Patient Selection
The research was approved by the Clinic Institutional Review Board, and complied with the Declaration of Helsinki. Informed consent was obtained from each participating subject. We enrolled 437 CSCR patients and 510 control patients in the original population. All patients were recruited from the Eye Center of the People’s Hospital of Peking University. Enrollees’ demographic details are shown in Table 1.
All subjects underwent a comprehensive ophthalmic examination, including best-corrected visual acuity measurement, slit-lamp biomicroscopy, dilated fundoscopy, color fundus photographs, optical coherence tomography, flurorescein angiography, and indocyanine green angiography.
Our criteria for CSCR patient selection were as follows: serous subretinal fluid on optical coherence tomography, focal leakage spot (ink blot) or smokestack pattern or ≧1 area of multifocal diffuse leakage, or presence of irregular retinal pigment epithelium window defects on fluorescein angiography and corresponding hyperfluorescence on indocyanine green angiography[28,29]. Participants with uveitis, diabetic retinopathy, age-related macular degeneration, polypoidal choroidal vasculopathy, other retinal and choroidal vascular diseases, pathologic myopia were excluded.
Genotyping
Five milliliters of peripheral blood was collected from each participant and stored at -80℃ prior to DNA extraction. Genomic DNA was extracted from venous blood leukocytes, using a genomic extraction kit (Beijing eBios Biotechnology, Beijing, China). Genotyping was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as previously described [11]. The primers used for CFH SNPs (rs800292, rs1061170, rs3753396, rs2284664, rs1329428, and rs1065489) were as follows:
SNP-1-F: CAGAAGGCACCCAGGCTATC
SNP-1-R: TGGCAATAGTGATATAATTCAGGCA
SNP-2-F: TCCTGAACTCCTCAATGGGAA
SNP-2-R: ACAGGTACTCTCCTCCACTATGT
SNP-3-F: TGTTTGCGTCATTAGATACCCT
SNP-3-R: TACAACCTTTACAGTTACCTCC
SNP-4-F: CTATTTGCTTTACGATCTATGG
SNP-4-R: CAGTGATACATCCAGGTACATTA
SNP-5-F: GTTGGTGACAGTCCGATAGACA
SNP-5-R: ATTTCCCACAGCAGTCCAGAATA
SNP-6-F: AGGGTTTCTTCTTGAAAATCACAGG
SNP-6-R: GAGTAGTGTACTTACTGACACGGA
Following amplification, the PCR products were used for locus-specific single-base extension reactions. The resulting products were desalted and transferred to a 384 SpectroCHIP array (Sequenom, San Diego, CA). Allele detection was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The mass spectrograms were analyzed using MassARRAY Typer software version 4.0 (Sequenom, San Diego, CA).
Statistical Analysis
The data were analyzed using SPSS (version 16.0; SPSS Science, Chicago, IL). All identified polymorphisms were assessed for Hardy-Weinberg equilibrium. Allelic association was evaluated for each SNP by Chi-square tests on 2 × 2 contingency tables. All odds ratio (OR) and corresponding 95% confidence interval (CI) were calculated with respect to minor alleles in the controls. For the allelic association tests, we used a Bonferroni correction for multiple testing, in which nominal P values were multiplied by 6 (the number of SNPs). Values of p < 0.05 were considered statistically significant.
To begin the meta-analysis, we searched PubMed, CNKI, and Wanfang for studies on CFH and CSCR. We identified five articles, only three of which accorded with our inclusion criteria requiring case-control studies showing P values and 95% CIs. Both a fixed-effect model and a random-effect model were fitted to the data in each study. Cochran’s Q statistic and the companying I2 index was used to access inter-cohort heterogeneity. For heterogeneity P values > 0.05, we assumed the effects were heterogeneous and used the fixed-effect model was selected; otherwise, we used the random-effect model. All statistical calculations were conducted using Stata version 13.0.