Functional Analysis of Candidate Genes in Primary T Cell Immunodeficiencies

描述（由申请人提供）：原发性T细胞免疫缺陷候选基因的功能分析。追踪严重联合免疫缺陷（SCID）或其他T淋巴细胞减少疾病（SCID变异型和联合免疫缺陷，CID）患者的分子病因已经导致许多在正常发育中起关键作用的新基因的鉴定。随着下一代测序（NGS）的出现，SCID致病突变的鉴定变得更加容易实现；然而，在某些情况下，NGS识别出许多候选突变，必须筛选以确定致病突变。在这种情况下，区分这些候选对象过于费力，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这种突变。正如我们和其他人所表明的那样，斑马鱼的造血功能是高度保守的，因此，由基本斑马鱼基因消除引起的发育异常可以通过它们的哺乳动物同源物来补充。因此，我们建议通过以下方法筛选新的候选SCID基因：1)降低它们在斑马鱼中的表达，以确定这是否会阻断T细胞的发育；2)确定重新表达野生型而非突变型的哺乳动物同源基因是否能恢复发育。我们最近证明，这可以快速有效地完成（Zhang et al., 2013）。在通过这种方法鉴定出可能的疾病基因后，我们将采用锌指核酸酶诱变技术创建缺乏鉴定基因的敲除小鼠模型和复制患者突变的敲除小鼠模型。这个项目将与Puck博士共同努力

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.