Reticular macular disease is associated with multilobular geographic atrophy in age-related macular degeneration.

PURPOSE: To investigate the incidence of reticular macular disease (RMD), a subphenotype of age-related macular degeneration, in multilobular geographic atrophy (GA) and its relation to GA progression. METHODS: One hundred and fifty-seven eyes of 99 subjects with age-related macular degeneration, primary GA, and good quality autofluorescence, and/or infrared images were classified into unilobular GA (1 lesion) or multilobular GA (≥ 2 distinct and/or coalescent lesions). Thirty-four subjects (50 eyes) had serial imaging. The authors determined the spatiotemporal relationships of RMD to GA and GA progression rates in five macular fields. RESULTS: 91.7% eyes (144 of 157) had multilobular GA, 95.8% of which exhibited RMD. In subjects with serial imaging, the mean GA growth rate significantly differed between the unilobular and multilobular groups (0.40 vs. 1.30 mm2/year, P < 0.001). Of the macular fields in these eyes, 77.1% of fields with RMD at baseline showed subsequent GA progression, while 53.4% of fields without RMD showed progression (P < 0.001). Percentage of fields with RMD significantly correlated with GA progression rate (P = 0.01). CONCLUSION: Autofluorescence and infrared imaging demonstrates that RMD is nearly always present with multilobular GA in age-related macular degeneration. Furthermore, GA lobules frequently develop in areas of RMD, suggesting progression of a single underlying disease process.

Overt cleft palate phenotype and TBX1 genotype correlations in velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome patients.

Velo-cardio-facial syndrome/DiGeorge syndrome, also known as 22q11.2 deletion syndrome (22q11DS) is the most common microdeletion syndrome, with an estimated incidence of 1/2,000-1/4,000 live births. Approximately 9-11% of patients with this disorder have an overt cleft palate (CP), but the genetic factors responsible for CP in the 22q11DS subset are unknown. The TBX1 gene, a member of the T-box transcription factor gene family, lies within the 22q11.2 region that is hemizygous in patients with 22q11DS. Inactivation of one allele of Tbx1 in the mouse does not result in CP, but inactivation of both alleles does. Based on these data, we hypothesized that DNA variants in the remaining allele of TBX1 may confer risk to CP in patients with 22q11DS. To test the hypothesis, we evaluated TBX1 exon sequencing (n = 360) and genotyping data (n = 737) with respect to presence (n = 54) or absence (n = 683) of CP in patients with 22q11DS. Two upstream SNPs (rs4819835 and rs5748410) showed individual evidence for association but they were not significant after correction for multiple testing. Associations were not identified between DNA variants and haplotypes in 22q11DS patients with CP. Overall, this study indicates that common DNA variants in TBX1 may be nominally causative for CP in patients with 22q11DS. This raises the possibility that genes elsewhere on the remaining allele of 22q11.2 or in the genome could be relevant.

Genotype and cardiovascular phenotype correlations with TBX1 in 1,022 velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome patients.

Haploinsufficiency of TBX1, encoding a T-box transcription factor, is largely responsible for the physical malformations in velo-cardio-facial /DiGeorge/22q11.2 deletion syndrome (22q11DS) patients. Cardiovascular malformations in these patients are highly variable, raising the question as to whether DNA variations in the TBX1 locus on the remaining allele of 22q11.2 could be responsible. To test this, a large sample size is needed. The TBX1 gene was sequenced in 360 consecutive 22q11DS patients. Rare and common variations were identified. We did not detect enrichment in rare SNP (single nucleotide polymorphism) number in those with or without a congenital heart defect. One exception was that there was increased number of very rare SNPs between those with normal heart anatomy compared to those with right-sided aortic arch or persistent truncus arteriosus, suggesting potentially protective roles in the SNPs for these phenotype-enrichment groups. Nine common SNPs (minor allele frequency, MAF > 0.05) were chosen and used to genotype the entire cohort of 1,022 22q11DS subjects. We did not find a correlation between common SNPs or haplotypes and cardiovascular phenotype. This work demonstrates that common DNA variations in TBX1 do not explain variable cardiovascular expression in 22q11DS patients, implicating existence of modifiers in other genes on 22q11.2 or elsewhere in the genome.

Corneal Optical Coherence Tomography Speckle in Crosslinked and Untreated Rabbit Eyes in Response to Elevated Intraocular Pressure.

Purpose: To ascertain the influence of intraocular pressure (IOP) on corneal optical coherence tomography (OCT) speckle in untreated and ultraviolet A-riboflavin induced corneal collagen crosslinked rabbit eyes. Methods: Left corneas of eight rabbits were de-epithelialized and crosslinked by applying riboflavin and 30-minute ultraviolet A light exposure. After enucleation (6 months after treatment), each eyeball (treated and untreated) was mounted in a measurement setup, in which IOP was increased from 15 to 45 mm Hg in steps of 5 mm Hg. At each IOP value, single B-scans of the central cornea were acquired three times with the spectral-domain OCT Copernicus-HR. Then, three regions of interest, including the anterior, posterior, and entire corneal stroma, were automatically extracted. Five different probability distributions were used as a model for the corneal speckle and the one with the best goodness of fit was chosen for further analysis. Results: The generalized gamma distribution achieved the best goodness of fit and its scale (a) and shape (v) parameters statistically significantly changed with increasing IOP in the three regions of analysis (two-way repeated measures analysis of variance, all P < 0.05). The statistically significant difference between untreated and crosslinked eyes was observed for the shape parameters of the posterior and entire corneal stroma. Conclusions: Corneal OCT speckle is influenced by IOP and shows to be significantly different in untreated and crosslinked eyes. Corneal OCT speckle analysis has the potential to be indirectly used for assessing changes in corneal stroma in ex vivo and in vivo studies. Translational Relevance: Investigation of corneal OCT speckle statistics can offer additional diagnostic biomarkers related to changes in the corneal stroma after ocular surgeries.

Genome-Wide Association Study to Find Modifiers for Tetralogy of Fallot in the 22q11.2 Deletion Syndrome Identifies Variants in the GPR98 Locus on 5q14.3.

BACKGROUND: The 22q11.2 deletion syndrome (22q11.2DS; DiGeorge syndrome/velocardiofacial syndrome) occurs in 1 of 4000 live births, and 60% to 70% of affected individuals have congenital heart disease, ranging from mild to severe. In our cohort of 1472 subjects with 22q11.2DS, a total of 62% (n=906) have congenital heart disease and 36% (n=326) of these have tetralogy of Fallot (TOF), comprising the largest subset of severe congenital heart disease in the cohort. METHODS AND RESULTS: To identify common genetic variants associated with TOF in individuals with 22q11.2DS, we performed a genome-wide association study using Affymetrix 6.0 array and imputed genotype data. In our cohort, TOF was significantly associated with a genotyped single-nucleotide polymorphism (rs12519770, P=2.98×10-8) in an intron of the adhesion GPR98 (G-protein-coupled receptor V1) gene on chromosome 5q14.3. There was also suggestive evidence of association between TOF and several additional single-nucleotide polymorphisms in this region. Some genome-wide significant loci in introns or noncoding regions could affect regulation of genes nearby or at a distance. On the basis of this possibility, we examined existing Hi-C chromatin conformation data to identify genes that might be under shared transcriptional regulation within the region on 5q14.3. There are 6 genes in a topologically associated domain of chromatin with GPR98, including MEF2C (Myocyte-specific enhancer factor 2C). MEF2C is the only gene that is known to affect heart development in mammals and might be of interest with respect to 22q11.2DS. CONCLUSIONS: In conclusion, common variants may contribute to TOF in 22q11.2DS and may function in cardiac outflow tract development.

Quantitative fundus autofluorescence in mice: correlation with HPLC quantitation of RPE lipofuscin and measurement of retina outer nuclear layer thickness.

PURPOSE: Our study was conducted to establish procedures and protocols for quantitative autofluorescence (qAF) measurements in mice, and to report changes in qAF, A2E bisretinoid concentration, and outer nuclear layer (ONL) thickness in mice of different genotypes and age. METHODS: Fundus autofluorescence (AF) images (55° lens, 488 nm excitation) were acquired in albino Abca4(-/-), Abca4(+/-), and Abca4(+/+) mice (ages 2-12 months) with a confocal scanning laser ophthalmoscope (cSLO). Gray levels (GLs) in each image were calibrated to an internal fluorescence reference. The bisretinoid A2E was measured by quantitative high performance liquid chromatography (HPLC). Histometric analysis of ONL thicknesses was performed. RESULTS: The Bland-Altman coefficient of repeatability (95% confidence interval) was ±18% for between-session qAF measurements. Mean qAF values increased with age (2-12 months) in all groups of mice. qAF was approximately 2-fold higher in Abca4(-/-) mice than in Abca4(+/+) mice and approximately 20% higher in heterozygous mice. HPLC measurements of the lipofuscin fluorophore A2E also revealed age-associated increases, and the fold difference between Abca4(-/-) and wild-type mice was more pronounced (approximately 3-4-fold) than measurable by qAF. Moreover, A2E levels declined after 8 months of age, a change not observed with qAF. The decline in A2E levels in the Abca4(-/-) mice corresponded to reduced photoreceptor cell viability as reflected in ONL thinning beginning at 8 months of age. CONCLUSIONS: The qAF method enables measurement of in vivo lipofuscin and the detection of genotype and age-associated differences. The use of this approach has the potential to aid in understanding retinal disease processes and will facilitate preclinical studies.

Fundus autofluorescence imaging in a patient with rapidly developing scotoma.

PURPOSE: To evaluate the findings in a case of acute macular neuroretinopathy involving sudden development of scotomas accompanied by rapid focal increases in fundus autofluorescence. METHODS: The clinical presentation of the patient was documented by color fundus photographs, fundus autofluorescence, infrared imaging, and high-resolution spectral domain optical coherence tomography. The scotomas were assessed by Humphrey visual field 10-2 and MP-1 microperimetry. RESULTS: Visual field defects exhibited spatial correspondence with wedge-shaped lesions demonstrable in color fundus photographs and infrared imaging. It was notable that the lesions exhibited increased intensity on autofluorescence images obtained within 3 weeks of presentation. Optical coherence tomography revealed focal loss of photoreceptor inner segment/outer segment junctions in both eyes. CONCLUSION: This case was distinguished by the relative rapidity with which the lesions became hyperautofluorescent in fundus autofluorescence images. Given that the bisretinoids that are the source of autofluorescence form in photoreceptor cells and are transferred to retinal pigment epithelium secondarily, the rapid increase in autofluorescence is unlikely to only reflect retinal pigment epithelium status and is more likely to be indicative of photoreceptor cell dysfunctioning and loss of structural integrity.

Fundus autofluorescence findings in a mouse model of retinal detachment.

PURPOSE: Fundus autofluorescence (fundus AF) changes were monitored in a mouse model of retinal detachment (RD). METHODS: RD was induced by transscleral injection of hyaluronic acid (Healon) or sterile balanced salt solution (BSS) into the subretinal space of 4-5-day-old albino Abca4 null mutant and Abca4 wild-type mice. Images acquired by confocal scanning laser ophthalmoscopy (Spectralis HRA) were correlated with spectral domain optical coherence tomography (SD-OCT), infrared reflectance (IR), fluorescence spectroscopy, and histologic analysis. Results. In the area of detached retina, multiple hyperreflective spots in IR images corresponded to punctate areas of intense autofluorescence visible in fundus AF mode. The puncta exhibited changes in fluorescence intensity with time. SD-OCT disclosed undulations of the neural retina and hyperreflectivity of the photoreceptor layer that likely corresponded to histologically visible photoreceptor cell rosettes. Fluorescence emission spectra generated using flat-mounted retina, and 488 and 561 nm excitation, were similar to that of RPE lipofuscin. With increased excitation wavelength, the emission maximum shifted towards longer wavelengths, a characteristic typical of fundus autofluorescence. CONCLUSIONS: In detached retinas, hyper-autofluorescent spots appeared to originate from photoreceptor outer segments that were arranged within retinal folds and rosettes. Consistent with this interpretation is the finding that the autofluorescence was spectroscopically similar to the bisretinoids that constitute RPE lipofuscin. Under the conditions of a RD, abnormal autofluorescence may arise from excessive production of bisretinoid by impaired photoreceptor cells.

The bisretinoids of retinal pigment epithelium.

The retina exhibits an inherent autofluorescence that is imaged ophthalmoscopically as fundus autofluorescence. In clinical settings, fundus autofluorescence examination aids in the diagnosis and follow-up of many retinal disorders. Fundus autofluorescence originates from the complex mixture of bisretinoid fluorophores that are amassed by retinal pigment epithelial (RPE) cells as lipofuscin. Unlike the lipofuscin found in other cell-types, this material does not form as a result of oxidative stress. Rather, the formation is attributable to non-enzymatic reactions of vitamin A aldehyde in photoreceptor cells; transfer to RPE occurs upon phagocytosis of photoreceptor outer segments. These fluorescent pigments accumulate even in healthy photoreceptor cells and are generated as a consequence of the light capturing function of the cells. Nevertheless, the formation of this material is accelerated in some retinal disorders including recessive Stargardt disease and ELOVL4-related retinal degeneration. As such, these bisretinoid side-products are implicated in the disease processes that threaten vision. In this article, we review our current understanding of the composition of RPE lipofuscin, the structural characteristics of the various bisretinoids, their related spectroscopic features and the biosynthetic pathways by which they form. We will revisit factors known to influence the extent of the accumulation and therapeutic strategies being used to limit bisretinoid formation. Given their origin from vitamin A aldehyde, an isomer of the visual pigment chromophore, it is not surprising that the bisretinoids of retina are light sensitive molecules. Accordingly, we will discuss recent findings that implicate the photodegradation of bisretinoid in the etiology of age-related macular degeneration.

Drusen analysis in a human-machine synergistic framework.

OBJECTIVES: To demonstrate how human-machine intelligence can be integrated for efficient image analysis of drusen in age-related macular degeneration and to validate the method in 2 large, independently graded, population-based data sets. METHODS: We studied 358 manually graded color slides from the Netherlands Genetic Isolate Study. All slides were digitized and analyzed with a user-interactive drusen detection algorithm for the presence and quantity of small, intermediate, and large drusen. A graphic user interface was used to preprocess the images, choose a region of interest, select appropriate corrective filters for images with photographic artifacts or prominent choroidal pattern, and perform drusen segmentation. Weighted κ statistics were used to analyze the initial concordance between human graders and the drusen detection algorithm; discordant grades from 177 left-eye slides were subjected to exhaustive analysis of causes of disagreement and adjudication. To validate our method further, we analyzed a second data set from our Columbia Macular Genetics Study. RESULTS: The graphical user interface decreased the time required to process images in commercial software by 60.0%. After eliminating borderline size disagreements and applying corrective filters for photographic artifacts and choroidal pattern, the weighted κ values were 0.61, 0.62, and 0.76 for small, intermediate, and large drusen, respectively. Our second data set demonstrated a similarly high concordance. CONCLUSIONS: Drusen identification performed by our user-interactive method presented fair to good agreement with human graders after filters for common sources of error were applied. This approach exploits a synergistic relationship between the intelligent user and machine computational power, enabling fast and accurate quantitative retinal image analysis.

Complement factor H 402H variant and reticular macular disease.

OBJECTIVE: To determine the association of high-risk alleles in the complement factor H (CFH; Y402H, rs1061170) and age-related maculopathy susceptibility (ARMS2; A69S, rs10490924) genes with reticular macular disease (RMD), a major clinical subphenotype of age-related macular degeneration (AMD). METHODS: Using retinal images from the Columbia Macular Genetics Study, we identified 67 subject individuals with RMD. A comparison group of 64 subjects with AMD without RMD was matched by ethnicity, age, sex, and AMD clinical stage. RESULTS: In the RMD group, 53 of 67 subjects (79.1%) were female, the mean age was 83 years, and 47 of 67 (70.1%) had late AMD, with closely matched values in the non-RMD group. The frequencies of the CFH 402H allele were 39.6% in the RMD group (53 of 134 individuals) and 58.6% in the non-RMD group (75 of 128 individuals) (χ(2) = 8.8; P = .003; odds ratio, 0.46 [95% confidence interval, 0.28-0.76]). The corresponding frequencies of the risk allele for ARMS2 were 44.0% (40 of 128 individuals) and 31.3% (40 of 128 individuals), respectively (χ(2) = 4.0; P = .045; odds ratio, 1.73 [95% confidence interval, 1.04-2.90]). Homozygosity for 402H was particularly associated with the absence of RMD, occurring in 8 of 67 subjects (11.9%) with RMD vs 24 of 64 subjects (37.5%) without RMD (P < .001). Retinal macular disease also was associated with hypertension among male patients. CONCLUSIONS: The AMD-associated CFH 402H risk variant is significantly associated with the absence of RMD but enhanced risk for RMD is conferred by the ARMS2 69S AMD risk allele. These results are consistent with the hypothesis that 402H may confer a survival benefit against certain infections, some of which may cause RMD. CLINICAL RELEVANCE: Reticular macular disease may be genetically distinct from the rest of AMD.