Microphthalmia, anophthalmia and coloboma (MAC) and retinoic acid pathway genes

SUMMARY Microphthalmia, anophthalmia and coloboma (MAC) are a group of clinically and genetically related eye defects that cause significant visual impairment. MAC can be caused by pathogenic variants in transcription factors and other genes involved in eye development and by environmental factors such as maternal vitamin A deficiency

(VAD). Vitamin A is critical for retinoic acid (RA) synthesis and pathogenic variants causing loss or gain of function for the genes in the RA pathway, such as STRA6, can result in severe MAC. We hypothesize that genetic variation in the RA pathway genes and genes involved in retinol metabolism can result in a predisposition, or lower

threshold, for the deleterious effects of VAD on eye development that contributes to the phenotypic severity of MAC. This gene-environment interaction is highly relevant for countries where VAD is common and remains a public health concern, such as India. In addition, the downstream genes that are dysregulated by lack of vitamin

A are poorly understood and are amenable to study in animal models. To identify the full spectrum of genetic variation in the RA pathway and other genes in patients with MAC, our first Aim will recruit patients with MAC and perform detailed phenotyping, environmental screening, and imaging so that we can generate accurate

phenotype genotype correlations. We will generate, analyze and compare whole genome sequencing (GS) data from our collaborators, Dr. Tibrewal and Dr. Kumar, in patients of Asian ancestry with our studies using GS in patients with MAC of predominantly European ancestry. We will also prospectively recruit patients with MAC

diagnosed in the newborn period from centers in the USA, so that we can obtain retinol levels from umbilical cord blood in babies with MAC and retinol levels in maternal plasma around the time of delivery. With this data, we will compare the severity of MAC with sequence variants that we observe in the RA pathway genes and in other

eye developmental genes, for patients of different ancestries, with and without evidence of VAD from symptoms or from low levels of retinol. In our second Aim, we will use gene-editing with Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 to determine the effects of altered function for stra6 and other genes in the RA

pathway on ocular morphogenesis in zebrafish. All crispants will have detailed phenotyping at different timepoints during eye development, including gene expression studies with single cell RNA-Seq, to determine the downstream genes that are regulated by RA. We will also investigate if we can rescue, or partially rescue, the

deleterious effects of genetic variants in stra6 and other genes causing MAC on eye morphogenesis by titrating levels of RA and retinol during periods of ocular development in Danio rerio. With this Aim, we will use an animal model to generate data regarding downstream genes, tissues and developmental timepoints that are affected by

deficiencies of RA and vitamin A. Our overall purpose is to improve our understanding of the pathogenesis of MAC to develop targeted prevention strategies and therapeutics for children affected by these devastating birth defects.