Paul G. Sanfilippo

Genome-wide association identifies ATOH7 as a major gene determining human optic disc size

Optic nerve assessment is important for many blinding diseases, with cup-to-disc ratio (CDR) assessments commonly used in both diagnosis and progression monitoring of glaucoma patients. Optic disc, cup, rim area and CDR measurements all show substantial variation between human populations and high heritability estimates within populations. To identify loci underlying these quantitative traits, we performed a genome-wide association study in two Australian twin cohorts and identified rs3858145, P = 6.2 × 10−10, near the ATOH7 gene as associated with the mean disc area. ATOH7 is known from studies in model organisms to play a key role in retinal ganglion cell formation. The association with rs3858145 was replicated in a cohort of UK twins, with a meta-analysis of the combined data yielding P = 3.4 × 10−10. Imputation further increased the evidence for association for several SNPs in and around ATOH7 (P = 1.3 × 10−10 to 4.3 × 10−11, top SNP rs1900004). The meta-analysis also provided suggestive evidence for association for the cup area at rs690037, P = 1.5 × 10−7, in the gene RFTN1. Direct sequencing of ATOH7 in 12 patients with optic nerve hypoplasia, one of the leading causes of blindness in children, revealed two novel non-synonymous mutations (Arg65Gly, Ala47Thr) which were not found in 90 unrelated controls (combined Fishers exact P = 0.0136). Furthermore, the Arg65Gly variant was found to have very low frequency (0.00066) in an additional set of 672 controls.

The Heritability of Ocular Traits

Heritability is the proportion of phenotypic variation in a population that is attributable to genetic variation among individuals. Many ophthalmic disorders and biometric traits are known to have a genetic basis and consequently much work has been published in the literature estimating the heritability of various ocular parameters. We collated and summarized the findings of heritability studies conducted in the field of ophthalmology. We grouped the various studies broadly by phenotype as follows: refraction, primary open-angle glaucoma, age-related macular degeneration (AMD), cataract, diabetic retinopathy, and others. A total of 82 articles were retrieved from the literature relating to estimation of heritability for an ocular disease or biometric trait; of these, 37 papers were concerned with glaucoma, 28 with refraction, 4 with AMD, 5 with diabetic retinopathy, and 4 with cataract. The highest reported heritability for an ophthalmic trait is 0.99 for the phenotype ≥ 20 small hard drusen, indicating that observed variation in this parameter is largely governed by genetic factors. Over 60% of the studies employed a twin study design and a similar percentage utilized variance components methods and structural equation modeling (SEM) to derive their heritability values. Using modern SEM techniques, heritability estimates derived from twin subjects were generally higher than those from family data. Many of the estimates are in the moderate to high range, but to date the majority of genetic variants accounting for these findings have not been uncovered, hence much work remains to be undertaken to elucidate fully their molecular etiology.

Twins Eye Study in Tasmania (TEST): Rationale and Methodology to Recruit and Examine Twins

Visual impairment is a leading cause of morbidity and poor quality of life in our community. Unravelling the mechanisms underpinning important blinding diseases could allow preventative or curative steps to be implemented. Twin siblings provide a unique opportunity in biology to discover genes associated with numerous eye diseases and ocular biometry. Twins are particularly useful for quantitative trait analysis through genome-wide association and linkage studies. Although many studies involving twins rely on twin registries, we present our approach to the Twins Eye Study in Tasmania to provide insight into possible recruitment strategies, expected participation rates and potential examination strategies that can be considered by other researchers for similar studies. Five separate avenues for cohort recruitment were adopted: (1) piggy-backing existing studies where twins had been recruited, (2) utilizing the national twin registry, (3) word-of-mouth and local media publicity, (4) directly approaching schools, and finally (5) collaborating with other research groups studying twins.

Meta-analysis of Genome-Wide Association Studies Identifies Novel Loci Associated With Optic Disc Morphology

Primary open‐angle glaucoma is the most common optic neuropathy and an important cause of irreversible blindness worldwide. The optic nerve head or optic disc is divided in two parts: a central cup (without nerve fibers) surrounded by the neuroretinal rim (containing axons of the retinal ganglion cells). The International Glaucoma Genetics Consortium conducted a meta‐analysis of genome‐wide association studies consisting of 17,248 individuals of European ancestry and 6,841 individuals of Asian ancestry. The outcomes of the genome‐wide association studies were disc area and cup area. These specific measurements describe optic nerve morphology in another way than the vertical cup‐disc ratio, which is a clinically used measurement, and may shed light on new glaucoma mechanisms. We identified 10 new loci associated with disc area (CDC42BPA, F5, DIRC3, RARB, ABI3BP, DCAF4L2, ELP4, TMTC2, NR2F2, and HORMAD2) and another 10 new loci associated with cup area (DHRS3, TRIB2, EFEMP1, FLNB, FAM101, DDHD1, ASB7, KPNB1, BCAS3, and TRIOBP). The new genes participate in a number of pathways and future work is likely to identify more functions related to the pathogenesis of glaucoma.

New insights into the genetics of primary open-angle glaucoma based on meta-analyses of intraocular pressure and optic disc characteristics

Primary open-angle glaucoma (POAG), the most common optic neuropathy, is a heritable disease. Siblings of POAG cases have a ten-fold increased risk of developing the disease. Intraocular pressure (IOP) and optic nerve head characteristics are used clinically to predict POAG risk. We conducted a genome-wide association meta-analysis of IOP and optic disc parameters and validated our findings in multiple sets of POAG cases and controls. Using imputation to the 1000 genomes (1000G) reference set, we identified 9 new genomic regions associated with vertical cup-disc ratio (VCDR) and 1 new region associated with IOP. Additionally, we found 5 novel loci for optic nerve cup area and 6 for disc area. Previously it was assumed that genetic variation influenced POAG either through IOP or via changes to the optic nerve head; here we present evidence that some genomic regions affect both IOP and the disc parameters. We characterized the effect of the novel loci through pathway analysis and found that pathways involved are not entirely distinct as assumed so far. Further, we identified a novel association between CDKN1A and POAG. Using a zebrafish model we show that six6b (associated with POAG and optic nerve head variation) alters the expression of cdkn1a. In summary, we have identified several novel genes influencing the major clinical risk predictors of POAG and showed that genetic variation in CDKN1A is important in POAG risk.

What is the appropriate age cut-off for cycloplegia in refraction?

To investigate the age range for which cycloplegia provides additional information compared with non‐cycloplegic refraction in teenagers and young adults.

Vitreous biomarkers in diabetic retinopathy: a systematic review and meta-analysis.

The aim of this study was to perform a systematic meta-analysis of biomarkers investigated with diabetic retinopathy (DR) in the vitreous, and to explore the molecular pathway interactions of these markers found to be consistently associated with DR. Relevant databases [PubMed and ISI web of science] were searched for all published articles investigating molecular biomarkers of the vitreous associated with DR. Based on set exclusion/inclusion criteria available data from studies with human vitreous samples were extracted and used for our meta-analysis. The interactions of significant biomarkers in DR were investigated via STRING and KEGG pathway analysis. Our meta-analysis of DR identifies eleven biomarkers as potential therapeutic candidates alternate to current anti-VEGF therapy. Four of these are deemed viable therapeutic targets for PDR; ET receptors (ET A and ET B), anti-PDGF-BB, blocking TGF-β using cell therapy and PEDF. The identification of supplementary or synergistic therapeutic candidates to anti VEGF in the treatment of DR may aid in the development of future treatment trials.

Pellucid marginal degeneration and scleroderma

Systemic scleroderma is a progressive multi‐system connective tissue disease. Ocular involvement includes keratoconjunctivitis sicca, progressive shallowing of conjunctival fornices, peripheral ulcerative keratitis and eyelid tightness. No association has been reported between scleroderma and pellucid marginal degeneration, which is a rare bilateral corneal ectasia. Pellucid marginal degeneration is characterised by noninflammatory and progressive peripheral corneal thinning inferiorly, often with high against‐the‐rule astigmatism. We report a case of a 55‐year‐old woman with systemic scleroderma who presented with rapidly progressing against‐the‐rule astigmatism. The differential diagnosis of peripheral corneal thinning and the challenge of the surgical management of pellucid marginal degeneration are briefly discussed.

Ophthalmic Phenotypes and the Representativeness of Twin Data for the General Population

PURPOSE To compare the distributional parameters for a series of ocular biometric traits between twins and their singleton siblings, to evaluate the generalizability of twin data, as used in heritability analyses to the general population. METHODS A series of birth, anthropometric, and 13 ocular biometric traits were selected for analysis: interpupillary distance (IPD), visual acuity (logMAR), spherical equivalent refractive error, corneal curvature, axial length, anterior chamber depth (ACD), central corneal thickness (CCT), intraocular pressure (IOP), optic disc, cup and rim areas, and measures of retinal vessel caliber; central retinal arteriolar equivalent (CRAE), and central retinal venular equivalent (CRVE). Structural equation modeling was used to test the assumption that the means and variances for each trait did not differ between twins and their siblings. RESULTS Significant differences in log-likelihood for birth weight and gestational age were observed between twins and siblings, with the latter being both heavier and closer to full-term at birth. Siblings were also found to have larger IPD and axial length, and better visual acuity compared with their twin counterparts. Refractive error, corneal curvature, ACD, CCT, optic disc parameters, and retinal vascular calibers did not differ significantly between the two groups. CONCLUSIONS Twins are representative of the general population for some but not all measures of ocular biometry. Consequently, care should be taken when extrapolating twin data for these traits in heritability and other genetic studies. Birth weight differences between twins and siblings do not appear to account for the differences in ocular biometry observed in this study.

A geometric morphometric assessment of the optic cup in glaucoma.

The morphologic appearance of the optic disc is of interest in glaucoma. In contrast to descriptive classification systems that are currently used, a quantitative approach to the analysis of optic disc morphology is required. Our goal was to determine the optimal method for quantifying optic cup shape by comparing traditional (ovality, form-factor and neuroretinal rim (NRR) width ratio) and geometric morphometric approaches. Left optic disc stereophotographs of 160 (80 normal and 80 glaucomatous (stratified by severity)) subjects were examined. The optic cup margins were stereoscopically delineated with a custom tracing system and saved as a series of discrete points. The geometric morphometric methods of elliptic Fourier analysis (EFA) and sliding semi-landmark analysis (SSLA) were used to eliminate variation unrelated to shape (e.g. size) and yield a series of shape variables. Differences in optic cup shape between normal and glaucoma groups were investigated. Discriminant functions were computed and the sensitivity and specificity of each technique determined. Receiver operator characteristic (ROC) curves were calculated for all methods and evaluated in their potential to discriminate between normal and glaucomatous eyes based on the shape variables. All geometric morphometric methods revealed differences between normal and glaucomatous eyes in optic cup shape, in addition to the traditional parameters of ovality, form-factor and NRR width ratio (p<0.0005). SSLA (minimum bending energy criterion--18 points) had the best sensitivity (83%) and area under the curve (AUC) (0.91). EFA (72 points) performed similarly well (74%, 0.89) as did the set of traditional shape-based variables (76%, 0.86). This study demonstrated that a geometric morphometric approach for discriminating between normal and glaucomatous eyes in optic cup shape is superior to that provided by traditional single parameter shape measures. Such analytical techniques could be incorporated into future automated optic disc screening modalities.