Purpose
To determine whether the complement factor H ( CFH ) tyrosine 402 histidine (Y402H) variant, recently shown to be associated with age-related macular degeneration (AMD) and multifocal choroiditis, is associated with specific ocular sarcoidosis clinical phenotypes in black and white persons.
Design
Case-control study.
Methods
The CFH Y402H polymorphism (rs1061170) was genotyped in 41 subjects with ocular sarcoidosis and 393 control subjects. Allele frequencies in the ocular sarcoidosis cases were compared with controls using chi-square score tests. Genotypic model-based (dominant, recessive, and additive) associations of the rs1061170 allele were tested using multivariate logistic regression. Bayesian information criteria were used to formalize model selection. Genotypes were correlated with disease characteristics and severity of ocular inflammation.
Results
The C allele (rs1061170) was found in 35% of controls, but occurred with a significantly higher frequency (48.7%) in ocular sarcoidosis cases (odds ratio, 1.72; 95% confidence interval, 1.09 to 2.78; P = .018). Logistic regression demonstrated an association between rs1061170 and ocular sarcoidosis in 2 of 3 genetic models (additive, P = .0078; recessive, P = .0018). Posterior uveitis and panuveitis were overrepresented significantly in cases with the homozygous variant genotype (CC, 91%; P = .047). The population-attributable risk related to this CFH risk variant was 20%.
Conclusions
The Y402H polymorphism of CFH seems to be associated with ocular sarcoidosis in black and white persons. Carriage of the CFH Y402H polymorphism in both alleles is associated with an increased risk for posterior uveitis and panuveitis presentation. The prognostic importance of this genotype will require prolonged follow-up studies.
Sarcoidosis is a systemic inflammatory disease characterized by the presence of noncaseating granulomas in multiple organ tissues. Ocular manifestations have been described in 25% to 60% of sarcoidosis patients, are the initial presenting sign in 10% to 20% of patients, and are responsible for 3% to 8% of all uveitis cases. The pathogenesis of sarcoidosis is not fully known, but familial clustering, racial variation, clinical heterogeneity, and genetic studies support the likelihood that the disorder is triggered by unidentified antigenic exposures in genetically susceptible hosts.
Recent investigations suggest age-related macular degeneration (AMD), a degenerative disorder of diseased retinal pigment epithelium (RPE), may result from dysregulation of the local inflammatory response in which complement may play an inciting role. Factor H is a serum glycoprotein that downregulates complement activation. The tyrosine 402 histidine (Y402H) single nucleotide polymorphism (SNP), a coding variant of the complement factor H ( CFH ) gene (the C allele of rs1061170), is the most significant known contributor to AMD disease risk.
CFH polymorphisms occur in a variety of immune-mediated diseases, including glomerulonephritis, atypical hemolytic uremic syndrome, Alzheimer (in ApoE4 risk allele carriers), atherosclerosis, and lupus. Recently, it has been shown that multifocal choroiditis, an ocular inflammatory disease of unknown origin, and AMD share the same Y402H high-risk polymorphism. Ocular sarcoidosis, like multifocal choroiditis and AMD, can affect the RPE and the choroid. Additionally, ocular sarcoidosis and AMD share alterations in monocyte and macrophage function. We aimed to evaluate whether these shared clinical features suggest a common genetic basis for disease. We used a candidate gene approach to investigate the association between the CFH SNP Y402H and ocular sarcoidosis.
Methods
Subjects
This case-control study was approved by the Neuroscience Institutional Review Board of the National Institutes of Health and conformed to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all patients. The ocular sarcoidosis cohort consisted of 41 consecutive patients recruited in the National Eye Institute Clinic (mean age, 50.4 years; male-to-female ratio, 42%:58%) during a 26-month period (from July 2009 through September 2011). The control cohort included 393 sarcoidosis-free subjects. Ninety-one control samples were from persons seen in the National Eye Institute Clinic with non–sarcoidosis-related diagnoses of uveitis. Three hundred two control samples were collected from volunteers with no self-reported personal history of sarcoidosis or uveitis at the National Institutes of Health Blood Bank and American Red Cross Blood Services.
All cases had undergone an extensive systemic evaluation, followed by complete ophthalmic examination before their enrollment in the study. Uveitis was categorized according to the anatomic location of the inflammatory process as defined by the Standardization of Uveitis Nomenclature criteria. Ocular sarcoidosis was defined as definitive in patients with positive histopathologic results of biopsy tissue in the presence of compatible uveitis. Ocular sarcoidosis cases were considered presumed in patients with any of the following 7 ocular signs in the presence of radiographic bilateral hilar lymphadenopathy (supported by X-ray or computed tomography scan) and 1 positive laboratory finding (elevated angiotensin-converting enzyme or elevated serum lysozyme level): mutton-fat keratic precipitates; trabecular meshwork nodules, peripheral anterior synechiae, or both; snowball vitreous opacities; chorioretinal peripheral lesions; nodular periphlebitis and/or retinal macroaneurysm; optic disc nodules or granulomas; and bilaterality of ocular inflammation.
Genomic DNA Extraction From Whole Blood
Genomic DNA was extracted from the peripheral blood of each individual using a DNA extraction and purification kit (Qiagen Blood DNA Mini Kit, Valencia, California, USA), according to the manufacturer’s instructions. DNA was stored at −80 C until polymerase chain reaction analyses.
Single Nucleotide Polymorphism Genotyping
The CFH gene was amplified by polymerase chain reaction. Genotyping was carried out by directional sequencing using Big Dye Terminator Ready reaction mix according to manufacturer instructions (Applied Biosystems, Foster City, California, USA). Sequencing was performed on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). Sequence traces were analyzed using Mutation Surveyor (Soft Genetics Inc, State College, Pennsylvania, USA) and the Seqman program of DNASTAR Software (DNASTAR Inc, Madison, Wisconsin, USA).
Statistical Analyses
All data were analyzed using Golden Helix SVS software suite 7 (Golden Helix, Bozeman, Montana, USA), Excel 2011 (Microsoft, Redmond, Washington, USA), and Graphpad Prism (Graphpad Software, San Diego, California, USA). Demographic characteristics of the study population were compared using chi-square tests. Deviations of the genotype distribution from Hardy-Weinberg equilibrium (HWE) were assessed with the HWE exact test. Allele frequencies were compared between patients and controls using the Pearson chi-square test for independence. The CFH variant adjusted odds ratio (OR) and corresponding 95% confidence interval (CI) were determined in multivariate logistic regression models that included sex, race, and a CFH × race interaction term. We examined 3 potential genetic models: dominant, recessive, and additive. Correction for multiple comparisons was carried out using a Bonferroni adjustment. We computed the log likelihood (LL, probability of the observed results given the parameter estimates) for an initial constrained model in which only the constant was included. This was used as the baseline against which the full models were assessed. The difference between the −2LL of the full and constrained model (the likelihood ratio) has a chi-square distribution with degrees of freedom equal to the difference in the number of parameters between the 2 competing models. To determine the best-fitting genetic model, we used Bayesian information criterion (BIC). The BIC for any model is equal to −2LL + Ln(sample size) × (number of estimated parameters). We used the BIC to compare all models and to select the best-fitting genetic model. Patients were grouped by genotype according to the genetic model with the lowest value of the BIC (the most parsimonious of the best-fitting models) and were compared for disease characteristics and severity using a one-way analysis of variance and Freeman-Halton extensions of Fisher exact tests. Population attributable risk, which is the reduction in prevalence that would be observed if the risk allele were completely removed from the current population, was calculated using the formula (P(OR − 1)/[P(OR − 1) + 1]) × 100, where P is the prevalence of ocular sarcoidosis in the study population exposed to the risk variant and OR is the odds ratio.
Results
Baseline Characteristics of Study Participants
A total of 434 subjects were enrolled in this study, including 41 patients with ocular sarcoidosis and 393 sarcoidosis-free control individuals. Sixty-three percent (26/41) of the ocular sarcoidosis cases were biopsy proven and therefore were considered definitive. There was no statistically significant difference in mean age or gender between the ocular sarcoidosis cases and the control cohort. The percentage of cases in black persons was significantly different from the percentage of black controls ( P = .001).
Complement Factor H Allele Distribution
The frequency of the C allele of rs1061170 (Y402H) in each race is summarized in Table 1 . The difference between the C allele frequency (rs1061170 or Y402H) in black and white controls did not show statistical significance ( P = .5). These allele frequencies were consistent with prior published studies.
| Study | All Cases | Whites | Blacks | ||
|---|---|---|---|---|---|
| Cases | Controls | Cases | Controls | ||
| Current study | 0.487 | 0.625 | 0.346 | 0.431 | 0.369 |
| Edwards and associates | 0.34 | — | |||
| Klein and associates | 0.35 | — | |||
| Haines and associates | 0.33 | — | |||
| Hageman and associates | 0.34 | — | |||
| Grassi and associates | — | 0.35 | |||
Main Effect of Case-Control Analysis
The genotyping data of rs1061170 on chromosome 1q31 did not show any significant deviations from HWE tests in each group ( P = .492, HWE for cases; P = .439, HWE for controls). Rs1061170 was found to be significantly associated with ocular sarcoidosis (OR, 1.72; 95% CI, 1.09 to 2.78; P = .018; Table 2 ). This corresponded to a population attributable risk fraction of 20% (formula provided in Methods).
| SNP Phenotype | CFH Y402H | |
|---|---|---|
| Case (n = 41) | Control (n = 393) | |
| Risk allele frequency | 48.7% (C) | 35.6% (C) |
| Allelic P value a | P = .018 b | |
| Odds ratio (95% confidence interval) | 1.72 (1.09 to 2.78) | |
a Allelic P value is derived from the Pearson chi-square test.
Genotype-Based Association Tests for Models of Inheritance
The genotype-specific associations of the CFH variant with ocular sarcoidosis are summarized in Table 3 . Rs1061170 remained significantly associated with ocular sarcoidosis in an additive model and a recessive model ( P genotypic = .008 and P genotypic = .002, respectively), but not in a dominant model ( P genotypic = .217). The data were best fit by an additive model (additive: BIC, 269.619; chi-square, 20.04; degrees of freedom, 3; P = .004), with significant main effects of race, genotype, and race × genotype ( Table 4 ).
| Genotype | No. of Cases (%) | No. of Controls (%) | Additive Model a , b | Recessive Model a , b | Dominant Model a , b | |||
|---|---|---|---|---|---|---|---|---|
| OR (95% CI) | P Value | OR (95% CI) a | P Value | OR (95% CI) a | P Value | |||
| CC | 11 (27) | 53 (13) | ||||||
| CT | 18 (44) | 174 (44) | 3.08 (1.31 to 7.24) | .008 c | 7.18 (2.24 to 24.97) | 0.002 c | 2.22 (0.58 to 8.44) | .217 |
| TT | 12 (29) | 166 (42) | ||||||
a Analyses for model-based ORs were adjusted by the following covariates: sex, race, and race × genotype interaction.
b Model-based P values are derived from the likelihood ratio test that compares the full model with a model that includes only the adjusting factors and thereby tests if the complement factor H genotype is a predictor for susceptibility in a given model.
c P < .0167 (Bonferroni-corrected significance level, 0.05/3 for 3 model specific tests).
| Parameters | Model | ||
|---|---|---|---|
| Additive OR a | Dominant OR a | Recessive OR a | |
| Rs1061170 | 3.08 | — | 7.18 |
| Race | 7.13 | 3.79 | 5.32 |
| Sex | — | — | — |
| Rs1061170 × race | 0.42 | — | 0.16 |
| Log likelihood | 125.715 | 130.222 | 126.497 |
| P value (Rs1061170) b | .008 | .217 | .002 |
| BIC | 269.619 | 272.590 | 271.213 |
a ORs were determined using multivariate logistic regression. ORs for covariates that changed the adjusted RS1061170 OR by more than 10% were reported.
b P values are derived from the likelihood ratio test and are adjusted for the following covariates: race, sex, race × genotype.
Clinical Features and Genotype
Using an additive model (based on the BIC from the genotype analysis), the clinical characteristics and severity of illness associated with genotype CC, CT, and TT were compared using the following categories: (1) history of cystoid macular edema, (2) biopsy-positive disease (definitive ocular sarcoidosis), (3) bilateral involvement, (4) number of extraocular organ involvement, and (5) anatomic site of ocular inflammation according to Standardization of Uveitis Nomenclature criteria. Analysis of clinical data revealed no significant differences between genotypes in categories 1 through 4 (see Table 5 ). Significant differences between genotypes were found when comparing the sites of ocular inflammation. Ninety-one percent of patients (10 of the 11 patients) with a homozygous variant genotype had panuveitis at presentation, suggesting that rs1071160 may be associated with greater severity of disease in a genotype-dependent fashion.
| CC, n = 11 (%) | CT, n = 18 (%) | TT, n = 12 (%) | P Value | |
|---|---|---|---|---|
| Positive biopsy results | 8 (73) | 13 (72) | 5 (42) | .19 a |
| Bilaterality | 9 (82) | 15 (83) | 9 (75) | .88 a |
| History of macular edema | 6 (55) | 7 (39) | 5 (42) | .71 a |
| No. involved organ systems | 2.27 | 1.94 | 2.16 | .51 b |
| Posterior uveitis or panuveitis | 10 (91) | 8 (44) | 7 (58) | .047 a |
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