Molecular Genetics of the Telomere Biology Disorders

端粒生物学障碍（TBD）先天性角化不良（DC）及其严重变体是儿童期发病的， 由于染色体末端（端粒）的维护受损而引起的多系统疾病。骨 骨髓衰竭（BMF）是对生命的主要威胁，在30岁之前发生在50-80%的受影响个体中。很长的- 该项目的长期目标是阐明与以下疾病相关的基因突变的机制： TBD导致端粒缩短，并发现如何减缓或更好地逆转这种缩短。 有了这些知识，我们将实现我们的长期目标，即确定针对端粒的新治疗途径 治疗这些危及生命的疾病到目前为止，已经有15个基因与TBD相关， 这是开发通用治疗靶点的挑战。编码TIN 2的TIN 2基因突变 蛋白质是儿童DC的第二大常见原因。TINF 2的突变如何导致 显著的端粒缩短不明确。我们的初步数据表明，TIN 2-DC突变蛋白具有新的 或者比正常蛋白质更强大的功能，然而，关于蛋白质的功能， 这种功能获得的分子方面。除了复杂的遗传学之外， 该领域缺乏临床前模型系统来开发和测试干预措施。通过协同 Bertuch和Hockemeyer实验室成立于2018年，已经开发出多能和造血干细胞 培养疾病模型以及复制端粒缩短相关的人源化小鼠模型， TINF 2-DC基因突变在这里，我们建议使用这些独特的干细胞为基础的系统，以检查的影响， TINF 2-DC突变对维持端粒长度和造血的影响，并测试治疗性 治疗相关BMF的方法。总的来说，我们将实现两个相辅相成的目标：目标1将 研究TIN 2-DC突变蛋白导致短端粒的分子决定因素，例如如何 它与端粒掩蔽蛋白复合物蛋白TRF 1和TRF 2以及端粒相互作用。我们将确定是否 TIN 2-DC突变蛋白对端粒长度的毒性作用需要与TRF 1相互作用，TIN 2-DC突变蛋白的作用需要与TRF 1相互作用。 TRF 2在端粒缩短中的相互作用。最后，我们将确定关键绑定事件是否 通过DC簇区域的突变改变。在目标2中，我们将确定药理学和遗传学机制， 恢复端粒长度并挽救受损的TINF 2-DC突变细胞的适应性。我们将建立基因 在我们的干细胞模型中端粒缩短的表达特征， 健身我们将测试达那唑的效果，达那唑是一种治疗DC BMF的药物， 影响端粒长度、TERT和造血潜能。最后，我们将制定一项遗传战略， TINF 2-DC突变患者造血干细胞中的端粒延长。这两个目标建立在数据基础上 由Bertuch和Hockemeyer实验室独立开发，并通过他们的合作努力， 发挥互补的专业知识，对TBD领域产生持续的影响。

The telomere biology disorder (TBD) dyskeratosis congenita (DC) and its severe variants are childhood onset, multisystem disorders caused by impaired maintenance of the ends of chromosomes known as telomeres. Bone marrow failure (BMF) is a major threat to life, occurring in 50-80% of affected individuals by age 30. The long- term objectives of this project are to elucidate the mechanisms by which mutations in the genes associated with the TBDs lead to telomere shortening and to discover how this shortening can be slowed or, better yet, reversed. With this knowledge, we will meet our long-term goal of identifying novel treatment avenues that target telomeres for these life-threatening disorders. To date, 15 genes have been associated with the TBDs, a number that poses a challenge to developing general therapeutic targets. Mutation of the TINF2 gene, which encodes the TIN2 protein, is the second most common cause of DC in children. How mutations in TINF2, which cluster, lead to marked telomere shortening is ill-defined. Our preliminary data suggest the TIN2-DC mutant protein has a new or more robust function than the normal protein, however, many critical gaps in knowledge remain as to the molecular aspects of this gain-of-function. In addition to the complex genetics, a second major challenge in the field is the lack of preclinical model systems to develop and test interventions. Through a collaboration established in 2018, the Bertuch and Hockemeyer labs have developed pluripotent and hematopoietic stem cell culture disease models as well as a humanized mouse model that reproduce the telomere shortening associated with TINF2-DC mutations. Here we propose to use these unique stem cell-based systems to examine the impact of TINF2-DC mutations on the maintenance of telomere length and hematopoiesis, and test therapeutic approaches to treat the associated-BMF. Overall, we will undertake two complementary Aims: Aim 1 will investigate the molecular determinants by which TIN2-DC mutant protein leads to short telomeres, such as how it interacts with the telomere shelterin complex proteins TRF1 and TRF2, and telomeres. We will determine if TIN2-DC mutant protein’s toxic effect on telomere length requires interaction with TRF1 and the role of TIN2- TRF2 interaction in the telomere shortening it induces. Lastly, we will determine if critical binding events are altered by mutation of DC cluster region. In Aim 2, we will determine pharmacologic and genetic mechanisms to restore telomere length and rescue the impaired fitness of TINF2-DC mutant cells. We will establish the gene expression signature of telomere shortening in our stem cell models to derive insight into the pathways impairing fitness. We will test the effect of danazol, which is a treatment for DC BMF, to assess the relationships between impacts on telomere length, TERT, and hematopoietic potential. Lastly, we will develop a genetic strategy to elongate telomeres in TINF2-DC mutant patient hematopoietic stem cells. The two Aims build upon data developed independently by the Bertuch and Hockemeyer labs as well as through their collaborative efforts and bring to bear the complementary expertise to bring a sustained impact on the TBD field.