Eintracht, Jonathan; (2023) Patient-specific stem cell-derived optic vesicles reveal novel pathogenic variants and common disease mechanisms in microphthalmia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Human eye development is tightly regulated by gene regulatory networks (GRNs) that guide early eye structures through morphogenetic events from optic vesicle formation to the maturation of complex specialised cell types in the retina that is only completed several months after birth. Disruptions to these GRNs and key regulatory genes can result in severe ocular malformation on a spectrum known as microphthalmia, anophthalmia and coloboma (MAC). Over 90 genes are associated with MAC, with the most frequently mutated genes encoding early eye transcription factors (EFTFs) that are responsible for most syndromic forms of MAC. These forms, alongside with bilateral cases, are more likely to receive a genetic diagnosis than unilateral cases. However, genetic diagnosis rates for MAC are still low (~20-30%) and the role of many associated genes in eye development is not clearly understood. Identifying novel variants in unsolved cases as well as dissecting common disease mechanism between patients with similar phenotypes but different genotypes will broaden the diagnostic scope for more extensive MAC clinical diagnosis. This thesis aims to identify a causative mutation in an unsolved multi-generational family with phenotypic variability and common disrupted pathways between the proband (known as BM) and an unrelated PAX6-associated microphthalmia p.(Asn124Lys) patient known as PX. The proband (BM) was diagnosed with bilateral microphthalmia and coloboma and his father (known as UM) was diagnosed with unilateral microphthalmia and coloboma. Using whole genome sequencing, a novel nonsense variant was identified in TEAD2 (c.47G>A; p.Trp16*) that segregated with disease phenotype. Additionally, TEAD2 expression was lost in patient optic vesicles at day 20. To investigate changes to developmental pathways in MAC, hiPSC-derived optic vesicles were generated from patient fibroblasts. At day 20 and 35, patient optic vesicles exhibited significant changes in known mRNA/protein early ocular markers such as transcription factors PAX6, MITF and VSX2. RNA-seq analysis showed disruptions to ocular developmental pathways modulated through NR2F1, suggesting possible mechanisms for ocular malformation in this family. Immunohistochemistry revealed reduced expression and altered expression patterns of key transcription factors and regulators RAX and YAP1 at day 20 and VSX2 and MITF at day 35 in patient optic vesicles. These data suggest molecular disruption of ocular development due to a novel TEAD2 nonsense variant. Shared disruptions in microphthalmia patients were investigated using optic vesicles derived from BM and a PAX6-associated microphthalmia p.(Asn124Lys) patient known as PX. At day 35, vesicle diameter was significantly reduced in both microphthalmia models compared to healthy controls. TUNEL staining revealed a significant increase in apoptotic cells in patient vesicles compared to healthy controls while PH3 staining revealed decreased cell proliferation. Upregulation of pro-apoptotic genes was identified in patient optic vesicles through RNA-seq analysis. A caspase-8 assay also highlighted increased expression of apoptosis initiator caspase 8 in microphthalmia optic vesicles. Additionally, RNA-seq analysis identified increased production of extracellular matrix (ECM) components such as collagen and laminin in microphthalmia optic vesicles that resulted in ECM abnormalities. The overproduction of ECM may overly constrict the growing eye, resulting in ocular malformation such as microphthalmia and coloboma that are dependent on biophysical cues guiding eye morphogenesis.

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