Multi-organism platform for functional assessment of human birth defect associated genomic variants

出生缺陷每年发生在美国出生的婴儿中的3%-5%，是导致儿童死亡和 反复住院，给家庭和社会带来很大负担。出生缺陷通常是由罕见的 基因变化，但确定哪个基因变异导致表型和疾病仍然具有挑战性， 尽管在下一代测序和分析方面取得了重大进展。而大约4,000个人类基因 与单基因罕见疾病有关，据估计，额外的6,000-13,000个罕见疾病基因 仍有待确定，其中许多可能是出生缺陷的原因；这突出表明有必要采取有效措施 评估相关变异的功能效应的策略。 在这一应用中，我们提出了一种多生物方法来连接患者的表现和基因 以前与疾病有关(不确定意义的基因，GUS)。专注于智力和 发育障碍和结构性出生缺陷，我们将首先筛查与出生缺陷相关的GUS 利用线虫、果蝇或斑马鱼的实验可追踪性，然后评估患者- 脊椎动物(斑马鱼、小鼠或已建立的人类胚胎干细胞系)的相关表型 (HESCs))这些基因的一个子集。目的1、家系临床外显子组序列的生物信息学分析 将进一步确定可能导致患者症状的高概率候选基因变异(S) 学习。在目标2中，将在蠕虫、苍蝇或鱼类中筛选提名的候选基因变体，以获得体内功能 支持不同因果关系的数据。功能信息将有助于确定该基因变体是否为 如果在特定组织中需要该基因，以及观察到的遗传机制(例如， 发育迟缓、显性否定等。)与患者的遗传特征相一致。此外，这些实验可能 阐明受基因变异干扰的分子或细胞生物学机制(例如，干扰 细胞骨架等)。我们将利用不同模式生物的优势--CRISPR编辑 对于线虫，在果蝇中的组织特异性RNAi，以及用于斑马鱼的mRNA和CRISPR胚胎注射 基因筛查方法。目标2将在拨款范围内对84名出生缺陷儿童进行筛查 句号。通常，在蠕虫或苍蝇中，同源基因的相应破坏的表型效应是 与在人类身上观察到的表型没有明显的关系。因此，在目标3中，对于基因子集(23) 在目标2中，我们将检查斑马鱼、小鼠、 或hESC系统，以促进我们对疾病表型和进展的理解，这在 简单的模型生物体或与病人。这些研究将利用我们在疾病基因建模方面的经验 以及我们与临床合作者的工作，以了解表型、遗传、分子和细胞生物学 每个病人的疾病的基础。这一创新的多生物实验平台将显著加快 识别导致出生缺陷的基因，并将为诊断、预防和治疗开辟道路。

Birth defects, which occur in 3 - 5% of US-born infants per year, are a leading cause of childhood mortality and repeated hospitalization and are a large burden to families and society. Birth defects typically result from rare genetic changes, but determining which gene-variant causes a phenotype and disease remains challenging, despite significant advances in next-generation sequencing and analysis. Whereas ~4,000 human genes have been linked to monogenic, rare diseases, it has been estimated that 6,000-13,000 additional rare disease genes remain to be identified, many of which are likely causal for birth defects; this underscores a need for effective strategies to assess the functional effects of associated variants. In this application, we propose a multi-organism approach to connect patient presentation with genes not previously associated with disease (genes of uncertain significance, GUS). Focusing on intellectual and developmental disorders and structural birth defects, we will first screen patient birth-defect associated GUS leveraging experimental tractability of C. elegans, Drosophila, or zebrafish, followed by assessment of patient- related phenotypes in vertebrate organisms (zebrafish, mouse, or established human embryonic stem cell lines (hESCs)) for a subset of these genes. In Aim 1, bioinformatic analysis of family based clinical exome sequence will identify high probability candidate gene-variants that may be causal for the patient’s symptom(s) for further study. In Aim 2, nominated candidate gene-variants will be screened in worm, fly or fish to obtain in vivo functional data in support of variant causality. Functional information will help determine whether the gene-variant is damaging, if the gene is required in a specific tissue, and whether the observed genetic mechanism (e.g., hypomorph, dominant negative, etc.) is consistent with patient genetics. Additionally, these experiments may illuminate the molecular or cell biological mechanism disrupted by the gene-variant (e.g., disruption of cytoskeleton, etc.). We will take advantage of the strengths of the different model organisms—CRISPR editing for C. elegans, tissue-specific RNAi in Drosophila, and mRNA and CRISPR embryo injections for zebrafish—in the genetic screening approach. A total of 84 candidate birth defect GUS will be screened in Aim 2 over the grant period. Often, the phenotypic effects of the corresponding disruption of the orthologous gene in worm or fly are not obviously related to the phenotype observed in humans. Therefore, in Aim 3, for a subset of genes (23) identified from the screen as likely disease-causing in Aim 2, we will examine phenotypes in zebrafish, mouse, or hESC systems, to advance our understanding of disease phenotype and progression that is not possible in simple model organisms or with the patient. These studies will leverage our experience in disease gene modeling and our work with clinical collaborators to understand the phenotypic, genetic, molecular, and cell biological basis of each patient’s disease. This innovative multi-organism experimental platform will significantly accelerate identification of birth defect-causing genes and will open avenues to diagnosis, prevention, and therapies.