Molecular characterization of TUBB4 mutations

我们最近发现β微管蛋白4（TUBB 4或TUBB 4A）基因突变是DYT 4肌张力障碍的原因。DYT 4的特征是突出的耳语发音困难和全身性肌张力障碍。随着在肌张力障碍微管蛋白基因突变的检测，一个新的途径，即。e.细胞骨架的破坏与肌张力障碍的病理生理学有关。在本提案中，我们旨在阐明TUBB 4突变的谱和功能后果，具有以下三个具体目标：（1）为了确定TUBB 4突变引起的肌张力障碍表型谱，我们将对约2000名患有不同形式的肌张力障碍/肌张力障碍综合征的患者的所有编码外显子进行测序。(2)为了确定TUBB 4中已知和新突变在分子水平上的影响，我们将分析三种已知和最多两种新鉴定的突变体和野生型TUBB 4的mRNA水平、蛋白质稳定性和亚细胞定位。为此，我们将主要使用生物学相关的细胞模型。即从我们的突变携带者产生的诱导多能干细胞（iPSC）衍生的神经元，以及TALEN校正的同基因对照系。(3)为了研究iPSC衍生的神经元中的神经元形态和轴突运输，我们将分析线粒体运输和神经突生长，并测试微管蛋白稳定剂拯救TUBB 4相关神经元功能障碍的能力。

We recently identified mutations in the beta Tubulin 4 (TUBB4 or TUBB4A) gene as the cause of DYT4 dystonia. DYT4 is characterized by prominent whispering dysphonia and generalized dystonia. With the detection of mutations in a tubulin gene in dystonia, a novel pathway, i. e. disruption of the cytoskeleton, has been implicated in the pathophysiology of dystonia warranting functional characterization. In the present proposal, we aim to elucidate the spectrum and functional consequences of TUBB4 mutations with the following three specific aims: (1) To determine the spectrum of dystonic phenotypes caused by mutations in TUBB4, we will sequence all coding exons in ~2000 patients with different forms of dystonia/dystonic syndromes. (2) To identify the impact of known and novel mutations in TUBB4 on the molecular level, we will analyze mRNA levels, protein stability, and subcellular localization of the three known and up to two newly identified mutants and wildtype TUBB4. For this, we will primarily use a biologically relevant cell model. i.e. induced pluripotent stem cell (iPSC)-derived neurons generated from our mutation carriers, as well as TALEN-corrected isogenic control lines. (3) To study neuronal morphology and axonal transport in iPSC-derived neurons, we will analyze mitochondrial transport and neurite outgrowth and test the ability of a tubulin-stabilizing agent to rescue TUBB4-related neuronal dysfunction.