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

描述（由申请人提供）：x连锁网状色素紊乱（XLPDR）是一种罕见的疾病，其特征是反复感染和一系列皮肤表现。利用全基因组测序，我们已经确定XLPDR的原因是一个内含子突变导致POLA1剪接错误，POLA1是一个重要的细胞复制基因，编码DNA聚合酶α的催化亚基。此外，我们发现XLPDR会导致明显的自然杀伤细胞（NK）缺陷，这与复发性感染的临床病史一致。XLPDR的皮肤和免疫变化与NF-？B激活基因NEMO，免疫功能的中枢调节因子。事实上，我们发现xldr衍生的成纤维细胞表现出严重受损的NF-？B激活，这是由RNAi沉默POLA1再现的，这表明POLA1与NF-？B通路。然而，这种普遍表达的基因突变导致XLPDR特异性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NF-？B激活，XLPDR及其相关免疫缺陷是由选择细胞群中POLA1表达减少引起的。为了实现这些目标，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减少的POLA1蛋白优先发生在选择的细胞系中。为此，我们将在患者外周血白细胞群中检查这种可能性。此外，利用剪接报告系统，我们将分析XLPDR突变对多个组织起源细胞系中POLA1剪接的影响，并详细定义在相应的小鼠内含子中重现POLA1剪接错误所需的元素。(2)明确NF-?缺陷的生化基础。B在XLPDR中的活化：除了NF-？B依赖基因在XLPDR中的表达，我们发现POLA1和NEMO蛋白之间存在物理相互作用。有趣的是，NF-？B复合物不受XLPDR突变的影响，这表明缺陷存在于NF-？B激活。在这个目的中，我们将研究POLA1- nemo复合物的组装和调控机制，以及POLA1参与NF-？B依赖基因诱导。(3)了解XLPDR的NK细胞缺陷：在本研究中，我们将仔细评估不同家族患者的NK细胞功能，并检验在野生型NK细胞中直接沉默POLA1是否足以概括XLPDR的细胞表型并生成XLPDR的细胞模型。总之，这些罕见和孤儿免疫疾病的研究提供了一个独特的机会，以获得对许多学科具有重要意义的免疫调节和信号通路的显着和新颖的见解。

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.