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Purpose: Keratoconus (KC) represents one of the leading causes of corneal transplantation worldwide. Detecting subclinical KC would lead to better management to avoid the need for corneal grafts, but the condition is clinically challenging to diagnose. We wished to compare eight commonly used machine learning algorithms using a range of parameter combinations by applying them to our KC dataset and build models to better differentiate subclinical KC from non-KC eyes., Methods: Oculus Pentacam was used to obtain corneal parameters on 49 subclinical KC and 39 control eyes, along with clinical and demographic parameters. Eight machine learning methods were applied to build models to differentiate subclinical KC from control eyes. Dominant algorithms were trained with all combinations of the considered parameters to select important parameter combinations. The performance of each model was evaluated and compared., Results: Using a total of eleven parameters, random forest, support vector machine and k-nearest neighbors had better performance in detecting subclinical KC. The highest area under the curve of 0.97 for detecting subclinical KC was achieved using five parameters by the random forest method. The highest sensitivity (0.94) and specificity (0.90) were obtained by the support vector machine and the k-nearest neighbor model, respectively., Conclusions: This study showed machine learning algorithms can be applied to identify subclinical KC using a minimal parameter set that are routinely collected during clinical eye examination., Translational Relevance: Machine learning algorithms can be built using routinely collected clinical parameters that will assist in the objective detection of subclinical KC., Competing Interests: Disclosure: K. Cao, None; K. Verspoor, None; S. Sahebjada, None; P.N. Baird, None, (Copyright 2020 The Authors.)

Importance: This is the first study to estimate the lifetime costs associated with keratoconus based on a questionnaire completed by patients and highlights the significant economic burden of the disease. As keratoconus affects individuals from a young age, the study highlights keratoconus as a public health concern., Background: Keratoconus is a disorder characterized by corneal steepening and thinning, leading to reduced visual acuity. To date, there have been no studies evaluating the economic costs of keratoconus from a patient's perspective., Design: A randomized cross-sectional study undertaken in Australia where keratoconus subjects were recruited from public and private ophthalmology and optometry clinics., Participants: A total of 100 participants completed the questionnaire: median age was 31 years and 57% were males., Methods: A keratoconus health expenditure questionnaire was designed to assess direct and indirect expenditures for each individual., Main Outcome Measures: Total direct and indirect costs associated with the condition were calculated along with the estimated lifetime per capita costs., Results: The total cost related to direct and indirect care was estimated to be AUD 3365. By applying our cost data to keratoconus prevalence data for the Australian population, the total cost is estimated to be approximately AUD 44.7 million per year in Australia., Conclusions and Relevance: Our results show that the costs associated with the diagnosis and management of keratoconus represent a significant cost to patients. An understanding of this is important not only to individuals and their families, but also health care providers, health insurers and the wider health system., (© 2019 Royal Australian and New Zealand College of Ophthalmologists.)

Corneal curvature, a highly heritable trait, is a key clinical endophenotype for myopia - a major cause of visual impairment and blindness in the world. Here we present a trans-ethnic meta-analysis of corneal curvature GWAS in 44,042 individuals of Caucasian and Asian with replication in 88,218 UK Biobank data. We identified 47 loci (of which 26 are novel), with population-specific signals as well as shared signals across ethnicities. Some identified variants showed precise scaling in corneal curvature and eye elongation (i.e. axial length) to maintain eyes in emmetropia (i.e. HDAC11/FBLN2 rs2630445, RBP3 rs11204213); others exhibited association with myopia with little pleiotropic effects on eye elongation. Implicated genes are involved in extracellular matrix organization, developmental process for body and eye, connective tissue cartilage and glycosylation protein activities. Our study provides insights into population-specific novel genes for corneal curvature, and their pleiotropic effect in regulating eye size or conferring susceptibility to myopia.

Refractive errors, including myopia, are the most frequent eye disorders worldwide and an increasingly common cause of blindness. This genome-wide association meta-analysis in 160,420 participants and replication in 95,505 participants increased the number of established independent signals from 37 to 161 and showed high genetic correlation between Europeans and Asians (>0.78). Expression experiments and comprehensive in silico analyses identified retinal cell physiology and light processing as prominent mechanisms, and also identified functional contributions to refractive-error development in all cell types of the neurosensory retina, retinal pigment epithelium, vascular endothelium and extracellular matrix. Newly identified genes implicate novel mechanisms such as rod-and-cone bipolar synaptic neurotransmission, anterior-segment morphology and angiogenesis. Thirty-one loci resided in or near regions transcribing small RNAs, thus suggesting a role for post-transcriptional regulation. Our results support the notion that refractive errors are caused by a light-dependent retina-to-sclera signaling cascade and delineate potential pathobiological molecular drivers.

Purpose: To identify genes and genetic markers associated with corneal astigmatism., Methods: A meta-analysis of genome-wide association studies (GWASs) of corneal astigmatism undertaken for 14 European ancestry (n=22,250) and 8 Asian ancestry (n=9,120) cohorts was performed by the Consortium for Refractive Error and Myopia. Cases were defined as having >0.75 diopters of corneal astigmatism. Subsequent gene-based and gene-set analyses of the meta-analyzed results of European ancestry cohorts were performed using VEGAS2 and MAGMA software. Additionally, estimates of single nucleotide polymorphism (SNP)-based heritability for corneal and refractive astigmatism and the spherical equivalent were calculated for Europeans using LD score regression., Results: The meta-analysis of all cohorts identified a genome-wide significant locus near the platelet-derived growth factor receptor alpha ( PDGFRA ) gene: top SNP: rs7673984, odds ratio=1.12 (95% CI:1.08-1.16), p=5.55×10 -9 . No other genome-wide significant loci were identified in the combined analysis or European/Asian ancestry-specific analyses. Gene-based analysis identified three novel candidate genes for corneal astigmatism in Europeans-claudin-7 ( CLDN7 ), acid phosphatase 2, lysosomal ( ACP2 ), and TNF alpha-induced protein 8 like 3 ( TNFAIP8L3 )., Conclusions: In addition to replicating a previously identified genome-wide significant locus for corneal astigmatism near the PDGFRA gene, gene-based analysis identified three novel candidate genes, CLDN7 , ACP2 , and TNFAIP8L3 , that warrant further investigation to understand their role in the pathogenesis of corneal astigmatism. The much lower number of genetic variants and genes demonstrating an association with corneal astigmatism compared to published spherical equivalent GWAS analyses suggest a greater influence of rare genetic variants, non-additive genetic effects, or environmental factors in the development of astigmatism.

Purpose: Presently, 52 independent nuclear single nucleotide polymorphisms (nSNPs) have been associated with age-related macular degeneration (AMD) but their effects do not explain all its variance. Genetic interactions between the nuclear and mitochondrial (mt) genome may unearth additional genetic loci previously unassociated with AMD risk., Methods: Joint effects of nSNPs and selected mtSNPs were analyzed by two degree of freedom (2df) joint tests of association in the International AMD Genomics Consortium (IAMDGC) dataset (17,832 controls and 16,144 advanced AMD cases of European ancestry). Subjects were genotyped on the Illumina HumanCoreExome array. After imputation using MINIMAC and the 1000 Genomes Project Phase I reference panel, pairwise linkage disequilibrium pruning, and quality control, 3.9 million nSNPs were analyzed for interaction with mtSNPs chosen based on association in this dataset or publications: A4917G, T5004C, G12771A, and C16069T., Results: Novel locus TRPM1 was identified with genome-wide significant joint effects (P < 5.0 × 10-8) of two intronic TRPM1 nSNPs and AMD-associated nonsynonymous MT-ND2 mtSNP A4917G. Stratified analysis by mt allele identified an association only in 4917A (major allele) carriers (P = 4.4 × 10-9, odds ratio [OR] = 0.90, 95% confidence interval [CI] = 0.87-0.93). Intronic and intergenic ABHD2/RLBP1 nSNPs demonstrated genome-wide significant joint effects (2df joint test P values from 1.8 × 10-8 to 4.9 × 10-8) and nominally statistically significant interaction effects with MT-ND5 synonymous mtSNP G12771A. Although a positive association was detected in both strata, the association was stronger in 12771A subjects (P = 0.0020, OR = 2.17, 95% CI = 1.34-3.60)., Conclusions: These results show that joint tests of main effects and gene-gene interaction reveal associations at some novel loci that were missed when considering main effects alone.

Myopia is the most common human eye disorder and it results from complex genetic and environmental causes. The rapidly increasing prevalence of myopia poses a major public health challenge. Here, the CREAM consortium performs a joint meta-analysis to test single-nucleotide polymorphism (SNP) main effects and SNP × education interaction effects on refractive error in 40,036 adults from 25 studies of European ancestry and 10,315 adults from 9 studies of Asian ancestry. In European ancestry individuals, we identify six novel loci (FAM150B-ACP1, LINC00340, FBN1, DIS3L-MAP2K1, ARID2-SNAT1 and SLC14A2) associated with refractive error. In Asian populations, three genome-wide significant loci AREG, GABRR1 and PDE10A also exhibit strong interactions with education (P<8.5 × 10(-5)), whereas the interactions are less evident in Europeans. The discovery of these loci represents an important advance in understanding how gene and environment interactions contribute to the heterogeneity of myopia.

Myopia can cause severe visual impairment. Here, we report a two-stage genome-wide association study for three myopia-related traits in 9,804 Japanese individuals, which was extended with trans-ethnic replication in 2,674 Chinese and 2,690 Caucasian individuals. We identify WNT7B as a novel susceptibility gene for axial length (rs10453441, Pmeta=3.9 × 10(-13)) and corneal curvature (Pmeta=2.9 × 10(-40)) and confirm the previously reported association between GJD2 and myopia. WNT7B significantly associates with extreme myopia in a case-control study with 1,478 Asian patients and 4,689 controls (odds ratio (OR)meta=1.13, Pmeta=0.011). We also find in a mouse model of myopia downregulation of WNT7B expression in the cornea and upregulation in the retina, suggesting its possible role in the development of myopia.

To identify genetic variants associated with refractive astigmatism in the general population, meta-analyses of genome-wide association studies were performed for: White Europeans aged at least 25 years (20 cohorts, N = 31,968); Asian subjects aged at least 25 years (7 cohorts, N = 9,295); White Europeans aged <25 years (4 cohorts, N = 5,640); and all independent individuals from the above three samples combined with a sample of Chinese subjects aged <25 years (N = 45,931). Participants were classified as cases with refractive astigmatism if the average cylinder power in their two eyes was at least 1.00 diopter and as controls otherwise. Genome-wide association analysis was carried out for each cohort separately using logistic regression. Meta-analysis was conducted using a fixed effects model. In the older European group the most strongly associated marker was downstream of the neurexin-1 (NRXN1) gene (rs1401327, P = 3.92E-8). No other region reached genome-wide significance, and association signals were lower for the younger European group and Asian group. In the meta-analysis of all cohorts, no marker reached genome-wide significance: The most strongly associated regions were, NRXN1 (rs1401327, P = 2.93E-07), TOX (rs7823467, P = 3.47E-07) and LINC00340 (rs12212674, P = 1.49E-06). For 34 markers identified in prior GWAS for spherical equivalent refractive error, the beta coefficients for genotype versus spherical equivalent, and genotype versus refractive astigmatism, were highly correlated (r = -0.59, P = 2.10E-04). This work revealed no consistent or strong genetic signals for refractive astigmatism; however, the TOX gene region previously identified in GWAS for spherical equivalent refractive error was the second most strongly associated region. Analysis of additional markers provided evidence supporting widespread genetic co-susceptibility for spherical and astigmatic refractive errors.

Refractive error (RE) is a complex, multifactorial disorder characterized by a mismatch between the optical power of the eye and its axial length that causes object images to be focused off the retina. The two major subtypes of RE are myopia (nearsightedness) and hyperopia (farsightedness), which represent opposite ends of the distribution of the quantitative measure of spherical refraction. We performed a fixed effects meta-analysis of genome-wide association results of myopia and hyperopia from 9 studies of European-derived populations: AREDS, KORA, FES, OGP-Talana, MESA, RSI, RSII, RSIII and ERF. One genome-wide significant region was observed for myopia, corresponding to a previously identified myopia locus on 8q12 (p = 1.25×10(-8)), which has been reported by Kiefer et al. as significantly associated with myopia age at onset and Verhoeven et al. as significantly associated to mean spherical-equivalent (MSE) refractive error. We observed two genome-wide significant associations with hyperopia. These regions overlapped with loci on 15q14 (minimum p value = 9.11×10(-11)) and 8q12 (minimum p value 1.82×10(-11)) previously reported for MSE and myopia age at onset. We also used an intermarker linkage- disequilibrium-based method for calculating the effective number of tests in targeted regional replication analyses. We analyzed myopia (which represents the closest phenotype in our data to the one used by Kiefer et al.) and showed replication of 10 additional loci associated with myopia previously reported by Kiefer et al. This is the first replication of these loci using myopia as the trait under analysis. "Replication-level" association was also seen between hyperopia and 12 of Kiefer et al.'s published loci. For the loci that show evidence of association to both myopia and hyperopia, the estimated effect of the risk alleles were in opposite directions for the two traits. This suggests that these loci are important contributors to variation of refractive error across the distribution.

Age-related macular degeneration (AMD) causes severe vision impairment in aged individuals. The health impact and cost of the disease will dramatically increase over the years, with the increase in the aging population. Currently, antivascular endothelial growth factor agents are routinely used for managing late-stage AMD, and recent data have shown that up to 15%-33% of patients do not respond to this treatment. Henceforth, there is a need to develop better treatment options. One avenue is to investigate the role proteases and inflammatory molecules might have in regulating and being regulated by vascular endothelial growth factor. Moreover, emerging data indicate that proteases and inflammatory molecules might be critical in the development and progression of AMD. This article reviews recent literature that investigates proteases and inflammatory molecules involved in the development of AMD. Gaining insights into the proteolytic and inflammatory pathways associated with the pathophysiology of AMD could enable the development of additional or alternative drug strategies for the treatment of AMD., (Copyright © 2014 Mosby, Inc. All rights reserved.)

The last 10 years have seen an unprecedented explosion in our knowledge regarding the genomic basis of age-related macular degeneration. This has come about through major advances in computing power, microfabrication of large numbers of molecular markers on chips and improved statistical algorithms for analysis. In tandem, it has become clear that age-related macular degeneration appears to be a multifactorial disease with influences from genetic and structural variants, as well as epigenetic involvement. The combination of these factors with known environmental determinants indicates the highly complex nature of this disease, but at the same time also offers insights into risk prediction and disease stratification through genotype profiling., (© 2013 Royal Australian and New Zealand College of Ophthalmologists.)

Refractive errors are common eye disorders of public health importance worldwide. Ocular axial length (AL) is the major determinant of refraction and thus of myopia and hyperopia. We conducted a meta-analysis of genome-wide association studies for AL, combining 12,531 Europeans and 8,216 Asians. We identified eight genome-wide significant loci for AL (RSPO1, C3orf26, LAMA2, GJD2, ZNRF3, CD55, MIP, and ALPPL2) and confirmed one previously reported AL locus (ZC3H11B). Of the nine loci, five (LAMA2, GJD2, CD55, ALPPL2, and ZC3H11B) were associated with refraction in 18 independent cohorts (n = 23,591). Differential gene expression was observed for these loci in minus-lens-induced myopia mouse experiments and human ocular tissues. Two of the AL genes, RSPO1 and ZNRF3, are involved in Wnt signaling, a pathway playing a major role in the regulation of eyeball size. This study provides evidence of shared genes between AL and refraction, but importantly also suggests that these traits may have unique pathways., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Visual refractive errors (REs) are complex genetic traits with a largely unknown etiology. To date, genome-wide association studies (GWASs) of moderate size have identified several novel risk markers for RE, measured here as mean spherical equivalent (MSE). We performed a GWAS using a total of 7280 samples from five cohorts: the Age-Related Eye Disease Study (AREDS); the KORA study ('Cooperative Health Research in the Region of Augsburg'); the Framingham Eye Study (FES); the Ogliastra Genetic Park-Talana (OGP-Talana) Study and the Multiethnic Study of Atherosclerosis (MESA). Genotyping was performed on Illumina and Affymetrix platforms with additional markers imputed to the HapMap II reference panel. We identified a new genome-wide significant locus on chromosome 16 (rs10500355, P = 3.9 × 10(-9)) in a combined discovery and replication set (26 953 samples). This single nucleotide polymorphism (SNP) is located within the RBFOX1 gene which is a neuron-specific splicing factor regulating a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins.

Narrow arterioles in the retina have been shown to predict hypertension as well as other vascular diseases, likely through an increase in the peripheral resistance of the microcirculatory flow. In this study, we performed a genome-wide association study in 18,722 unrelated individuals of European ancestry from the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium and the Blue Mountain Eye Study, to identify genetic determinants associated with variations in retinal arteriolar caliber. Retinal vascular calibers were measured on digitized retinal photographs using a standardized protocol. One variant (rs2194025 on chromosome 5q14 near the myocyte enhancer factor 2C MEF2C gene) was associated with retinal arteriolar caliber in the meta-analysis of the discovery cohorts at genome-wide significance of P-value <5×10(-8). This variant was replicated in an additional 3,939 individuals of European ancestry from the Australian Twins Study and Multi-Ethnic Study of Atherosclerosis (rs2194025, P-value = 2.11×10(-12) in combined meta-analysis of discovery and replication cohorts). In independent studies of modest sample sizes, no significant association was found between this variant and clinical outcomes including coronary artery disease, stroke, myocardial infarction or hypertension. In conclusion, we found one novel loci which underlie genetic variation in microvasculature which may be relevant to vascular disease. The relevance of these findings to clinical outcomes remains to be determined.

Purpose: The ARMS2/HTRA1 genes at the 10q26 locus have been associated with risk of age-related macular degeneration (AMD), with the most significantly associated variants being A69S (rs10490924), del443ins54 (EU427539) and rs11200638. We wished to explore the association of the del443ins54 in two ethnically different populations from India and Australia., Methods: The del443ins54 was screened in a large cohort of ~1500 subjects from these two populations by a combination of PCR-based agarose gel electrophoresis and validated by resequencing. Statistical analysis comprised the calculations of allele, genotype and haplotype frequencies along with their p values and corresponding odds ratios (OR), and 95% confidence intervals (95% CI) and measures of linkage disequilibrium (LD)., Results: The del443ins54 was significantly associated with AMD in both the Indian (p=1.74 × 10(-13); OR = 2.80, 95%CI, 2.12-3.70) and Australian cohorts (p = 2.78 × 10(-30); OR = 3.15, 95%CI, 2.58-3.86). These associations were similar to those previously identified for the A69S and the rs11200638 variant in these populations that also exhibited high degrees of LD (D' of 0.87-0.99). A major risk haplotype of "T-indel-A" (p = 5.7 × 10(-16); OR = 3.16, 95%CI, 2.34-4.19 and p=6.33 × 10(-30); OR = 3.15, 95%CI, 2.57-3.85) and a protective haplotype of "G-wild type-G" (p=2.35 × 10(-11); OR = 0.39, 95%CI, 0.29-0.52 and p=1.02 × 10(-30); OR = 0.31, 95%CI, 0.25-0.38) were identified in the Indian and Australian cohorts, respectively., Conclusions: These data provide an independent replication of the association of del443ins54 variant in two different ethnicities, despite differences in allele and haplotype frequencies between them. High levels of LD in both populations limit further genetic dissection of this region in AMD.

Age-related macular degeneration (AMD) is a common cause of blindness in older individuals. To accelerate the understanding of AMD biology and help design new therapies, we executed a collaborative genome-wide association study, including >17,100 advanced AMD cases and >60,000 controls of European and Asian ancestry. We identified 19 loci associated at P < 5 × 10(-8). These loci show enrichment for genes involved in the regulation of complement activity, lipid metabolism, extracellular matrix remodeling and angiogenesis. Our results include seven loci with associations reaching P < 5 × 10(-8) for the first time, near the genes COL8A1-FILIP1L, IER3-DDR1, SLC16A8, TGFBR1, RAD51B, ADAMTS9 and B3GALTL. A genetic risk score combining SNP genotypes from all loci showed similar ability to distinguish cases and controls in all samples examined. Our findings provide new directions for biological, genetic and therapeutic studies of AMD.

Refractive error is the most common eye disorder worldwide and is a prominent cause of blindness. Myopia affects over 30% of Western populations and up to 80% of Asians. The CREAM consortium conducted genome-wide meta-analyses, including 37,382 individuals from 27 studies of European ancestry and 8,376 from 5 Asian cohorts. We identified 16 new loci for refractive error in individuals of European ancestry, of which 8 were shared with Asians. Combined analysis identified 8 additional associated loci. The new loci include candidate genes with functions in neurotransmission (GRIA4), ion transport (KCNQ5), retinoic acid metabolism (RDH5), extracellular matrix remodeling (LAMA2 and BMP2) and eye development (SIX6 and PRSS56). We also confirmed previously reported associations with GJD2 and RASGRF1. Risk score analysis using associated SNPs showed a tenfold increased risk of myopia for individuals carrying the highest genetic load. Our results, based on a large meta-analysis across independent multiancestry studies, considerably advance understanding of the mechanisms involved in refractive error and myopia.

Myopia is a complex genetic disorder and a common cause of visual impairment among working age adults. Genome-wide association studies have identified susceptibility loci on chromosomes 15q14 and 15q25 in Caucasian populations of European ancestry. Here, we present a confirmation and meta-analysis study in which we assessed whether these two loci are also associated with myopia in other populations. The study population comprised 31 cohorts from the Consortium of Refractive Error and Myopia (CREAM) representing 4 different continents with 55,177 individuals; 42,845 Caucasians and 12,332 Asians. We performed a meta-analysis of 14 single nucleotide polymorphisms (SNPs) on 15q14 and 5 SNPs on 15q25 using linear regression analysis with spherical equivalent as a quantitative outcome, adjusted for age and sex. We calculated the odds ratio (OR) of myopia versus hyperopia for carriers of the top-SNP alleles using a fixed effects meta-analysis. At locus 15q14, all SNPs were significantly replicated, with the lowest P value 3.87 × 10(-12) for SNP rs634990 in Caucasians, and 9.65 × 10(-4) for rs8032019 in Asians. The overall meta-analysis provided P value 9.20 × 10(-23) for the top SNP rs634990. The risk of myopia versus hyperopia was OR 1.88 (95 % CI 1.64, 2.16, P < 0.001) for homozygous carriers of the risk allele at the top SNP rs634990, and OR 1.33 (95 % CI 1.19, 1.49, P < 0.001) for heterozygous carriers. SNPs at locus 15q25 did not replicate significantly (P value 5.81 × 10(-2) for top SNP rs939661). We conclude that common variants at chromosome 15q14 influence susceptibility for myopia in Caucasian and Asian populations world-wide.

In genome-wide association studies (GWASs) of colorectal cancer, we have identified two genomic regions in which pairs of tagging-single nucleotide polymorphisms (tagSNPs) are associated with disease; these comprise chromosomes 1q41 (rs6691170, rs6687758) and 12q13.13 (rs7163702, rs11169552). We investigated these regions further, aiming to determine whether they contain more than one independent association signal and/or to identify the SNPs most strongly associated with disease. Genotyping of additional sample sets at the original tagSNPs showed that, for both regions, the two tagSNPs were unlikely to identify a single haplotype on which the functional variation lay. Conversely, one of the pair of SNPs did not fully capture the association signal in each region. We therefore undertook more detailed analyses, using imputation, logistic regression, genealogical analysis using the GENECLUSTER program and haplotype analysis. In the 1q41 region, the SNP rs11118883 emerged as a strong candidate based on all these analyses, sufficient to account for the signals at both rs6691170 and rs6687758. rs11118883 lies within a region with strong evidence of transcriptional regulatory activity and has been associated with expression of PDGFRB mRNA. For 12q13.13, a complex situation was found: SNP rs7972465 showed stronger association than either rs11169552 or rs7136702, and GENECLUSTER found no good evidence for a two-SNP model. However, logistic regression and haplotype analyses supported a two-SNP model, in which a signal at the SNP rs706793 was added to that at rs11169552. Post-GWAS fine-mapping studies are challenging, but the use of multiple tools can assist in identifying candidate functional variants in at least some cases.

Visual impairment and blindness impose substantial morbidity and premature mortality on the population. The direct costs for vision disorders have been shown to be more than the cost of coronary heart disease, stroke, arthritis or depression and were estimated to be $9.85 billion in 2004 in Australia. Hence it is important to identify the causes of common eye diseases and understand their aetiology which in turn would allow determination of better management strategies and treatment options. Age related Macular Degeneration, Cataract, Diabetic Retinopathy, Glaucoma and uncorrected refractive errors represent the majority of the visual impairment and blindness in Australia and various parts of the world. This article reviews the gene patents available for these eye conditions and highlights the important discoveries that have so far contributed to our understanding of these diseases and provides valuable information as to where research will be heading in the future.

Age-related macular degeneration (AMD) is the most common cause of incurable visual impairment in high-income countries. Previous studies report inconsistent associations between AMD and apolipoprotein E (APOE), a lipid transport protein involved in low-density cholesterol modulation. Potential interaction between APOE and sex, and smoking status has been reported. We present a pooled analysis (n = 21,160) demonstrating associations between late AMD and APOε4 (odds ratio [OR] = 0.72 per haplotype; confidence interval [CI]: 0.65-0.74; P = 4.41×10(-11) ) and APOε2 (OR = 1.83 for homozygote carriers; CI: 1.04-3.23; P = 0.04), following adjustment for age group and sex within each study and smoking status. No evidence of interaction between APOE and sex or smoking was found. Ever smokers had significant increased risk relative to never smokers for both neovascular (OR = 1.54; CI: 1.38-1.72; P = 2.8×10(-15) ) and atrophic (OR = 1.38; CI: 1.18-1.61; P = 3.37×10(-5) ) AMD but not early AMD (OR = 0.94; CI: 0.86-1.03; P = 0.16), implicating smoking as a major contributing factor to disease progression from early signs to the visually disabling late forms. Extended haplotype analysis incorporating rs405509 did not identify additional risks beyond ε2 and ε4 haplotypes. Our expanded analysis substantially improves our understanding of the association between the APOE locus and AMD. It further provides evidence supporting the role of cholesterol modulation, and low-density cholesterol specifically, in AMD disease etiology., (© 2011 Wiley-Liss, Inc.)

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.

Predictive tests for estimating the risk of developing late-stage neovascular age-related macular degeneration (AMD) are subject to unique challenges. AMD prevalence increases with age, clinical phenotypes are heterogeneous and control collections are prone to high false-negative rates, as many control subjects are likely to develop disease with advancing age. Risk prediction tests have been presented previously, using up to ten genetic markers and a range of self-reported non-genetic variables such as body mass index (BMI) and smoking history. In order to maximise the accuracy of prediction for mainstream genetic testing, we sought to derive a test comparable in performance to earlier testing models but based purely on genetic markers, which are static through life and not subject to misreporting. We report a multicentre assessment of a larger panel of single nucleotide polymorphisms (SNPs) than previously analysed, to improve further the classification performance of a predictive test to estimate the risk of developing choroidal neovascular (CNV) disease. We developed a predictive model based solely on genetic markers and avoided inclusion of self-reported variables (eg smoking history) or non-static factors (BMI, education status) that might otherwise introduce inaccuracies in calculating individual risk estimates. We describe the performance of a test panel comprising 13 SNPs genotyped across a consolidated collection of four patient cohorts obtained from academic centres deemed appropriate for pooling. We report on predictive effect sizes and their classification performance. By incorporating multiple cohorts of homogeneous ethnic origin, we obtained >80 per cent power to detect differences in genetic variants observed between cases and controls. We focused our study on CNV, a subtype of advanced AMD associated with a severe and potentially treatable form of the disease. Lastly, we followed a two-stage strategy involving both test model development and test model validation to present estimates of classification performance anticipated in the larger clinical setting. The model contained nine SNPs tagging variants in the regulators of complement activation (RCA) locus spanning the complement factor H (CFH), complement factor H-related 4 (CFHR4), complement factor H-related 5 (CFHR5) and coagulation factor XIII B subunit (F13B) genes; the four remaining SNPs targeted polymorphisms in the complement component 2 (C2), complement factor B (CFB), complement component 3 (C3) and age-related maculopathy susceptibility protein 2 (ARMS2) genes. The pooled sample size (1,132 CNV cases, 822 controls) allowed for both model development and model validation to confirm the accuracy of risk prediction. At the validation stage, our test model yielded 82 per cent sensitivity and 63 per cent specificity, comparable with metrics reported with earlier testing models that included environmental risk factors. Our test had an area under the curve of 0.80, reflecting a modest improvement compared with tests reported with fewer SNPs.

Purpose: Anti-vascular endothelial growth factor (anti-VEGF) drugs have dramatically improved the treatment of neovascular AMD. In pivotal studies, almost 90% of patients maintain vision, with approximately 30% showing significant improvement. Despite these successes, 10% to 15% of patients continue to lose vision, even with treatment. It has been reported that variants in some AMD-associated genes influence treatment outcome. This study showed an association of treatment outcome with variants in the apolipoprotein E (APOE) gene., Methods: One hundred ninety-two patients receiving anti-VEGF treatment for subfoveal choroidal neovascularization secondary to AMD were enrolled. Information on demographics, lesion characteristics, delay until treatment, visual acuity (VA), and number of treatments was collected, and variants of APOE were assessed in all patients at baseline. Best corrected logarithm of the minimum angle of resolution (logMAR) VA was recorded in all patients., Results: The presence of the APOE ε4 allele was associated with improved treatment outcome at 3 (P = 0.02) and 12 (P = 0.06) months, compared with the presence of the ε2 allele, after adjustment for baseline acuity, treatment delay after first symptoms, age, and sex. Patients with an APOE ε4 allele had an odds ratio (OR) of 4.04 (95% confidence interval [CI], 1.11-14.70) for a 2-line gain in vision from baseline at 3 months (P = 0.03) and an OR of 2.54 (95% CI, 0.61-10.52; P = 0.20) at 12 months after treatment, based on multivariate analysis., Conclusions: In patients with neovascular AMD, the presence of the APOE ε4 allele conferred significantly better visual outcomes after anti-VEGF treatment than did the ε2 allele. These findings suggest a possible role for a personalized approach to treatment with anti-VEGF.

Genome-wide association studies (GWAS) have identified 14 tagging single nucleotide polymorphisms (tagSNPs) that are associated with the risk of colorectal cancer (CRC), and several of these tagSNPs are near bone morphogenetic protein (BMP) pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3), BMP4 (14q22.2), and BMP2 (20p12.3) using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10)) and BMP2 (rs4813802, P = 4.65×10(-11)). Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8)) and rs11632715 (P = 2.30×10(-10)). As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases., Competing Interests: The authors have declared that no competing interests exist.

Diseases of the retina are the leading causes of blindness in the industrialized world. The recognition that animals develop retinal diseases with similar traits to humans has led to not only a dramatic improvement in our understanding of the pathogenesis of retinal disease but also provided a means for testing possible treatment regimes and successful gene therapy trials. With the advent of genetic and molecular biological tools, the association between specific gene mutations and retinal signs has been made. Animals carrying natural mutations usually in one gene now provide well-established models for a host of inherited retinal diseases, including retinitis pigmentosa, Leber congenital amaurosis, inherited macular degeneration, and optic nerve diseases. In addition, the development of transgenic technologies has provided a means by which to study the effects of these and novel induced mutations on retinal structure and function. Despite these advances, there is a paucity of suitable animal models for complex diseases, including age-related macular degeneration (AMD) and diabetic retinopathy, largely because these diseases are not caused by single gene defects, but involve complex genetics and/or exacerbation through environmental factors, epigenetic, or other modes of genetic influence. In this review, we outline in detail the available animal models for inherited retinal diseases and how this information has furthered our understanding of retinal diseases. We also examine how transgenic technologies have helped to develop our understanding of the role of isolated genes or pathways in complex diseases like AMD, diabetes, and glaucoma., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recent imaging studies have suggested that macular pigment is decreased centrally in macular telangiectasia type 2 (MT2). The uptake of xanthophyll pigment into the macula is thought to be facilitated by a xanthophyll-binding protein (XBP). The Pi isoform of glutathione S-transferase (GSTP1) represents one such XBP with high binding affinity. This case-control study aimed to determine whether two common single-nucleotide polymorphisms (SNPs) of GSTP1 were associated with MT2. DNA samples from 39 cases and 21 controls were collected. Two polymorphic sites of Ile105Val and Ala114Val in exons 5 and 6 respectively, of the GSTP1 gene were analysed. Comparison of alleles and genotypes between cases and controls indicated that there were no statistically significant differences for either the Ile105Val SNP (P=0.43) or the Ala114Val SNP (P=0.85), or for any combinations; however, the homozygous at-risk genotype (GG) of the Ile105Val SNP was present in 8% of cases but absent in controls. This study found no statistically significant association between two common GSTP1 SNPs and MT2; however, a trend towards a greater frequency of the GG genotype of the Ile105Val SNP in cases is of great interest. The biological plausibility of disturbed macular pigment uptake in MT2 makes GSTP1 an excellent candidate gene. Further investigation is warranted in future studies of MT2.

Purpose: There is still a debate as to whether the LOC387715 or HTRA1 genes represent the key significant association identified with age-related macular degeneration (AMD) on the long arm of chromosome 10, region 26., Methods: An Australian patient cohort was genotyped by using tagged single nucleotide polymorphisms (tSNPs) to identify a causal SNP within this region., Results: Multiple tSNPs across the region showed association with AMD with the tSNP rs3793917 (odds ratio [OR], 3.45; 95% confidence interval [CI], 2.36-5.05, P = 2.8 × 10(-13)) having the highest association with AMD. This tSNP occurred in the intergenic region between the LOC387715 and HTRA1 genes. A second tSNP rs2672587 (OR, 2.92; 95% CI, 2.04-4.17; P = 7.7 × 10(-11)) located in intron 1 of the HTRA1 gene had the second highest association with AMD. After logistic regression analysis, the only tSNP to survive covariate testing was rs3793917, which occurred in the same LD block as the HTRA1 promoter SNP rs11200638 (r(2) = 0.88, D' = 0.97)., Conclusions: The findings indicate that the intergenic region between the tSNP rs3793917 and the SNP rs11200638 in the HTRA1 gene is the most likely site explaining the significant association with AMD.

Background: Retinal ganglion cell loss is considered to be a cause of visual impairment in Alzheimer;s patients. Alterations in amyloid precursor protein (APP) processing and amyloid-beta (Abeta) accumulation, key molecules associated with Alzheimer;s disease pathogenesis, may therefore contribute to retinal damage. We therefore investigated retinal APP processing and eye morphology in Alzheimer;s transgenic mouse models., Methods: Eyes and brain samples of 2- to 18-month-old transgenic mice expressing human APP with the double Swedish mutation (APPswe) (APP K595N/M596L)(Tg2576) were compared with eyes and brain tissue from wild-type background C57BL6xSJL controls. In addition, 6- to 12-month-old double transgenic mice over-expressing human APPswe and mutant presenilin 1 with exon 9 deletion (APPswe/PS1-dE9) were compared with background controls of C57BL6xC3H strain. Tissue samples were fixed in formalin for immunohistochemistry, and dissected retinal and cerebellar extracts were frozen for Western blotting and enzyme-linked immunosorbent assay (ELISA). Monoclonal antibodies 1E8 and WO2 were used for immunohistochemical detection of APP and Abeta, whereas Abeta 42/40 levels were assayed by ELISA. APP and processed fragments were detected biochemically by Western blotting with domain-specific antibodies, using antibody WO2 (Abeta) and rabbit antibody 369 to the C-terminal domain of APP., Results: Immunocytochemistry revealed strong cytoplasmic expression of APP and possibly Abeta in retinal ganglion cells and inner nuclear layer cells, and in lens and corneal epithelia for APP transgenic mice. Retinas from the APP transgenic mouse strains contained 18 to 70 kDa APP proteolytic products that were not detected in the cerebellum. We found a higher proportion of APP alpha-secretase generated C-terminal fragments in transgenic retinal tissues than beta-secretase-generated C-terminal fragments. Very low level Abeta was detected in transgenic retinas by ELISA; retinal Abeta 42 was 75 times less than for transgenic brain. Abeta was not detected in mouse retina by Western blotting in our study, indicating much less generation of Abeta in retina than brain tissue., Conclusions: Alzheimer's mouse model retinas present with different APP proteolytic products and have a significantly lower production of amyloidogenic Abeta than found in brain.

Age Related Macular Degeneration (AMD) is a complex neurodegenerative disorder accounting for 50% of blind registrations in the western world. Its substantial impact on quality of life has been a main driver in research to understand its etiology, which up until recently was mostly unknown. In the last three years our understanding of the molecular pathology of AMD has increased dramatically with the identification of two major AMD loci comprising of, Complement Factor H (CFH) and a chromosome 10q26 locus consisting of the Heat Shock Serine Protease (HTRA1) and LOC387715 genes. These two loci have been described as associated with over 50% of disease in certain ethnicities. The rapid pace in our understanding of the complex biology of this disease has placed a large emphasis on gene patenting, especially with the licensing of the CFH and chromosome 10 patents to a private life science company called Optherion Inc. The patents discussed in this review highlight the important discoveries that have contributed to our understanding of AMD and provide valuable information as to where research in this area will be heading in the future.

Purpose: Transforming growth beta-induced factor (TGIF) has been identified as a candidate gene for high myopia through genetic linkage studies and through its role in ocular growth in animal studies. However, the association of single nucleotide polymorphisms (SNPs), based solely on myopia refraction, has so far been inconclusive. This is the first study conducted to investigate the association of TGIF with refraction and ocular biometric measurements., Methods: Twelve tag SNPs (tSNPs) encompassing the TGIF gene and 2 kb upstream of its promoter region were used to evaluate the association between TGIF variants with both ocular biometric measures and refraction. A total of 257 cases of myopia (spherical equivalent [SE] worse than -0.50 D) and 294 control subjects (no myopia) were genotyped. Genotype frequencies were analyzed by chi(2) test and one-way ANOVA., Results: Two tSNPs showed significant association with biometric measures, with the SNP rs8082866 being associated with both axial length (P = 0.013) and corneal curvature (P = 0.007) and the SNP rs2020436 being associated with corneal curvature (P = 0.022). However, these associations became nonsignificant after multiple testing (Bonferroni correction)., Conclusions: Findings of this study suggest that the TGIF gene is unlikely to play a major role in either ocular biometric measures or refraction in a Caucasian population. Future studies should focus on other genes in the MYP2 linkage region or other linked regions to identify myopia-causing genes.

Purpose: Age-related macular degeneration (AMD) is the leading cause of poor vision in the developed world, and its pathogenesis remains unknown. The most devastating form of end stage disease is neovascular or wet AMD, where there is abnormal growth of new blood vessels under the retina. Vascular endothelial growth factor (VEGF) is thought to be a major player in the stimulus of this abnormal growth of blood vessels. We undertook a case-control association study to investigate the VEGF-A gene, a known angiogenic gene that has previously been associated with AMD., Methods: We recruited 577 individuals with AMD (early, atrophic, and neovascular AMD) and 173 ethnically matched controls for our study. We employed a tag-single nucleotide polymorphisms (tSNP) approach to investigate this gene using a series of seven tSNPs that encompassed the coding region of the VEGF gene as well as its promoter. Alleles were determined by a MALDI-TOF based approach followed by statistical analysis., Results: One SNP (rs3024997) showed evidence of departure from Hardy-Weinberg equilibrium in only the AMD cases. Therefore, it was retained for further analysis. All other SNPs in our study showed no departure from Hardy-Weinberg equilibrium. No association was found between any of the VEGF tSNPs analyzed in our study and AMD nor any of its sub-types., Conclusions: Using a tSNP approach, we found no evidence of an association of these SNPs within the VEGF-A gene being associated with either AMD or any of its subtypes in our population.

Purpose: Myopia (short sightedness) is a complex trait influenced by as yet unidentified genetic factors. To date, there have been four myopia susceptibility loci (MYP7 to -10) identified in twin studies, but these are yet to be independently verified. In an independent yet ethnically and phenotypically similar twin cohort, linkage to these chromosomal regions was sought., Methods: Participants were 223 dizygotic twin pairs from the Australian Twin Registry who were assessed for evidence of linkage, by using polymorphic microsatellite markers spanning MYP7-10. Data were analyzed by using Haseman-Elston regression analysis., Results: No evidence of linkage of myopia or its underlying biological components such as eye length to the MYP7-10 regions was found in this twin cohort., Conclusions: This is the first study to assess for linkage in a secondary myopia twin cohort and highlights the problems associated with applying linkage results from complex traits to the other populations.

Purpose: Myopia is a common disorder with a large public health impact. Although 12 myopia loci have been reported and heterogeneity for high myopia loci have been demonstrated, replication of high-myopia loci with a common myopia phenotype has not been successful. This study reports the successful replication of MYP12 in three large, multigenerational families with autosomal dominant (AD) common myopia (spherical equivalent [SphE]

Background: Whole blood provides one of the most common sources of both high-quality DNA and high-quantity DNA for molecular biological purposes. Typically, blood storage at 4 degrees C is short term, which ranges from a few days to a few weeks. However, long-term storage usually involves blood being frozen, with a resultant loss in DNA yield. The authors examined the effects of long-term storage at 4 degrees C., Methods: Duplicate blood samples were collected from 301 participants (aged 20-98 years) enrolled as part of ongoing studies. Samples were stored at 4 degrees C for between 11 days and 922 days, and DNA was subsequently extracted using a phenol/chloroform procedure., Results: A negative correlation of the number of storage days existed at 4 degrees C with DNA yield. The main determinant on DNA yield was the age of the participant in the study, with older persons having a lower DNA yield., Conclusions: Long-term storage of blood at 4 degrees C does have a detrimental effect on DNA yield, but this effect seemed less significant than the age of a person. The impact of age of a person or storage time has a minimal impact on DNA quality. Therefore, storage of blood at 4 degrees C offers an acceptable alternative to frozen storage.

Purpose: To date, of all the genes studied in relation to age-related macular degeneration (AMD), the alleles of the apolipoprotein (apoE) gene have been the most consistently associated with disease. However, not all apoE studies have found an association, and among these the associations differ. The current study was conducted to investigate further the association of this gene in AMD., Methods: Three hundred twenty-two unrelated individuals with diagnosed AMD and 123 unrelated but ethnically matched control subjects were analyzed. All subjects completed a standard questionnaire and were given a fundus examination. A blood sample was collected for DNA extraction. The common allelic variants of apoE were screened through the use of polymerase chain reaction (PCR) and restriction enzyme digestion followed by statistical analysis., Results: Individuals with the epsilon3 epsilon4 genotype of apoE had an approximate halving of disease risk for late (end-stage) AMD (odds ratio [OR] 0.58, 95% confidence interval [CI] 0.34-0.98) relative to the epsilon3 epsilon3 genotype at age of ascertainment. Stratification of late AMD into atrophic and neovascular disease revealed that the greatest protective effect for the epsilon3 epsilon4 genotype was in individuals with atrophic disease (OR 0.35, 95% CI 0.13-0.92). Men with the epsilon3 epsilon4 genotype also showed almost a threefold reduction in risk of disease in late AMD (OR 0.36, 95% CI 0.16-0.82). However, individuals with late AMD and the epsilon2 epsilon3 genotype had a significantly earlier mean age of diagnosis of disease (3.4 years, P = 0.015) compared with those with the epsilon3 epsilon3 genotype, and this was most evident in women (3.9 years, P = 0.011) and in individuals with neovascular disease (4.7 years, P = 0.003)., Conclusions: The alleles of apoE appear to have a role in the etiology of AMD, with the epsilon4 allele being protective, or at the very least, delaying the age of diagnosis of disease, whereas the epsilon2 allele appears to have a modifier effect by bringing forward the mean age of disease diagnosis.