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

总结 小眼症、无眼症和眼缺损（MAC）是一组与临床和遗传相关的眼部缺陷 会导致严重的视力障碍MAC可由转录因子中的致病性变体引起， 其他与眼睛发育有关的基因，以及环境因素，如母亲缺乏维生素A， （VAD）。维生素A对维甲酸（RA）的合成和致病性变体造成的功能丧失或获得至关重要 因为RA通路中的基因，如STRA 6，可能导致严重的MAC。我们假设遗传变异 在RA途径中，基因和涉及视黄醇代谢的基因可导致易感性，或降低 阈值，用于VAD对眼发育的有害影响，其有助于表型严重性 Mac.这种基因-环境相互作用对于VAD常见的国家具有高度相关性，并且仍然是一个重要的因素。 公共卫生问题，如印度。此外，由于缺乏维生素而失调的下游基因， A是知之甚少，适合在动物模型中研究。为了鉴定出所有的遗传 在MAC患者中RA通路和其他基因的变异，我们的第一个目标是招募MAC患者 并进行详细的表型分析、环境筛选和成像， 表型基因型相关我们将生成、分析和比较全基因组测序（GS）数据 来自我们的合作者Tibrewal博士和Kumar博士，在亚洲血统的患者中使用GS进行研究， 主要为欧洲血统的MAC患者。我们还将前瞻性招募MAC患者 在新生儿时期从美国的中心诊断，这样我们就可以从脐带中获得视黄醇水平 分娩时婴儿血液中MAC和母亲血浆中视黄醇水平。有了这些数据，我们 我们将比较MAC的严重程度与我们在RA通路基因和其他基因中观察到的序列变异。 眼睛发育基因，对于不同祖先的患者，有和没有症状的VAD证据 或低水平的视黄醇。在我们的第二个目标中，我们将使用基因编辑和规则间隔 短回文重复序列/Cas9以确定RA中stra 6和其他基因的功能改变的影响 斑马鱼眼形态发生的途径。所有酥脆剂将在不同时间点进行详细的表型分析 包括用单细胞RNA-Seq进行的基因表达研究，以确定 受RA调控的下游基因。我们还将调查我们是否可以拯救或部分拯救， 通过滴定法测定导致MAC的stra 6和其他基因中的遗传变异对眼形态发生的有害影响 斑马鱼眼发育过程中RA和视黄醇的水平。有了这个目标，我们将使用一种动物 模型，以生成关于受影响的下游基因、组织和发育时间点的数据， 缺乏RA和维生素A。我们的总体目的是提高我们对MAC发病机制的认识 为受这些毁灭性出生缺陷影响的儿童制定有针对性的预防战略和治疗方法。

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.