Aaron Y. Lee

Genome-wide association study of advanced age-related macular degeneration identifies a role of the hepatic lipase gene (LIPC)

Advanced age-related macular degeneration (AMD) is the leading cause of late onset blindness. We present results of a genome-wide association study of 979 advanced AMD cases and 1,709 controls using the Affymetrix 6.0 platform with replication in seven additional cohorts (totaling 5,789 unrelated cases and 4,234 unrelated controls). We also present a comprehensive analysis of copy-number variations and polymorphisms for AMD. Our discovery data implicated the association between AMD and a variant in the hepatic lipase gene (LIPC) in the high-density lipoprotein cholesterol (HDL) pathway (discovery P = 4.53e-05 for rs493258). Our LIPC association was strongest for a functional promoter variant, rs10468017, (P = 1.34e-08), that influences LIPC expression and serum HDL levels with a protective effect of the minor T allele (HDL increasing) for advanced wet and dry AMD. The association we found with LIPC was corroborated by the Michigan/Penn/Mayo genome-wide association study; the locus near the tissue inhibitor of metalloproteinase 3 was corroborated by our replication cohort for rs9621532 with P = 3.71e-09. We observed weaker associations with other HDL loci (ABCA1, P = 9.73e-04; cholesterylester transfer protein, P = 1.41e-03; FADS1-3, P = 2.69e-02). Based on a lack of consistent association between HDL increasing alleles and AMD risk, the LIPC association may not be the result of an effect on HDL levels, but it could represent a pleiotropic effect of the same functional component. Results implicate different biologic pathways than previously reported and provide new avenues for prevention and treatment of AMD.

Common Variants near FRK/COL10A1 and VEGFA are Associated with Advanced Age-related Macular Degeneration

Despite significant progress in the identification of genetic loci for age-related macular degeneration (AMD), not all of the heritability has been explained. To identify variants which contribute to the remaining genetic susceptibility, we performed the largest meta-analysis of genome-wide association studies to date for advanced AMD. We imputed 6 036 699 single-nucleotide polymorphisms with the 1000 Genomes Project reference genotypes on 2594 cases and 4134 controls with follow-up replication of top signals in 5640 cases and 52 174 controls. We identified two new common susceptibility alleles, rs1999930 on 6q21-q22.3 near FRK/COL10A1 [odds ratio (OR) 0.87; P = 1.1 × 10−8] and rs4711751 on 6p12 near VEGFA (OR 1.15; P = 8.7 × 10−9). In addition to the two novel loci, 10 previously reported loci in ARMS2/HTRA1 (rs10490924), CFH (rs1061170, and rs1410996), CFB (rs641153), C3 (rs2230199), C2 (rs9332739), CFI (rs10033900), LIPC (rs10468017), TIMP3 (rs9621532) and CETP (rs3764261) were confirmed with genome-wide significant signals in this large study. Loci in the recently reported genes ABCA1 and COL8A1 were also detected with suggestive evidence of association with advanced AMD. The novel variants identified in this study suggest that angiogenesis (VEGFA) and extracellular collagen matrix (FRK/COL10A1) pathways contribute to the development of advanced AMD.

Pharmacogenetics of Complement Factor H (Y402H) and treatment of exudative age-related macular degeneration with ranibizumab

Aims: To determine whether complement factor H (CFH) genotypes have a pharmacogenetic effect on the treatment of exudative age-related macular degeneration (AMD) with ranibizumab. Methods: A retrospective study of 156 patients with exudative AMD treated with intravitreal ranibizumab monotherapy was conducted. AMD phenotypes were characterised by clinical examination, visual acuity, fundus photography, fluorescein angiography and injection timing. Patients received intravitreal ranibizumab injections as part of routine ophthalmological care and were followed for a minimum of 9 months. Each patient was genotyped for the single nucleotide polymorphism rs1061170 (Y402H) in the CFH gene. Results: Baseline lesion size and angiographic type, as well as mean visual acuities at baseline, 6 months, and 9 months were similar among the three CFH genotypes. Over 9 months, patients with both risk alleles received approximately one more injection (p = 0.09). In a recurrent event analysis, patients homozygous for the CFH Y402H risk allele had a 37% significantly higher risk of requiring additional ranibizumab injections (p = 0.04). Conclusions: In this study cohort, the response to treatment of AMD with ranibizumab differed according to CFH genotype, suggesting that determining patients’ CFH genotype may be helpful in the future in tailoring treatment for exudative AMD with intravitreal ranibizumab.

HLA-B27 and human β2-microglobulin affect the gut microbiota of transgenic rats

The HLA-B27 gene is a major risk factor for clinical diseases including ankylosing spondylitis, acute anterior uveitis, reactive arthritis, and psoriatic arthritis, but its mechanism of risk enhancement is not completely understood. The gut microbiome has recently been shown to influence several HLA-linked diseases. However, the role of HLA-B27 in shaping the gut microbiome has not been previously investigated. In this study, we characterize the differences in the gut microbiota mediated by the presence of the HLA-B27 gene. We identified differences in the cecal microbiota of Lewis rats transgenic for HLA-B27 and human β2-microglobulin (hβ2m), compared with wild-type Lewis rats, using biome representational in situ karyotyping (BRISK) and 16S rRNA gene sequencing. 16S sequencing revealed significant differences between transgenic animals and wild type animals by principal coordinates analysis. Further analysis of the data set revealed an increase in Prevotella spp. and a decrease in Rikenellaceae relative abundance in the transgenic animals compared to the wild type animals. By BRISK analysis, species-specific differences included an increase in Bacteroides vulgatus abundance in HLA-B27/hβ2m and hβ2m compared to wild type rats. The finding that HLA-B27 is associated with altered cecal microbiota has not been shown before and can potentially provide a better understanding of the clinical diseases associated with this gene.

Heritability and Genome-Wide Association Study to Assess Genetic Differences between Advanced Age-Related Macular Degeneration Subtypes

PURPOSE To investigate whether the 2 subtypes of advanced age-related macular degeneration (AMD), choroidal neovascularization (CNV), and geographic atrophy (GA) segregate separately in families and to identify which genetic variants are associated with these 2 subtypes. DESIGN Sibling correlation study and genome-wide association study (GWAS). PARTICIPANTS For the sibling correlation study, 209 sibling pairs with advanced AMD were included. For the GWAS, 2594 participants with advanced AMD subtypes and 4134 controls were included. Replication cohorts included 5383 advanced AMD participants and 15 240 controls. METHODS Participants had the AMD grade assigned based on fundus photography, examination, or both. To determine heritability of advanced AMD subtypes, a sibling correlation study was performed. For the GWAS, genome-wide genotyping was conducted and 6 036 699 single nucleotide polymorphisms (SNPs) were imputed. Then, the SNPs were analyzed with a generalized linear model controlling for genotyping platform and genetic ancestry. The most significant associations were evaluated in independent cohorts. MAIN OUTCOME MEASURES Concordance of advanced AMD subtypes in sibling pairs and associations between SNPs with GA and CNV advanced AMD subtypes. RESULTS The difference between the observed and expected proportion of siblings concordant for the same subtype of advanced AMD was different to a statistically significant degree (P = 4.2 × 10(-5)), meaning that in siblings of probands with CNV or GA, the same advanced subtype is more likely to develop. In the analysis comparing participants with CNV to those with GA, a statistically significant association was observed at the ARMS2/HTRA1 locus (rs10490924; odds ratio [OR], 1.47; P = 4.3 × 10(-9)), which was confirmed in the replication samples (OR, 1.38; P = 7.4 × 10(-14) for combined discovery and replication analysis). CONCLUSIONS Whether CNV versus GA develops in a patient with AMD is determined in part by genetic variation. In this large GWAS meta-analysis and replication analysis, the ARMS2/HTRA1 locus confers increased risk for both advanced AMD subtypes, but imparts greater risk for CNV than for GA. This locus explains a small proportion of the excess sibling correlation for advanced AMD subtype. Other loci were detected with suggestive associations that differ for advanced AMD subtypes and deserve follow-up in additional studies.

Deep Learning Is Effective for Classifying Normal versus Age-Related Macular Degeneration OCT Images

Objective The advent of Electronic Medical Records (EMR) with large electronic imaging databases along with advances in deep neural networks with machine learning has provided a unique opportunity to achieve milestones in automated image analysis. Optical coherence tomography (OCT) is the most commonly obtained imaging modality in ophthalmology and represents a dense and rich dataset when combined with labels derived from the EMR. We sought to determine if deep learning could be utilized to distinguish normal OCT images from images from patients with Age-related Macular Degeneration (AMD). Design EMR and OCT database study. Subjects Normal and AMD patients who had a macular OCT. Methods Automated extraction of an OCT imaging database was performed and linked to clinical endpoints from the EMR. OCT macula scans were obtained by Heidelberg Spectralis, and each OCT scan was linked to EMR clinical endpoints extracted from EPIC. The central 11 images were selected from each OCT scan of two cohorts of patients: normal and AMD. Cross-validation was performed using a random subset of patients. Receiver operator curves (ROC) were constructed at an independent image level, macular OCT level, and patient level. Main outcome measure Area under the ROC. Results Of a recent extraction of 2.6 million OCT images linked to clinical datapoints from the EMR, 52,690 normal macular OCT images and 48,312 AMD macular OCT images were selected. A deep neural network was trained to categorize images as either normal or AMD. At the image level, we achieved an area under the ROC of 92.78% with an accuracy of 87.63%. At the macula level, we achieved an area under the ROC of 93.83% with an accuracy of 88.98%. At a patient level, we achieved an area under the ROC of 97.45% with an accuracy of 93.45%. Peak sensitivity and specificity with optimal cutoffs were 92.64% and 93.69% respectively. Conclusions Deep learning techniques achieve high accuracy and is effective as a new image classification technique. These findings have important implications in utilizing OCT in automated screening and the development of computer aided diagnosis tools in the future.Purpose The advent of electronic medical records (EMRs) with large electronic imaging databases along with advances in deep neural networks with machine learning has provided a unique opportunity to achieve milestones in automated image analysis. Optical coherence tomography (OCT) is the most common imaging modality in ophthalmology and represents a dense and rich data set when combined with labels derived from the EMR. We sought to determine whether deep learning could be utilized to distinguish normal OCT images from images from patients with age-related macular degeneration (AMD). Design EMR and OCT database study. Subjects Normal and AMD patients who underwent macular OCT. Methods Automated extraction of an OCT database was performed and linked to clinical end points from the EMR. Optical coherence tomography scans of the macula were obtained by Heidelberg Spectralis, and each OCT scan was linked to EMR clinical end points extracted from EPIC. The central 11 images were selected from each OCT scan of 2 cohorts of patients: normal and AMD. Cross-validation was performed using a random subset of patients. Receiver operating characteristic (ROC) curves were constructed at an independent image level, macular OCT level, and patient level. Main Outcome Measure Area under the ROC curve. Results Of a recent extraction of 2.6 million OCT images linked to clinical data points from the EMR, 52 690 normal macular OCT images and 48 312 AMD macular OCT images were selected. A deep neural network was trained to categorize images as either normal or AMD. At the image level, we achieved an area under the ROC curve of 92.78% with an accuracy of 87.63%. At the macula level, we achieved an area under the ROC curve of 93.83% with an accuracy of 88.98%. At a patient level, we achieved an area under the ROC curve of 97.45% with an accuracy of 93.45%. Peak sensitivity and specificity with optimal cutoffs were 92.64% and 93.69%, respectively. Conclusions The deep learning technique achieves high accuracy and is effective as a new image classification technique. These findings have important implications in utilizing OCT in automated screening and the development of computer-aided diagnosis tools in the future.

The effect of genetic variants in SERPING1 on the risk of neovascular age-related macular degeneration

Purpose Genetic factors influence an individuals risk for developing age-related macular degeneration (AMD), a leading cause of irreversible vision loss. Previous studies investigating the potential association between all AMD subtypes and the SERPING1 gene, which encodes a key regulator of the classic complement pathway, have yielded conflicting results. The purpose of this study is to determine whether variations in SERPING1 are associated with neovascular AMD. Methods A total of 556 patients with neovascular AMD and 256 ethnically matched controls were genotyped for polymorphisms in SERPING1. A tagging single nucleotide polymorphism (tSNP) approach was used to cover the SERPING1 gene plus 2 kb on each side, spanning the promoter and the 3′ untranslated regions. Ten SNPs with a minor allele frequency of 0.10 were covered by three tSNPs (rs1005510, rs11603020, rs2511989). Results SERPING1 SNPs rs1005510 and rs2511989 were significantly associated with neovascular AMD in our cohort, with rs1005510 conferring an adverse risk effect (OR 1.49, 95% CI 1.18 to 1.88) and rs2511989 conferring a protective effect (OR 0.73, 95% CI 0.59 to 0.90). For both tSNPs, logistic regression of individual genotypes demonstrated statistically significant stepwise changes in the risk of developing AMD. Combined analysis of rs1005510 with variants in CFH and HTRA1 confirmed an independent risk effect. The rs11603020 variant had no effect on AMD susceptibility in this study (OR 0.98, 95% CI 0.78 to 1.24). Conclusions The SERPING1 gene is comprehensively investigated in this study (using three tSNPs), and its genetic variants are evaluated in the largest neovascular AMD cohort to date. The hypothesis that SERPING1 has a modest effect on the risk of neovascular AMD is supported by our results.

IgG4-associated orbital and ocular inflammation

BackgroundIgG4-associated orbital and ocular inflammation is a relatively unknown entity characterized by sclerosing inflammation with infiltration of IgG4-positive plasma cells. Some so-called idiopathic inflammation syndromes are being re-classified as IgG4-associated inflammation with histopathologic evaluation.FindingsWe report three cases with differing manifestations of IgG4-associated ocular and orbital inflammation: a case of recurrent, treatment-refractory sclero-uveitis that was diagnosed as granulomatosis with polyangiitis with an IgG4-related component, a case of pachymeningitis with optic neuritis that resulted in permanent visual loss, and a case of orbital inflammatory pseudotumor. All three would have been incompletely diagnosed without thorough histopathologic evaluation (including immunohistochemistry).ConclusionsIgG4-associated disease is an idiopathic, multi-organ inflammatory state that can manifest as chronic, relapsing, sclerosing inflammation in virtually any organ system. There is a wide range of presentations in ocular and orbital inflammation. Ophthalmologists should keep IgG4-associated inflammation in mind when examining chronic, sclerofibrosing inflammation with multi-system involvement. The histology of biopsy specimens is crucial in making the correct diagnosis. Timely assessment may lead to fewer diagnostic tests and more targeted therapy.

UK AMD EMR USERS GROUP REPORT V: benefits of initiating ranibizumab therapy for neovascular AMD in eyes with vision better than 6/12.

Background/aims To study the effectiveness and clinical relevance of eyes treated with good (better than 6/12 or >70 Early Treatment Diabetic Retinopathy Study letters) visual acuity (VA) when initiating treatment with ranibizumab for neovascular age-related macular degeneration (nAMD) in the UK National Health Service. Currently eyes with VA better than (>) 6/12 are not routinely funded for therapy. Methods Multicentre national nAMD database study on patients treated 3–5 years prior to the analysis. Anonymised structured data were collected from 14 centres. The primary outcome was the mean VA at year 1, 2 and 3. Secondary measures included the number of clinic visits and injections. Results The study included 12 951 treatment-naive eyes of 11 135 patients receiving 92 976 ranibizumab treatment episodes. A total of 754 patients had baseline VA better than 6/12 and at least 1-year of follow up. Mean VA of first treated eyes with baseline VA>6/12 at year 1, 2, 3 were 6/10, 6/12, 6/15, respectively and those with baseline VA 6/12 to >6/24 were 6/15, 6/17, 6/20, respectively (p values <0.001 for comparing differences between 6/12 and 6/12–6/24 groups). For the second eyes with baseline VA>6/12, mean VA at year 1, 2, 3 were 6/9, 6/9, 6/10 and those with baseline VA 6/12 to >6/24 were 6/15, 6/15, 6/27, respectively (p values <0.001–0.005). There was no significant difference in the average number of clinic visits or injections between those with VA better and worse than 6/12. Conclusions All eyes with baseline VA>6/12 maintained better mean VA than the eyes with baseline VA 6/12 to >6/24 at all time points for at least 2 years. The significantly better visual outcome in patients who were treated with good baseline VA has implications on future policy regarding the treatment criteria for nAMD patients’ funding.

Paucibacterial microbiome and resident DNA virome of the healthy conjunctiva

Purpose To characterize the ocular surface microbiome of healthy volunteers using a combination of microbial culture and high-throughput DNA sequencing techniques. Methods Conjunctival swab samples from 107 healthy volunteers were analyzed by bacterial culture, 16S rDNA gene deep sequencing (n = 89), and biome representational in silico karyotyping (BRiSK; n = 80). Swab samples of the facial skin (n = 42), buccal mucosa (n = 50), and environmental controls (n = 27) were processed in parallel. 16S rDNA gene quantitative PCR was used to calculate the bacterial load in each site. Bacteria were characterized by site using principal coordinate analysis of metagenomics data. BRiSK data were analyzed for presence of fungi and viruses. Results Corynebacteria, Propionibacteria, and coagulase-negative Staphylococci were the predominant organisms identified by all three techniques. Quantitative 16S PCR demonstrated approximately 0.1 bacterial 16S rDNA/human actin copy on the ocular surface compared with greater than 10 16S rDNA/human actin copy for facial skin or the buccal mucosa. The conjunctival bacterial community structure is distinct compared with the facial skin (R = 0.474, analysis of similarities P = 0.0001), the buccal mucosa (R = 0.893, P = 0.0001), and environmental control samples (R = 0.536, P = 0.0001). 16S metagenomics revealed substantially more bacterial diversity on the ocular surface than other techniques, which appears to be artifactual. BRiSK revealed presence of torque teno virus (TTV) on the healthy ocular surface, which was confirmed by direct PCR to be present in 65% of all conjunctiva samples tested. Conclusions Relative to adjacent skin or other mucosa, healthy ocular surface microbiome is paucibacterial. Its flora are distinct from adjacent skin. Torque teno virus is a frequent constituent of the ocular surface microbiome. (ClinicalTrials.gov number, NCT02298881.)