Guy Hughes

Complement factor H genotypes impact risk of age-related macular degeneration by interaction with oxidized phospholipids

The rs1061170T/C variant encoding the Y402H change in complement factor H (CFH) has been identified by genome-wide association studies as being significantly associated with age-related macular degeneration (AMD). However, the precise mechanism by which this CFH variant impacts the risk of AMD remains largely unknown. Oxidative stress plays an important role in many aging diseases, including cardiovascular disease and AMD. A large amount of oxidized phospholipids (oxPLs) are generated in the eye because of sunlight exposure and high oxygen content. OxPLs bind to the retinal pigment epithelium and macrophages and strongly activate downstream inflammatory cascades. We hypothesize that CFH may impact the risk of AMD by modulating oxidative stress. Here we demonstrate that CFH binds to oxPLs. The CFH 402Y variant of the protective rs1061170 genotype binds oxPLs with a higher affinity and exhibits a stronger inhibitory effect on the binding of oxPLs to retinal pigment epithelium and macrophages. In addition, plasma from non-AMD subjects with the protective genotype has a lower level of systemic oxidative stress measured by oxPLs per apolipoprotein B (oxPLs/apoB). We also show that oxPL stimulation increases expression of genes involved in macrophage infiltration, inflammation, and neovascularization in the eye. OxPLs colocalize with CFH in drusen in the human AMD eye. Subretinal injection of oxPLs induces choroidal neovascularization in mice. In addition, we show that the CFH risk allele confers higher complement activation and cell lysis activity. Together, these findings suggest that CFH influences AMD risk by modulating oxidative stress, inflammation, and abnormal angiogenesis.

Assessing susceptibility to age-related macular degeneration with genetic markers and environmental factors

OBJECTIVES To evaluate the independent and joint effects of genetic factors and environmental variables on advanced forms of age-related macular degeneration (AMD), including geographic atrophy and choroidal neovascularization, and to develop a predictive model with genetic and environmental factors included. METHODS Demographic information, including age at onset, smoking status, and body mass index, was collected for 1844 participants. Genotypes were evaluated for 8 variants in 5 genes related to AMD. Unconditional logistic regression analyses were performed to generate a risk predictive model. RESULTS All genetic variants showed a strong association with AMD. Multivariate odds ratios were 3.52 (95% confidence interval, 2.08-5.94) for complement factor H, CFH rs1061170 CC, 4.21 (2.30-7.70) for CFH rs2274700 CC, 0.46 (0.27-0.80) for C2 rs9332739 CC/CG, 0.44 (0.30-0.66) for CFB rs641153 TT/CT, 10.99 (6.04-19.97) for HTRA1/LOC387715 rs10490924 TT, and 2.66 (1.43-4.96) for C3 rs2230199 GG. Smoking was independently associated with advanced AMD after controlling for age, sex, body mass index, and all genetic variants. CONCLUSION CFH confers more risk to the bilaterality of geographic atrophy, whereas HTRA1/LOC387715 contributes more to the bilaterality of choroidal neovascularization. C3 confers more risk for geographic atrophy than choroidal neovascularization. Risk models with combined genetic and environmental factors have notable discrimination power. CLINICAL RELEVANCE Early detection and risk prediction of AMD could help to improve the prognosis of AMD and to reduce the outcome of blindness. Targeting high-risk individuals for surveillance and clinical interventions may help reduce disease burden.

High Temperature Requirement Factor A1 (HTRA1) Gene Regulates Angiogenesis through Transforming Growth Factor-β Family Member Growth Differentiation Factor 6

Background: Genetic variants of high temperature requirement factor A1 (HTRA1) associate with AMD risk. Results: Growth differentiation factor 6 (GDF6) gene polymorphism significantly associated with AMD. HTRA1 knock-out mice display reduced blood vessel in retina and up-regulation of GDF6. Conclusion: HTRA1 regulates angiogenesis via TGF-β signaling by GDF6, a novel disease gene. Significance: This novel pathway of HTRA1 in regulation of vascularization is critical for understanding AMD pathogenesis. Genome-wide association study (GWAS) has identified genetic variants in the promoter region of the high temperature requirement factor A1 (HTRA1) gene associated with age-related macular degeneration (AMD). As a secreted serine protease, HTRA1 has been reported to interact with members of the transforming growth factor-β (TGF-β) family and regulate their signaling pathways. Growth differentiation factor 6 (GDF6), a member of the TGF-β family, is involved in ectoderm patterning and eye development. Mutations in GDF6 have been associated with abnormal eye development that may result in microphthalmia and anophthalmia. In this report, we identified a single nucleotide polymorphism (SNP) rs6982567 A/G near the GDF6 gene that is significantly associated with AMD (p value = 3.54 × 10−8). We demonstrated that the GDF6 AMD risk allele (rs6982567 A) is associated with decreased expression of the GDF6 and increased expression of HTRA1. Similarly, the HTRA1 AMD risk allele (rs10490924 T) is associated with decreased GDF6 and increased HTRA1 expression. We observed decreased vascular development in the retina and significant up-regulation of GDF6 gene in the RPE layer, retinal and brain tissues in HTRA1 knock-out (htra1−/−) mice as compared with the wild-type counterparts. Furthermore, we showed enhanced SMAD signaling in htra1−/− mice. Our data suggests a critical role of HTRA1 in the regulation of angiogenesis via TGF-β signaling and identified GDF6 as a novel disease gene for AMD.

Production of ELOVL4 transgenic pigs: a large animal model for Stargardt-like macular degeneration

Background Truncation mutations in the elongation of very long chain fatty acids-4 (AF277094, MIM #605512) (ELOVL4) gene cause Stargardt-like macular dystrophy type 3 (STGD3). Mice expressing truncated ELOVL4 develop rapid retinal degeneration, but are poor STGD3 models since mice lack a macula. Photoreceptor topography in the pig retina is more similar to that in humans as it includes the cone rich, macula-like area centralis. The authors generated transgenic pigs expressing human disease-causing ELOVL4 mutations to better model the pathobiology of this macular disease. Methods Pronuclear DNA microinjection and somatic cell nuclear transfer were used to produce transgenic pigs for two different ELOVL4 mutations: the 5 base pair deletion (5 bpdel) and the 270 stop mutation (Y270terEYFP). Retinal transgene expression, morphology and electrophysiology were examined. Results The authors obtained four lines of Y270terEYFP and one line of 5 bpdel transgenic animals. Direct fluorescence microscopy indicated that the Y270terEYFP protein is expressed in photoreceptors and mislocalised within the cell. Immunohistochemical examination of transgenic pigs showed photoreceptor loss and disorganised inner and outer segments. Electroretinography demonstrated diminished responses in both transgenic models. Conclusions These transgenic pigs provide unique animal models for examining macular degeneration and STGD3 pathogenesis.

Essential role of ELOVL4 protein in very long chain fatty acid synthesis and retinal function.

Background: Phospholipids containing very long chain polyunsaturated fatty acids (VLC-PUFAs) are enriched in retina. Results: Specific ELOVL4 rod or cone photoreceptor conditional knock-outs cause decreases in retinal VLC-PUFAs. Conclusion: ELOVL4 is critical for the synthesis of phosphatidylcholine-containing sn-1 VLC-PUFAs and vision. Significance: ELOVL4 mutations are implicated in Stargardt disease, a type of juvenile macular degeneration. Very long chain polyunsaturated fatty acid (VLC-PUFA)-containing glycerophospholipids are highly enriched in the retina; however, details regarding the specific synthesis and function of these highly unusual retinal glycerophospholipids are lacking. Elongation of very long chain fatty acids-4 (ELOVL4) has been identified as a fatty acid elongase protein involved in the synthesis of VLC-PUFAs. Mutations in ELOVL4 have also been implicated in an autosomal dominant form of Stargardt disease (STGD3), a type of juvenile macular degeneration. We have generated photoreceptor-specific conditional knock-out mice and used high performance liquid chromatography-mass spectrometry (HPLC-MS) to examine and analyze the fatty acid composition of retinal membrane glycerophosphatidylcholine and glycerophosphatidylethanolamine species. We also used immunofluorescent staining and histology coupled with electrophysiological data to assess retinal morphology and visual response. The conditional knock-out mice showed a significant decrease in retinal glycerophospholipids containing VLC-PUFAs, specifically contained in the sn-1 position of glycerophosphatidylcholine, implicating the role of Elovl4 in their synthesis. Conditional knock-out mice were also found to have abnormal accumulation of lipid droplets and lipofuscin-like granules while demonstrating photoreceptor-specific abnormalities in visual response, indicating the critical role of Elovl4 for proper rod or cone photoreceptor function. Altogether, this study demonstrates the essential role of ELOVL4 in VLC-PUFA synthesis and retinal function.

Genes of the Unfolded Protein Response Pathway Harbor Risk Alleles for Primary Open Angle Glaucoma

The statistical power of genome-wide association (GWA) studies to detect risk alleles for human diseases is limited by the unfavorable ratio of SNPs to study subjects. This multiple testing problem can be surmounted with very large population sizes when common alleles of large effects give rise to disease status. However, GWA approaches fall short when many rare alleles may give rise to a common disease, or when the number of subjects that can be recruited is limited. Here, we demonstrate that this multiple testing problem can be overcome by a comparative genomics approach in which an initial genome-wide screen in a genetically amenable model organism is used to identify human orthologues that may harbor risk alleles for adult-onset primary open angle glaucoma (POAG). Glaucoma is a major cause of blindness, which affects over 60 million people worldwide. Several genes have been associated with juvenile onset glaucoma, but genetic factors that predispose to adult onset primary open angle glaucoma (POAG) remain largely unknown. Previous genome-wide analysis in a Drosophila ocular hypertension model identified transcripts with altered regulation and showed induction of the unfolded protein response (UPR) upon overexpression of transgenic human glaucoma-associated myocilin (MYOC). We selected 16 orthologous genes with 62 polymorphic markers and identified in two independent human populations two genes of the UPR that harbor POAG risk alleles, BIRC6 and PDIA5. Thus, effectiveness of the UPR in response to accumulation of misfolded or aggregated proteins may contribute to the pathogenesis of POAG and provide targets for early therapeutic intervention.