Peter Charbel Issa

Complement factor H binds malondialdehyde epitopes and protects from oxidative stress.

Oxidative stress and enhanced lipid peroxidation are linked to many chronic inflammatory diseases, including age-related macular degeneration (AMD). AMD is the leading cause of blindness in Western societies, but its aetiology remains largely unknown. Malondialdehyde (MDA) is a common lipid peroxidation product that accumulates in many pathophysiological processes, including AMD. Here we identify complement factor H (CFH) as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice. The CFH polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy for AMD and other chronic inflammatory diseases.

High-Resolution Spectral Domain-OCT Imaging in Geographic Atrophy Associated with Age-Related Macular Degeneration

PURPOSE To describe morphologic variations in outer retinal layers in eyes with atrophic age-related macular degeneration (AMD) using high-resolution, spectral-domain optical coherence tomography (SD-OCT). METHODS SD-OCT scans were obtained with a combined confocal scanning laser ophthalmoscope (cSLO) and SD-OCT for simultaneous tomographic and topographic in vivo imaging. A total of 81 eyes of 56 patients (mean age, 77.8 +/- 7.4 years) with geographic atrophy (GA) were examined. Morphologic alterations were analyzed and classified in the perilesional zone, at the junction between GA and nonatrophic retina, and in the atrophic area itself. RESULTS In the perilesional zone, distinct morphologic alterations included elevations of the outer retinal layers, thickening, and spikes of the outer hyperreflective band as well as clumps at different neurosensory retinal levels. At the junction, highly variable transitions of the outer retinal layers were present with different degrees of loss of the normal hyperreflective bands. Within the actual GA, hyperreflective clumps at different retinal levels, segmented plaques of the outer band and elevations with variable reflectivity were visualized. CONCLUSIONS SD-OCT imaging in eyes with GA revealed a wide spectrum of morphologic alterations, both in the surrounding retinal tissue and in the atrophic area. These alterations may reflect different disease stages or, alternatively, heterogeneity on a cellular and molecular level. Longitudinal studies using in vivo SD-OCT imaging may allow evaluation of the relevance of these phenotypic changes as potential predictive markers for the progression of disease (i.e., enlargement rates of GA over time) and may be used for monitoring of future therapeutic interventions.

A novel gene for Usher syndrome type 2: mutations in the long isoform of whirlin are associated with retinitis pigmentosa and sensorineural hearing loss

Usher syndrome is an autosomal recessive condition characterized by sensorineural hearing loss, variable vestibular dysfunction, and visual impairment due to retinitis pigmentosa (RP). The seven proteins that have been identified for Usher syndrome type 1 (USH1) and type 2 (USH2) may interact in a large protein complex. In order to identify novel USH genes, we followed a candidate strategy, assuming that mutations in proteins interacting with this “USH network” may cause Usher syndrome as well. The DFNB31 gene encodes whirlin, a PDZ scaffold protein with expression in both hair cell stereocilia and retinal photoreceptor cells. Whirlin represents an excellent candidate for USH2 because it binds to Usherin (USH2A) and VLGR1b (USH2C). Genotyping of microsatellite markers specific for the DFNB31 gene locus on chromosome 9q32 was performed in a German USH2 family that had been excluded for all known USH loci. Patients showed common haplotypes. Sequence analysis of DFNB31 revealed compound heterozygosity for a nonsense mutation, p.Q103X, in exon 1, and a mutation in the splice donor site of exon 2, c.837+1G>A. DFNB31 mutations appear to be a rare cause of Usher syndrome, since no mutations were identified in an additional 96 USH2 patients. While mutations in the C-terminal half of whirlin have previously been reported in non-syndromic deafness (DFNB31), both alterations identified in our USH2 family affect the long protein isoform. We propose that mutations causing Usher syndrome are probably restricted to exons 1–6 that are specific for the long isoform and probably crucial for retinal function. We describe a novel genetic subtype for Usher syndrome, which we named USH2D and which is caused by mutations in whirlin. Moreover, this is the first case of USH2 that is allelic to non-syndromic deafness.

Reversal of end-stage retinal degeneration and restoration of visual function by photoreceptor transplantation

One strategy to restore vision in retinitis pigmentosa and age-related macular degeneration is cell replacement. Typically, patients lose vision when the outer retinal photoreceptor layer is lost, and so the therapeutic goal would be to restore vision at this stage of disease. It is not currently known if a degenerate retina lacking the outer nuclear layer of photoreceptor cells would allow the survival, maturation, and reconnection of replacement photoreceptors, as prior studies used hosts with a preexisting outer nuclear layer at the time of treatment. Here, using a murine model of severe human retinitis pigmentosa at a stage when no host rod cells remain, we show that transplanted rod precursors can reform an anatomically distinct and appropriately polarized outer nuclear layer. A trilaminar organization was returned to rd1 hosts that had only two retinal layers before treatment. The newly introduced precursors were able to resume their developmental program in the degenerate host niche to become mature rods with light-sensitive outer segments, reconnecting with host neurons downstream. Visual function, assayed in the same animals before and after transplantation, was restored in animals with zero rod function at baseline. These observations suggest that a cell therapy approach may reconstitute a light-sensitive cell layer de novo and hence repair a structurally damaged visual circuit. Rather than placing discrete photoreceptors among preexisting host outer retinal cells, total photoreceptor layer reconstruction may provide a clinically relevant model to investigate cell-based strategies for retinal repair.

Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.

Macular telangiectasia type 2

Macular telangiectasia type 2 is a bilateral disease of unknown cause with characteristic alterations of the macular capillary network and neurosensory atrophy. Its prevalence may be underestimated and has recently been shown to be as high as 0.1% in persons 40 years and older. Biomicroscopy may show reduced retinal transparency, crystalline deposits, mildly ectatic capillaries, blunted venules, retinal pigment plaques, foveal atrophy, and neovascular complexes. Fluorescein angiography shows telangiectatic capillaries predominantly temporal to the foveola in the early phase and a diffuse hyperfluorescence in the late phase. High-resolution optical coherence tomography (OCT) may reveal disruption of the photoreceptor inner segment-outer segment border, hyporeflective cavities at the level of the inner or outer retina, and atrophy of the retina in later stages. Macular telangiectasia type 2 shows a unique depletion of the macular pigment in the central retina and recent therapeutic trials showed that such depleted areas cannot re-accumulate lutein and zeaxanthin after oral supplementation. There have been various therapeutic approaches with limited or no efficacy. Recent clinical trials with compounds that block vascular endothelial growth factor (VEGF) have established the role of VEGF in the pathophysiology of the disease, but have not shown significant efficacy, at least for the non-neovascular disease stages. Recent progress in structure-function correlation may help to develop surrogate outcome measures for future clinical trials. In this review article, we summarize the current knowledge on macular telangiectasia type 2, including the epidemiology, the genetics, the clinical findings, the staging and the differential diagnosis of the disease. Findings using retinal imaging are discussed, including fluorescein angiography, OCT, adaptive optics imaging, confocal scanning laser ophthalmoscopy, and fundus autofluorescence, as are the findings using visual function testing including visual acuity and fundus-controlled microperimetry. We provide an overview of the therapeutic approaches for both non-neovascular and neovascular disease stages and provide a perspective of future directions including animal models and potential therapeutic approaches.

Genetic control of the alternative pathway of complement in humans and age-related macular degeneration

Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues.

Clinical evaluation of simultaneous confocal scanning laser ophthalmoscopy imaging combined with high‐resolution, spectral‐domain optical coherence tomography

Acta Ophthalmol. 2010: 88: 842–849

Abnormal macular pigment distribution in type 2 idiopathic macular telangiectasia

Purpose: To determine the distribution of macular pigment in type 2 idiopathic macular telangiectasia (IMT). Methods: Twenty-two eyes of 12 patients with type 2 IMT were examined by means of best-corrected visual acuity testing, fundus biomicroscopy, fundus photography, fluorescein angiography, and optical coherence tomography. Macular pigment optical density (MPOD) was assessed using a modified confocal scanning laser ophthalmoscope whereby MPOD was calculated from fundus autofluorescence images acquired at two different excitation wavelengths (488 and 514 nm). The results were verified with a method that provides density maps after digital subtraction of log fundus reflectance maps (four patients) and by means of heterochromatic flicker photometry (four patients). Results: MOPD distribution showed an abnormal pattern for all patients with type 2 IMT. In correspondence to the late-phase hyperfluorescent areas shown by fluorescein angiography, MPOD was reduced in the macular area, while there was preserved MPOD at 5° to 7° eccentricity. Conclusions: The central depletion of macular pigment represents a novel phenotypic characteristic of type 2 IMT. Recording of macular pigment distribution may prove useful in the diagnosis of type 2 IMT and implicates an impaired trafficking or storage of lutein and zeaxanthin in the disease process.

Tracking Progression with Spectral-Domain Optical Coherence Tomography in Geographic Atrophy Caused by Age-Related Macular Degeneration

PURPOSE To investigate, with the use of spectral-domain optical coherence tomography (SD-OCT), microstructural alterations over time in eyes with progressive geographic atrophy (GA) due to age-related macular degeneration. METHODS Forty-six eyes of 26 patients (median age, 77.9 years [interquartile range (IQR), 71.8-81.0]) with GA without evidence of active or previous neovascular disease at baseline were examined by simultaneous confocal scanning laser ophthalmoscopy (cSLO) and SD-OCT. Serial examinations with alignment of follow-up to baseline scans were performed over a median period of 12.2 months (IQR, 10.2-15.3). Longitudinal SD-OCT variations were evaluated, including quantification of retinal thickness (RT) change and lateral spread of GA (LSGA) at a temporal, nasal, inferior, and superior GA border-section in each eye. RESULTS GA-enlargement was characterized by progressive loss of the outer hyperreflective SD-OCT bands and by thinning of the outer nuclear layer with subsequent approach of the outer plexiform layer toward Bruchs membrane. In the perilesional zone, various dynamic changes were recorded, including migration of hyperreflective material and changes in drusen height. At the borders, there was a median RT change of -14.09 microm/y (IQR -26.21 to -7.48 microm/y). The median LSGA was 106.90 microm/y (IQR, 55.44-161.70 microm/y). Both parameters showed only moderate intraocular agreement (RT change: intraclass correlation coefficient [ICC], 0.54; 95% CI, 0.39-0.67; LSGA: ICC, 0.49; 95% CI, 0.34-0.64) and no statistical significant difference for one location (RT change, P = 0.125; LSGA, P = 0.516; likelihood ratio test). CONCLUSIONS Combined cSLO and SD-OCT imaging provides unprecedented insight into dynamic microstructural changes of GA enlargement that may help to better understand the pathogenesis of the disease. Quantitative progression data indicate local factors may exist that drive progression in junctional areas (ClinicalTrials.gov number, NCT00393692).