Imaging diabetic retinal disease: clinical imaging requirements.

Diabetic retinopathy (DR) is a sight-threatening complication of diabetes mellitus (DM) and it contributes substantially to the burden of disease globally. During the last decades, the development of multiple imaging modalities to evaluate DR, combined with emerging treatment possibilities, has led to the implementation of large-scale screening programmes resulting in improved prevention of vision loss. However, not all patients are able to participate in such programmes and not all are at equal risk of DR development and progression. In this review, we discuss the relevance of the currently available imaging modalities for the evaluation of DR: colour fundus photography (CFP), ultrawide-field photography (UWFP), fundus fluorescein angiography (FFA), optical coherence tomography (OCT), OCT angiography (OCTA) and functional testing. Furthermore, we suggest where a particular imaging technique of DR may aid the evaluation of the disease in different clinical settings. Combining information from various imaging modalities may enable the design of more personalized care including the initiation of treatment and understanding the progression of disease more adequately.

Analysis of hemopexin plasma levels in patients with age-related macular degeneration.

Purpose: A protein quantitative trait locus (pQTL) analysis recently revealed a strong association between hemopexin (HPX) levels and genetic variants at the complement factor H (CFH) locus. In this study, we aimed to determine HPX plasma levels in patients with age-related macular degeneration (AMD) and to compare them with those in controls. We also investigated whether genetic variants at the CFH locus are associated with HPX plasma levels. Methods: HPX levels were quantified in 200 advanced AMD cases and 200 controls using an enzyme-linked immunosorbent assay and compared between the two groups. Furthermore, HPX levels were analyzed per genotype group of three HPX-associated variants (rs61818956, rs10494745, and rs10801582) and four AMD-associated variants (rs794362 [proxy for rs187328863], rs570618, rs10922109, and rs61818924 [proxy for rs61818925]) at the CFH locus. Results: HPX levels were similar in the control group compared with the AMD group. The three variants at the CFH locus, which were previously associated with the HPX levels, showed no association with the HPX levels in our data set. No significant differences in HPX levels were detected between the different genotype groups of AMD-associated variants at the CFH locus. Conclusions: In this study, HPX levels were not associated with AMD or AMD-associated variants at the CFH locus. The finding of a previous pQTL study that variants at the CFH locus were associated with HPX levels was also not confirmed in this study.

Loss of the AMD-associated B3GLCT gene affects glycosylation of TSP1 without impairing secretion in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) has been associated with protective genetic variants in the ß1-3 glucosyltransferase (B3GLCT) locus through genome-wide association studies. B3GLCT mediates modification of proteins with thrombospondin type I repeats (TSR) that contain O-linked glucose ß1-3 fucose and C-linked mannose glycosylation motifs. B3GLCT-mediated modification is required for proper secretion of TSR-containing proteins. We aimed to start understanding the role of B3GLCT in AMD by evaluating its effect on glycosylation and secretion of proteins from retinal pigment epithelium (RPE) cells. We generated B3GLCT knockout (KO) RPE cells and analyzed glycosylation and secretion of thrombospondin 1 (TSP1), a protein involved in cellular processes highly relevant to AMD. Glycopeptide analysis confirmed the presence of the glucose-ß1,3-fucose product of B3GLCT on TSP1 in wildtype (WT) cells and its absence in KO cells. C-mannosylation was variably present on WT TSP1 and increased on TSR domains 1 and 3 in KO cells. Secretion of TSP1 was not affected by the absence of B3GLCT, even not when TSP1 was upregulated by TNFα treatment or when TSP1 was overexpressed in HEK293T cells. Future research is needed to elucidate the effect of the observed glycosylation defects in the context of AMD, which might involve functional loss of TSP1 or effects on other TSR proteins.

Micro-costing diagnostics in oncology: from single-gene testing to whole- genome sequencing.

Purpose: Predictive diagnostics play an increasingly important role in personalized medicine for cancer treatment. Whole-genome sequencing (WGS)-based treatment selection is expected to rapidly increase worldwide. This study aimed to calculate and compare the total cost of currently used diagnostic techniques and of WGS in treatment of non-small cell lung carcinoma (NSCLC), melanoma, colorectal cancer (CRC), and gastrointestinal stromal tumor (GIST) in the Netherlands.Methods: The activity-based costing (ABC) method was conducted to calculate total cost of included diagnostic techniques based on data provided by Dutch pathology laboratories and the Dutch-centralized cancer WGS facility. Costs were allocated to four categories: capital costs, maintenance costs, software costs, and operational costs.Results: The total cost per cancer patient per technique varied from € 58 (Sanger sequencing, three amplicons) to € 2925 (paired tumor-normal WGS). The operational costs accounted for the vast majority (over 90%) of the total per cancer patient technique costs.Conclusion: This study outlined in detail all costing aspects and cost prices of current and new diagnostic modalities used in treatment of NSCLC, melanoma, CRC, and GIST in the Netherlands. Detailed cost differences and value comparisons between these diagnostic techniques enable future economic evaluations to support decision-making.

Long-term outcomes of vitrectomy for proliferative diabetic retinopathy.

PURPOSE: To investigate the long-term outcomes of patients who underwent vitrectomy for proliferative diabetic retinopathy. METHODS: Cumulative incidences were calculated for low vision (<0.3), re-vitrectomy in the study eye and fellow eye vitrectomy. To identify potential prognostic factors that associate with these outcomes, we used multivariable Cox regression models. RESULTS: In a total of 217 patients, we found 1-, 5- and 10-year cumulative incidences of low vision in the study eye of 24%, 31% and 39%, respectively. For both eyes, these rates were, respectively, 10%, 14% and 14%. Low vision in both eyes was associated with higher age and worse contralateral visual acuity. The 1-, 5- and 10-year cumulative incidences for re-vitrectomy in the study eye were 16%, 27% and 27%, respectively, and for a vitrectomy in the fellow eye 24%, 40% and 54%, respectively. Re-vitrectomy of the study eye was associated with worse contralateral visual acuity, while vitrectomy of the fellow eye was associated with shorter diabetes duration, worse contralateral visual acuity, higher HbA1c level and worse diabetic retinopathy severity stage of the fellow eye. CONCLUSION: Functional visual acuity in at least one eye was achieved or preserved in most patients. After 10 years, about a quarter of all patients underwent a re-vitrectomy, while more than half of the patients needed a vitrectomy of the fellow eye. Knowledge of these long-term outcomes is essential when counselling patients for a vitrectomy.

Validation of a model for the prediction of retinopathy in persons with type 1 diabetes.

BACKGROUND/AIM: To validate a previously developed model for prediction of diabetic retinopathy (DR) for personalised retinopathy screening in persons with type 1 diabetes. METHODS: Retrospective medical data of persons with type 1 diabetes treated in an academic hospital setting were used for analysis. Sight-threatening retinopathy (STR) was defined as the presence of severe non-proliferative DR, proliferative DR or macular oedema. The presence and grade of retinopathy, onset of diabetes, systolic blood pressure, and levels of haemoglobin A1c were used to calculate an individual risk estimate and personalised screening interval. In persons with STR, the occurrence was compared with the calculated date of screening. The model's predictive performance was measured using calibration and discrimination techniques. RESULTS: Of the 268 persons included in our study, 24 (9.0%) developed STR during a mean follow-up of 4.6 years. All incidences of STR occurred after the calculated screening date. By applying the model, the mean calculated screening interval was 30.5 months, which is a reduction in screening frequency of 61% compared with annual screening and 21% compared with biennial screening. The discriminatory ability was good (Harrell's C-statistic=0.82, 95% CI 0.74 to 0.90), and calibration showed an overestimation of risk in persons who were assigned to a higher risk for STR. CONCLUSION: This validation study suggests that a screening programme based on the previously developed prediction model is safe and efficient. The use of a personalised screening frequency could improve cost-effectiveness of diabetic eye care.

Effect of rare coding variants in the CFI gene on Factor I expression levels.

Factor I (FI) is one of the main inhibitors of complement activity, and numerous rare coding variants have been reported in patients with age-related macular degeneration, atypical hemolytic uremic syndrome and C3 glomerulopathy. Since many of these variants are of unknown clinical significance, this study aimed to determine the effect of rare coding variants in the complement factor I (CFI) gene on FI expression. We measured FI levels in plasma samples of carriers of rare coding variants and in vitro in the supernatants of epithelial cells expressing recombinant FI. FI levels were measured in 177 plasma samples of 155 individuals, carrying 24 different rare coding variants in CFI. In carriers of the variants p.Gly119Arg, p.Leu131Arg, p.Gly188Ala and c.772G>A (r.685_773del), significantly reduced FI plasma levels were detected. Furthermore, recombinant FI expression levels were determined for 126 rare coding variants. Of these variants 68 (54%) resulted in significantly reduced FI expression in supernatant compared to wildtype (WT). The recombinant protein expression levels correlated significantly with the FI level in plasma of carriers of CFI variants. In this study, we performed the most comprehensive FI expression level analysis of rare coding variants in CFI to date. More than half of CFI variants lead to reduced FI expression, which might impair complement regulation in vivo. Our study will aid the interpretation of rare coding CFI variants identified in clinical practice, which is in particular important in light of patient inclusion in ongoing clinical trials for CFI gene supplementation in AMD.

Complement Activation Levels Are Related to Disease Stage in AMD.

Purpose: To study the levels of complement activation in different disease stages of AMD and the influence of genetic polymorphisms in complement genes. Methods: We included 797 patients with AMD and 945 controls from the European Genetic Database. Patients were grouped into five AMD stages: early AMD, intermediate AMD, central geographic atrophy, active choroidal neovascularization or inactive choroidal neovascularization. Differences in complement activation, as defined by the systemic C3d/C3 ratio, between AMD stages were evaluated using general linear modeling. In addition, we evaluated the influence of 18 genetic AMD polymorphisms in complement genes and their effect on complement activation. Differences in complement activation between stages were evaluated stratifying by complement associated haplotypes. Results: Complement activation levels differed significantly between AMD disease stages. As compared with controls, the C3d/C3 ratio was higher in patients with intermediate AMD (P < 0.001) and central geographic atrophy (P = 0.001). Two polymorphisms in CFH (rs10922109 and rs570618) and one in CFB (rs116503776) were significantly associated with complement activation. The association between AMD disease stage and complement activation was more pronounced in patients with haplotypes associated with the highest complement activation. Conclusions: In general, consecutive AMD disease stages showed increasing levels of complement activation, especially in individuals with a genetic burden in complement genes. These findings contribute to the discussion on the pathogenesis of AMD in relation to complement activation and might suggest refinement in patient selection and the optimum window of treatment with complement inhibitors. Prospective studies are needed to confirm these results.

Major Predictive Factors for Progression of Early to Late Age-Related Macular Degeneration.

INTRODUCTION: We present a prediction model for progression from early/intermediate to advanced age-related macular degeneration (AMD) within 5.9 years. OBJECTIVES: To evaluate the combined role of genetic, nongenetic, and phenotypic risk factors for conversion from early to late AMD over ≥5 years. METHODS: Baseline phenotypic characteristics were evaluated based on color fundus photography, spectral-domain optical coherence tomography, and infrared images. Genotyping for 36 single-nucleotide polymorphisms as well as systemic lipid and complement measurements were performed. Multivariable backward logistic regression resulted in a final prediction model. RESULTS AND CONCLUSIONS: During a mean of 5.9 years of follow-up, 22.4% (n = 52) of the patients (n = 232) showed progression to late AMD. The multivariable prediction model included age, CFH variant rs1061170, pigment abnormalities, drusenoid pigment epithelial detachment (DPED), and hyperreflective foci (HRF). The model showed an area under the curve of 0.969 (95% confidence interval 0.948-0.990) and adequate calibration (Hosmer-Lemeshow test, p = 0.797). In addition to advanced age and carrying a CFH variant, pigment abnormalities, DPED, and HRF are relevant imaging biomarkers for conversion to late AMD. In clinical routine, an intensified monitoring of patients with a high-risk phenotypic profile may be suitable for the early detection of conversion to late AMD.

Increased circulating levels of Factor H-Related Protein 4 are strongly associated with age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness. Genetic variants at the chromosome 1q31.3 encompassing the complement factor H (CFH, FH) and CFH related genes (CFHR1-5) are major determinants of AMD susceptibility, but their molecular consequences remain unclear. Here we demonstrate that FHR-4 plays a prominent role in AMD pathogenesis. We show that systemic FHR-4 levels are elevated in AMD (P-value = 7.1 × 10-6), whereas no difference is seen for FH. Furthermore, FHR-4 accumulates in the choriocapillaris, Bruch's membrane and drusen, and can compete with FH/FHL-1 for C3b binding, preventing FI-mediated C3b cleavage. Critically, the protective allele of the strongest AMD-associated CFH locus variant rs10922109 has the highest association with reduced FHR-4 levels (P-value = 2.2 × 10-56), independently of the AMD-protective CFHR1-3 deletion, and even in those individuals that carry the high-risk allele of rs1061170 (Y402H). Our findings identify FHR-4 as a key molecular player contributing to complement dysregulation in AMD.

GENETIC RISK FACTORS IN SEVERE, NONSEVERE AND ACUTE PHENOTYPES OF CENTRAL SEROUS CHORIORETINOPATHY.

PURPOSE: To study genetic predispositions and differences between severe chronic central serous chorioretinopathy (cCSC), nonsevere cCSC, and acute central serous chorioretinopathy (aCSC). METHODS: One hundred seventy-three severe cCSC patients, 272 nonsevere cCSC patients, 135 aCSC patients, and 1,385 control individuals were included. Eight single-nucleotide polymorphisms were genotyped in the ARMS2 (rs10490924), CFH (rs800292, rs1061170, rs1065489, rs1329428, rs2284664, rs3753394), and NR3C2 (rs2070951). Additionally, C4B gene copy numbers were analyzed. RESULTS: A significant association in 5 single-nucleotide polymorphisms in the CFH gene could be reproduced among severe cCSC patients, including rs800292 (P = 0.0014; odds ratio [OR] = 1.93; 95% confidence interval [CI] = 1.51-2.47), rs1065489 (P = 2.22 × 10; OR = 0.49; 95% CI = 0.34-0.72), rs1329428 (P = 0.001; OR = 1.89; 95% CI = 1.49-2.40), rs2284664 (P = 1.21× 10; OR = 1.65; 95% CI = 1.28-2.13), and rs3753394 (P = 6.10× 10; OR = 0.61; 95% CI = 0.46-0.81). Carrying three C4B copies was protective for severe cCSC (P = 0.001; OR = 0.29; 95% CI = 0.14-0.61). No significant differences in allele frequencies could be found among the CSC phenotypes. CONCLUSION: Acute CSC, nonsevere cCSC, and severe cCSC all showed a similar association with the CFH and C4B genes, and the three phenotypes could not be distinguished based on the genetics. This shows that despite the differences in clinical presentation and severity, there is an overlap in the genetic predisposition of different CSC phenotypes. Nongenetic factors may play a more important role in determining the clinical course of CSC.

RETINAL HYPERREFLECTIVE FOCI IN TYPE 1 DIABETES MELLITUS.

PURPOSE: To investigate hyperreflective foci (HF) on spectral-domain optical coherence tomography in patients with Type 1 diabetes mellitus across different stages of diabetic retinopathy (DR) and diabetic macular edema (DME) and to study clinical and morphological characteristics associated with HF. METHODS: Spectral-domain optical coherence tomography scans and color fundus photographs were obtained of 260 patients. Spectral-domain optical coherence tomography scans were graded for the number of HF and other morphological characteristics. The distribution of HF across different stages of DR and DME severity were studied. Linear mixed-model analysis was used to study associations between the number of HF and clinical and morphological parameters. RESULTS: Higher numbers of HF were found in patients with either stage of DME versus patients without DME (P < 0.001). A trend was observed between increasing numbers of HF and DR severity, although significance was only reached for moderate nonproliferative DR (P = 0.001) and proliferative DR (P = 0.019). Higher numbers of HF were associated with longer diabetes duration (P = 0.029), lower high-density lipoprotein cholesterol (P = 0.005), and the presence of microalbuminuria (P = 0.005). In addition, HF were associated with morphological characteristics on spectral-domain optical coherence tomography, including central retinal thickness (P = 0.004), cysts (P < 0.001), subretinal fluid (P = 0.001), and disruption of the external limiting membrane (P = 0.018). CONCLUSION: The number of HF was associated with different stages of DR and DME severity. The associations between HF and clinical and morphological characteristics can be of use in further studies evaluating the role of HF as a biomarker for disease progression and treatment response.

Genome-wide association analyses identify two susceptibility loci for pachychoroid disease central serous chorioretinopathy.

The recently emerged pachychoroid concept has changed the understanding of age-related macular degeneration (AMD), which is a major cause of blindness; recent studies attributed AMD in part to pachychoroid disease central serous chorioretinopathy (CSC), suggesting the importance of elucidating the CSC pathogenesis. Our large genome-wide association study followed by validation studies in three independent Japanese and European cohorts, consisting of 1546 CSC samples and 13,029 controls, identified two novel CSC susceptibility loci: TNFRSF10A-LOC389641 and near GATA5 (rs13278062, odds ratio = 1.35, P = 1.26 × 10-13; rs6061548, odds ratio = 1.63, P = 5.36 × 10-15). A T allele at TNFRSF10A-LOC389641 rs13278062, a risk allele for CSC, is known to be a risk allele for AMD. This study not only identified new susceptibility genes for CSC, but also improves the understanding of the pathogenesis of AMD.

Elevated Steroid Hormone Levels in Active Chronic Central Serous Chorioretinopathy.

Purpose: Chronic central serous chorioretinopathy (cCSC) is characterized by fluid accumulation between photoreceptors and the retinal pigment epithelium of which the cause is unknown. Associations with steroid use, stress, pregnancy, and the male sex suggest a role for the steroid hormone system in the disease. Here, we performed a comprehensive analysis of the steroid hormone system in active cCSC. Methods: Serum hormone levels of 17 steroid hormones were measured in 46 male Caucasian patients with active cCSC and 46 male Caucasian age-matched controls using the AbsoluteIDQ stero17 kit. Results: Elevated levels of androsterone, estrone, etiocholanolone, and androstenedione were observed in cCSC patients compared with controls. Median hormone levels in cCSC patients versus controls, respectively, were as follows: androsterone, 0.84 ng/mL (interquartile range [IQR] = 0.61-1.06) versus 0.69 ng/mL (IQR = 0.48-0.96, P = 0.031); estrone, 0.12 ng/mL (IQR = 0.10-0.15) versus 0.10 ng/mL (IQR = 0.08-0.11; P = 0.0048); etiocholanolone, 0.19 ng/mL (IQR = 0.15-0.29) versus 0.13 ng/mL (IQR = 0.099-0.20, P = 0.0061). Mean levels of androstenedione were 3.10 ng/ml (SD = 1.03) versus 2.55 ng/mL (SD = 0.95), in cCSC patients versus controls, respectively. Additionally, Spearman's correlations between aldosterone and 11-deoxycortisol, androsterone, DHEA, DHEAS, and E1 differed between cCSC patients and controls, as well as between andosterone and E1, and between DHT and 17OHP. Conclusions: We present a comprehensive overview of the status of the steroid hormone system in active cCSC. Levels of four hormones were elevated in cCSC patients compared with controls, and the relationships between steroid hormones was altered, indicating that the balance in the steroid hormone system is altered in cCSC patients.

Metabolomics in serum of patients with non-advanced age-related macular degeneration reveals aberrations in the glutamine pathway.

Age-related macular degeneration (AMD) is a common, progressive multifactorial vision-threatening disease and many genetic and environmental risk factors have been identified. The risk of AMD is influenced by lifestyle and diet, which may be reflected by an altered metabolic profile. Therefore, measurements of metabolites could identify biomarkers for AMD, and could aid in identifying high-risk individuals. Hypothesis-free technologies such as metabolomics have a great potential to uncover biomarkers or pathways that contribute to disease pathophysiology. To date, only a limited number of metabolomic studies have been performed in AMD. Here, we aim to contribute to the discovery of novel biomarkers and metabolic pathways for AMD using a targeted metabolomics approach of 188 metabolites. This study focuses on non-advanced AMD, since there is a need for biomarkers for the early stages of disease before severe visual loss has occurred. Targeted metabolomics was performed in 72 patients with early or intermediate AMD and 72 control individuals, and metabolites predictive for AMD were identified by a sparse partial least squares discriminant analysis. In our cohort, we identified four metabolite variables that were most predictive for early and intermediate stages of AMD. Increased glutamine and phosphatidylcholine diacyl C28:1 levels were detected in non-advanced AMD cases compared to controls, while the rate of glutaminolysis and the glutamine to glutamate ratio were reduced in non-advanced AMD. The association of glutamine with non-advanced AMD corroborates a recent report demonstrating an elevated glutamine level in early AMD using a different metabolomics technique. In conclusion, this study indicates that metabolomics is a suitable method for the discovery of biomarker candidates for AMD. In the future, larger metabolomics studies could add to the discovery of novel biomarkers in yet unknown AMD pathways and expand our insights in AMD pathophysiology.

Genetic risk score has added value over initial clinical grading stage in predicting disease progression in age-related macular degeneration.

Several prediction models for progression of age-related macular degeneration (AMD) have been developed, but the added value of using genetic information in those models in addition to clinical characteristics is ambiguous. In this prospective cohort study, we explored the added value of genetics using a genetic risk score (GRS) based on 52 AMD-associated variants, in addition to the clinical severity grading at baseline as quantified by validated drusen detection software, to predict disease progression in 177 AMD patients after 6.5 years follow-up. The GRS was strongly associated with the drusen coverage at baseline (P < 0.001) and both the GRS and drusen coverage were associated with disease progression. When the GRS was added as predictor in addition to the drusen coverage, R2 increased from 0.46 to 0.56. This improvement by the GRS was predominantly seen in patients with a drusen coverage <15%. In patients with a larger drusen coverage, the GRS had less added value to predict progression. Thus, genetic information has added value over clinical characteristics in predicting disease progression in AMD, but only in patients with a less severe disease stage. Patients with a high GRS should be made aware of their risk and could be selected for clinical trials for arresting progression.

Exome sequencing in patients with chronic central serous chorioretinopathy.

Chronic central serous chorioretinopathy (cCSC) is a multifactorial eye disease characterized by subretinal fluid accumulation that leads to vision loss. Clinically, cCSC is associated with stress, hypercortisolism and corticosteroid use, and is more frequent in males (80%) than in females (20%). Current genetic studies on cCSC have thus far focussed on common variants, but familial occurrence of cCSC also suggests a role for rare variants in the disease susceptibility. Therefore, in this study, we performed exome sequencing of cCSC patients to elucidate the role of rare (protein-altering) variants in the disease. Exome sequencing was performed on 269 cCSC patients and 1,586 controls. Data were processed according to the Genome-Analysis-Toolkit (GATK) best practices. Principal component analysis was performed to check for genetic ancestry and only unrelated subjects of European descent were retained. Burden, SKAT and SKAT-O tests were performed using 2 different grouping criteria. One group included protein-altering variants only, while the other contained synonymous and splice site variants as well. The gene-based analyses were performed using the SKAT R-package correcting for two principal components using two approaches; (1) on the entire cohort correcting for sex and (2) on males and females separately. Additionally, the gene-based associations of genes at previously reported cCSC loci were investigated. After filtering, the dataset contained 263 cCSC patients (208 males [79%]) and 1352 controls (671 males [50%]) carrying 197,915 protein-altering variants in 16,370 genes and 330,689 exonic variants in 18,173 genes. Analysis stratified by sex identified significant associations with the PIGZ (PSKAT = 9.19 × 10-7 & PSKAT-O = 2.48 × 10-6), DUOX1 (PSKAT = 1.03 × 10-6), RSAD1 (PSKAT = 1.92 × 10-7 & PSKAT-O = 8.57 × 10-8) and LAMB3 (PBurden = 1.40 × 10-6 & PSKAT-O = 1.14 × 10-6) genes in female cCSC patients, after correction for multiple testing. The number of rare variant carriers in these genes was significantly higher in the female cCSC cohort compared to female controls (45,5% vs. 18.5%, P = 1.92 × 10-6, OR = 3.67 [95% CI = 2.09-6.46]). No significant associations were identified in the entire cohort nor in the male patients. In this exome study on cCSC patients, we have identified PIGZ, DUOX1, RSAD1 and LAMB3 as potential new candidate genes for cCSC in females. The sex-specific associations identified here suggest a possible interaction between rare genetic factors and sex for cCSC, but replication of these findings in additional cohorts of cCSC patients is necessary.

Exome sequencing in families with chronic central serous chorioretinopathy.

BACKGROUND: Central serous chorioretinopathy (CSC) is a chorioretinal disease characterized by fluid accumulation between the neuroretina and retinal pigment epithelium with unknown etiology. Family studies have suggested a heritable component for CSC with an autosomal dominant inheritance pattern. Therefore, exome sequencing was performed on familial cCSC to indentify the genetic components contributing to familial cCSC. METHODS: Exome sequencing was performed on 72 individuals of 18 families with CSC. In these families, we determined whether rare genetic variants (minor allele frequency < 1%) were segregated with CSC and also performed familial gene-burden analysis. RESULTS: In total, 11 variants segregated in two out of 18 families. One of these variants, c.4145C>T; p.T1382I (rs61758735) in the PTPRB gene, was also associated with CSC in a large case-control cohort sequenced previously (p = 0.009). Additionally, in 28 genes two or more different heterozygous variants segregated in two or more families, but no gene showed consistent associations in both the family gene-burden results and gene-burden analysis in the case-control cohort. CONCLUSION: We identified potential candidate genes for familial CSC and managed to exclude Mendelian inheritance of variants in one or a limited number of genes. Instead, familial CSC may be a heterogeneous Mendelian disease caused by variants in many different genes, or alternatively CSC may represent a complex disease to which both environmental factors and genetics contribute.

Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes.

BACKGROUND: Age-related macular degeneration (AMD) is a degenerative disorder of the central retina and the foremost cause of blindness. The retinal pigment epithelium (RPE) is a primary site of disease pathogenesis. The genetic basis of AMD is relatively well understood; however, this knowledge is yet to yield a treatment for the most prevalent non-neovascular disease forms. Therefore, tissue-specific epigenetic mechanisms of gene regulation are of considerable interest in AMD. We aimed to identify differentially methylated genes associated with AMD in the RPE and differentiate local DNA methylation aberrations from global DNA methylation changes, as local DNA methylation changes may be more amenable to therapeutic manipulation. METHODS: Epigenome-wide association study and targeted gene expression profiling were carried out in RPE cells from eyes of human donors. We performed genome-wide DNA methylation profiling (Illumina 450k BeadChip array) on RPE cells from 44 human donor eyes (25 AMD and 19 normal controls). We validated the findings using bisulfite pyrosequencing in 55 RPE samples (30 AMD and 25 normal controls) including technical (n = 38) and independent replicate samples (n = 17). Long interspersed nucleotide element 1 (LINE-1) analysis was then applied to assess global DNA methylation changes in the RPE. RT-qPCR on independent donor RPE samples was performed to assess gene expression changes. RESULTS: Genome-wide DNA methylation profiling identified differential methylation of multiple loci including the SKI proto-oncogene (SKI) (p = 1.18 × 10-9), general transcription factor IIH subunit H4 (GTF2H4) (p = 7.03 × 10-7), and Tenascin X (TNXB) (p = 6.30 × 10-6) genes in AMD. Bisulfite pyrosequencing validated the differentially methylated locus cg18934822 in SKI, and cg22508626 within GTF2H4, and excluded global DNA methylation changes in the RPE in AMD. We further demonstrated the differential expression of SKI, GTF2H4, and TNXB in the RPE of independent AMD donors. CONCLUSIONS: We report the largest genome-wide methylation analysis of RPE in AMD along with associated gene expression changes to date, for the first-time reaching genome-wide significance, and identified novel targets for functional and future therapeutic intervention studies. The novel differentially methylated genes SKI and GTF2H4 have not been previously associated with AMD, and regulate disease pathways implicated in AMD, including TGF beta signaling (SKI) and transcription-dependent DNA repair mechanisms (GTF2H4).

FAMILIAL CENTRAL SEROUS CHORIORETINOPATHY.

PURPOSE: To assess ophthalmologic characteristics in patients and unaffected individuals in families with multiple members affected by central serous chorioretinopathy (CSC), both at presentation and long-term follow-up. METHODS: In 103 subjects from 23 families with at least 2 affected patients with CSC per family, prospective extensive ophthalmologic examination was performed, including best-corrected visual acuity, indirect ophthalmoscopy, digital color fundus photography, optical coherence tomography, fundus autofluorescence, and fluorescein angiography imaging. From these, 24 individuals from 6 families had undergone extensive ophthalmologic examination in either 1994 or 1995 and were followed up in this study. RESULTS: Subretinal fluid accumulation on optical coherence tomography and/or "hot spots" of leakage on fluorescein angiography indicative of CSC were detected in 45 of 103 phenotyped subjects (44%). Findings suggestive of CSC, but without the presence of subretinal fluid on optical coherence tomography and/or "hot spots" of leakage on fluorescein angiography, were observed in an additional 27 family members (26%). In 4 of 17 previously nonaffected subjects (24%) from the 24 individuals that were followed up after more than 20 years, we found more severe abnormalities. CONCLUSION: Extensive ophthalmologic phenotyping resulted in the detection of (suggestive) CSC in 52% of family members of patients with CSC. Genetic factors may play an important role in these specific CSC cases. Moreover, during follow-up, progressive disease can occur in a noteworthy number of patients.