Ocular maldevelopment is responsible for more than one-third of blindness and severe visual impairment in children worldwide. It encompasses whole eyeball abnormalities e.g. microphthalmia [abnormal small eye], anophthalmia [complete absence of the eye] and ocular coloboma [cleft of the eye], collectively known as MAC. It is the most common cause of childhood sight impairment certification in England and Wales, accounting for 18.4%. The UK incidence of MAC is 10.4 per 100,000 live births, with only 2% considered related to environmental factors e.g. maternally vitamin A deficiency and teratogenic exposure.
There are approximately 90 genes known to cause non-syndromic and syndromic MAC, accounting for only 6% of genetic diagnoses. SOX2 and OTX2 are responsible for 60% of the severe bilateral cases of anophthalmia and microphthalmia. Little is known about the dysfunction of these key genes acting between 4-7 weeks gestation, as access to human ocular tissue is near impossible at this critical stage of early eye development. As a result there have been no comprehensive human studies investigating normal and abnormal ocular development as no suitable model exists. Most work is undertaken on animal models but these are suboptimal for several reasons: (i) the eye develops differently, for example the zebrafish optic and lens vesicles are solid structures that cavitate, unlike the human equivalent which are hollow, (ii) some genes fail to generate a comparable phenotype when disrupted due to potential redundancy, or (ii) cause embryonic lethality if knocked out preventing further study.
Of the families with a positive genetic diagnosis there is significant inter-familial phenotypic variability e.g. some affected members display a unilateral anophthalmia, others a bilateral microphthalmia and some unaffected family members harbour the mutation but with non-penetrance. This proposal aims to discover and understand the role of genetic modifiers in ocular maldevelopment. We will replace the use of zebrafish and mouse models and optimise the use of induced pluripotent stem cell (iPSC) technology to derive human in vitro 3D optic vesicles. We will dissect the molecular pathways disrupted by an OTX2 mutation (c.97+1delG intron 3) in a three-generation family displaying extremes of phenotypic variability. We will use CRISPR/Cas9 gene editing to correct the mutation in one patient’s iPSC to observe reversal of the phenotype in the human model to determine whether the mutation is unequivocally causative or not. Comparative analysis of the transcriptome in optic vesicles will provide information on the gene expression profiles and help to identify potential predisposing/protective genetic modifiers that could be exploited to develop a therapy and immediately inform genetic counselling for families.
Identifying genetic modifiers will allow us to investigate the interplay between genotype, gene expression and phenotype in early human eye development and provide useful genetic tools for future human organoid cultures. It will demonstrate the efficacy of an in vitro system to study gene function. Mouse, zebrafish and xenopus geneticists and developmental biologists will be encouraged to use organoid cultures as replacement for in vivo studies for a more accurate representation of human eye development.