R. Theodore Smith

Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration

Age-related macular degeneration (AMD) is the most common form of irreversible blindness in developed countries. Variants in the factor H gene (CFH, also known as HF1), which encodes a major inhibitor of the alternative complement pathway, are associated with the risk for developing AMD. Here we test the hypothesis that variation in genes encoding other regulatory proteins of the same pathway is associated with AMD. We screened factor B (BF) and complement component 2 (C2) genes, located in the major histocompatibility complex class III region, for genetic variation in two independent cohorts comprising ∼900 individuals with AMD and ∼400 matched controls. Haplotype analyses identify a statistically significant common risk haplotype (H1) and two protective haplotypes. The L9H variant of BF and the E318D variant of C2 (H10), as well as a variant in intron 10 of C2 and the R32Q variant of BF (H7), confer a significantly reduced risk of AMD (odds ratio = 0.45 and 0.36, respectively). Combined analysis of the C2 and BF haplotypes and CFH variants shows that variation in the two loci can predict the clinical outcome in 74% of the affected individuals and 56% of the controls. These data expand and refine our understanding of the genetic risk for AMD.

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.

Reticular Macular Disease

PURPOSE To present a unified description of reticular macular disease (RMD), a common clinical entity that includes reticular pseudodrusen (RPD) and confers high-risk of progression to advanced age-related macular degeneration. DESIGN Population-based, retrospective, cross-sectional study. Forty-two patients with reticular findings in at least one imaging method, of whom 21 were followed up. METHODS RMD was defined as RPD in color or red-free photography, in a reticular pattern on scanning laser ophthalmoscope imaging (autofluorescence scans, infrared photographs, or indocyanine green angiography), or both. Color and red-free images were contrast-enhanced, and color photographs were examined in green and blue channels. Image registration in different methods allowed comparison of areas involved and assessment of lesion colocalization. RESULTS RMD generally was present in both photography and scanning laser ophthalmoscope imaging. When present in two image methods, areas of RMD either largely overlapped or one fell within the other. Individual lesions had high spatial correspondence. Serial imaging showed faded to absent findings in eyes in which choroidal neovascularization developed. CONCLUSIONS RMD is a single disease entity with stereotypical presentations in multiple imaging methods, of which RPD is one. Autofluorescence, infrared imaging, and indocyanine green angiography suggest that it involves the retinal pigment epithelium and choriocapillaris, whereas photographic patterns implicate the inner choroid. Infrared imaging, unlike other methods, can demonstrate RMD in the central macula. RMD is associated with progression to advanced age-related macular degeneration, perhaps on an inflammatory basis. RMD deserves wider recognition among clinicians caring for elderly patients.

Quantitative measurements of autofluorescence with the scanning laser ophthalmoscope.

PURPOSE To evaluate the feasibility and reliability of a standardized approach for quantitative measurements of fundus autofluorescence (AF) in images obtained with a confocal scanning laser ophthalmoscope (cSLO). METHODS AF images (30°) were acquired in 34 normal subjects (age range, 20-55 years) with two different cSLOs (488-nm excitation) equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The gray levels (GLs) of each image were calibrated to the reference, the zero GL, and the magnification, to give quantified autofluorescence (qAF). Images from subjects and fixed patterns were used to test detector linearity with respect to fluorescence intensity, the stability of qAF with change in detector gain, field uniformity, effect of refractive error, and repeatability. RESULTS qAF was independent of detector gain and laser power over clinically relevant ranges, provided that detector gain was adjusted to maintain exposures within the linear detection range (GL < 175). Field uniformity was better than 5% in a central 20°-diameter circle but decreased more peripherally. The theoretical inverse square magnification correction was experimentally verified. Photoreceptor bleaching for at least 20 seconds was performed. Repeatability (95% confidence interval) for same day and different-day retests of qAF was ±6% to ±14%. Agreement (95% confidence interval) between the two instruments was <11%. CONCLUSIONS Quantitative AF imaging appears feasible. It may enhance understanding of retinal degeneration, serve as a diagnostic aid and as a sensitive marker of disease progression, and provide a tool to monitor the effects of therapeutic interventions.

A Partial Intensity Invariant Feature Descriptor for Multimodal Retinal Image Registration

Detection of vascular bifurcations is a challenging task in multimodal retinal image registration. Existing algorithms based on bifurcations usually fail in correctly aligning poor quality retinal image pairs. To solve this problem, we propose a novel highly distinctive local feature descriptor named partial intensity invariant feature descriptor (PIIFD) and describe a robust automatic retinal image registration framework named Harris-PIIFD. PIIFD is invariant to image rotation, partially invariant to image intensity, affine transformation, and viewpoint/perspective change. Our Harris-PIIFD framework consists of four steps. First, corner points are used as control point candidates instead of bifurcations since corner points are sufficient and uniformly distributed across the image domain. Second, PIIFDs are extracted for all corner points, and a bilateral matching technique is applied to identify corresponding PIIFDs matches between image pairs. Third, incorrect matches are removed and inaccurate matches are refined. Finally, an adaptive transformation is used to register the image pairs. PIIFD is so distinctive that it can be correctly identified even in nonvascular areas. When tested on 168 pairs of multimodal retinal images, the Harris-PIIFD far outperforms existing algorithms in terms of robustness, accuracy, and computational efficiency.

A Comparison of Fundus Autofluorescence and Retinal Structure in Patients with Stargardt Disease

PURPOSE To improve the understanding of Stargardt disease by comparing structural changes seen on spectral domain optical coherence tomography (SD-OCT) to those visible on fundus autofluorescence (FAF). METHODS FAF and SD-OCT were performed on 22 eyes of 11 patients with Stargardt disease. SD-OCT images were obtained at the fovea and at the eccentric preferred retinal locus (PRL). The diameters of absent (hypoautofluorescence) and abnormal FAF areas were measured. The extent of the transverse defect of the junction between the inner and outer segments of the photoreceptors (IS-OS) was measured in the foveal area. The PRL was evaluated with fundus photography and microperimetry. RESULTS Twenty-one of 22 eyes showed defective FAF. In 17 eyes, FAF was absent in the fovea and in four eyes, FAF was abnormal. All eyes showed disorganization and/or loss of the IS-OS junction in the foveal area on SD-OCT. The diameter of the absent FAF area was smaller than the measurement of the IS-OS junction loss; the latter was closer to the diameter of the abnormal FAF area. Seventeen eyes had an eccentric PRL associated with a retinal area with no defects on FAF. CONCLUSIONS In the majority of eyes, changes shown by SD-OCT correlated well with changes in FAF. However, in three patients, photoreceptor abnormalities were seen in the fovea on SD-OCT without an equivalent abnormality on FAF. This result suggests that in these patients, the structural integrity of the photoreceptors may be affected earlier than changes in the RPE at least as detected by FAF.

Quantitative Autofluorescence and Cell Density Maps of the Human Retinal Pigment Epithelium

PURPOSE Lipofuscin (LF) accumulation within RPE cells is considered pathogenic in AMD. To test whether LF contributes to RPE cell loss in aging and to provide a cellular basis for fundus autofluorescence (AF) we created maps of human RPE cell number and histologic AF. METHODS Retinal pigment epithelium-Bruchs membrane flat mounts were prepared from 20 donor eyes (10 ≤ 51 and 10 > 80 years; postmortem: ≤4.2 hours; no retinal pathologies), preserving foveal position. Phalloidin-binding RPE cytoskeleton and LF-AF (488-nm excitation) were imaged at up to 90 predefined positions. Maps were assembled from 83,330 cells in 1470 locations. From Voronoi regions representing each cell, the number of neighbors, cell area, and total AF intensity normalized to an AF standard was determined. RESULTS Highly variable between individuals, RPE-AF increases significantly with age. A perifoveal ring of high AF mirrors rod photoreceptor topography and fundus-AF. Retinal pigment epithelium cell density peaks at the fovea, independent of age, yet no net RPE cell loss is detectable. The RPE monolayer undergoes considerable lifelong re-modeling. The relationship of cell size and AF, a surrogate for LF concentration, is orderly and linear in both groups. Autofluorescence topography differs distinctly from the topography of age-related rod loss. CONCLUSIONS Digital maps of quantitative AF, cell density, and packing geometry provide metrics for cellular-resolution clinical imaging and model systems. The uncoupling of RPE LF content, cell number, and photoreceptor topography in aging challenges LFs role in AMD.

Image registration and multimodal imaging of reticular pseudodrusen

PURPOSE To characterize reticular pseudodrusen (RPD) by using a point-to-point comparison of the reticular pattern on infrared reflectance (IR), autofluorescence (AF), and red-free (RF) images registered with en face sections of the choroid from spectral domain optical coherence tomography (SD-OCT) scans. METHODS A cross-sectional, retrospective study of all patients with the diagnosis of AMD who presented to the Doheny Retina Institute between December 2007 and November 2009 was conducted to identify patients with RPD. IR, AF, and RF images were obtained using confocal scanning laser ophthalmoscopy and were manually registered to OCT choroidal sections to study the location of RPD. The main outcome measured was point-to-point localization of RPD across multiple imaging modalities. RESULTS Of the 153 patients with AMD, 51 had RPD. In all 51 patients (97 eyes), RPD appeared as areas of hypoautofluorescence and hyporeflectance on AF and IR imaging, respectively, and as hyporeflective interlacing networks on RF. Reticular lesions on AF, IR, and RF images consistently colocalized with stromal regions between large choroidal vessels on registered en face choroidal sections. In contrast, outer retinal changes and subretinal deposits tended to localize immediately adjacent to the RPD. CONCLUSIONS Point-to-point correlation of registered IR, AF, and RF images consistently localizes the reticular pattern to the intervascular choroidal stroma on en face OCT sections. In contrast, subretinal deposits and disturbances of the inner outer segment on OCT did not colocalize with the RPD, and may represent secondary mechanical or biologic disturbances in the overlying RPE and outer retina.

Quantitative comparison of drusen segmented on SD-OCT versus drusen delineated on color fundus photographs.

PURPOSE Spectral domain-optical coherence tomography (SD-OCT) may be useful for efficient measurement of drusen in patients with age-related macular degeneration (AMD). Areas identified as drusen from semiautomated segmentation of drusen on SD-OCT were compared to those identified from review of digital color fundus photographs (CFPs). METHODS Twelve eyes with nonneovascular AMD were prospectively imaged with digital CFP and SD-OCT. For each eye, areas on CFP in which at least two of three retina specialists agreed on drusen presence produced the composite CFP drusen map. Automated image analysis produced another CFP map. Areas identified as drusen by segmentation on SD-OCT B-scans were plotted as the SD-OCT drusen map. The CFP and SD-OCT maps were compared and agreement was quantified. Disagreement was characterized into distinct types, and the frequency of each type was quantified. RESULTS There was general agreement between CFP and SD-OCT in identifying presence and absence of drusen, with mean agreement in 82% ± 9% of total image pixels. Most disagreement (80% ± 15%) occurred at drusen margins. There was a trend toward greater detection of drusen with SD-OCT in eyes with larger drusen and with hyperpigmentation. There was a trend toward greater detection of smaller drusen by CFP. CONCLUSIONS Good agreement was demonstrated in drusen detection between CFP and SD-OCT. Areas of disagreement underscore limitations of CFP-based measurement of drusen, particularly in the sizing of large, soft drusen. SD-OCT shows great promise as an adjunctive tool for assessing drusen burden in AMD. (ClinicalTrials.gov number, NCT00734487.).