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

出生缺陷，发生在3 - 5%的美国出生的婴儿每年，是一个主要的原因，儿童死亡率和 反复住院，给家庭和社会带来很大负担。出生缺陷通常由罕见的 遗传变化，但确定哪种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。然而，大约4,000个人类基因 与单基因的罕见疾病有关，据估计，还有6000 - 13000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;这强调了需要有效的 评估相关变异的功能效应的策略。 在这个应用中，我们提出了一个多生物体的方法来连接病人的表现与基因，而不是 以前与疾病相关的基因（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，我们将首先筛选患者出生缺陷相关的GUS 利用C.线虫、果蝇或斑马鱼，然后评估患者- 脊椎动物生物体（斑马鱼、小鼠或已建立的人胚胎干细胞系）中的相关表型 （hESC））中的一部分。目的1：对家系临床外显子序列进行生物信息学分析 将识别可能导致患者症状的高概率候选基因变异，以进一步 study.在目标2中，将在蠕虫、蝇或鱼中筛选提名的候选基因变体，以获得体内功能性的基因变体。 支持变量因果关系的数据。功能信息将有助于确定基因变异是否是 如果特定组织需要该基因，以及观察到的遗传机制（例如， 亚型、显性阴性等）与病人的基因相符此外，这些实验可能 阐明被基因变体破坏的分子或细胞生物学机制（例如，破坏 细胞骨架等）。我们将利用不同模式生物的优势--CRISPR编辑 梭线虫，果蝇中的组织特异性RNAi，以及斑马鱼中的mRNA和CRISPR胚胎注射 基因筛选方法。在目标2中，共有84名候选出生缺陷GUS将在补助金范围内进行筛选 期通常，蠕虫或苍蝇中相应的正向同源基因破坏的表型效应是 与在人类中观察到的表型无明显相关。因此，在目标3中，对于基因的子集（23）， 在目标2中从筛选中鉴定为可能致病的，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，这是不可能的， 简单的模式生物或患者。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合作，以了解表型，遗传，分子和细胞生物学 根据每个病人的病情。这一创新的多生物实验平台将显著加快 这将有助于确定导致出生缺陷的基因，并为诊断、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.