Uncovering the molecular basis of SEPT2-associated Tyshchenko syndrome

Tyshchenko综合征(TS)于2011年由Tyshchenko等人首次描述。作为一种常染色体显性遗传综合征，表现为明显的面部特征、特定的畸形和学习障碍。这些微小的异常类似于Baraitser-温特脑额面部综合征(BWCFF)。虽然已知非肌肉肌动蛋白基因ACTB或ACTG1的突变导致BWCFF，但尚未找到TS的潜在原因。通过整个外显子组测序，我们在Tyshchenko等人描述的两个原始家族中的一个中发现了SEPT2错义突变。此外，在另外两名面部特征相同的无关患者中，我们发现SEPT2发生了从头开始的错义突变。在人类Septin 2的三维蛋白质模型中，所有三个突变都位于空间上非常接近的位置。Septin 2和其他Septin一样，在包括胞质分裂在内的各种细胞过程中发挥着重要作用。间隔蛋白是细胞骨架的重要组成部分，是细胞内的支架和扩散屏障。它们都具有具有GTPase活性的鸟嘌呤核苷酸结合域和NC界面，即它们的N-端和C-端延伸。通过这两个结构域，Septin相互作用形成线性杂化低聚物，最常见的是Septin 2位于其中心，作为络合物形成的启动者。该项目的主要目的是阐明导致SEPT2相关TS的分子机制，并揭示Septin-肌动蛋白相互作用的分子机制，这些分子机制导致TS和BWCFF具有相似但不同的临床表现。为此，我们将进行体外实验，利用重组蛋白和蛋白结构域的免疫沉淀来演示SEPT2突变对Septin寡聚的影响。由于在TS患者中发现的Septin 2的所有突变都位于NC界面，我们推测TS患者Sept2的同源二聚或与其他Septin的异源寡聚可能受到干扰。此外，我们还将研究在胞质分裂过程中SEPT2突变对Septin功能的影响。我们还将通过免疫染色和使用纯化的重组蛋白来检测Septin-肌动蛋白的相互作用，以建立TS和BWCFF之间的功能联系。最终，我们希望加深对不同细胞骨架组件之间相互作用的洞察，并拓宽我们对细胞骨架的形成和功能的知识。通过这一点，我们希望为患者带来最终的好处，了解他们的情况，预测未来的并发症，并促进治疗可能性的发展。

Tyshchenko syndrome (TS) was first described in 2011 by Tyshchenko et al. as an autosomal-dominant syndrome presenting with distinct facial features, specific malformations and learning disabilities. The minor anomalies resemble Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF). Whereas mutations in either non-muscular Actin gene ACTB or ACTG1 are known to cause BWCFF, no underlying cause of TS could be found.Via whole exome sequencing, we identified a missense mutation in SEPT2 in one of the two original families described by Tyshchenko et al. In addition, in two other unrelated patients with the same striking facial features we found de novo missense mutations in SEPT2. In a 3-D protein model of human Septin 2, all three mutations are located in close spatial proximity.Septin 2, like other Septins, plays a major role in a variety of cell processes including cytokinesis. Septins are important components of the cytoskeleton and serve as scaffolds and diffusion barriers in the cell. They all possess a guanine nucleotide binding domain with GTPase activity and an NC interface, that is their N- and C-terminal extensions. Via both domains, Septins interact forming linear hetero-oligomers, most commonly with Septin 2 located is its centre, functioning as an initiator of the complex formation. Amongst others, filamentous Septins also closely co-localize with Actin and control F-Actin assembly and Actin organization.The main goal of this project is to delineate the molecular mechanisms leading to SEPT2-related TS and to unravel the molecular mechanisms in Septin-Actin interaction, that lead to a similar yet differing clinical presentation of TS and BWCFF.To do so, we will perform in vitro experiments to demonstrate the effect of SEPT2 mutations on Septin-oligomerization using immunoprecipitation of recombinant proteins and protein-domains. As all of the mutations found in TS patients in Septin 2 are located in the NC interface, we hypothesize that the homodimerization of Sept2 or the hetero-oligomerization with other Septins might be disturbed in TS patients. In addition, we will investigate the effects of SEPT2 mutations on the Septin function during cytokinesis. We will also measure Septin-Actin interactions via immunostaining and using purified recombinant proteins in order to establish a functional link between TS and BWCFF. Eventually, we want to deepen our insights into the interaction between different cytoskeletal components and broaden our knowledge of the formation and function of the cytoskeleton. Through this, we want to lead to an ultimate benefit to the patients, understand their condition, anticipate future complications and facilitate the development of therapeutic possibilities.