Autosomal dominant OTULIN deficiency and staphylococcal disease

项目总结 金黄色葡萄球菌是影响人类健康的主要致病菌。然而，尽管大多数受试者携带S。 在金黄色葡萄球菌中，只有一小部分人会患上危及生命的葡萄球菌疾病。我们假设严重的或复发的 葡萄球菌感染，至少在一些患者中，可由单基因先天性免疫错误(IEI)引起。 我们已经证明，与NF-kB和TLR2相关的基因中的IEI易感于葡萄球菌感染。AS 作为该项目的基础，100名患有严重或反复金黄色葡萄球菌感染的儿童和年轻人组成的队列 已经使用完整外显子组测序(WES)对其进行了遗传分析，这是实验室为 IEIS。在全基因组范围内寻找候选基因类型，通过测试各种遗传模型，我们 在6个家系的7名患者中发现OTULIN罕见的杂合性非同义变异。奥图林，一个 脱泛素酶是已知的调节炎症的酶，但不能控制抗葡萄球菌免疫。双等位基因 OTULIN的有害变异导致一种称为OTULIN相关自炎的严重早发性疾病 综合征(ORAS)。有趣的是，OTULIN位于5号染色体的短臂上，经常被删除 在5P-(Cri-Du-Chat)综合征中，这是一种患病率为1/30,000的遗传病。患有5P综合征的患者 容易受到细菌感染。我们假设常染色体显性遗传性OTULIN缺乏症是通过 单倍体功能不全是葡萄球菌疾病的基础。初步结果显示，这6名患者的等位基因是 4例杂合子携带者真皮成纤维细胞功能丧失与OTULIN表达 OTULIN变异体和来自1名5P综合征患者(OTULIN副本缺失)的大约一半，当 与健康对照组比较。此外，还观察到线性泛素链在真皮中的积累。 来自3名患者的成纤维细胞，包括1名5P综合征患者。最后，我们注意到洞穴的堆积， 已知可促进ADAM10的募集，ADAM10是主要葡萄球菌毒力因子a-的受体- 溶血素。我们始终如一地观察到患者成纤维细胞对a-溶血素的高度选择性敏感性。 细胞毒性。因此，本申请的目的是更深入地探索OTULIN的具体作用 在OTULIN有害变异杂合子患者的成纤维细胞和单核细胞中。我们会调查的 奥图林对真皮成纤维细胞线性泛素体的影响，特别是对核因子-kB和 使用转录和蛋白质组学方法检测TLR2下游的炎症标志物。我们还将 研究患者白细胞亚群的先天免疫反应。最后，我们将研究内在的 患者对包括a-溶血素在内的细菌毒素的免疫力，与小窝调节失调和 ADAM10表达式。总体而言，这项研究将支持细菌感染可以 在遗传免疫缺陷的情况下优先引发严重疾病。这个项目将给我们带来新的启示 金黄色葡萄球菌病的发病机制及OTULIN的生物学，为新的预防方法铺平道路 以及针对OTULIN突变患者的治疗措施，包括那些患有5P综合征的患者。

PROJECT SUMMARY Staphylococcus aureus is a major bacterial pathogen impacting human health. Yet, while most subjects carry S. aureus, only a minority develop life-threatening staphylococcal disease. We hypothesize that severe or recurrent staphylococcal infections, at least in some patients, can result from single-gene inborn errors of immunity (IEI). We have already shown that IEIs in NF-kB- and TLR2-related genes predispose to staphylococcal infections. As a basis for this project, a cohort of 100 children and young adults with severe or recurrent S. aureus infections has been analyzed genetically using whole exome sequencing (WES), which has been pioneered in the lab for IEIs. Searching for candidate genotypes in a genome-wide manner, by testing various genetic models, we discovered heterozygous rare non-synonymous variants in OTULIN in 7 patients from 6 kindreds. OTULIN, a deubiquitinase, is known to regulate inflammation but not to govern anti-staphylococcal immunity. Bi-allelic deleterious variants in OTULIN cause a severe early-onset condition termed OTULIN-related autoinflammatory syndrome (ORAS). Interestingly, OTULIN is located on the short arm of the chromosome 5 and is often deleted in the 5p- (Cri-du-Chat) Syndrome, a genetic disease with a prevalence of 1/30,000. Patients with 5p- Syndrome are susceptible to bacterial infections. We hypothesize that autosomal dominant OTULIN deficiency operates by haploinsufficiency and underlies staphylococcal disease. Preliminary results show that the 6 patients’ alleles are loss-of-function and that expression of OTULIN in dermal fibroblasts from 4 patients carrying heterozygous OTULIN variants and from 1 patient with 5p- Syndrome (with a missing copy of OTULIN) is about half, when compared with healthy controls. Moreover, accumulation of linear ubiquitin chains was observed in dermal fibroblasts from 3 patients, including one with the 5p- Syndrome. Finally, we noticed an accumulation of Caveolin, which is known to facilitate recruitment of ADAM10, the receptor for the major staphylococcal virulence factor a- hemolysin. Consistently, we observed a high and selective susceptibility of patients’ fibroblasts to a-hemolysin cytotoxicity. The aim of the present application is therefore to explore in more depth the specific role of OTULIN in fibroblasts and monocytes from patients heterozygous for deleterious variants in OTULIN. We will investigate the impact of OTULIN on the linear ubiquitinome in dermal fibroblasts, with a special emphasis on NF-kB and markers of inflammation downstream TLR2 using transcriptomic and proteomics approaches. We will also investigate the innate immune responses in patients’ leukocyte subsets. Finally, we will investigate the intrinsic immunity of patients to bacterial toxins, including a-hemolysin, in the context of Caveolin dysregulation and ADAM10 expression. Overall, this study will give weight to the emerging notion that bacterial infection can preferentially trigger severe disease in the context of genetically deficient immunity. This project will shed light on the pathogenesis of staphylococcal disease and biology of OTULIN, while paving the way for novel preventive and therapeutic measures for OTULIN-mutated patients, including those with the 5p- Syndrome.