Identification of Candidate Disease-Causing Variants

包括严重联合免疫缺陷（SCID）在内的几种原发性免疫缺陷的特征是 T细胞缺陷。阐明T细胞缺陷的病因提供了了解T细胞生物学的机会。 细胞发育，并为T细胞缺乏症患者的诊断和治疗提供信息。电流 外显子组方法解决了<50%的T细胞缺陷病例。全基因组测序（WGS）提供了一个 有机会开发一种更全面的变异发现方法，具有上级外显子覆盖率， 以及对非编码序列的新的访问，允许对基因组调节变异的询问。 WGS的解释是通过与特定疾病相关的已知基因和调控区域实现的 表型为此，在目标1中，我们将预测与T细胞相关的推定基因和调控区域， 紊乱这些将在项目2中通过CRISPR筛选进行评估，并在项目3中进行更详细的研究。 综合结果将为免疫学领域提供资源，并将直接告知我们的诊断。 WGS解释。 对于目标2，我们将确定受影响个体中的候选致病变异，并将其分层， 实验验证和询问。利用目标1中的资源，我们将建立一个分析 解决先证者WGS病例的管道。我们将根据程度对结论进行分层。 在计算研究的信心，以通知不同的实验项目2和3或目标3。 该管道将具有定量综合评分，并将考虑蛋白质改变和调节 单核苷酸变异和小插入缺失，以及结构变异（特别是缺失）。我们将 根据预测效应的严重程度、与T细胞缺陷的基因相关性和一致性对变体进行优先排序 先证者表型（来自核心C）。“令人信服”的情况下，有可能验证致病变异， 可能会在项目3中进行分子机制的研究，而“有趣”的案例产生了数十个 变体需要通过项目2中的中等规模研究进行测试。“神秘”案件， 未知的重要性将首先通过目标3 RNA-seq进行检查，然后进行项目2和3。 对于目标3，我们将整合trio RNA-seq以鉴定与T细胞相关的调控剪接和表达变体， 缺乏症大多数假定的调节变体的影响今天不能可靠地预测。 然而，转录组分析，它揭示了RNA表达水平和剪接事件，是一种有效的方法。 方法来评估遗传变异的监管后果。来自父母的转录组 当T细胞因疾病而不可用时，研究其以推断先证者的T细胞。我们将确定 与改变的剪接相关的变体，以及与等位基因特异性改变的基因相关的调节变体， 表情这些数据将被纳入Aim 2基因组解释管道，以识别变体 在项目2和3中进行进一步调查。

Several primary immunodeficiencies, including severe combined immunodeficiency (SCID), are characterized by T cell deficits. Elucidating the etiology of T cell deficits provides opportunities to understand the biology of T cell development and inform diagnosis and treatment of individuals with T cell deficiency disorders. Current exome approaches solve <50% of cases of T cell deficiencies. Whole genome sequencing (WGS) offers an opportunity to develop a more comprehensive approach for variant discovery, with superior coverage of exons, as well as new access to noncoding sequences, allowing for interrogation of genomic regulatory variation. Interpretation of WGS is enabled by known genes and regulatory regions associated with particular disease phenotypes. To this end, in Aim 1 we will predict putative genes and regulatory regions associated with T cell disorders. These will be evaluated by CRISPR screens in Project 2 and more detailed studies in Project 3. The integrated results will provide a resource for the field of immunology, and will directly inform our diagnostic WGS interpretation. For Aim 2, we will identify candidate disease-causing variants in affected individuals, and stratify these for experimental validation and interrogation. Drawing upon the resource in Aim 1, we will establish an analysis pipeline that solves cases from probands' WGS. We will stratify our conclusions based on degree of confidence in the computational studies, in order to inform different experiments for Projects 2 and 3 or Aim 3. This pipeline will have quantitative integrative scoring, and will consider both protein altering and regulatory single nucleotide variants and small indels, as well as structural variations (especially deletions). We will prioritize variants based on severity of predicted effect, gene relevance to T cell deficiencies, and consistency with proband phenotype (from Core C). “Compelling” cases where validation of causative variants is likely, may proceed to investigations of molecular mechanism in Project 3, while “intriguing” cases yielding dozens of variants need testing via medium-scale studies in Project 2. “Mysterious” cases with numerous variants of unknown significance will first be examined through Aim 3 RNA-seq, and then proceed to Projects 2 and 3. For Aim 3, we will integrate trio RNA-seq to identify regulatory splicing and expression variants related to T cell deficiency disease. The impact of most putative regulatory variants cannot be reliably predicted today. However, transcriptome profiling, which reveals both RNA expression level and splicing events, is an effective method to assess the regulatory consequences of genetic variations. Transcriptomes from parents will be studied for inferring those of the probands, when T cells are not available due to the disease. We will identify variants associated with altered splicing, as well as regulatory variants associated with allele-specific altered expression. These data will be incorporated into the Aim 2 genome interpretation pipeline, to identify variants for further investigations in Projects 2 and 3.