Harding, Philippa; (2022) Cellular modelling of human optic vesicles to dissect the genetic basis of microphthalmia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

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.

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