Genomic analysis of microphthalmia, anophthalmia and coloboma

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SUMMARY Microphthalmia, anophthalmia and coloboma (MAC) are highly important birth defects because of the lifelong effects of severely reduced vision. MAC can be caused by pathogenic sequence variants and copy number variants affecting transcription factors and other genes involved in eye development. Patients with MAC typically undergo genetic testing with microarrays, gene panels and exome sequencing, but a genetic etiology is not identified for many patients. A complete catalogue of the genomic variation underlying MAC is critical for optimizing patient care, facilitating future therapies, and providing information to support research into the developmental biology of eye defects. In this proposal, we will use whole genome sequencing (WGS) and in vitro and in vivo functional studies to advance our understanding of the genomic architecture of MAC. Our first Aim involves the use of WGS to obtain complete genomic data, including coding and non-coding sequence variants, copy number variants and structural cytogenetic variants, in a minimum of 200 patients with MAC and their biological parents. We will prioritize patients with MAC who have had negative prior genetic testing, multiple affected family members, consanguineous parents and bilateral eye involvement to increase our changes of identifying novel variants and genes. This aim will enable us to investigate the full range of genomic variation in MAC and to determine the contribution of non-coding variation and structural cytogenetic variation to the genetic etiology of these birth defects. In our second Aim, we will use zebrafish with gene editing and CRISPR/Cas9 to investigate the effects of loss and gain of function for novel candidate genes for MAC and to determine the effects of specific sequence variants on eye development. We will comprehensively phenotype crispant and control larvae and perform RNA-Seq to study alterations in downstream gene expression. We will also study non-coding variation by performing bioinformatic studies, minigene assays, RT-PCR in patient cells as appropriate, and dual-color reporter transgenesis assays in zebrafish. Our results will generate comprehensive data on the genomic variation that can cause MAC and will provide functional validation of novel genes and variants. This proposal will substantially improve our understanding of eye development, in addition to generating rich resources for future investigations and collaborations between clinicians, developmental biologists and eye researchers.