Inherited IRF9 deficiency: a novel genetic etiology of severe influenza

项目摘要 甲型流感病毒（IAV）是一种人畜共患病原体，每年引起季节性流行， 造成全球性的流行病危及生命的肺炎只发生在极少数感染者中， 个人（0.04 - 0.4%）。先前存在的肺部疾病和获得性免疫缺陷- 已知的风险因素。然而，绝大多数严重的流感病例仍然无法解释，特别是在 孩子有趣的是，典型的原发性免疫缺陷并不容易导致严重的流感。我们最近 报道了一名健康儿童因遗传性常染色体隐性（AR）IRF7而患严重流感 缺乏导致浆细胞样树突状细胞（pDC）和 诱导多能干细胞（iPSC）衍生的肺上皮细胞（PEC）。这是第一 证明流感易感性是可以遗传的。我们假设，额外的严重病例 儿童和年轻人的流感可能是由单基因先天免疫缺陷引起的， 表现出完全的恍惚状态我们最近在一个25人的队列中进行了全外显子组测序（WES）。 患者及其父母（三重设计），患有危及生命的流感，无继发性细菌性肺炎。 我们在一名患有危及生命的流感的儿童中发现了一种纯合子IRF9突变， 接种MMR疫苗的反应。IRF9与STAT1和STAT2一起，是ISGF 3的三个亚基之一， 转录复合物驱动抗病毒I型IFN应答。这种突变是这个家族的私有， 影响剪接位点的一个重要核苷酸我们打算测试这种突变是否有害， 表达和功能。我们还将测试患者的细胞对I型IFN的反应及其对 流感病毒在I型IFN的存在下。最后，我们将比较全基因组转录组 对患者细胞中I型IFN的应答与来自其他常染色体隐性遗传患者的应答， IFNAR1、IFNAR2、STAT 1和STAT 2的完全缺陷。我们的初步数据是令人信服的和强有力的，因为 我们发现剪接突变不是泄漏的，编码的蛋白质是ISGF3表达的缺失， 功能此外，患者的细胞似乎对流感病毒的控制也很差，即使在存在 外源性I型干扰素最后，我们从患有I型先天性缺陷的所有类型的患者中收集细胞， IFN应答通路。因此，我们的项目是高度创新的，但有强有力的初步证据支持。 总的来说，我们的项目将深入研究一种新的原发性肝癌的分子和细胞基础。 免疫缺陷，常染色体隐性IRF9缺陷，从而记录了第二个遗传和 儿童重症流感免疫学病因学研究这项研究将强调新出现的概念， 严重的儿童流感可由单基因先天免疫缺陷引起。我们的研究也将有 新的重要生物学意义，首次分析IRF 9依赖性和非依赖性 I型干扰素的反应。总的来说，我们的发现将具有生物学和临床意义。

Project Summary Influenza A virus (IAV) is a zoonotic pathogen that causes seasonal epidemics annually and has the potential to cause worldwide pandemics. Life-threatening pneumonia occurs in only a small minority of infected individuals (0.04-0.4% of cases). Pre-existing pulmonary disease and acquired immunodeficiencies are well- known risk factors. The vast majority of severe influenza cases, however, remain unexplained, especially in children. Intriguingly, classical primary immunodeficiencies do not predispose to severe influenza. We recently reported an otherwise healthy child with severe influenza due to inherited autosomal recessive (AR) IRF7 deficiency resulting in impaired interferon (IFN)-α/β production in both plasmacytoid dendritic cells (pDCs) and induced pluripotent stem cell (iPSC)-derived pulmonary epithelial cells (PECs). This was the first demonstration that influenza susceptibility could be heritable. We hypothesize that additional cases of severe influenza in children and young adults may result from single-gene inborn errors of immunity, not necessarily displaying complete penetrance. We recently performed whole-exome sequencing (WES) in a cohort of 25 patients and their parents (trio design) with life-threatening influenza without secondary bacterial pneumonia. We discovered a homozygous IRF9 mutation in a child with life-threatening influenza and a history of adverse reaction to MMR vaccination. IRF9, with STAT1 and STAT2, is one of the three subunits of ISGF3, the main transcriptional complex that drives antiviral type I IFN responses. The mutation is private to this kindred and affects an essential nucleotide at a splice site. We intend to test whether the mutation is deleterious, in terms of expression and function. We will also test the patient's cells for response to type I IFN and their control of influenza virus in the presence of type I IFNs. Finally, we will compare the genome-wide transcriptome responses to type I IFNs in the patient's cells with those from other patients with autosomal recessive, complete defects in IFNAR1, IFNAR2, STAT1, and STAT2. Our preliminary data are convincing and strong, as we showed that the splice mutation is not leaky and the encoded protein is loss of ISGF3 expression and function. Moreover, the patient's cells seem to control influenza virus poorly even in the presence of exogenous type I IFNs. Finally, we have collected cells from patients with all types of inborn errors of the type I IFN responsive pathway. Our project is thus highly innovative, yet supported by strong preliminary evidence. Collectively, our project will characterize, in depth, the molecular and cellular basis of a new primary immunodeficiency, autosomal recessive IRF9 deficiency, thereby documenting a second genetic and immunological etiology of severe influenza of childhood. This study will give weight to the emerging notion that severe influenza of childhood can be caused by single-gene inborn errors of immunity. Our study will also have new and important biological implications, providing the first analysis of IRF9-dependent and -independent responses to type I IFNs in humans. Overall, our discovery will have both biological and clinical implications.