Functional Analysis of Candidate Genes in Primary T Cell Immunodeficiencies

描述（由申请方提供）：原发性T细胞免疫缺陷中候选基因的功能分析。追踪严重联合免疫缺陷（SCID）或其他T淋巴细胞减少性疾病（变体SCID和联合免疫缺陷，CID）患者的分子病因学，导致许多新的基因，在正常发育中发挥关键作用的鉴定。随着下一代测序（NGS）的出现，SCID中致病突变的鉴定变得更容易实现;然而，在某些情况下，NGS鉴定了许多候选突变，这些候选突变必须被筛选以鉴定致病突变。在这种情况下，区分这些候选者过于劳动密集型，无法使用小鼠模型完成。因此，我们建议在斑马鱼中使用快速筛选过程来识别此类突变。正如我们和其他人已经表明的那样，斑马鱼的造血是高度保守的，因此由消除必需的斑马鱼基因引起的发育异常可以由其哺乳动物直系同源物补充。因此，我们提出通过以下方式筛选新的候选SCID基因：1）敲低它们在斑马鱼中的表达以确定这是否阻断T细胞发育;和2）确定野生型而非突变体哺乳动物直系同源物的再表达是否恢复发育。我们最近已经证明，这可以快速有效地完成（Zhang et al.，2013年）。在通过这种方法鉴定可能的疾病基因后，我们将采用锌指核酸酶诱变来创建缺乏所鉴定基因的敲除小鼠模型和复制患者突变的敲入模型。这个项目将作为一个共同努力， （UCSF），其具有T淋巴细胞减少症患者的库，对于这些患者，已经鉴定了许多候选疾病基因。这些患者是通过加州SCID新生儿筛查项目确定的，她是该项目的免疫学顾问。如果在疾病由于感染而变得明显之后开始，那么SCID的治疗效果要差得多。然而，由于超过80%的SCID病例没有既往家族史，诊断症状前的SCID患者特别具有挑战性。为了解决这个问题，Puck博士和其他人开发了一种有效的基于人群的筛查测试来识别T淋巴细胞减少症。该测试需要对从已经用于新生儿筛查的干血斑中提取的DNA中的T细胞受体切除环（TRECs）进行定量。在对加州的130万名婴儿进行了32个月的筛查（T细胞淋巴细胞减少症的发病率约为1/20，000）后，Puck博士确定了10名T淋巴细胞减少症患者，这些患者没有已知的SCID基因型，NGS已经确定了许多候选基因。我们建议从这10名患者以及在24个月的赠款过程中确定的其他患者中筛选候选基因的功能。

DESCRIPTION (provided by applicant): Functional analysis of candidate genes in primary T cell immunodeficiencies. Tracing the molecular etiologies of patients with severe combined immunodeficiency (SCID) or other T lymphopenic diseases (variant SCID and combined immunodeficiency, CID) has led to the identification of many novel genes that play critical roles in normal development. With the advent of Next Gen Sequencing (NGS), identification of the causative mutations in SCID is becoming more readily achievable; however, in some cases NGS identifies numerous candidate mutations that must be screened to identify the causative mutation. Distinguishing among these candidates in such cases is too labor-intensive to accomplish using mouse models. Consequently, we propose to identify such mutations using a rapid screening process in zebrafish. As we and others have shown, hematopoiesis in zebrafish is highly conserved, such that developmental abnormalities caused by the elimination of essential zebrafish genes can be complemented by their mammalian orthologs. Accordingly, we propose to screen novel candidate SCID genes by: 1) knocking down their expression in zebrafish to determine if this blocks T cell development; and 2) determining if re- expression of the wild type, but not mutant, mammalian ortholog, restores development. We have recently demonstrated that this can be done rapidly and effectively (Zhang et al., 2013). After identification of a likely disease gene by this means, we will employ zinc-finger nuclease mutagenesis to create knockout mouse models lacking the identified genes and knockin models replicating the patients' mutation(s). This project will be pursued as a joint effort with Dr. Puck (UCSF), who has a repository of T lymphopenic patients for whom numerous candidate disease genes have been identified. These patients were identified through the California SCID newborn screening program, for which she serves as Immunology Consultant. Treatment of SCID is far less effective if initiated after the disease becomes manifest due to infections. However, because more than 80% of SCID cases have no prior family history, diagnosing pre-symptomatic SCID patients is particularly challenging. To circumvent this problem, Dr. Puck and others have developed an effective population-based, screening test to identify T lymphopenia. This test entails the quantitation of T cell receptor excision circles (TRECs) in DNA extracted from the dried blood spots already used for universal newborn screening. After 32 months of screening 1.3 million infants in California, (with an incidence of T cell lymphopenia about 1/20,000 births), Dr. Puck has identified 10 T lymphopenic patients without a known SCID genotype for whom numerous candidate genes have been identified by NGS. We propose to screen the function of candidate genes from these 10 patients as well as additional patients identified during the 24 month course of this grant.