Molecular and cellular basis of epidermodysplasia verruciformis

项目摘要 疣状表皮发育不良(EV)是第一个被描述的原发性免疫缺陷(PID)。到1946年，它是 在描述之前，Lutz显示为嗜皮性病毒的常染色体隐性遗传(AR)易感性 Kostmann(1950)的先天性中性粒细胞减少症。它缺乏相关的免疫表型，长期以来阻止了它的 被认为是一个PID。EV的特征是弥漫和持续的扁平疣，通常会演变成皮肤。 癌症。这些皮损是由人乳头瘤病毒β属的E5和E8缺陷成员引起的 (HPV)，它只存在于角质形成细胞中，在普通人群中保持沉默。电动汽车通常会罢工 其他健康的人(“隔离的EV”)，或很少发生在其他传染病的背景下 (“综合症EV”)。2002年，在中国发现了编码Ever1和EveR2的TMC6和TMC8双等位基因突变。 单纯性EV患者，T细胞正常。CIB1双等位基因突变于2018年在其他 单纯性EV患者。值得注意的是，CIB1、Ever1和EveR2形成了一个与E5和E8结合的复合体。这 复合体是角质形成细胞对β-HPV固有免疫的限制因子。从2012年起， 在综合征EV患者中发现了RhoH、STK4和其他T细胞基因的突变。我们假设 对β-HPV的皮肤免疫的另一个相关的单基因先天错误，是其他患者EV的基础。 因此，这一应用程序的目标是更深入地分析分离的和 综合征型EV。首先，我们将发现新的EV基因病因，这要归功于不断招募无关的 EV家族，通过全基因组连锁(GWL)和全外显子组测序(WES)相结合。第二，我们将 通过研究分离和患者体内的突变蛋白来表征新的基因类型 细胞，包括它们与已知的EV致病基因产物的关系，如Ever-CIB1 角质形成细胞中的复合体和T细胞中的RhoH或STK4。第三，我们将建立HPV感染角质形成细胞的模型。 利用角质形成细胞系、包皮角质形成细胞、 患者的角质形成细胞，或诱导多能步进细胞(IPSC)来源的角质形成细胞，将由 CRISPR/CAS9.我们的项目是高度创新的，但得到了强大的初步数据的支持。事实上，我们有 招募了52个新的家系，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OX40)，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。我们的研究为皮肤对β-HPV的免疫机制提供了新的见解。 强调角质形成细胞内源性免疫和T细胞获得性免疫的双重贡献。我们的研究 使EV患者和家庭受益，开发新的诊断方法，包括基因 咨询，并促进基于理性理解的新治疗方法的开发 对发病机制的认识。最后，EV的研究是分析其他粘膜和皮肤疾病的一个富有成效的模型。 由其他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.