A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants.

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.3448Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.We thank all participants of all the studies included for enabling this research by their participation in these studies. Computer resources for this project have been provided by the high-performance computing centers of the University of Michigan and the University of Regensburg. Group-specific acknowledgments can be found in the Supplementary Note. The Center for Inherited Diseases Research (CIDR) Program contract number is HHSN268201200008I. This and the main consortium work were predominantly funded by 1X01HG006934-01 to G.R.A. and R01 EY022310 to J.L.H

Synthesis and cation-binding abilities of novel polyquinane crown ethers containing a bis-acetal ether functionality

New macrocyclic ether hosts bearing a 'bis-acetal ether' functionality and a lipophilic posterior have been synthesised from the readily available cis, syn, cis-triquinane dione 1 and ethylene glycols in one step. Complexation characteristics and ability of these host molecules to translocate cations across phospholipid bilayer vesicles or liposomes has been studied

Synthesis of a new isoxazolidine from diacetone glucose

The synthesis of a new furo-pyran-based isoxazolidine, making use of an intramolecular oxime-olefin cycloaddition (IOOC) reaction, is reported here, starting from diacetone glucose

Effect of posterior donor lenticule thickness on optical abberations and visual outcome with time after descemet stripping endothelial Kkeratoplasty (DSEK)

Purpose: : To evaluate optical and visual outcomes of DSEK as a function of time. Methods: : Fifty-one eyes from 50 consecutive patients who underwent DSEK for Fuchs’ endothelial dystrophy and pseudophakic bullous keratopathy and had been followed up for &gt; 12 months (mean=12.43 months) were identified retrospectively. All donor lenticules were prepared manually. Pre and post surgical data analysed included best corrected LogMAR acuity, manifest refraction, central corneal thickness, endothelial graft thickness (Zeiss Visante AS-OCT), corneal wavefront ocular aberrometry (root mean square values for higher order, lower order and total astigmatism in the central 4mm, 5mm and 6mm areas using Zeiss-Atlas Omni corneal topography) and total aberration using Shack-Hartman wavefront analysis (AMO-VisX Wavescan). Statistical analysis was performed using SPSS v17. Results: Mean age of our cohort was 73.4 years (n=50). Mean spherical and cylindrical error did not change significantly post DSEK (0.77 ± 1.44 DS and -1.75 ± 1.81 DC versus 1.06 ± 2.7 DS and -1.80 ± 2.5 DC pre DSEK). Visual acuity improved from a mean of 0.97 ± 0.54 logMAR to 0.66 ± 0.55 logMAR (P&lt;0.0001). Post DSEK root mean square values for total aberrations ranged from 1.22 µm to 2.68 µm, while values for higher order abberations ranged from 0.61 µm to 1.73 µm (n=19). Corneal thickness reduced from a mean of 637 µ to 525 µ (P&lt;0.0001). Median donor lenticule thickness was 269 µ at month 1, 247 µ at month 3, 232 µ at month 6 and 143 µ at last follow up (range = 52µ to 290µ) with an average diameter of 9.04 ± 0.41 mm. No statistical correlation could be found between donor lenticule thickness and ocular abberations or between lenticule thickness and visual outcomes. Conclusions: This is the first study to examine posterior lenticule thickness using AS-OCT and explore the relationship with corneal HOA and refractive and visual outcomes over time. DSEK donor lenticular thickness varied significantly with time. Correlation with refractive metrics including corneal aberration, total HOA and refractive status are discussed. Defocus and astigmatism did not change significantly one year post DSEK compared with pre-operative values whilst visual acuity improved significantly. Lenticule thickness measured by AS-OCT after one year in-situ did not correlate with optical aberration, refractive or visual outcome

Age related macular degeneration is associated with the HLA Cw*0701 genotype and the Natural Killer cell receptor AA haplotype

PURPOSE. To determine the association of Human Leucocyte Antigen - HLA C and its cognate killer-cell immunoglobulin-like receptor (KIR) ligands with age related macular degeneration (AMD). Methods: HLA class I allele groups including the HLA C principal alleles were genotyped in a cohort of 104 AMD cases and 93 controls using the PCR-SSP.METHOD. This cohort was then genotyped for 16 KIR genes by PCR-SSP. Frequencies of the tested HLA / KIR alleles were then compared between AMD patients and normal controls. HLA C1, Cw*07, Cw*0701 genotypes and their combinations with KIR genotypes/haplotypes were tested for association with AMD. P values were obtained using a 2-tailed chi-squared test and Bonferroni corrections applied for multiple testing (Pc).RESULTS. The HLA Cw*0701 allele in combination with the inhibitory KIR AA haplotype was associated with AMD following logistic regression analysis (P=0.006, Pc=0.036, OR= 4.35, 95% CI =1.41-13.44).CONCLUSIONS. The HLA Cw*0701 allele and KIR haplotype AA are associated with AMD. This genotype combination suggests that Natural killer cells have a role in the pathogenesis of AMD. Replication studies are required to confirm these novel HLA-KIR associations with AMD.<br/

AAV2/8 anti-angiogenic gene therapy using single-chain antibodies inhibits murine choroidal neovascularization

While anti-angiogenic therapies for wet age-related macular degeneration (AMD) are effective for many patients, they require multiple injections, are expensive and prone to complications. Gene therapy could be an elegant solution for this problem by providing a long-term source of anti-angiogenic proteins after a single administration. Another potential issue with current therapeutic proteins containing a fragment crystallizable (Fc) domain (such as whole antibodies like bevacizumab) is the induction of an unwanted immune response. In wet AMD a low level of inflammation is already present, so to avoid exacerbation of disease by the therapeutic protein, we propose single-chain fragment variable antibodies (scFv, which lack the Fc domain) as a safer alternative. To investigate the feasibility of this, anti-vascular endothelial growth factor (VEGF) blocking antibodies in two formats were produced and tested in vitro and in vivo. The scFv transgene was then cloned into an adeno-associated virus (AAV) vector.A therapeutic effect in a mouse model of choroidal neovascularization (CNV) was demonstrated with antibodies in both scFv and immunoglobulin G1 (IgG1) formats (p&lt;0.04). Importantly, the scFv anti-VEGF antibody expressed from an AAV vector also had a significant beneficial effect (p=0.02), providing valuable preclinical data for future translation to the clinic

A lasered mouse model of retinal degeneration displays progressive outer retinal pathology providing insights into early geographic atrophy

Early stages of geographic atrophy (GA) age-related macular degeneration is characterised by the demise of photoreceptors, which precedes the loss of underlying retinal pigment epithelial (RPE) cells. Sight-loss due to GA has no effective treatment; reflecting both the complexity of the disease and the lack of suitable animal models for testing potential therapies. We report the development and characterisation of a laser-induced mouse model with early GA-like pathology. Retinas were lasered at adjacent sites using a 810nm laser (1.9J/spot), resulting in the development of confluent, hypopigmented central lesions with well-defined borders. Optical Coherence Tomography over 2-months showed progressive obliteration of photoreceptors with hyper-reflective outer plexiform and RPE/Bruch’s membrane (BrM) layers within lesions, but an unaffected inner retina. Light/electron microscopy after 3-months revealed lesions without photoreceptors, leaving the outer plexiform layer apposed to the RPE. We observed outer segment debris, hypo/hyperpigmented RPE, abnormal apical-basal RPE surfaces and BrM thickening. Lesions had wedge-shaped margins, extended zones of damage, activated Müller cells, microglial recruitment and functional retinal deficits. mRNA studies showed complement and inflammasome activation, microglial/macrophage phagocytosis and oxidative stress providing mechanistic insights into GA. We propose this mouse model as an attractive tool for early GA studies and drug-discovery

A transcriptome-wide association study based on 27 tissues identifies 106 genes potentially relevant for disease pathology in age-related macular degeneration

Genome-wide association studies (GWAS) for late stage age-related macular degeneration (AMD) have identified 52 independent genetic variants with genome-wide significance at 34 genomic loci. Typically, such an approach rarely results in the identification of functional variants implicating a defined gene in the disease process. We now performed a transcriptome-wide association study (TWAS) allowing the prediction of effects of AMD-associated genetic variants on gene expression. The TWAS was based on the genotypes of 16,144 late-stage AMD cases and 17,832 healthy controls, and gene expression was imputed for 27 different human tissues which were obtained from 134 to 421 individuals. A linear regression model including each individuals imputed gene expression data and the respective AMD status identified 106 genes significantly associated to AMD variants in at least one tissue (Q-value &lt; 0.001). Gene enrichment analysis highlighted rather systemic than tissue- or cell-specific processes. Remarkably, 31 of the 106 genes overlapped with significant GWAS signals of other complex traits and diseases, such as neurological or autoimmune conditions. Taken together, our study highlights the fact that expression of genes associated with AMD is not restricted to retinal tissue as could be expected for an eye disease of the posterior pole, but instead is rather ubiquitous suggesting processes underlying AMD pathology to be of systemic nature.</p

Pathway Analysis Integrating Genome-Wide and Functional Data Identifies PLCG2 as a Candidate Gene for Age-Related Macular Degeneration

PURPOSE. Age-related macular degeneration (AMD) is the worldwide leading cause of blindness among the elderly. Although genome-wide association studies (GWAS) have identified AMD risk variants, their roles in disease etiology are not well-characterized, and they only explain a portion of AMD heritability. METHODS. We performed pathway analyses using summary statistics from the International AMD Genomics Consortium's 2016 GWAS and multiple pathway databases to identify biological pathways wherein genetic association signals for AMD may be aggregating. We determined which genes contributed most to significant pathway signals across the databases. We characterized these genes by constructing protein-protein interaction networks and performing motif analysis. RESULTS. We determined that eight genes (C2, C3, LIPC, MICA, NOTCH4, PLCG2, PPARA, and RAD51B) drive'' the statistical signals observed across pathways curated in the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and Gene Ontology (GO) databases. We further refined our definition of statistical driver gene to identify PLCG2 as a candidate gene for AMD due to its significant gene-level signals (P < 0.0001) across KEGG, Reactome, GO, and NetPath pathways. CONCLUSIONS. We performed pathway analyses on the largest available collection of advanced AMD cases and controls in the world. Eight genes strongly contributed to significant pathways from the three larger databases, and one gene (PLCG2) was central to significant pathways from all four databases. This is, to our knowledge, the first study to identify PLCG2 as a candidate gene for AMD based solely on genetic burden. Our findings reinforce the utility of integrating in silico genetic and biological pathway data to investigate the genetic architecture of AMD