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

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，各种生物化学和细胞生物学分析可以应用于患者来源的细胞和/或建立的细胞模型。功能研究对于破译未表征的开放阅读框的功能尤其重要，其中致病性变异导致疾病。在这个项目中，我们将对三个基因的致病变异进行功能研究。我们鉴定了新的FBXW11错义变体c.1139C>T/p。（Ser380Phe）在颅缝紧闭，小头畸形，三叶草头骨和面部畸形患者。在不同表型的个体中，已经报道了7个与疾病相关的FBXW11错义变异。FBXW11编码一种F-box蛋白，该蛋白作为s期激酶相关蛋白(SKP)-cullin - 1-F-box蛋白泛素连接酶复合物的底物适配器，参与各种底物的蛋白酶体降解。为了深入了解FBXW11变体的不同功能效应，我们将通过确定ha标记的FBXW11野生型和突变体在细胞系统中的结合伙伴集，研究p.（Ser380Phe）和四种已发表的变体对FBXW11底物结合的影响。与FBXW11突变体结合减弱或增强的底物将被进一步研究，以确定它们的周转率、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变体影响的特定途径和/或细胞功能。我们发现C20orf204编码一种特征不明显的蛋白，是一种新的常染色体隐性疾病基因，可导致严重的免疫缺陷。我们的初步数据显示C20orf204在naïve T细胞中表达，并预测其作为细胞因子或细胞因子抑制剂的功能。为了表征C20orf204在T细胞中的生物学作用，我们将与相关领域的专家合作。我们将在真核细胞中制备和纯化重组C20orf204蛋白。然后，我们将通过分析C20orf204是否通过白细胞介素7受体发出信号，C20orf204是否作为胸腺基质淋巴生成素的负调节因子，以及C20orf204在naïve T细胞存活和维持中的作用，来研究C20orf204在控制T细胞稳态中的可能功能。我们在小头型原始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/引物酶复合体来启动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背后的生物过程的理解。

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.