Molecular and cellular basis of epidermodysplasia verruciformis

项目摘要 疣状表皮发育不良（EV）是最早发现的原发性免疫缺陷（PID）。到1946年， Lutz显示，在描述皮肤嗜性病毒之前， 先天性中性粒细胞减少症，Kostmann（1950）。由于缺乏相关的免疫表型， 识别为PID。EV的特征是播散性和持续性扁平疣，通常演变为皮肤 癌病变是由人乳头瘤病毒β属E5和E8缺陷成员引起的 HPV（人类乳头状瘤病毒），其仅存在于角质形成细胞中并且在一般人群中保持沉默。电动汽车通常罢工 其他健康个体（“孤立EV”），或很少在其他传染病背景下发生 （“综合征EV”）。2002年，在TMC 6和TMC 8中发现了编码EVER 1和EVER 2的双等位基因突变， 分离EV患者，其T细胞正常。2018年在其他研究中报告了CIB 1的双等位基因突变 孤立性EV患者。值得注意的是，CIB 1，EVER 1和EVER 2形成了一个与E5和E8结合的复合物。这 复合物作为控制角化细胞对β-HPV的内在免疫的限制因子起作用。从2012年开始， 在EV综合征患者中发现了RHOH、STK 4和其他T细胞基因的突变。我们假设 其他相关的单基因先天性皮肤抗β-HPV免疫缺陷是其他患者EV的基础。 因此，本申请的目标是更深入地分析分离的和 EV综合征首先，我们将发现新的EV遗传病因，这要归功于正在招募的无关的 EV家族，通过结合全基因组连锁（GWL）和全外显子组测序（WES）。二是 通过研究分离的突变蛋白和患者的突变蛋白， 细胞，包括它们与已知EV致病基因产物的关系，如EVER-CIB 1 复合物和T细胞中的RhoH或STK 4。第三，我们将对HPV感染的角质形成细胞进行建模， 使用角质形成细胞系，包皮角质形成细胞， 患者的角质形成细胞或诱导多能性步进细胞（iPSC）衍生的角质形成细胞，其将由 CRISPR/Cas9.我们的项目具有高度创新性，但有强大的初步数据支持。我确已 招募了52个新的家庭，确定了三种新的遗传病因，潜在的孤立（RBPJ突变）或 综合征EV（ITGAL和OX 40），并开始阐明它们与角质形成细胞和T细胞的机制联系 细胞，分别。我们的研究为β-HPV的皮肤免疫机制提供了新的见解， 突出了角质形成细胞内在免疫和T细胞适应性免疫的双重作用。我们的研究 随着新诊断方法的发展，包括遗传学， 咨询，并促进基于理性理解的新型治疗方法的发展 的发病机制。最后，EV的研究是分析其他粘膜和皮肤疾病的富有成效的模型 其他HPV引起的。

Project Summary Epidermodysplasia verruciformis (EV) was the first described primary immunodeficiency (PID). By 1946, it was shown by Lutz to be an autosomal recessive (AR) predisposition to skin-tropic viruses, prior to the description of congenital neutropenia by Kostmann (1950). Its lack of associated immunological phenotypes long prevented its recognition as a PID. EV is characterized by disseminated and persistent flat warts, which often evolve into skin cancer. The lesions are caused by E5- and E8-deficient members of the β genus of human papillomaviruses (HPVs), which exclusively reside in keratinocytes and remain silent in the general population. EV typically strikes otherwise healthy individuals (“isolated EV”), or rarely occurs in the context of other infectious diseases (“syndromic EV”). In 2002, bi-allelic mutations in TMC6 and TMC8, encoding EVER1 and EVER2, were found in patients with isolated EV, whose T cells were normal. Bi-allelic mutations in CIB1 were reported in 2018 in other patients with isolated EV. Remarkably, CIB1, EVER1, and EVER2 form a complex that binds to E5 and E8. This complex operates as a restriction factor governing keratinocyte-intrinsic immunity to β-HPVs. From 2012 onward, mutations in RHOH, STK4, and other T cell genes were found in patients with syndromic EV. We hypothesize that other, related single-gene inborn errors of cutaneous immunity against β-HPVs, underlie EV in other patients. The goal of this application is thus to analyze in greater depth the molecular and cellular basis of isolated and syndromic EV. First, we will discover new genetic etiologies of EV thanks to the ongoing recruitment of unrelated EV families, by combining genome-wide linkage (GWL) and whole exome sequencing (WES). Second, we will functionally characterize the novel genotypes by studying the mutant proteins in isolation and in the patients’ cells, including their relationship with the products of the known EV-causing genes, such as the EVER-CIB1 complex in keratinocytes and RhoH or STK4 in T cells. Third, we will model HPV infection of keratinocytes in the presence of T cells in vitro, with viral proteins and particles, using keratinocyte cell lines, foreskin keratinocytes, the patients’ keratinocytes, or induced pluripotent step cell (iPSC)-derived keratinocytes, which will be edited by CRISPR/Cas9. Our project is highly innovative yet supported by strong preliminary data. Indeed, we have recruited 52 novel families, identified three novel genetic etiologies, underlying isolated (mutations in RBPJ) or syndromic EV (ITGAL and OX40), and began elucidating their mechanistic connection with keratinocytes and T cells, respectively. Our research provides novel insights into the mechanisms of cutaneous immunity to β-HPVs, highlighting the dual contribution of keratinocyte-intrinsic immunity and T-cell adaptive immunity. Our research benefits EV patients and families, with the development of novel diagnostic approaches, including genetic counseling, and facilitating the development of novel therapeutic approaches based on a rational understanding of the pathogenesis. Finally, the study of EV is a fruitful model to analyze other mucosal and cutaneous illnesses caused by other HPVs.