 45 s with a final
extension of 728C for 5 min. The PCR products were digested
with ExoSAP-IT (GE Healthcare, Chalfont St Giles, UK),
sequenced using the BigDye Terminator V.3.1 Cycle Sequencing
Kit and resolved on an ABI3130xl Genetic Analyzer (Applied
Biosystems, Warrington, UK). The sequencing primers were
CFH-R, VEGF-F and HTRA1-F, respectively. Chromatograms
were viewed using Sequencing Analysis 5.2 software (Applied
Biosystems) to identify SNPs.
To test the statistical significance of potential predictor vari-
ables, multiple regression analyses were conducted, using both
2 of 5
a linear model with actual letter score change and a binary
logistic model with a gain of >5 ETDRS letters. The following
potential predictor variables were included in both models: sex
(male or female), smoking status (current smoker, ex-smoker
and never smoked), number of injections received (<4 or $4 in
the first 6 months), lesion type (RAP or other angiographic
classification), baseline acuity (<54 letters or $54 letters), VEGF
genotype (TT, TC or CC), HTRA1 genotype (AA, GA or GG) and
CFH genotype (TT, TC or CC). All p values reported refer to
significance tests of the potential predictor variables derived
from these multiple regression models. The predetermined level
of statistical significance was p<0.01.
RESULTS
The baseline demographics, lesion characteristics and ETDRS
letter score for the initial cohort of 104 Caucasian participants
without prior treatment are described in table 1. Mean baseline
acuity was 51.5 ETDRS letters (SD ¼15.7), and subjects received
an average of 4.0 injections (SD ¼0.9) before the month 6 visit.
The mean change in acuity at the month 6 visit was a gain of
+5.2 letters (SD ¼13.6). Visual loss of $15 letters was avoided
in 98 eyes (94.2%). A gain of at least 1 letter was seen in 68 eyes
(65.4%) and gains of $5 letters and $15 letters were seen in 57
(54.8%) and 27 eyes (26.0%), respectively.
The association of sex, smoking status, lesion type, number of
injections and baseline visual acuity with ETDRS letter score
change at month 6 is shown in table 2. Of these variables, only
baseline visual acuity had a significant association (p<0.01) with
visual acuity change at month 6 when included in the multiple
regression analyses. Eyes with baseline acuity <54 letters had
a mean change of +9.9 letters while those with 54 letters or
more at baseline had a change of +0.4 letters. A gain of >5
ETDRS letters was seen in 65% of the eyes with baseline acuity
<54 letters and only 37% of those with 54 or more letters at
baseline.
The CFH Y402H, VEGF and HTRA1 genotype frequencies,
baseline ETDRS letter score, letter score change after 6 months
and number of injections within the first 6 months are shown in
table 3. In the multivariate analysis, non-significant trends,
suggesting a more favourable visual acuity outcome with
possession of one or more high-risk alleles, were seen for CFH,
VEGF and HTRA1. For the CFH genotype, mean letter score
change after 6 months was +1.6 letters for the lower risk TT
genotype and +5.9 and +7.2 for the higher risk TC and CC
genotype (p¼0.18, 0.71 and 0.17 for TC, CC and combined TC
Table 1 Baseline demographics and lesion characteristics
Age
Mean (SD) 81.5 years (6.3)
Range 65e96
Sex
Male 46 (44.2%)
Female 58 (55.8%)
Smoking status
Current smokers 12 (11.5%)
Ex-smokers* 48 (46.2%)
Non-smokers 44 (42.3%)
Lesion type
Classic only 20 (19.2%)
Predominantly classic 8 (7.7%)
Minimally classic 14 (13.5%)
Occult only 49 (47.1%)
RAP 13 (12.5%)
*Not smoked for at least 12 months prior to the first injection.
McKibbin M, Ali M, Bansal S, et al. Br J Ophthalmol (2011). doi:10.1136/bjo.2010.193680
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Clinical science

Table 2 Visual acuity change in baseline and clinical subgroups

Potential variable p Values
Sex Male Female
Number of cases 46 58
Mean baseline BCVA letter score (SD) 54.6 (16.0) 49.1 (15.2) 0.08
Mean BCVA letter score change (SD) +3.1 (12.4) +6.8 (14.5) 0.17
>5 letter gain (%) 19 (41.3%) 34 (58.6%) 0.12

Smoking status Never smoked Ex-smokers Current smokers

Number of cases 44 48 12
Mean baseline BCVA letter score (SD) 49.4 (16.4) 52.7 (16.2) 54.3 (10.5) 0.32*, 0.33*
Mean BCVA letter score change (SD) +7.0 (13.2) +4.0 (13.4) +3.2 (16.6) 0.27*, 0.40*
>5 letter gain (%) 24 (54.5%) 23 (47.9%) 6 (50%) 0.67*, 1.00*

Lesion characteristics RAP Other lesion type

Number of cases 13 91
Mean baseline BCVA letter score (SD) 44.2 (13.1) 52.5 (15.8) 0.07
Mean BCVA letter score change (SD) +11.0 (11.9) +4.3 (13.7) 0.10
>5 letter gain (%) 9 (69.2%) 44 (48.4%) 0.27

Baseline visual acuity <54 letters ‡54 letters

Number of cases 52 52
Mean baseline BCVA letter score (SD) 38.3 (9.7) 64.7 (7.2)
Mean BCVA letter score change (SD) +9.9 (13.4) +0.4 (12.3) 0.0002
>5 letter gain (%) 34 (65.4%) 19 (36.5%) 0.006

Number of injections <4 injections ‡4 injections

Number of cases 34 70
Mean baseline BCVA letter score (SD) 54.7 (14.1) 49.9 (16.3) 0.14
Mean BCVA letter score change (SD) +6.8 (10.3) +4.3 (15.0) 0.39
>5 letter gain (%) 18 (52.9%) 35 (50%) 0.94
*‘Never smoked’ is the reference category.
BCVA, best corrected visual acuity; RAP, retinal angiomatous proliferation.

or CC genotypes compared with TT). A gain of >5 ETDRS
letters was seen in 34.6%, 56.6% and 56% of eyes with the TT,
TC and CC genotypes (p¼0.04, 0.4 and 0.06 for TC, CC or
combined TC or CC genotypes compared with TT).
For VEGF, mean ETDRS letter score change at month 6 was
a gain of +1.3, +5.8 and +7.4 letters for the lower risk TT and
higher risk TC and CC genotypes, respectively (p¼0.11, 0.11 and
0.09 for the TC, CC and combined TC or CC genotypes
compared with TT). A gain of >5 letters was seen in 40%, 55.8%
and 51.9% of eyes with the TT, TC and CC genotypes (p¼0.08,
0.38 and 0.13 for TC, CC and combined TC or CC genotypes
compared with TT).

Table 3 Visual acuity change from baseline, number of injections and genotype
p Values
CFH genotype TT TC CC
Frequency (%) 26 (25%) 53 (50.9%) 25 (24.1%)
Mean baseline BCVA letter score (SD) 53.5 (18.0) 51.3 (14.1) 49.8 (16.9)
Mean BCVA letter score change after 6 months (SD) +1.6 (13.1) +5.9 (14.6) +7.2 (11.7) 0.18*, 0.71*
Number with >5 BCVA letter score gain after 6 months (%) 9 (34.6%) 30 (56.6%) 14 (56%) 0.04*, 0.4*
Mean number of injections in 6 months (SD) 4.1 (0.8) 4.1 (0.9) 3.7 (1.1)

HTRA1 genotype GG GA AA
Frequency (%) 27 (26%) 55 (52.9%) 22 (21.1%)
Mean baseline BCVA letter score (SD) 51.0 (14.4) 50.7 (15.6) 54.1 (18.0)
Mean BCVA letter score change after 6 months (SD) +2.2 (14.4) +7.5 (13.2) +2.9 (13.4) 0.03*, 0.55*
Number with >5 BCVA letter score gain after 6 months (%) 13 (48.1%) 30 (54.5%) 10 (45.5%) 0.16*, 0.59*
Mean number of injections in 6 months (SD) 4.0 (0.9) 4.0 (1.0) 4.2 (1.0)

VEGF genotype TT TC CC
Frequency (%) 25 (24.1%) 52 (50%) 27 (25.9%)
Mean baseline BCVA letter score (SD) 51.3 (15.7) 52.6 (14.2) 49.5 (18.6)
Mean BCVA letter score change after 6 months (SD) +1.3 (12.7) +5.8 (14.8) +7.4 (11.8) 0.11*, 0.11*
Number with >5 BCVA letter score gain after 6 months (%) 10 (40%) 29 (55.8%) 14 (51.9%) 0.08*, 0.38*
Mean number of injections in 6 months (SD) 4.2 (1.0) 3.9 (0.9) 4.1 (1.0)
*Compared with the lower risk CFH TT, HTRA1 GG and VEGF TT genotypes in multivariate analysis.
BCVA, best corrected visual acuity; CFH, complement factor H; HTRA1, high-temperature requirement factor A-1; VEGF, vascular endothelial growth factor.

McKibbin M, Ali M, Bansal S, et al. Br J Ophthalmol (2011). doi:10.1136/bjo.2010.193680 3 of 5

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Clinical science
For HTRA1, mean ETDRS letter score change at month 6 was
a gain of +2.2, +7.5 and +2.9 letters for the lower risk GG and
higher risk GA and AA genotypes, respectively (p¼0.03, 0.55 and
0.07 for GA, AA and combined GA or AA genotypes compared
with GG). No association was seen in the binary visual acuity
change model.
DISCUSSION
In the ANCHOR and MARINA studies, the response to treat-
ment seemed to be consistent despite baseline variation in lesion
size, visual acuity and lesion characteristics.10e12 Although
current evidence still suggests that regular monthly treatment is
associated with the optimum visual acuity response, a number
of other studies have suggested that a proportion of patients
may not need such regular dosage after the initial loading phase
to maintain the early gain in visual acuity.13 14 In the PIER
study, 40% of patients maintained their initial gain with quar-
terly treatment after the initial loading phase although 60% lost
their initial gain.13 We hypothesised that the variable acuity
responses seen in these trials may be related to genotype. The
results of our pilot study suggest that the visual acuity change
after 6 months of ranibizumab therapy may indeed be influ-
enced by polymorphisms in three genes that are known to
increase the risk of developing neovascular AMD.
Prior studies have investigated a possible association between
the CFH genotype and response to verteporfin photodynamic
therapy (PDT) and both intravitreal bevacizumab and ranibi-
zumab therapies. For PDT, the results are conflicting. Goverdhan
reported greater loss of visual acuity after PDT in patients with
the CC and CT CFH genotypes and Feng reported a trend
towards an association between a negative PDT response and
the CC genotype.15 16 However, Brantley identified a worse
outcome with the TT CFH genotype and Seitsonen found
a lower proportion of PDT responders in the TT genotype
group.17 18 With intravitreal bevacizumab, Brantley and Nischler
reported a trend towards reduced visual acuity and a lower
percentage of subjects with improved acuity for the CC geno-
type.19 20 Lee identified that patients with the CC genotype
required one more ranibizumab injection over the first 9 months,
but the CFH genotype was not significantly associated with
post-treatment visual acuity at either 6 or 9 months, after
adjusting for pretreatment acuity.21 In our study, we did not find
an association between CFH genotype and the number of
injections in the first 6 months, but we did find a trend towards
a better visual acuity response with the higher risk TC or CC
genotypes.
Although the VEGF gene does not appear to be a major
genetic contributor to the development of neovascular AMD,
the +674 C/T polymorphism has been most strongly associated
with neovascular AMD in a Caucasian population.7 8 McKay has
also suggested that VEGF polymorphisms may influence the
development of certain anatomic types of neovascular AMD.22
Given the role of VEGF in the angiogenesis and hyper-
permeability that characterise neovascular AMD, this poly-
morphism was considered to be another potential factor that
might influence the response to blockage of the biological effect
of VEGF within the eye. In the linear acuity change model alone,
there was a trend suggesting a better outcome with the higher
risk TC or CC genotypes. To our knowledge, no other group has
investigated the influence of this polymorphism on the response
to ranibizumab therapy for AMD. However, Immonen has
recently identified a potential link between other intronic and
promoter site VEGF polymorphisms and the anatomic response
to PDT.23 These SNPs cover the entire VEGF gene and will
4 of 5 McKibbin M, Ali M, Bansal S, et al. Br J Ophthalmol (2011). doi:10.1136/bjo.2010.193680
therefore detect the segregation of common haplotypes
containing the +674 polymorphism.
Polymorphisms in the promoter region of the HTRA1 (high-
temperature requirement factor A-1) gene have been shown to
increase susceptibility to AMD, especially the neovascular form,
in Caucasian and other populations.3 4 Possession of the high-
risk A allele is associated with increased levels of the HTRA1
protein in drusen, retinal pigment epithelium and choroidal
neovascular membranes of eyes with AMD.3 4 Chowers found
no association between the HTRA1 promoter genotype and the
visual acuity outcome or number of injections after PDT.24 In
our series, a trend towards better visual acuity outcome after
6 months of intravitreal ranibizumab therapy was seen for the
increasing AMD risk HTRA1 genotypes, using the linear model
alone. Although the total number of injections was similar for all
three HTRA1 genotypes, there was a large gain in visual acuity in
the individuals who were heterozygous for the high AMD risk A
allele and a more modest gain in homozygotes. The apparent
advantage of heterozygotes over homozygotes for either HTRA1
genotype is unexpected. However, heterozygote advantage is
a documented phenomenon in many biological systems and is
not unprecedented in autoimmune disease, which some consider
to be the basis of AMD.25 A similar, but non-significant, trend
was also seen in the binary acuity outcome model. In this
study, the allele frequency of the A allele was 0.48, higher than in
other published Caucasian AMD series.3 24 26 The higher
frequency of this allele in our study population of AMD patients
from Yorkshire may have provided the opportunity to identify
this effect.
In this pilot study, we have identified preliminary evidence of
associations between visual acuity outcome after 6 months of
intravitreal ranibizumab and polymorphisms in the CFH, VEGF
and HTRA1 genes. For each gene, a trend towards a better
outcome was seen with the polymorphisms considered to
increase the risk of developing neovascular or advanced AMD.
Although the mechanism by which these polymorphisms
contribute to AMD susceptibility is not known, these findings
may imply that neovascular AMD is not one disease but rather
several different diseases with a common end point. There may
be a common form of AMD that results from the action of one
or more high-risk alleles at HTRA1, CFH and VEGF. This form of
AMD may be characterised by high levels of intraocular VEGF
and may respond favourably to ranibizumab therapy. Another
form of AMD, secondary to other susceptibility genes, may not
be characterised by high intraocular VEGF and may respond
differently to treatment.27
Our original hypothesis was that genotype may be associated
with either visual acuity change or the number of additional
injections required. The 6-month time point provided the
opportunity to investigate both these effects.13 28 The initial
response to treatment for the entire group is in keeping with
data from multicentre clinical trials, suggesting that the associ-
ation with genotype is real and not the result of selection bias. In
addition, the allele frequencies for the CFH and VEGF genes are
similar to other published series and there are no minor alleles
with a frequency below 10%. Despite these strengths, there are
potential limitations to the findings of our study. As the treat-
ment was given with a loading phase of three consecutive
injections, followed by a maintenance phase with repeated
injection only as required, the results may not be applicable to
patients treated with regular monthly injections. The modest
number of subjects in this initial analysis means that we are
liable to detect only large associations between genotype and
visual outcome and that none of the potential associations met
 Downloaded from bjo.bmj.com on September 28, 2011 - Published by group.bmj.com
the predetermined level of significance. The study population
indicates that the results are only applicable to other Caucasian
populations and that the high risk HTRA1 A allele was partic-
ularly common in the local population. The association with
visual acuity was not supported by an increased number of
injections.
In this study, we have identified trends suggesting a more
favourable visual acuity outcome after 6 months of intravitreal
ranibizumab therapy with the higher AMD risk CFH, VEGF and
HTRA1 genotypes. These findings may imply that the mecha-
nisms underlying the more common form(s) of AMD, due largely
to the action of risk alleles at the loci tested in this study, are
those best targeted by anti-VEGF therapy. Neovascular AMD in
individuals without these high-risk alleles may in part be due to
mechanisms that respond less favourably to anti-VEGF therapy.
Funding This project was supported by a grant from Yorkshire Eye Research and by
an investigator-initiated research grant from Novartis Pharmaceuticals UK. The funding
organisations had no role in either the design or the conduct of the research.
Competing interests None.
Ethics approval This study was conducted with the approval of the Leeds East
Research Ethics Committee.
Provenance and peer review Not commissioned; externally peer reviewed.
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CFH, VEGF and HTRA1 promoter genotype

may influence the response to intravitreal
ranibizumab therapy for neovascular
age-related macular degeneration
Martin McKibbin, Manir Ali, Shveta Bansal, et al.

Br J Ophthalmol published online May 10, 2011

doi: 10.1136/bjo.2010.193680

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