Functional Analysis of Variants Underlying T Cell Defects

该计划项目的总体目标是整合其成员的专业知识， 利用生物信息学和基因组学的进步，不仅能够识别致病变异， 通过基于人群的新生儿重症联合免疫缺陷（SCID）筛查发现，但 也将开发基因组编辑作为个性化治疗方法。全外显子组测序（WES）和 全基因组测序（WGS）确定多个候选变体（项目1;核心B和C）， 然后筛选以鉴定导致T细胞不足的致病性变体。项目二采用 在正常人类造血祖细胞中进行基于CRISPR的筛选以识别重要基因 对于T细胞发育，项目3将把该项目的所有发现整合到一个统一的人类T细胞发育模型中。 T细胞发育。研究人员布伦纳，帕克和韦斯特已经合作整合生物信息学 在斑马鱼和人类造血细胞中进行功能验证以鉴定BCL 11B作为 新的SCID基因，并研究其作用模式（Punwani et al，NEJM，2016）。这种方法将是 扩增以进行数百种变体的高通量分析。项目3目标1将建立一个分子 通过表征原代人造血干细胞分化的人T细胞发育图 和祖细胞（HSPC）。分子图谱将通过使用 功能丧失分析以评估已知的SCID基因和另外的新的SCID基因在T细胞发育中的作用。 由Project 2确定的基因在人类T细胞发育中发挥重要作用。我们将使用 Perturb-seq是一种将单个基因的功能丧失与单细胞表达特征联系起来的新方法， 分化的连续阶段。这种方法不仅提供了发展的精确定义， 逮捕的基础上表达签名阶段，但也洞察到逮捕的方式， 超越了由异质造血干细胞的流式细胞术分析所提供的有限分辨率。 中间体（Adamson et al，Cell，2016）。事实上，Perturb-seq将使我们能够建立基因组， 在T细胞发育过程中共表达，并测试这些基因之间的上位性关系， 发育阶段在目标2中，我们将对候选致病编码变体进行功能分析 使用斑马鱼和人类HSPC模型。我们将采用斑马鱼胚胎模型来确定， 一个特定的编码变体实际上破坏了基因产物的功能，足以阻断T细胞增殖， 体内发育，以及其他器官是否也受到影响。此外，我们将深入开展机械化 分析3-4个最高优先级的变体，因为从该分析中获得的见解将有助于告知变体 项目1中的提名程序。总的来说，这些努力将大大促进我们对人类的理解。 T细胞的发展，这将推动优化的发现，机制的理解和治疗， 人SCID和相关疾病。

The overall aim of this Program Project is to integrate the expertise of its members in a comprehensive effort to exploit bioinformatic and genomic advances to enable not only identification of disease-causing variants discovered through population-based newborn screening for severe combined immunodeficiency (SCID), but also to develop genome editing as a personalized approach to treatment. Whole exome sequencing (WES) and whole genome sequencing (WGS) identify multiple candidate variants (Project 1; Cores B and C) that must then be screened to identify the pathogenic variant(s) responsible for T cell insufficiency. After Project 2 employs CRISPR-based screening in normal human hematopoietic progenitor cells to identify genes that are important for T cell development, Project 3 will integrate all of the findings from the program into a unifying model of human T cell development. Investigators Brenner, Puck, and Wiest have already collaborated to integrate bioinformatic variant calling with functional validation in zebrafish and human hematopoietic cells to identify BCL11B as a novel SCID gene and investigate its mode of action (Punwani et al, NEJM, 2016). This approach will be amplified to perform high-throughput analysis of hundreds of variants. Project 3 Aim 1 will establish a molecular map of human T cell development by characterizing the differentiation of primary human hematopoietic stem and progenitor cells (HSPC) in vitro using single-cell RNASeq. The molecular map will then be enriched by using loss-of-function analysis to assess the role in T cell development of known SCID genes and additional, novel genes determined by Project 2 to play an essential role in human T cell development. We will do so using Perturb-seq, a novel method that links loss-of-function of individual genes to single cell expression signatures at sequential stages of differentiation. This approach provides not only a precise definition of the developmental stage of arrest based on the expression signature, but also insight into the mechanism of arrest in a manner that transcends the limited resolution afforded by flow cytometry analysis of the heterogeneous hematopoietic intermediates (Adamson et al, Cell, 2016). Indeed, Perturb-seq will enable us to establish groups of genes that are co-expressed during T cell development, and to test the epistatic relationships between these genes at each developmental stage. In Aim 2, we will perform functional analysis on candidate disease-causing coding variants using both the zebrafish and human HSPC models. We will employ the zebrafish embryo model to determine if a particular coding variant actually damages the function of a gene product sufficiently to block T cell development in vivo, and whether other organs are also affected. In addition, we will perform in depth mechanistic analysis on the 3-4 highest priority variants, as insight gained from this analysis will help to inform the variant nomination process in Project 1. Collectively, these efforts will markedly advance our understanding of human T cell development, which will drive optimization of the discovery, mechanistic understanding and treatment of human SCID and related diseases.