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Association Analysis of CFH, C2, BF, and HTRA1 Gene
Polymorphisms in Chinese Patients with Polypoidal
Choroidal Vasculopathy
Kelvin Y. Lee,
1,2
Eranga N. Vithana,
2
Ranjana Mathur,
1
Victor H. Yong,
2
Ian Y. Yeo,
1
Anbupalam Thalamuthu,
3
Mun-Wai Lee,
1
Adrian H. Koh,
1
Marcus C. Lim,
1,2
Alicia C. How,
1,2
Doric W. Wong,
1
and Tin Aung
1,2,4
PURPOSE. Polypoidal choroidal vasculopathy (PCV) is a major
cause of serosanguinous maculopathy in Chinese patients with
age-related macular degeneration (AMD). Variants in the CFH
and HTRA1/LOC387715 genes are strongly associated with
AMD in Caucasians and Chinese. Variants in the C2 and BF
genes have been found to confer a significantly reduced risk of
AMD. This study was undertaken to determine whether these
associations occur in Chinese patients with PCV.
M
ETHODS. Patients of Chinese ethnicity with clinically and an-
giographically diagnosed PCV and normal control subjects
were recruited from the Singapore National Eye Centre. Five
single-nucleotide polymorphisms (SNPs) in the CFH gene, two
each within the C2 and BF genes and two variants located in
the LOC387715 and HTRA1 genes, were screened in all pa-
tients and control subjects.
R
ESULTS. Seventy-two patients with PCV and 93 normal control
subjects were studied. A significant association was noted with
CFH variants rs3753394 and rs800292 among the PCV cases
(P ⫽ 0.0015 and P ⫽ 0.0045, respectively). Individuals ho-
mozygous for the TT genotype of rs3753394 had a significantly
higher risk (P ⫽ 0.0076) of PCV (OR ⫽ 4.29; 95% CI: 1.47–
12.50) than those carrying a single copy of the T allele (P ⫽
0.3210; OR ⫽ 1.69; 95% CI: 0.60 –4.78), after adjustment for
such risk factors as age and sex. The genotype frequencies of
rs11200638 and rs10490924 in HTRA1 and LOC387715, re-
spectively, were also found to be significantly different be-
tween patients with PCV and normal control subjects (P ⫽
0.00032 and P ⫽ 0.003, respectively). The AA genotype of
rs11200638 and TT genotype of rs10490924 conferred a 4.9-
fold (95% CI: 1.85–12.95) and 4.89-fold (95% CI: 1.85–12.90)
increased risk of PCV, respectively, after adjustment for age
and sex. The Y402H variant of CFH (rs1061170) and the BF
and C2 variants were not significantly different in patients and
normal control subjects.
C
ONCLUSIONS. The SNPs rs3753394 and rs800292 of CFH and
rs11200638 of HTRA1 are significantly associated with the risk
of PCV in Chinese patients. (Invest Ophthalmol Vis Sci. 2008;
49:2613–2619) DOI:10.1167/iovs.07-0860
P
olypoidal choroidal vasculopathy (PCV) is a major cause of
serosanguinous maculopathy in elderly Chinese and Japa-
nese patients with choroidal neovascular anomaly
1,2
as a dis-
tinct form of age-related macular degeneration (AMD).
1–6
The
incidence of PCV in the Chinese and Japanese populations
with neovascular AMD has been reported to be as high as
24.5% and 54.7% respectively, compared with a much lower
incidence in Caucasians.
4,6,7–9
PCV is characterized on indocyanine green (ICG) angiography
by a branching vascular network with polypoidal structures at the
border of the choroidal vascular network.
10
These polypoidal
structures lead to recurrent serosanguinous retinal pigment epi-
thelial detachments and neurosensory retinal detachments with
associated lipid exudates.
7
Soft drusen associated with AMD are
often not seen in patients with PCV, and since disciform scarring
does not always occur despite recurrent leakage and bleeding
from the polypoidal structures, patients with PCV can often retain
useful vision.
7
However, severe cases of PCV maculopathy can
result in irreversible loss of vision.
The CFH gene has been shown to be strongly associated with
AMD through the Y402H variant (single-nucleotide polymor-
phism [SNP] rs1061170) and significantly increases the risk for
AMD in Caucasian and Taiwanese populations.
11–14
This gene,
however, has not been found to be associated with AMD in
Japanese patients.
15
Other variants in the CFH gene including the
CFH promoter (rs3753394), I62V (rs800292), and IVS15
(rs1329428) have also been reported to be associated with AMD
in Caucasians and Chinese.
16,17
Variants E318D (rs9332739) in
the complement component 2 gene (C2) and L9H (rs4151667)
and R32Q (rs641153) in the factor B gene (BF) have been found
to confer a significantly reduced risk of AMD in Caucasians.
18
All
these findings suggest a role of the complement system in the
molecular pathogenesis of AMD. Recently, SNPs rs10490924
(LOC387715 locus) and rs11200638 in the promoter region of
HTRA1, a serine protease gene on chromosome 10, area q26,
were found to be associated with wet AMD in Chinese, Cauca-
sians, and Japanese.
19–21
Our intention was to investigate whether these associations
occur in Chinese patients with PCV from Singapore.
METHODS
Subjects
Patients of Chinese ethnicity, with clinically and angiographically di-
agnosed PCV and normal control subjects were recruited from the
Singapore National Eye Centre. Written informed consent was ob-
tained from all patients, and the study had the approval of the Ethics
Committee of the Singapore Eye Research Institute and was performed
according to the tenets of the Declaration of Helsinki.
From the
1
Singapore National Eye Centre, Singapore; the
2
Singa-
pore Eye Research Institute, Singapore; the
3
Genome Institute of Sin-
gapore, Singapore; and the
4
Yong Loo Lin School of Medicine, National
University of Singapore, Singapore.
Supported by a grant from the Singapore Eye Research Institute.
Submitted for publication July 10, 2007; revised January 4 and
February 9, 2008; accepted April 7, 2008.
Disclosure: K.Y. Lee, None; E.N. Vithana, None; R. Mathur,
None; V.H. Yong, None; I.Y. Yeo, None; A. Thalamuthu, None;
M.-W. Lee, None; A.H. Koh, None; M.C. Lim, None; A.C. How, None;
D.W. Wong, None; T. Aung, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Tin Aung, Singapore National Eye Centre,
11 Third Hospital Avenue, Singapore 168751; tin11@pacific.net.sg.
Investigative Ophthalmology & Visual Science, June 2008, Vol. 49, No. 6
Copyright © Association for Research in Vision and Ophthalmology
2613
All patients were examined by retina-fellowship–trained ophthal-
mologists, and detailed characterization of each patient’s phenotype
was performed with fundus photography, fundus fluorescein angiog-
raphy (FFA), and ICG angiography (Fig. 1). Clinical features of patients
with clearly defined PCV phenotypes revealed the presence of (1)
subretinal red or orange nodules and hemorrhagic pigment epithe-
lial detachment (PED) and (2) characteristic saccular vascular ab-
normalities in the inner choroid, as visualized on ICG angiography
(Figs. 1C, 1D).
Subjects were excluded if they did not meet the criteria for diag-
nosis of PCV or if there was presence of choroidal neovascularization
(CNV) on FFA and ICG angiography or drusen. Thus, all subjects
included only had PCV, which we consider to be a neovascular form of
AMD distinct from CNV in our population. Chinese subjects with
normal results in a macular examination and without clinical signs of
retinal disease, PCV, or AMD were recruited as the control.
Genotyping
Genomic DNA was extracted from peripheral white blood cells. The
SNPs in the CFH gene (rs3753394, rs800292, rs1061170, rs2274700,
and rs1329428), C2 gene (rs9332739 and rs547154), BF gene
(rs4151667, rs12614 and rs641153), LOC387715 locus (rs10490924),
and HTRA1 gene (rs11200638) were amplified by polymerase chain
reaction (PCR; Thermocycler 9700; Applied Biosystems, Inc. [ABI],
Foster City, CA). PCR reactions were performed in 50-
L reaction
volumes containing 10 mM Tris HCl (pH 8.9), 50 mM KCl, 1.5 mM
MgCl
2
, 25 picomoles of each primer, 200
M of each dNTP, 50 to 100
ng of patient genomic DNA, and 0.7 units of Taq thermostable DNA
polymerase (Promega, Madison, WI). Cycling parameters were 3 min-
utes at 95°C, followed by 35 cycles of 30 seconds at 95°C, 30 seconds
at the melting temperature (T
m
) of the primers (52°C–62°C), and 30
seconds to 1 minute at 72°C, with a final 5-minute extension at 72°C.
PCR products were purified using PCR clean-up columns (GFX; GE
Healthcare, Piscataway, NJ). Sequence variations were identified by
automated bidirectional sequencing (BigDye terminator chemistry,
ver. 3.1; ABI). An automated DNA sequencer (Prism 3100; ABI) was
used. Primers for sequence reactions were the same as those for the
PCR reaction.
Statistical Analysis
Fisher exact tests were used to test the allelic and genotypic associa-
tions of all the SNPs with PCV. Logistic regression was used to test
genotypic associations adjusted for age and sex. Conditional analysis
using logistic regression was also performed to examine the indepen-
dent effect of an SNP conditional on another SNP. The Hardy-Weinberg
equilibrium of the genotypic frequencies among cases and separately
among the control subjects was also examined. All the analyses were
performed with the software R.
22
Haploview was used to compute the
LD statistics and the LD plot.
23
Haplotype association analysis was
performed with the software package WHAP.
24
Joint associations of all
the haplotypes and haplotype-specific and sole variant associations
were determined with this program. A haplotype-specific test of asso-
ciation is used to examine the independent effect of any specific
haplotype. For this test, under the null model none of the haplotypes
is used, and under the alternative model the specific haplotype effect
is entered. A likelihood-ratio test is then constructed to assess the
significance of the haplotype-specific effect. Additional details regard-
ing the haplotype associations implemented in the program can be
found in the reference article cited.
24
RESULTS
Demographics of Subjects
Seventy-two patients with PCV and 93 normal control subjects
were recruited for the study. There were 26 female and 46
male patients with PCV, and all were Chinese. The mean age of
the patients was 63.8 ⫾ 7.6 years (range, 42– 84), and the mean
age of normal control subjects (53 females and 40 males) was
67.2 ⫾ 4.60 years (range, 60 –85). For the age- and sex-adjusted
analysis, age was categorized into three groups: ⬍65 (40%),
65–70 (36%), and ⬎75 (24%) years.
Clinical Phenotype
There were 10 patients who had a diagnosis of bilateral dis-
ease. All patients had angiographically diagnosed PCV, with
characteristic polypoidal choroidal lesions on ICG angiogra-
phy. Symptoms at presentation included blurring of vision,
decreased central vision, metamorphopsia, a black patch in the
field of vision, and floaters. The duration of presenting symp-
toms ranged from 1 week to 3 years. Best corrected Snellen’s
visual acuity at presentation ranged from 6/6 to hand motions
(HM). Patients with foveal involvement from the exudative
lesion had poorer presenting visual acuity. Focal laser was the
treatment of choice in patients with extrafoveal lesions,
whereas patients with subfoveal lesions received photody-
namic therapy.
SNP Analysis
The allelic and genotype frequency of the SNPs investigated are
shown in Tables 1, 2, and 3. The genotype frequencies of the
controls and cases followed Hardy-Weinberg equilibrium. In
the analysis of CFH gene variants between PCV and control
subjects, significant allelic associations were detected only
with the CFH variants rs3753394 and rs800292 but not with
rs1061170, rs2274700, or rs1329428. The frequency of the C
risk allele at Y402H was 6.9% in PCV cases and 5.4% in con-
trols, with no significant difference (P ⫽ 0.64). In contrast the
T allele of rs3753394 and G allele of rs800292 conferred a
2.1-fold (95% CI: 1.30 –3.43) and 2.0-fold (95% CI: 1.21–3.36)
increased likelihood of PCV, respectively. The association sig-
FIGURE 1. (A) Color fundus photograph of a patient with serosangui-
nous maculopathy. There was a hemorrhagic pigment epithelial de-
tachment with hard exudates, and an orange lesion was visible in the
macula. (B) FFA showing the notched pigment epithelial detachment
(PED) with a large area of blocked fluorescence from the hemorrhage.
(C, D) The diagnosis of PCV was confirmed with ICG angiography. The
polypoidal lesions (arrow) were best visualized with ICG angiography.
Saccular outpouchings of the choroidal vessel in the same location as
the orange lesion in (A) were seen. The typical polypoidal lesions were
more distinct in the later ICG film (D), as the dye was slowly washed
from the choroidal system.
2614 Lee et al. IOVS, June 2008, Vol. 49, No. 6
nal with rs800292 was weaker than that for the CFH promoter
variant rs3753394. Individuals homozygous for the TT geno-
type of the CFH variant rs3753394 had a significantly higher
risk (P ⫽ 0.0055) of PCV (OR ⫽ 4.05; 95% CI: 1.39–13.13) than
those carrying a single copy of the T allele (P ⫽ 0.3494; OR ⫽
1.72, 95% CI: 0.60 –5.42). The significant association of the TT
genotype was consistent (P ⫽ 0.0076; OR ⫽ 4.29, 95% CI:
1.47–12.50) even after adjustment for the risk factors age and
sex (Table 4). Similarly, the GG homozygote of rs800292 was
significantly associated (P ⫽ 0.0095), conferring a 5.2-fold
increased risk (95% CI: 1.5–18.2), compared with the homozy-
gous nonrisk genotype after adjustment for age and sex. We
also used logistic regression to determine whether the effect at
rs3753394 might explain the association at rs800292 and vice
versa (Table 5). After conditioning on rs3753394, rs800292
was found not to be significantly associated with disease and
vice versa, indicating that both SNPs are highly correlated and
represent the same association at this locus.
The SNPs rs11200638 and rs10490924 in HTRA1 and
LOC387715, respectively, showed significant association to
PCV with OR ⫽ 2.24, P ⫽ 0.0004 for allele A of rs11200638,
and OR ⫽ 1.98, P ⫽ 0.0027 for allele T of rs10490924. The
TABLE 1. SNPs of the CFH Gene Investigated in Patients with PCV
SNP ID Designation
Allele Distribution (%)
Allele
Association
(P)
Odds Ratio
(95% CI)
Genotype Distribution
(%)
Genotype
Association
P-value
P Odds Ratio
(95% CI)Cases Controls Cases Controls
rs3753394 CFH T 101 (70.1) 98 (52.7) 0.0015 2.10 TT* 36 (50.0) 25 (26.9) 0.0054 0.0055
Promoter (1.30–3.43) 4.05 (1.38–13.13)⫹
(⫺257) C 43 (29.9) 88 (47.3) CT 29 (40.3) 48 (51.6) 0.35
1.72 (0.60–5.42)⫹⫹
CC 7 (9.7) 20 (21.5) 0.016
2.37 (1.13–5.02)⫹⫹⫹
rs800292 CFH G 109 (75.7) 113 (60.8) 0.0045 2.01 GG* 41 (56.9) 36 (38.7) 0.0175 0.011
Exon 2 (1.21–3.36) 4.49 (1.29–20.15)⫹
162V A 35 (24.3) 73 (39.2) GA 27 (37.5) 41 (44.1) 0.1196
2.61 (0.73–11.88)⫹⫹
AA 4 (5.6) 16 (17.2) 0.133
1.72 (0.85–3.53)⫹⫹⫹
rs1061170 CFH C 10 (6.9) 10 (5.4) 0.6438 1.31 CC* 0 (0.0) 1 (1.1) NA NA
Exon 9 (0.47–3.62)
Y402H T 134 (93.1) 176 (94.6) CT 10 (13.9) 8 (8.6)
1q31
TT 62 (86.1) 84 (90.3)
rs2274700 CFH C 102 (70.8) 113 (60.8) 0.0628 1.57 CC* 33 (45.8) 31 (33.3) 0.1084 NS
Exon 10 (0.96–2.57)
T 42 (29.2) 73 (39.2) CT 36 (50.0) 51 (54.8)
TT 3 (3.2) 11 (11.8)
rs1329428 CFH C 95 (66.0) 106 (57.0) 0.1115 1.46 CC* 29 (40.3) 27 (29.0) 0.2041 NS
IVS15 (0.91–2.36)
T 49 (34.0) 80 (43.0) CT 37 (51.4) 52 (55.9)
TT 6 (8.3) 14 (15.1)
Cases n ⫽ 72; controls n ⫽ 93. ⫹, Comparing the likelihood of AMD in individuals with two copies of the risk allele versus individuals with
no copies of the risk allele; ⫹⫹, comparing the likelihood of PCV in individuals with one copy of risk allele versus no copies; ⫹⫹⫹, comparing
the likelihood of PCV in individuals with two copies of risk allele versus one copy. Bold odds ratios represent significance at P ⬍ 0.05/3 ⫽ 0.0167
(Bonferroni correction).
* Homozygous for the risk allele.
† Homozygous for the protective allele.
TABLE 2. SNPs within LOC387715 and HTRA1 Genes Investigated in Patients with PCV
SNP ID Designation
Allele Distribution (%)
Allele
Association
(P)
Odds Ratio
(95% CI)
Genotype Distribution
(%)
Genotype
Association
(P)
P Odds Ratio
(95% CI)Cases Control Cases Control
rs11200638 HTRA1 A 92 (63.9) 82 (44.1) 0.0004 2.24 AA* 32 (44.4) 15 (16.1) 0.0003 0.0011
Promoter (1.40–3.60) 4.53 (1.69–12.87)⫹
(⫺512) G 52 (36.1) 104 (55.9) AG 28 (38.9) 52 (55.9) 0.8358
10q26 1.16 (0.48–2.94)⫹⫹
GG 12 (16.7) 26 (28.0) 4 ⫻ 10
⫺4
3.92 (1.73–9.21)⫹⫹⫹
rs10490924 LOC387715 T 80 (55.6) 72 (38.7) 0.0027 1.98 TT* 25 (34.7) 12 (12.9) 0.0034 0.0025
10q26 (1.24–3.15) 3.97 (1.50–11.06)⫹
G 64 (44.4) 114 (61.3) TG 30 (41.7) 48 (51.6) 0.7078
1.21 (0.54–2.74)⫹⫹
GG 17 (23.6) 33 (35.5) 0.005
3.30 (1.36–8.37)⫹⫹⫹
For an explanation of the data, see the footnote to Table 1.
IOVS, June 2008, Vol. 49, No. 6 Gene Polymorphisms in Polypoidal Choroidal Vasculopathy 2615
genotype frequencies of rs11200638 and rs10490924 were
also found to be significantly different between patients with
PCV and normal control subjects (P ⫽ 0.0003 and P ⫽ 0.0034
respectively). The AA genotype of rs11200638 and TT geno-
type of rs10490924 conferred a 4.9-fold (95% CI: 1.85–12.95),
and 4.89-fold (95% CI: 1.85–12.90) of increased risk of PCV,
respectively, even after adjustment for age and sex (Table 4).
Conditional analysis showed that after conditioning on
rs10490924, rs11200638 was only marginally associated with
disease risk (P ⫽ 0.0449). Furthermore, after conditioning on
rs11200638, rs10490924 also did not remain significantly asso-
ciated with disease risk indicating that the effect of rs11200638
is mainly responsible for the association signal for PCV at the
HTRA1 locus and that these two SNPs are also correlated.
We also evaluated the role of epistasis between the CFH
SNPs rs3753394 and rs800292 and the two HTRA1 and
LOC387715 SNPs rs11200638 and rs10490924, which showed
associations in our study. Using logistic regression, we did not
observe statistically significant interaction terms between any
pair of these SNPs (data not shown). In support of this, the
association of rs11200638 with PCV was found to be signifi-
cant (P ⫽ 0.0028) when analyzed conditional on CFH SNP
rs3753394, and the association of rs3753394 when conditional
on rs11200638 was also found to be significant (P ⫽ 0.0077).
The BF and C2 variants investigated were not significantly
different in patients and normal control subjects. No patients
or normal subjects were homozygous for the protective alleles
in C2 and BF genes. As the frequencies of the protective alleles
were very low in the Chinese population, a larger sample size
may be needed to reveal the protective effect of these alleles.
Linkage Disequilibrium (LD) and Haplotype
Association Analysis
Within the CFH gene pair-wise LD analysis showed rs2274700
in high LD with rs1329428 (D⬘⫽0.97, 95% CI: 0.89–1.0; Fig.
TABLE 4. Distribution of Unadjusted and Adjusted Odds Ratio for Risk Genotypes in CFH and HTRA1
Locus (SNP) Genotype
OR (95% CI
Unadjusted) P
OR (95% CI
Adjusted)* P
CFH (rs3753394) TT 4.05 (1.39–13.13) 0.0055 4.29 (1.47–12.50) 0.0076
CT 1.72 (0.60–5.42) 0.3494 1.69 (0.60–4.78) 0.3210
CFH (rs800292) GG 4.49 (1.29–20.15) 0.011 5.22 (1.50–18.20) 0.0095
GA 2.61 (0.73–11.88) 0.1196 3.24 (0.91–11.50) 0.0687
HTRA1 (rs11200638) AA 4.53 (1.69–12.87) 0.0011 4.90 (1.85–12.95) 0.0014
AG 1.17 (0.48–2.94) 0.8358 1.01 (0.43–2.39) 0.9775
LOC387715 (rs10490924) TT 3.97 (1.50–11.06) 0.0025 4.89 (1.85–12.90) 0.0013
TG 1.21 (0.54–2.74) 0.7078 1.27 (0.57–2.80) 0.5591
* Adjusted for age and sex.
TABLE 3. SNPs of the C2 and BF Gene Investigated in Patients with PCV
SNP ID Designation
Allele Distribution (%)
Allele
Association
(P)
Odds Ratio
(95% CI)
Genotype Distribution
(%)
Genotype
Association
(P)
Odds Ratio
(95% CI)Cases Controls Cases Controls
rs9332739 C2 C 2 (1.4) 4 (2.2) 0.7000 1.56 CC† 0 (0.0) 0 (0.0) NA NA
E318D (0.22–17.45)
6p21 G 142 (98.6) 182 (97.8) CG 2 (2.8) 4 (4.3)
GG 70 (97.2) 89 (95.7)
rs547154 C2 T 5 (3.5) 9 (4.8) 0.5940 1.41 TT† 0 (0.0) 0 (0.0) NA NA
IVS10 (0.41–5.49)
G 139 (96.5) 177 (95.2) TG 5 (6.9) 9 (9.7)
GG 67 (93.1) 84 (90.3)
rs4151667 BF A 3 (2.1) 4 (2.2) 1.0000 1.03 AA† 0 (0.0) 0 (0.0) NA NA
Exon 1 (0.17–7.16)
L9H T 141 (97.9) 182 (97.8) AT 3 (4.2) 4 (4.3)
TT 69 (95.8) 89 (95.7)
rs12614 BF T 4 (2.8) 6 (3.2) 1.0000 1.17 TT† 0 (0.0) 0 (0.0) NA NA
Exon 2 (0.27–5.73)
R32W C 140 (97.2) 180 (96.8) TC 4 (5.6) 6 (6.5)
CC 68 (94.4) 87 (93.5)
rs641153 BF A 8 (5.6) 13 (7.0) 0.6552 1.28 AA† 0 (0.0) 0 (0.0) NA NA
Exon 2 (0.47–3.66)
R32Q G 136 (94.4) 173 (93.0) AG 8 (11.1) 13 (14.0)
GG 64 (88.9) 80 (86.0)
For an explanation of the data, see the footnote to Table 1.
2616 Lee et al. IOVS, June 2008, Vol. 49, No. 6
2). The rs3753394 was also in moderately high LD with
rs800292 (D⬘⫽0.82, 95% CI: 0.68 – 0.91). Haplotype analysis
using the five SNPs of CFH revealed nine different haplotypes
among the patient and control individuals, after the rare hap-
lotypes with frequencies less than 1% were trimmed. The
estimated haplotype frequencies are presented in Table 6. A
significant association was noted for the C-A-T-T-T (P ⫽ 0.0062)
haplotype that was present approximately two times higher
(29.8% vs. 17.1%) in controls than in cases, indicating that it
could be protective. The association of PCV with at-risk hap-
lotype T-G-T-C-C and protective haplotype C-A-T-T-T were only
marginally significant after adjustment for age and sex. When
only the two locus haplotype comprising the CFH SNPs
(rs3753394 and rs800292) were considered, the risk haplotype
(T-G) was found to be more strongly associated with disease
(P ⫽ 0.0007; OR ⫽ 2.27 (95% CI: 1.40 –3.68) even after adjust-
ing for age and sex (P ⫽ 0.0011; OR ⫽ 2.26; 95% CI: 1.37–
3.72).
The two SNPs rs11200638 and rs10490924 in HTRA1 and
LOC387715 were in strong LD with each other (D⬘⫽0.83,
95% CI: 0.71–0.91). Haplotype analysis of the two SNPs
rs11200638 and rs10490924 identified four haplotypes (Table
7). Consistent with individual SNP analysis, the haplotype T-A
conferred a 2.39-fold (95% CI: 1.48 –3.85) increased likelihood
of PCV (P ⫽ 0.0003) after adjustment for age and sex. This
association was further supported by a nonsignificant adjusted
probability (P ⫽ 0.069) in the sole-variant-haplotype test,
24
—a
test of association of all other haplotypes excluding this hap-
lotype, which indicated that the T-A haplotype contributes to
the global test of association.
DISCUSSION
Patients with PCV can develop recurrent serous and hemor-
rhagic detachments of the retinal pigment epithelium and the
neurosensory retina. The fundus typically lacks drusen, and
most cases are unilateral and occur more frequently in males
and at an earlier age than CNV occurs.
2,5,25
The pathogenesis
of the disorder is unknown; however, unlike CNV, PCV in-
volves primarily the choroidal vasculature with characteristic
lesions seen as vascular outpouchings of the normal choroidal
vessel, which may be seen as orange-reddish nodules or polyp-
like structures at the posterior pole.
25
In Singapore, PCV is
seen in more than 40% of cases of exudative AMD (unpub-
lished hospital data, July 2007) and is a major cause of visual
loss from serosanguinous maculopathy.
Our study shows that there is an association between CFH
and HTRA1 gene variants and PCV. The SNPs rs3753394 and
rs800292 of CFH, rs10490924 of LOC387715, and rs11200638
of HTRA1 were significantly associated with the risk of PCV in
our Chinese patients. After adjustment for age and sex, all these
SNPs showed a partially recessive effect on the association
with PCV. Our study is the first to detect association between
variants within the 10q locus (rs10490924 and rs11200638)
and PCV in the Chinese. These two SNPs also showed a highly
significant allelic association with PCV (rs10490924, P ⫽ 5.7 ⫻
10
⫺6
; rs11200638, P ⫽ 5.2 ⫻ 10
⫺6
) in a recent Japanese study
involving 76 PCV cases and 94 control subjects.
26
The risk
allele A of rs11200638 was present at a lower frequency
(38.3%) among the Japanese control samples than in our Chi-
nese control samples (44.1%), whereas the frequency of this
allele in PCV cases was similar between the Japanese (63.2%)
and the Chinese (63.9%). The frequency of the risk allele of
rs10490924 was also similar between Japanese and Chinese
control subjects but differed between the Japanese (62.5%)
and Chinese PCV cases (55.6%) of our study. These differences
in allele frequencies may explain the differences in association
TABLE 5. Association between CFH and HTRA1 Variants and PCV
SNP
Risk
Allele
Control
Frequency
(%)
Affected
Frequency
(%) P OR (95% CI)
Conditional on:
rs3753394 rs800292
P OR (95% CI) P OR (95% CI)
rs3753394 T 52.7 70.1 0.0015 2.10 (1.30–3.43) — — 0.0819 1.76 (0.93–3.32)
rs800292 G 60.8 75.7 0.0045 2.01 (1.21–3.36) 0.3976 1.33 (0.69–2.55) — —
SNP
Risk
Allele
Control
Frequency
(%)
Affected
Frequency
(%) P OR (95% CI)
Conditional on:
rs11200638 rs10490924
P OR (95% CI) P OR (95% CI)
rs11200638 A 44.1 63.9 0.0004 2.24 (1.4–3.6) — — 0.0449 1.99 (1.01–3.89)
rs10490924 T 38.7 55.6 0.0027 1.98 (1.24–3.15) 0.597 1.19 (0.62–2.29) — —
FIGURE 2. Analysis of pair-wise LD across the five CFH SNPs in the
Chinese cohort.
IOVS, June 2008, Vol. 49, No. 6 Gene Polymorphisms in Polypoidal Choroidal Vasculopathy 2617
signal strengths observed between our study and that of the
Japanese, even though both involved a similar number of cases
and controls.
In a previous study of Chinese exudative AMD cases, the
CFH variants rs3753394, rs800292, and rs1329428 were found
to be significantly associated with exudative AMD.
17
In our
Chinese PCV cohort, we found a significant association with
only two of the above SNPs (i.e., rs3753394 and rs800292).
However, after correcting for multiple comparisons, we found
that the genotype association signal with rs800292 was much
weaker. Pair-wise LD analysis also indicated a strong LD be-
tween rs3753394 and rs800292 and the risk haplotype (T-G)
was found to be strongly associated (P ⫽ 0.0011), conferring a
2.26-fold increased risk (95% CI: 1.37–3.72) of PCV after ad-
justment for age and sex. Our data therefore indicate
rs3753394 in the CFH promoter to be one of the major AMD
susceptibility polymorphisms in the Chinese population, al-
though it was found not be associated with AMD in two
previous studies conducted in Caucasians.
12,16
It is possible
that a larger sample cohort would give more power to the
association shown with the SNP rs800292, but it may not be so
for the rs1329428 variant. Hence, such genetic differences may
partly underlie the phenotypic variations/diversity observed
between the two AMD phenotypes in the Chinese. The Y402H
variant of CFH (rs1061170) was not associated with the risk of
PCV in our Chinese patients and variants in C2 and BF did not
confer a reduced risk of PCV. The frequency of the Y402H
variant in the Chinese and Japanese populations have been
reported to be low, and our study provides further evidence
that other variants in the CFH gene, such as rs3753394, may
play a more important role in AMD in Chinese popula-
tions.
14,15,17
Studies have shown that there are multiple AMD-
predisposing variants in the CFH gene.
27
Our study was limited
to only five CFH variants and therefore does not preclude the
possibility of other CFH variants that may play a role in the
pathogenesis of PCV in combination with environmental vari-
ables.
Our findings suggest a role of the complement system in the
molecular pathogenesis of PCV. CFH specifically inhibits the
alternative complement cascade and also regulates the com-
mon pathway.
28
It is hypothesized that variants in CFH, like
Y402H, result in an aberrant inflammatory process with inap-
propriate complement activation and damage to the Bruch’s
membrane, with resultant neovascularization.
11,16,29
The
HTRA1 gene encodes a heat shock serine protein and is acti-
vated by cellular stress.
30
It is hypothesized that overexpres
-
sion of HTRA1 alters the integrity of Bruch’s membrane, facil-
itating invasion of choroidal capillaries, as HTRA1 appears to
TABLE 6. Haplotype Analysis of SNPs in CFH
CFH Haplotypes Frequency*
OR
(95% CI
Unadjusted) P‡
OR
(95% CI
Adjusted)† P‡rs3753394 rs800292 rs1061170 rs2274700 rs1329428 Controls Cases
T G T C C 0.428 0.554 1.764 0.0210 1.789 0.0272
(1.09–2.86) (1.07–2.97)
C A T T T 0.298 0.171 0.467 0.0062 0.51 0.0214
(0.26–1.21) (0.28–0.92)
T G T T T 0.060 0.081 1.439 0.4473 1.32 0.5775
(0.57–3.63) (0.50–3.48)
C G C C C 0.045 0.065 1.45 0.4199 1.54 0.3964
(0.56–3.75) (0.57–4.16)
C G T C C 0.058 0.023 0.444 0.1562 0.37 0.0915
(0.13–1.49) (0.11–1.26)
T A T T T 0.035 0.030 0.865 0.8510 0.778 0.6532
(0.26–2.84) (0.22–2.69)
C A T C T 0.018 0.043 2.58 0.2166 2.53 0.2635
(0.60–11.04) (0.56–11.34)
C A T C C 0.042 0.010 0.168 0.0402 0.19 0.1119
(0.02–1.39) (0.02–1.57)
T G T C T 0.015 0.022 1.53 0.5873 1.84 0.4867
(0.27–8.64) (0.31–10.79)
* All haplotypes with frequency 1% in the combined case and control sample are shown.
† Adjusted for age and sex. Global unadjusted P ⫽ 0.0743; adjusted ⫽ 0.0712.
‡ P reported is the haplotype-specific test P obtained for 10,000 permutations.
TABLE 7. Haplotype Analysis of SNPs in LOC387715 and HTRA1
LOC387715
rs10490924
HTRA1
rs11200638
Frequency*
OR
(95% CI
Unadjusted) P‡
OR
(95% CI
Adjusted)† P‡Controls Cases
G G 0.525 0.360 0.49 0.0029 0.46 0.0015
(0.31–0.79) (0.28–0.75)
T A 0.353 0.554 2.22 0.0005 2.39 0.0003
(1.41–3.51) (1.48–3.85)
G A 0.088 0.085 0.97 0.8860 0.96 0.9018
(0.49–1.89) (0.48–1.92)
T G 0.034 0.002 NA NA NA NA
* All haplotypes with frequency ⬎1% in the combined case and control sample are shown.
† Adjusted for age and sex. Global test 0.0005; adjusted P ⫽ 0.0008.
‡ P is the haplotype-specific test P obtained for 10,000 permutations.
2618 Lee et al. IOVS, June 2008, Vol. 49, No. 6
regulate the degradation of extracellular matrix proteogly-
cans.
31
We hypothesize that both of these distinct pathways
are involved in the pathogenesis of PCV. That we did not
observe any statistically significant interaction between
rs3753394 of CFH and rs11200638 of HTRA1 also suggests that
these variants confer risk for PCV in an independent additive
fashion. This result reflects what has been observed previously
in other studies for AMD where CFH and HTRA1 variants were
found to confer independent risks.
32,33
In conclusion, we have found significant association be-
tween CFH variants (rs3753394 and rs800292) and variants
within the 10q locus (rs11200638 and rs10490924) and PCV in
Chinese subjects, even after adjustment for risk factors such as
age and sex. We note, however, that further studies in a larger
population should be performed to ascertain the effects of rare
variants and risk factors such as smoking, which has been
found to modify the association of CFH polymorphisms and
AMD.
34,35
Our findings suggest a role of two biological path
-
ways, each contributing to the pathogenesis of PCV. The ge-
netic risk factors (rs3753394, rs800292, rs11200638, and
rs10490924) common to both exudative AMD and PCV also
suggest that these two diseases have common pathogenic
mechanisms.
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