Functional Analysis of Candidate Genes in Primary T Cell Immunodeficiencies

原发性 T 细胞免疫缺陷候选基因的功能分析

• 批准号: 8914488
• 负责人: Jennifer M. Puck
• 金额: $ 25.62万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8914488
• 关键词: B-Lymphocytes; Biological Assay; Biological Markers; Biological Response Modifiers; Birth; Blood; California; Candidate Disease Gene; Cells; Characteristics; Complement; DNA; Data; Defect; Development; Diagnosis; Disease; Ectopic Expression; Embryo; Enrollment; Etiology; Excision; Family; Freedom; Genes; Genetic Models; Genotype; Grant; Health; Hematopoiesis; Hematopoietic stem cells; Human; Immune; Immunologic Deficiency Syndromes; Immunology; Immunophenotyping; In Vitro; Inborn Genetic Diseases; Incidence; Infant; Infection; Joints; Knock-out; Knockout Mice; Learning; Life; Lymphocyte; Lymphocyte Count; Lymphoid; Lymphopenia; Modeling; Molecular; Mus; Mutagenesis; Mutation; Neonatal Screening; Newborn Infant; Oligonucleotides; Orthologous Gene; Parents; Patients; Play; Process; Production; Proteins; Protocols documentation; Publishing; Recording of previous events; Resources; Ribosomal Proteins; Role; Severe Combined Immunodeficiency; Spottings; T-Cell Development; T-Cell Immunodeficiency; T-Cell Receptor; T-Lymphocyte; Testing; Tracer; Transgenic Organisms; Variant; Zebrafish; Zinc Fingers; adaptive immunity; disease-causing mutation; embryonic stem cell; exome; exome sequencing; in vivo; insight; knock-down; loss of function; medical complication; mouse model; mutant; next generation sequencing; novel; nuclease; offspring; population based; programs; repository; research study; screening; transmission process

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.

描述（由申请人提供）：原代T细胞免疫缺陷中候选基因的功能分析。追踪严重合并免疫缺陷（SCID）或其他T淋巴细胞减少性疾病（变异SCID和合并免疫缺陷，CID）的分子病因，导致了许多在正常发育中起关键作用的新型基因。随着下一代测序（NGS）的出现，SCID中的因果突变的鉴定变得越来越容易实现。但是，在某些情况下，NGS识别了许多必须筛选的候选突变，以鉴定因果突变。在这种情况下，这些候选者之间的区分太大了，无法使用小鼠模型来完成。因此，我们建议使用斑马鱼中的快速筛选过程来识别此类突变。正如我们和其他人所表明的那样，斑马鱼中的造血是高度保守的，因此可以通过其哺乳动物的直系同源物来补充由于消除必需斑马鱼基因而引起的发育异常。因此，我们建议通过以下方式筛选新的候选SCID基因，1）击倒它们在斑马鱼中的表达，以确定这是否阻止了T细胞的发育； 2）确定野生类型的表达（而不是突变体）是否会恢复发育。我们最近证明，这可以快速有效地完成（Zhang等，2013）。通过这种方式鉴定了可能的疾病基因后，我们将采用锌指核酸酶诱变来创建缺乏鉴定基因的基因敲除小鼠模型，并敲除敲除模型，以复制患者突变（S）。该项目将与Puck博士共同努力。 （UCSF），他的T淋巴细胞减少患者的存储库已被鉴定出许多候选疾病基因。这些患者是通过加利福尼亚SCID新生儿筛查计划确定的，她担任免疫学顾问。如果由于感染而显现出来，SCID的治疗效率远不如效率远不如效果。但是，由于超过80％的SCID病例没有先前的家族史，因此诊断症状前SCID患者尤其具有挑战性。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，以鉴定淋巴细胞减少症。该测试需要从已用于通用新生儿筛查的干血点中提取的DNA中T细胞受体切除圈（TREC）进行定量。在加利福尼亚州进行了32个月的130万婴儿筛查（T细胞淋巴细胞减少症的发生率约为1/20,000个出生），Puck博士确定了10名没有已知SCID基因型的T淋巴细胞减少患者，该患者已通过NGS鉴定出许多候选基因。我们建议从这10位患者中筛选候选基因的功能，以及在这笔赠款期间24个月期间确定的其他患者。