Functional Analysis of Candidate Genes in Primary T Cell Immunodeficiencies

描述(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(变异型SCID和联合免疫缺陷，CID)患者的分子病因，发现了许多在正常发育中发挥关键作用的新基因。随着下一代测序(NGS)的出现，鉴定SCID中的致病突变变得更加容易；然而，在某些情况下，NGS识别出许多候选突变，必须对这些候选突变进行筛选以鉴定致病突变。在这种情况下，区分这些候选对象太费力了，使用鼠标模型无法完成。因此，我们建议使用斑马鱼的快速筛选过程来鉴定这种突变。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，因此由于斑马鱼基本基因的消除而导致的发育异常可以用它们在哺乳动物中的同源基因来补充。因此，我们建议通过以下方法筛选新的候选SCID基因：1)下调它们在斑马鱼中的表达，以确定这是否阻止T细胞的发育；以及2)确定野生型而不是突变型哺乳动物同源基因的重新表达是否能恢复发育。我们最近证明，这可以快速而有效地完成(张等人，2013年)。在通过这种方法鉴定出一个可能的疾病基因后，我们将使用锌指核酸酶突变来创建缺乏已鉴定基因的敲除小鼠模型和复制患者突变的敲打模型(S)。这个项目将作为与帕克博士的共同努力而进行。 (加州大学旧金山分校)，该组织拥有T淋巴细胞减少患者的存储库，这些患者的许多候选疾病基因已被确定。这些患者是通过加州SCID新生儿筛查计划确定的，她担任该计划的免疫学顾问。如果在疾病因感染而变得明显之后开始治疗，则SCID的治疗效果要差得多。然而，由于80%以上的SCID病例没有既往家族史，诊断无症状的SCID患者尤其具有挑战性。为了绕过这个问题，帕克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。这项测试需要对从已用于普遍新生儿筛查的干血斑点提取的DNA中的T细胞受体切除环(TRECs)进行定量。在加利福尼亚州对130万名婴儿进行了32个月的筛查后(T淋巴细胞减少症的发病率约为万分之一)，帕克博士确定了10名没有已知SCID基因的T淋巴细胞减少症患者，NGS已经为这些患者确定了许多候选基因。我们建议从这10名患者以及在这项赠款的24个月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.