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Human eye development is coordinated through an extensive network of genetic signalling pathways. Disruption of key regulatory genes in the early stages of eye development can result in aborted eye formation, resulting in an absent eye (anophthalmia) or a small underdeveloped eye (microphthalmia) phenotype. Anophthalmia and microphthalmia (AM) are part of the same clinical spectrum and have high genetic heterogeneity, with >90 identified associated genes. By understanding the roles of these genes in development, including their temporal expression, the phenotypic variation associated with AM can be better understood, improving diagnosis and management. This review describes the genetic and structural basis of eye development, focusing on the function of key genes known to be associated with AM. In addition, we highlight some promising avenues of research involving multiomic approaches and disease modelling with induced pluripotent stem cell (iPSC) technology, which will aid in developing novel therapies.

Retinal photoreceptors are amongst the most metabolically active cells in the body, consuming more glucose as a metabolic substrate than even the brain. This ensures that there is sufficient energy to establish and maintain photoreceptor functions during and after their differentiation. Such high dependence on glucose metabolism is conserved across vertebrates, including zebrafish from early larval through to adult retinal stages. As the zebrafish retina develops rapidly, reaching an adult-like structure by 72 hours post fertilisation, zebrafish larvae can be used to study metabolism not only during retinogenesis, but also in functionally mature retinae. The interplay between rod and cone photoreceptors and the neighbouring retinal pigment epithelium (RPE) cells establishes a metabolic ecosystem that provides essential control of their individual functions, overall maintaining healthy vision. The RPE facilitates efficient supply of glucose from the choroidal vasculature to the photoreceptors, which produce metabolic products that in turn fuel RPE metabolism. Many inherited retinal diseases (IRDs) result in photoreceptor degeneration, either directly arising from photoreceptor-specific mutations or secondary to RPE loss, leading to sight loss. Evidence from a number of vertebrate studies suggests that the imbalance of the metabolic ecosystem in the outer retina contributes to metabolic failure and disease pathogenesis. The use of larval zebrafish mutants with disease-specific mutations that mirror those seen in human patients allows us to uncover mechanisms of such dysregulation and disease pathology with progression from embryonic to adult stages, as well as providing a means of testing novel therapeutic approaches.

Animal models have provided many insights into ocular development and disease, but they remain suboptimal for understanding human oculogenesis. Eye development requires spatiotemporal gene expression patterns and disease phenotypes can differ significantly between humans and animal models, with patient-associated mutations causing embryonic lethality reported in some animal models. The emergence of human induced pluripotent stem cell (hiPSC) technology has provided a new resource for dissecting the complex nature of early eye morphogenesis through the generation of three-dimensional (3D) cellular models. By using patient-specific hiPSCs to generate in vitro optic vesicle-like models, we can enhance the understanding of early developmental eye disorders and provide a pre-clinical platform for disease modelling and therapeutics testing. A major challenge of in vitro optic vesicle generation is the low efficiency of differentiation in 3D cultures. To address this, we adapted a previously published protocol of retinal organoid differentiation to improve embryoid body formation using a microwell plate. Established morphology, upregulated transcript levels of known early eye-field transcription factors and protein expression of standard retinal progenitor markers confirmed the optic vesicle/presumptive optic cup identity of in vitro models between day 20 and 50 of culture. This adapted protocol is relevant to researchers seeking a physiologically relevant model of early human ocular development and disease with a view to replacing animal models.

Social monogamy has evolved multiple times and is particularly common in birds. However, it is not well understood why some species live in long‐lasting monogamous partnerships while others change mates between breeding attempts. Here, we investigate mate fidelity in a sequential polygamous shorebird, the snowy plover (Charadrius nivosus), a species in which both males and females may have several breeding attempts within a breeding season with the same or different mates. Using 6 years of data from a well‐monitored population in Bahía de Ceuta, Mexico, we investigated predictors and fitness implications of mate fidelity both within and between years. We show that in order to maximize reproductive success within a season, individuals divorce after successful nesting and re‐mate with the same partner after nest failure. Therefore, divorced plovers, counterintuitively, achieve higher reproductive success than individuals that retain their mate. We also show that different mating decisions between sexes predict different breeding dispersal patterns. Taken together, our findings imply that divorce is an adaptive strategy to improve reproductive success in a stochastic environment. Understanding mate fidelity is important for the evolution of monogamy and polygamy, and these mating behaviors have implications for reproductive success and population productivity. In sequential polygamous snowy plover, with limited breeding time, individuals divorce after successful nesting and re‐mating to the same partner after nest failure to maximize reproductive success within a season.

Background/aimsMicrophthalmia, anophthalmia and coloboma (MAC) are clinically and genetically heterogenous rare developmental eye conditions, which contribute to a significant proportion of childhood blindness worldwide. Clear understanding of MAC aetiology and comorbidities is essential to providing patients with appropriate care. However, current management is unstandardised and molecular diagnostic rates remain low, particularly in those with unilateral presentation. To further understanding of clinical and genetic management of patients with MAC, we charted their real-world experience to ascertain optimal management pathways and yield from molecular analysis.MethodsA prospective cohort study of consecutive patients with MAC referred to the ocular genetics service at Moorfields Eye Hospital between 2017–2020.ResultsClinical analysis of 50 MAC patients (15 microphthalmia; 2 anophthalmia; 11 coloboma; and 22 mixed) from 44 unrelated families found 44% had additional ocular features (complex) and 34% had systemic involvement, most frequently intellectual/developmental delay (8/17). Molecular analysis of 39 families using targeted gene panels, whole genome sequencing and microarray comparative genomic hybridisation identified genetic causes in, 28% including novel variants in six known MAC genes (SOX2, KMT2D, MAB21L2, ALDH1A3, BCOR and FOXE3), and a molecular diagnostic rate of 33% for both bilateral and unilateral cohorts. New phenotypic associations were found for FOXE3 (bilateral sensorineural hearing loss) and MAB21L2 (unilateral microphthalmia).ConclusionThis study highlights the importance of thorough clinical and molecular phenotyping of MAC patients to provide appropriate multidisciplinary care. Routine genetic testing for both unilateral and bilateral cases in the clinic may increase diagnostic rates in the future, helping elucidate genotype–phenotype correlations and informing genetic counselling.

EPHA2 is a transmembrane tyrosine kinase receptor that, when disrupted, causes congenital and age-related cataracts. Cat-Map reports 22 pathogenic EPHA2 variants associated with congenital cataracts, variable microcornea, and lenticonus, but no previous association with microphthalmia (small, underdeveloped eye, ≥2 standard deviations below normal axial length). Microphthalmia arises from ocular maldevelopment with >90 monogenic causes, and can include a complex ocular phenotype. In this paper, we report two pathogenic EPHA2 variants in unrelated families presenting with bilateral microphthalmia and congenital cataracts. Whole genome sequencing through the 100,000 Genomes Project and cataract-related targeted gene panel testing identified autosomal dominant heterozygous mutations segregating with the disease: (i) missense c.1751C>T, p.(Pro584Leu) and (ii) splice site c.2826-9G>A. To functionally validate pathogenicity, morpholino knockdown of epha2a/epha2b in zebrafish resulted in significantly reduced eye size ± cataract formation. Misexpression of N-cadherin and retained fibre cell nuclei were observed in the developing lens of the epha2b knockdown morphant fish by 3 days post-fertilisation, which indicated a putative mechanism for microphthalmia pathogenesis through disruption of cadherin-mediated adherens junctions, preventing lens maturation and the critical signals stimulating eye growth. This study demonstrates a novel association of EPHA2 with microphthalmia, suggesting further analysis of pathogenic variants in unsolved microphthalmia cohorts may increase molecular diagnostic rates.

Name of the disease (synonyms)See Table 1, Column 1—“Name of disease” and Column 2—“Alternative names”.OMIM# of the diseaseSee Table 1, Column 3—“OMIM# of the disease”.Name of the analysed genes or DNA/chromosome segments and OMIM# of the gene(s)Core genes (irrespective of being tested by Sanger sequencing or next-generation sequencing):See Table 1, Column 4—“Cytogenetic location”, Column 5—“Associated gene(s)” and Column 6—“OMIM# of associated gene(s)”.Additional genes (if tested by next-generation sequencing, including Whole exome/genome sequencing and panel sequencing):See Table 2, Column 1—“Gene”, Column 2—“Alternative names”, Column 3—“OMIM# of gene” and Column 4—“Cytogenetic location”.Review of the analytical and clinical validity as well as of the clinical utility of DNA-based testing for mutations in the gene(s) in diagnostic, predictive and prenatal settings, and for risk assessment in relatives.

Human eye development is coordinated through an extensive network of signalling pathways. Disruption of key regulatory genes can perturb eye formation, resulting in microphthalmia (small, underdeveloped eye). High phenotypic and genetic heterogeneity is observed in patients, with associated variants in >90 genes. However, molecular diagnostic rates remain low (20-30%) with few recognised genotype-phenotype correlations. Moreover, the roles of many associated genes are poorly understood. Identifying additional molecular causes and clarifying complex genetic mechanisms could elucidate microphthalmia pathogenesis and explain clinical variation, improving diagnosis and management. This thesis aims to develop molecular understanding of microphthalmia through clinical and genetic evaluation of 50 MAC (microphthalmia, anophthalmia and coloboma) patients, as well as creation and analysis of a patient-derived cellular model. Using a combination of genetic testing techniques, molecular causes were detected in 33% of patients, including a novel association of EPHA2 with microphthalmia in two unrelated families, validated through zebrafish knockdowns. Another patient with a complex ocular phenotype including bilateral microphthalmia and aniridia was diagnosed with a heterozygous missense variant PAX6 c.372C>A p.(Asn124Lys). To investigate molecular pathways involved in PAX6-associated microphthalmia, 3D hiPSC optic vesicle-like models of early eye development were generated from patient fibroblasts. Patient-derived microphthalmia models displayed significant mRNA/protein disruption of established markers at 20 and 35 days of differentiation, including early eye transcription factors PAX6, RAX, OTX2 and SOX2 and neural retina progenitor marker VSX2, reflecting the early-stage disease phenotype observed in patients. Total RNA-seq transcriptome-wide profiling characterised global gene expression changes, identifying significant disruption in transcription factors, Notch signalling and cell cycle regulators, potentially causing the variant-specific microphthalmia phenotype in PAX6 p.(Asn124Lys) patients. In summary, this thesis presents an in vitro model of PAX6-related microphthalmia accompanied by transcriptomic analysis which provides novel insights into PAX6 regulation of eye development and the molecular basis of microphthalmia.

The human macula is a specialized, cone-rich region of the eye, critical for high-acuity vision, yet the pathways regulating its development remain poorly understood. RA-catabolizing enzyme CYP26A1 establishes the chick high-acuity area via upregulation of FGF8. However, detailed analysis of this pathway and its functions has not been performed in early human fetal tissue. Fluorescent in situ hybridization revealed striking biphasic CYP26A1 expression but little FGF8 in the presumptive macula region between post-conception weeks (PCW)6-17. Pharmacological RA signaling inhibition in human retinal organoids mimicking the two waves of CYP26A1 revealed early RA inhibition prompted early cell cycle exit and increased cone genesis, while late inhibition altered cone subtype specification. Conversely, recombinant FGF8 had no effect on photoreceptor fate. This work provides spatiotemporal examination of CYP26A1 across human macular development, as well as experimental evidence for the different roles of RA signaling inhibition in a human model of retinal development.

Aniridia is a rare, pan-ocular disease causing severe sight loss, with only symptomatic intervention offered to patients. Approximately 40% of aniridia patients present with heterozygous nonsense variants in PAX6, resulting in haploinsufficiency. Translational readthrough inducing compounds (TRIDs) have the ability to weaken the recognition of in-frame premature stop codons (PTCs), permitting full-length protein to be translated. We have established induced pluripotent stem cell (iPSC)-derived 3D optic cups and 2D limbal epithelial stem cell (LESC) models from an aniridia patient with a prevalent PAX6 nonsense mutation. Both in vitro models show reduced PAX6 protein levels, mimicking the disease. Repurposed TRIDs amlexanox and 2,6-diaminopurine (DAP), and positive control compounds ataluren and G418 were tested for their efficiency. Amlexanox was identified as the most promising TRID, increasing full-length PAX6 levels in both models, and rescuing the disease phenotype through normalization of VSX2 and cell proliferation in the optic cups and reduction of ABCG2 protein and SOX10 expression in LESC. This study highlights the significance of patient iPSC-derived cells as a new model system for aniridia and proposes amlexanox as a new putative treatment for nonsense-mediated aniridia. Competing Interest Statement The authors have declared no competing interest.