NF-kB Signaling Insights from a Rare X-linked Immunodeficiency Syndrome

来自罕见 X 连锁免疫缺陷综合征的 NF-kB 信号传导见解

• 批准号: 8891525
• 负责人: Ezra Burstein
• 金额: $ 39.75万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 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 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8891525
• 关键词: 5' Splice Site; Address; Affect; Alleles; Animal Disease Models; Biochemical; CD8B1 gene; Catalytic Domain; Cell Line; Cell Lineage; Cell model; Cell physiology; Cells; Cellular biology; Chronic; Clinical; Complex; Cutaneous; Cytotoxic T-Lymphocytes; DNA Polymerase I; DNA biosynthesis; DNA-Directed DNA Polymerase; Defect; Discipline; Disease; Ectodermal Dysplasia; Elements; Enterocolitis; Evaluation; Event; Exons; Family; Fibroblasts; Figs - dietary; Gene Activation; Gene Expression; Genes; Genetic; Goals; Human; Hyperpigmentation; Hypohidrosis; Immune; Immunologic Deficiency Syndromes; Immunologics; Infection; Interdisciplinary Study; Introns; Length; Leukocytes; Link; Messenger RNA; Molecular; Molecular Genetics; Mus; Mutation; NF-kappa B; Natural Killer Cells; Nonsense Codon; North America; Nuclear; Orphan; Pathway interactions; Patients; Phenotype; Play; Population; Positioning Attribute; Proteins; RNA Interference; RNA Splicing; Reagent; Recording of previous events; Recurrence; Regulation; Reporter; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Skin Manifestations; System; Time; Tissues; antibody-dependent cell cytotoxicity; base; clinical phenotype; congenital immunodeficiency; conserved helix-loop-helix ubiquitous kinase; cytotoxicity; gene induction; genome sequencing; immune function; insight; novel; patient population; peripheral blood; proband; protein expression; protein function

DESCRIPTION (provided by applicant): X-linked reticulate pigmentary disorder (XLPDR) is a rare condition characterized by recurrent infections and a range of skin manifestations. Utilizing whole genome sequencing, we have identified that the cause of XLPDR is an intronic mutation resulting in missplicing of POLA1, an essential cell replication gene encoding the catalytic subunit of DNA polymerase alpha. Moreover, we have uncovered that XLPDR results in a prominent natural killer cell (NK) defect, consistent with the clinical history of recurrent infections. The cutaneous and immunologic changes seen in XLPDR are reminiscent of mutations of the NF-?B activating gene NEMO, a central regulator of immune function. Indeed, we find that XLPDR-derived fibroblasts display severely impaired NF-?B activation, which is recapitulated by RNAi silencing of POLA1, suggesting an unexpected connection between POLA1 and the NF-?B pathway. However, the mechanism by which a mutation in this ubiquitously expressed gene results in the specific manifestations of XLPDR remains unclear. Thus, the project's overall goal is to uncover the cellular and molecular basis of XLPDR and its associated immunodeficiency. Our hypothesis is that the POLA1 protein functions not only in DNA replication, but also plays a direct role in NF-?B activation, and that XLPDR and its associated immunodeficiency result from reduced POLA1 expression in select cell populations. To address these goals, we propose the following Specific Aims: (1) Determine the genetic mechanism of XLPDR: In order to explain the specific phenotype of XLPDR, we hypothesize that missplicing and reduced POLA1 protein occurs preferentially in select cell lineages. In this aim, we will examine this possibility in leukocyte populations from patients' peripheral blood. Furthermore, using a splicing reporter system we will analyze the effect of the XLPDR mutation on POLA1 splicing in cell lines of multiple tissue origins and define in detail the elements required to recapitulate POLA1 missplicing in the corresponding mouse intron. (2) Define the biochemical basis of defective NF-?B activation in XLPDR: In addition to the defect in NF-?B dependent gene expression in XLPDR, we have found a physical interaction between the POLA1 and NEMO proteins. Interestingly, the nuclear entry of NF-?B complexes is not affected by the XLPDR mutation, suggesting that the defect lies in nuclear events required for NF-?B activation. In this aim we will examine the mechanism of assembly and regulation of the POLA1-NEMO complex, and the participation of POLA1 in nuclear events required for NF-?B dependent gene induction. (3) Understand the NK cell deficiency of XLPDR: In this Aim, we will perform a careful evaluation of NK cell function in patients from different families and examine whether direct silencing of POLA1 in wild-type NK cells is sufficient to recapitulate the XLPDR cellular phenotype and generate a cellular model of XLPDR. Altogether, these studies of a rare and orphan immunologic disorder offer a unique opportunity to obtain remarkable and novel insights into immune regulation and signaling pathways of great significance to a number of disciplines.

描述(申请人提供)：X连锁网状色素性疾病(XLPDR)是一种罕见的疾病，以反复感染和一系列皮肤表现为特征。利用全基因组测序，我们已经确定XLPDR的原因是内含子突变导致编码DNA聚合酶α催化亚基的重要细胞复制基因POLA1的错接。此外，我们还发现XLPDR导致了一个显著的自然杀伤细胞(NK)缺陷，这与反复感染的临床病史一致。在XLPDR中看到的皮肤和免疫学变化让人想起免疫功能的中枢调节因子--核因子？B激活基因NEMO的突变。事实上，我们发现XLPDR来源的成纤维细胞表现出严重的NF-？B活性受损，这是由POLA1的RNAi沉默所概括的，这表明POLA1与NF-？B途径之间存在意想不到的联系。然而，这种普遍表达的基因突变导致XLPDR特殊表现的机制尚不清楚。因此，该项目的总体目标是揭示XLPDR及其相关免疫缺陷的细胞和分子基础。我们的假设是，POLA1蛋白不仅在DNA复制中起作用，而且在核因子-βB的激活中起直接作用，而XLPDR及其相关的免疫缺陷是由于特定细胞群体中POLA1表达减少所致。为了解决这些目标，我们提出了以下具体目标：(1)确定XLPDR的遗传机制：为了解释XLPDR的特定表型，我们假设错误剪接和POLA1蛋白减少优先发生在选定的细胞谱系中。在这个目标中，我们将在患者外周血中的白细胞群中检查这种可能性。此外，使用剪接报告系统，我们将分析XLPDR突变对多组织来源细胞系POLA1剪接的影响，并详细定义在相应的小鼠内含子中重现POLA1错误剪接所需的元件。(2)明确XLPDR中核因子-βB激活缺陷的生化基础：除了XLPDR中依赖于核因子-βB的基因表达缺陷外，我们还发现POLA1和NEMO蛋白之间存在物理相互作用。有趣的是，核因子-βB复合体的核进入不受XLPDR突变的影响，这表明缺陷存在于核因子-βB激活所需的核事件中。为此，我们将研究POLA1-NEMO复合体的组装和调控机制，以及POLA1参与核因子依赖基因诱导所需的核事件。(3)了解XLPDR的NK细胞缺陷：为此，我们将仔细评估来自不同家系的患者的NK细胞功能，并检查野生型NK细胞中POLA1的直接沉默是否足以概括XLPDR的细胞表型并建立XLPDR的细胞模型。总之，这些对一种罕见的孤儿免疫疾病的研究提供了一个独特的机会，以获得对免疫调节和信号通路的非凡和新颖的见解，这对许多学科具有重要意义。

项目成果

Defective DNA Polymerase α-Primase Leads to X-Linked Intellectual Disability Associated with Severe Growth Retardation, Microcephaly, and Hypogonadism.

有缺陷的 DNA 聚合酶 α-引物酶会导致与严重生长迟缓、小头畸形和性腺功能减退症相关的 X 连锁智力障碍。

• DOI: 10.1016/j.ajhg.2019.03.006
• 发表时间: 2019
• 期刊: American journal of human genetics
• 影响因子: 9.8
• 作者: VanEsch,Hilde;Colnaghi,Rita;Freson,Kathleen;Starokadomskyy,Petro;Zankl,Andreas;Backx,Liesbeth;Abramowicz,Iga;Outwin,Emily;Rohena,Luis;Faulkner,Claire;Leong,GaryM;Newbury-Ecob,RuthA;Challis,RachelC;Õunap,Katrin;Jaeken,Jacqu
• 通讯作者: Jaeken,Jacqu