HTRA1 promoter variant differentiates polypoidal choroidal vasculopathy from exudative age-related macular degeneration

Exudative age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) share similar abnormal choroidal vasculature, but responses to treatments are different. In this study, we sequenced the whole HTRA1 gene and its promoter by direct sequencing in a Hong Kong Chinese PCV cohort. We identified rs11200638, c.34delCinsTCCT, c.59C>T, rs1049331 and rs2293870 significantly associated with PCV. Notably, rs2672598 was significantly associated with exudative AMD (p = 1.31 × 10−4) than PCV (p = 0.11). Logistic regression indicated that rs2672598 (p = 2.27 × 10−3) remained significant after adjusting for rs11200638 in exudative AMD. Moreover, the rs11200638-rs2672598 joint genotype AA-CC conferred higher risk to exudative AMD (43.11 folds) than PCV (3.68 folds). Promoter analysis showed that rs2672598 C-allele showed higher luciferase expression than wildtype T-allele (p = 0.026), independent of rs11200638 genotype (p = 0.621). Coherently, vitreous humor HTRA1 expression with rs2672598 CC genotype was significantly higher than that with TT genotype by 2.56 folds (p = 0.02). Furthermore, rs2672598 C-allele was predicted to alter the transcription factor binding sites, but not rs11200638 A-allele. Our results revealed that HTRA1 rs2672598 is more significantly associated with exudative AMD than PCV in ARMS2/HTRA1 region, and it is responsible for elevated HTRA1 transcriptional activity and HTRA1 protein expression.

the entire ARMS2 gene by direct sequencing in our Hong Kong Chinese exudative AMD and PCV cohort 15 . Although significant differences in the genotypic distributions of 11 polymorphisms in ARMS2 gene were found between exudative AMD and PCV, all of the associated polymorphisms showed similar trends. Since the ARMS2 and HTRA1 genes are in complete linkage disequilibrium (LD), it is also important to delineate the association pattern of HTRA1 in PCV patients. Three HTRA1 haplotypes are associated individually with typical AMD and PCV in a Japanese study although no difference between them was reported 16 . Therefore, the differential association between exudative AMD and PCV remains equivocal. The association of other HTRA1 variants in PCV should be investigated. In this study, 9 exons, intron-exon junctions and the promoter region of HTRA1 gene were sequenced in our Hong Kong Chinese PCV cohort with a view to clarify the association of HTRA1 with PCV.
Joint genotype analysis. The 4 associated variants between exudative AMD and PCV were examined by joint genotype analysis SNP rs11200638 was used as a proxy because it was in perfect LD with other two SNPs (rs1049331 and rs2293870). Therefore, only rs11200638 and rs2672598 were included in the analysis. The results of the joint genotypes included homozygous carriers of rs2672598-C risk allele. All genotypes of rs11200638 were associated with exudative AMD (p AA-CC = 4.31 × 10 −8 , p GA-CC = 3.00 × 10 −3 and p GG-CC = 2.00 × 10 −3 , respectively). Significant differences were found between exudative AMD and PCV (p AA-CC = 4.00 × 10 −3 , OR = 11.70, 95% CI:

HTRA1 promoter analysis.
Cloning the HTRA1 promoter identified three rs11200638-rs2672598 haplotypes: G-T (wildtype), G-C and A-C. No A-T haplotype was found. Luciferase expression analysis showed that the rs11200638-rs2672598 haplotypes affect differential transcriptional activities of the HTRA1 promoter (Fig. 4). Comparing to G-T haplotype, elevated transcriptional activity was observed in G-C and A-C haplotypes  (p = 0.009, one-way ANOVA; Post-hoc tests adjusted by Tukey HSD: G-C vs G-T, p = 0.026, and A-C vs G-T, p = 0.009) with 1.78 ± 0.35 and 1.99 ± 0.30 fold increase, respectively. However, there was no difference in luciferase expression level between G-C and A-C haplotypes (p = 0.621), suggesting that A-allele of rs11200638 might not alter the transcriptional activity of the HTRA1 promoter. Therefore, rs2672598 should be the variant that contributed to the altered transcriptional activity of the HTRA1 promoter.
Our luciferase-reporter analysis showed that transcription activity increased with rs2672598 C-allele but not rs11200638 A-allele. This phenomenon may be due to influences of transcription factor binding sites within these regions. With G-T haplotype as a reference, one transcription factor E2F-1 binding site was predicted at rs11200638, and one each of STAT4, NFκ B, c-Ets-1, RelA, Elk-1 and WT1 binding sites were predicted at rs2672598 site (Fig. 5). When rs11200638 G-allele was replaced by A-allele, the transcription factor E2F-1 binding site was unchanged. In contrast, when rs2672598 T-allele was replaced by C-allele, all the transcription factor binding sites were abolished and changed to GR-alpha, AP-2α A and Sp1 binding sites. Bioinformatic analysis confirmed that rs2672598 contributed to the altered transcriptional activity of the HTRA1 promoter.   Table 3. Haplotype-based association analysis of HTRA1 polymorphisms. The haplotypes were defined by rs11200638, rs2672598, c.59C> T, rs1049331 and rs2293870. The p-value was corrected by permutation test (n = 10,000). Correlation of rs2672598 genotype and HTRA1 expression in vitreous humor. In order to validate the elevated transcriptional activity of rs2672598 C-allele, we performed the genotype-expression correlation analysis in vitreous humor collected from our previous study 18 , which HTRA1 protein expression has been determined. In this study, the rs2672598 genotype of the vitreous humor samples was determined, and 5 samples were in TT genotype, 23 samples in TC genotype and 27 samples in CC genotypes. The HTRA1 expression was higher in the vitreous humor with rs2672598 CC genotype than that with TT genotype by 2.56 folds (p = 0.009, one-way ANOVA; Post-hoc tests adjusted by Tukey HSD: CC vs TT, p = 0.02; Fig. 6). HTRA1 level of TC genotype is also higher than TT genotype by 2.03 folds but not statistically significant (p = 0.32). As we have previously shown than rs11200638 is not correlated with HTRA1 expression in vitreous humor 18 , this result confirmed that rs2672598 should be the variant leading to the differential HTRA1 expression in human ocular samples.

Discussion
PCV has been regarded as a different disease entity from AMD, with a distinct set of demographic, pathological and clinical characteristics 3 . However, emerging evidences suggest that PCV could be a variant of typical CNV 5 , sharing common genetic determinants and environmental risk factors 6 . Moreover, elevated serum C reactive protein and VEGF levels in CNV were also found in PCV 19,20 . In clinical practice, PCV usually responds better to PDT 8,11 , whereas exudative AMD responds better to anti-VEGF treatment 12,21 . Meanwhile, they share some common genetic determinants. ARMS2 rs10490924 and HTRA1 rs11200638 were associated with both exudative AMD and PCV in a Japanese study, in which neither allelic nor genotypic frequencies showed significant difference 22 . Similar association of rs10490924 with advanced AMD and PCV has also been reported in Caucasians 23 .
HTRA1 has its own role in both AMD and PCV 24,25 , governed by different molecular determinants. In this study, rs2672598, located in the promoter region of HTRA1, not only showed differential associations between AMD and PCV, but also altered the transcriptional activity of the HTRA1 promoter. Higher transcriptional activity of the HTRA1 promoter was observed in the constructs with rs2672598 C-allele rather than that with  rs11200638 A-allele (Fig. 4), suggesting that the rs2672598 C-allele, not rs11200638 A-allele, is responsible for the alteration in the transcriptional activity of the HTRA1 promoter. Our luciferase-reporter analysis agreed with the transcription factor binding site prediction analysis (Fig. 5). No alteration of transcription factor binding site was found in rs11200638 A-allele when compared to the wildtype G-allele. In contrast, the transcription factor binding sites in rs2672598 C-allele were totally different from that in the wildtype T-allele. This further confirmed that rs2672598 should be the variant contributed to the altered transcriptional activity of the HTRA1 promoter. In order to validate the results of in vitro transcriptional activity, we performed a correlation analysis on the HTRA1 protein expression in vitreous humor with the rs2672598 genotypes. Our results showed that HTRA1 expression was higher in the vitreous humor with rs2672598 CC genotype than that with TT genotype by 2.56 folds (p = 0.02; Fig. 6). As we previously showed than rs11200638 is not correlated with HTRA1 expression in vitreous humor 18 , the vitreous humor expression results in this study not only validate our findings in the promoter analysis, but also confirm that rs2672598 is responsible for the higher HTRA1 expression in human ocular samples. Our luciferase activity and vitreous humor expression results of rs11200638 are similar to previous reports that there is no significant change of HTRA1 gene expression level in human retina among genotypes of HTRA1 rs11200638, rs1049331 and rs2293870, and ARMS2 rs10490924 and the del443ins54 variant 26,27 .
In summary, our results revealed that a HTRA1 promoter variant, rs2672598, is more significantly associated with exudative AMD than PCV. It should be responsible for elevated transcriptional activity in the HTRA1 promoter.

Methods
Study subjects. We recruited 188 PCV patients from the Prince of Wales Hospital Eye Centre and the Hong Kong Eye Hospital in Hong Kong. Together with 195 exudative AMD patients and 183 normal controls reported in our previous study 28 , a total of 534 unrelated Han Chinese were included (Supplementary Table 2). For validation, we recruited 187 PCV patients and 190 control subjects from the Joint Shantou International Eye Center of Shantou University and The Chinese University of Hong Kong, Shantou, China. All study subjects, including patients and controls, were given comprehensive ophthalmic evaluations and examined by clinical ophthalmologists. Both fluorescein angiography (FA) and ICGA were performed in the exudative AMD and PCV patients. AMD was graded according to an international classification and grading system 29 . Patients with exudative AMD had non-drusenoid RPE detachment, choroidal neovascularization, serous or hemorrhagic retinal detachments, sub-retinal or sub-RPE hemorrhage or fibrosis. PCV patients had sub-retinal red or orange nodules and hemorrhagic pigment epithelial detachment and characteristic sacculated vascular abnormalities in the inner choroid as visualized on ICGA. The diagnosis of PCV was distinguished from AMD by FA and ICGA staining 30 . PCV patients with geographic atrophy or early signs of AMD were excluded, together with patients who were difficult to be clearly differentiated (at late stage of disease that possess fibrosis and disciform scar or at advanced stage that present with intensive hemorrhage). The control subjects were recruited from elderly people greater than 60 years. They did not have any identifiable signs of AMD, PCV or other major eye diseases, except for mild senile cataract and slight refractive errors ranging from − 1.5 to + 1.5 diopters. The study protocol, approved by the Ethics Committee for Human Research at the Chinese University of Hong Kong, is in accordance with the tenets of the Declaration of Helsinki. Informed consent was obtained from each study subject. Sequence analysis. Genomic DNA from whole blood was extracted (Qiagen QIAamp DNA Blood Mini kit, Qiagen, Hiden, Germany) according to the supplier's instructions. The 9 exons, intron-exon junctions of HTRA1 (ENSG00000166033) and its promoter region were screened by polymerase chain reaction (PCR) with specific primers followed by direct sequencing (BigDye Terminator Cycle Sequencing Reaction Kit, v3.1; Applied Biosystems, Foster City, CA) on a DNA sequencer (ABI 3130XL, Applied Biosystems) 28 .
Association analysis. All the identified polymorphisms were assessed for Hardy-Weinberg equilibrium using χ 2 analysis. Allelic and genotypic distributions for association between PCV and controls, between exudative AMD and controls, and between exudative AMD and PCV) were compared using the χ 2 test or Fisher's exact test (SPSS, version 16.0; SPSS Science, Chicago, IL). Logistic regression was used to examine the association profiles of the associated polymorphisms in HTRA1 between exudative AMD and PCV (SPSS). LD and haplotype-based association analyses were performed based on Haploview, version 4.2; http://www.broadinstitute.org/) 31 . Multiple testing correction was performed by permutation test (n = 10,000).
For the meta-analysis, the Review Manager software (RevMan, version 5.2, The Cochrane Collaboration, Copenhagen, Denmark) was used to generate the Z scores. The exact P values with the Z scores was then calculated using R (v3.0.0, http://cran.r-project.org/). The I2 statistic was used to assess the heterogeneity among studies, which corresponds to no (< 25%), low (25-50%), moderate (50-75%), and high heterogeneity (≥ 75%). We adopted the random effects model in the meta-analysis. Summary p < 0.05 was considered statistically significant.
Joint genotype analysis. The genotype combinations of rs11200638 and rs2672598 in exudative AMD, PCV and control subjects were counted. Their corresponding odds ratios (OR) were calculated by the χ 2 test (SPSS). The ORs were compared to the baseline genotype of the two genes that showed the lowest frequency of the disease risk alleles (homozygous G carrier of rs11200638 with homozygous T carrier of rs2672598). primer (5′ -TAATAGATCTGGGAGAGTGCAG GAGGG). Constructs with different rs11200638-rs2672598 haplotypes were generated. The cloned sequence of all constructs was verified by direct sequencing.

Promoter analysis.
Human retinal pigment epithelial cell line ARPE-19 (American Type Culture Collection, Manassas, VA) 32 was cultured in Dulbecco's modified Eagle's medium and F-12 nutrient mixture (DMEM/F-12) supplemented with 10% fetal bovine serum (Gibco BRL, Rockville, MD). Cells were plated in 60 mm tissue culture dishes at a density of 2-3 × 10 5 cells per dish one day before transfection. After 24 hours, cells were transfected with 2.5 μ g of luciferase constructs in 7.5 μ l FuGene HD transfection reagent (Roche, Indianapolis, IN) per dish. Empty pGL3 was used as negative control. At 72 hours after transfection, cell lysates were extracted by RIPA reagent (Sigma-Aldrich, St. Louis, MO) for detection of luciferase expression.
Luciferase expression was detected by immunoblotting according to our published method 33 . Briefly, the denatured cell lysates of the transfected cells were resolved on 10% SDS-polyacrylamide gel and electro-transferred to nitrocellulose membranes for probing with a mouse monoclonal primary antibody against firefly luciferase (LifeSpan BioSciences, Seattle, WA) and a secondary antibody against mouse IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX). Signals were detected by the enhanced chemiluminescence (ECL) system (Amersham Pharmacia, Cleveland, OH) and quantified by ChemiDoc (BioRad, Hercules, CA). Normalized luciferase intensities were calculated by dividing the quantified luciferase intensities by the housekeeping β -actin intensities. Two different clones of each haplotype were used in the transfection experiment. Triplicated experiments were performed. The wildtype rs11200638-rs2672598 haplotype (G-T) was set as a reference. One-way Analysis of Variance (ANOVA) with post-hoc tests adjusted by Tukey HSD (SPSS) was used to compare the means among different groups. Recombinant firefly luciferase (Promega) was used as a positive control.
DNA sequence of the cloned HTRA1 promoter was used to predict the transcription factor binding sites. We predicted the transcription factors that would bind to the region of rs11200638 and rs2672598 in the HTRA1 promoter by PROMO (version 3.0.2; http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB= TF_8.3) 34 . Predictions with different alleles of rs11200638 and rs2672598 were performed.
Correlation analysis of rs2672598 genotype and HTRA1 expression in vitreous humor. Fifty-five unrelated Chinese patients underwent ocular surgeries at the Prince of Wales Hospital in Hong Kong and the Joint Shantou International Eye Center (JSIEC) of Shantou University and the Chinese University of Hong Kong were recruited and given complete ophthalmoscopic examinations 18 . In all patients, a standard three-port pars plana vitrectomy was performed as a part of the regular surgical procedures. Undiluted vitreous humor samples (0.5-1 ml) were collected into sterile tubes at the time of surgery and aliquots were rapidly frozen at − 80 °C until assay.
HTRA1 protein expression has been previously determined 18 . Briefly, total protein concentrations in the vitreous samples were measured by Protein assay (BioRad, Hercules, CA). Equal amount of total protein (10 ug) for each denatured vitreous humor samples were resolved on 12.5% SDS-polyacrylamide gel and electro-transferred to nitrocellulose membranes for probing with the mouse monoclonal antibody against HTRA1 (R&D Systems Inc., Minneapolis, MN), and secondary antibody against mouse IgG conjugated with horseradish peroxidase (Jackson Immuno. Res., West Grove, PA). The signals were detected by enhanced chemiluminescence (ECL) (Amersham Pharmacia, Cleveland, OH) and the band intensities quantified by Quantity One ® Image Analysis software (BioRad).
DNA was extracted from all of the vitreous humor samples using a commercially available DNA extraction kit (Qiagen, Germany). The HTRA1 rs2672598 genotypes were determined in all 55 samples by polymerase chain reaction (PCR) and direct DNA sequencing as described above 28 .