Identification of Candidate Disease-Causing Variants

几种原发免疫缺陷，包括严重联合免疫缺陷(SCID)，被描述为 T细胞缺乏症。阐明T细胞缺陷的病因为理解T细胞的生物学提供了机会 细胞发育和T细胞缺乏症患者的诊断和治疗。当前 Exome方法解决了50%的T细胞缺陷病例。全基因组测序(WGS)提供了 有机会开发一种更全面的发现变异的方法，具有更好的外显子覆盖率， 以及对非编码序列的新访问，允许询问基因组调节变异。 对WGS的解释是由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细胞相关的假定基因和调节区 精神错乱。这些项目将通过项目2中的CRISPR筛选和项目3中的更详细研究进行评估。 综合结果将为免疫学领域提供资源，并将直接为我们的诊断提供信息 WGS解释。 对于目标2，我们将在受影响的个体中识别候选的致病变异，并对这些变异进行分层 实验验证和审问。利用目标1中的资源，我们将建立一个分析 解决先证者WGS的案件的渠道。我们将根据程度对我们的结论进行分层 对计算研究的信心，以便为项目2和3或目标3的不同实验提供信息。 这条管道将有量化的综合评分，并将同时考虑蛋白质改变和调控 单核苷酸变异和小插入，以及结构变异(特别是缺失)。我们会 根据预测效果的严重程度、与T细胞缺陷的基因相关性以及一致性对变体进行优先排序 具有先证者表型(来自核心C)。“令人信服的”案例，其中因果变异很可能得到确认， 可能会在项目3中继续分子机制的研究，同时“有趣”的案例产生了数十个 在项目2中，变种需要通过中等规模的研究进行测试。“神秘”案件中有许多变种 未知的意义将首先通过AIM 3RNA-seq进行检查，然后继续进行项目2和3。 对于目标3，我们将整合Trio RNA-seq以识别与T细胞相关的调控剪接和表达变体 缺乏症。目前还不能可靠地预测大多数假定的监管变种的影响。 然而，转录组分析是一种有效的方法，它同时揭示了RNA表达水平和剪接事件 评估基因变异的调控后果的方法。家长的成绩单将会是 研究用于推断先证者的T细胞因疾病而不可用时的T细胞。我们将确定 与剪接改变相关的变体，以及与等位基因特异性改变相关的调控变体 表情。这些数据将被合并到AIM 2基因组解释流水线中，以识别变异 以便在项目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.