Deciphering the pathomechanism of monogenic disorders associated with pathogenic variants in C20orf204, FBXW11 and WDHD1

破译与 C20orf204、FBXW11 和 WDHD1 致病变异相关的单基因疾病的病理机制

• 批准号: 511275754
• 负责人: Professorin Dr. Kerstin Kutsche
• 金额: --
• 依托单位: Institut für Humangenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/511275754?language=en
• 关键词: Deciphering; pathomechanism; monogenic; disorders; associated

Next-generation sequencing technologies have accelerated the discovery of novel disease genes for monogenic disorders. To prove pathogenicity of genomic variants and improve our understanding of disease biology and pathophysiology, a variety of bio¬che¬mical and cell biological assays can be applied on patient-derived cells and/or well-established cell models. Functional studies are particularly important to decipher the function of uncharacterized open reading frames in which pathogenic variants cause disease.Within this project, we will perform functional studies for pathogenic variants in three genes. We identified the de novo FBXW11 missense variant c.1139C>T/p.(Ser380Phe) in a patient with craniosynostosis, microcephaly, cloverleaf scull, and facial dysmorphism. Seven de novo disease-associated FBXW11 missense variants in individuals with diverse phenotypes have been reported. FBXW11 encodes an F-box protein that functions as substrate adaptor for the S-phase kinase-associated protein (SKP)-cullin 1-F-box protein ubiquitin ligase complexes involved in proteasomal degradation of various substrates. To gain insight into the differential functional effects of FBXW11 variants, we will study the impact of p.(Ser380Phe) and four published variants on FBXW11 substrate binding by determining the set of binding partners of HA-tagged FBXW11 wildtype and mutants in a cellular system. Substrates with decreased or enhanced binding to FBXW11 mutants will be investigated further to determine their turnover rate and steady-state and ubiquitination levels. Possible further experiments will focus on specific pathways and/or cellular functions affected by p.(Ser380Phe) and/or other FBXW11 variants. We identified C20orf204, encoding a poorly characterized protein, as a novel autosomal recessive disease gene for a severe immunodeficiency. Our preliminary data revealed expression of C20orf204 in naïve T cells and a predicted function as a cytokine or cytokine inhibitor. To characterize the biological role of C20orf204 in T cells, we will collaborate with experts in the respective field. We will produce and purify recombinant C20orf204 protein in eukaryotic cells. We will then study the possible function of C20orf204 in controlling T cell homeostasis by analyzing if C20orf204 signals through the interleukin 7 receptor, if C20orf204 is a negative regulator of thymic stromal lymphopoietin and by investigating the role of C20orf204 in the survival and maintenance of naïve T cells. We identified biallelic WDHD1 pathogenic variants in individuals with microcephalic primordial dwarfism. WDHD1 integrates the CMG helicase complex and DNA polymerase α/primase complex to initiate DNA replication. By using patient-derived fibroblasts, we will study the impact of WDHD1 variants on cell cycle and cell proliferation, DNA damage response, and the DNA replication machinery. Our studies will advance our understanding of biological processes underlying human health and disease.

下一代测序技术加速了单基因疾病新疾病基因的发现。为了证明基因组变异的致病性，并提高我们对疾病生物学和病理生理学的理解，可以在患者来源的细胞和/或成熟的细胞模型上应用各种生物化学和细胞生物学检测方法。功能研究对于破译致病变异导致疾病的未知开放阅读框架的功能尤为重要。在这个项目中，我们将对三个基因中的致病变异进行功能研究。我们在一名患有颅缝融合、小头畸形、三叶草畸形和面部畸形的患者中发现了新发现的FBXW11错义变体c.1139C&gt；T/p(Ser380Phe)。已有7个与新发疾病相关的FBXW11错义变异在具有不同表型的个体中被报道。FBXW11编码一个F-box蛋白，它作为底物适配器与S相激酶相关蛋白-cullin 1-F-box蛋白泛素连接酶复合体结合，参与多种底物的蛋白酶体降解。为了深入了解FBXW11突变体的不同功能效应，我们将通过确定细胞系统中HA标记的FBXW11野生型和突变体的结合伙伴集，研究P.(Ser380Phe)和四个已发表的突变体对FBXW11底物结合的影响。与FBXW11突变体结合减少或增强的底物将被进一步研究，以确定它们的周转率以及稳态和泛素化水平。可能的进一步实验将集中于受P.(Ser380Phe)和/或其他FBXW11变体影响的特定通路和/或细胞功能。我们鉴定C20orf204是一种新的常染色体隐性遗传病基因，编码一种特征不佳的蛋白，导致严重的免疫缺陷。我们的初步数据显示，C20orf204在幼稚T细胞中表达，并被预测具有细胞因子或细胞因子抑制物的功能。为了确定C20orf204在T细胞中的生物学作用，我们将与各自领域的专家合作。我们将在真核细胞中生产和纯化重组C20orf204蛋白。然后，我们将通过分析C20orf204是否通过白细胞介素7受体传递信号，C20orf204是否是胸腺基质淋巴生成素的负调节因子，以及通过研究C20orf204在原始T细胞存活和维持中的作用来研究C20orf204在控制T细胞动态平衡中的可能作用。我们在小头症原始性侏儒症患者中发现了双等位基因WDHD1致病变异。WDHD1整合CMG解旋酶复合体和DNA聚合酶α/Primase复合体，启动DNA复制。通过使用患者来源的成纤维细胞，我们将研究WDHD1变异对细胞周期和细胞增殖、DNA损伤反应和DNA复制机制的影响。我们的研究将促进我们对人类健康和疾病潜在的生物过程的理解。