Molecular Genetics of Dyskeratosis Congenita

先天性角化不良的分子遗传学

• 批准号: 9079942
• 负责人: Alison A Bertuch
• 金额: $ 52.26万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-15 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9079942
• 关键词: Acute Myelocytic Leukemia; Affect; Age; Alleles; Amino Acids; Binding; Biology; Blood Cell Count; C-terminal; Cell Cycle; Cell Line; Child; Childhood; Chromosomes; Cicatrix; Cirrhosis; Complex; DNA; DNA Damage; Data; Defect; Disease; Docking; Dyskeratosis Congenita; Dysmyelopoietic Syndromes; Enzymes; Gene Components; Generations; Genes; Genetic Anticipation; Genetic Complementation Test; Goals; Head and Neck Squamous Cell Carcinoma; Hematopoietic Stem Cell Transplantation; Hereditary Disease; Immunoprecipitation; In Vitro; Incidence; Individual; Inherited; Intervention; Lead; Length; Life; Light; Liver diseases; Lung; Malignant Neoplasms; Mass Spectrum Analysis; Medical; Methods; Missense Mutation; Molecular; Molecular Genetics; Mutate; Mutation; Nuclear; Pancytopenia; Pathogenesis; Patients; Phosphorylation; Phosphorylation Site; Predisposition; Process; Protein Binding Domain; Protein Isoforms; Protein Region; Proteins; Protocols documentation; Pulmonary Fibrosis; Reporting; Research; Risk; Role; Series; Site; Solid Neoplasm; Structure; Syndrome; TERF1 gene; TINF2 gene; Telomerase; Telomere Length Maintenance; Telomere Maintenance; Telomere Shortening; Telomeric Repeat Binding Protein 2; Testing; Work; autosomal dominant mutation; bone marrow failure syndrome; early onset; effective therapy; in vivo; kindred; knock-down; lifetime risk; lymphoblastoid cell line; mutant; novel; public health relevance; response; telomere

 DESCRIPTION (provided by applicant): Dyskeratosis congenita (DC) is a multisystem disorder caused by defective telomere maintenance. Bone marrow failure, myelodysplastic syndrome, pulmonary fibrosis, solid tumors, and liver disease are life-threatening manifestations of DC. The goal of our research is to understand the molecular genetics of DC, thereby opening avenues for targeted interventions that may be relevant not only to DC but also to other conditions associated with telomere shortening. Mutations in TINF2, which encodes the telomeric shelterin complex component, TIN2, are causes of DC. An outstanding question is why heterozygous and de novo mutations in TINF2 result in extreme telomere shortening and severe, early-onset disease whereas heterozygous mutations in telomerase component genes lead to more severe manifestations of disease and progressively earlier onset disease over generations in kindred's. All DC-associated TINF2 mutations reported to date, whether missense or truncating, cluster in a 34-amino acid segment located centrally within both the short (TIN2S1-354) and long (TIN2L1-451) TIN2 isoforms. Our research focuses on the unstudied TIN2L isoform, which either harbors missense mutations within the DC cluster or is not expressed when truncating mutations are present. Our preliminary data indicate that TIN2L and TIN2S are not equivalent components of shelterin and that the most common DC-associated missense mutation has deleterious effects on TIN2L but not TIN2S. Additionally, we found that TIN2L contains a conserved phosphorylation site that, when mutated, has the same functional effect on TIN2L as mutations within the DC cluster. Lastly, we found that the enhanced interaction between TIN2L and TRF2 requires TRF2-F120, a key residue within the TRF homology (TRFH) domain that serves as a docking site for various DNA-processing and DNA damage response factors. We hypothesize that TIN2L regulates the ability of TRFH domain-interacting proteins to access TRF2 and that altered TIN2L activity and enhanced recruitment of TIN2L to telomeres drives telomere shortening in patients. In Specific Aim (SA) 1 of this proposal, we will determine the relative impacts of TIN2S and TIN2L on telomere structure and function by generating an allelic series of mutant cell lines and performing complementation tests with patient-derived TINF2 mutant cell lines. We will also determine if TIN2L, compared to TIN2S, differentially interacts with other proteins in addition to TRF2 using a novel immunoprecipitation protocol followed by mass spectrometry sequencing methods. Finally, we will screen genetically uncharacterized patients with very short telomeres for specific mutations in TIN2L. In SA2, we will elucidate the function of TIN2L phosphorylation by identifying its impact on TRF2 binding and clarifying its relationship with the DC cluster through an array of in vitro and in vivo analyses. In SA3, we will determine the functional interplay between TIN2L and TRF2 TRFH domain- interacting proteins and identify how they are altered in TINF2 mutant cell lines. This work is expected to not only shed light on DC pathogenesis but also generate new paradigm shifts in telomere biology.

 描述（由申请人提供）：先天性角化不良（DC）是一种由端粒维持缺陷引起的多系统疾病，骨髓增生异常综合征、肺纤维化、实体瘤和肝脏疾病是 DC 的危及生命的表现。研究的目的是了解 DC 的分子遗传学，从而为有针对性的干预措施开辟途径，这些干预措施可能不仅与 DC 相关，而且还与端粒缩短相关的其他疾病相关。编码端粒庇护蛋白复合体成分 TIN2 的 TINF2 突变是 DC 的病因，一个突出的问题是为什么 TINF2 的杂合突变会导致端粒极度缩短和严重的早发性疾病，而端粒酶成分基因的杂合突变会导致这种情况。迄今为止报道的所有 DC 相关 TINF2 突变是否会导致更严重的疾病表现以及在亲属中逐渐提早的疾病发作。错义或截短，簇位于位于短 (TIN2S1-354) 和长 (TIN2L1-451) TIN2 亚型中心的 34 个氨基酸片段中。我们的研究重点是未经研究的 TIN2L 亚型，该亚型在 DC 内含有错义突变。当存在截短突变时，TIN2L 和 TIN2S 不表达。且最常见的 DC 相关错义突变对 TIN2L 但对 TIN2S 没有有害影响。此外，我们发现 TIN2L 包含一个保守的磷酸化位点，当突变时，该位点对 TIN2L 具有与 DC 簇内的突变相同的功能影响。最后，我们发现TIN2L和TRF2之间增强的相互作用需要TRF2-F120，TRF2-F120是TRF同源（TRFH）结构域内的一个关键残基，充当对接我们发现 TIN2L 调节 TRFH 结构域相互作用蛋白访问 TRF2 的能力，并改变 TIN2L 活性并增强 TIN2L 向端粒的募集，从而驱动患者端粒缩短。 SA) 1 该提案中，我们将通过生成突变细胞的等位基因系列来确定 TIN2S 和 TIN2L 对端粒结构和功能的相对影响细胞系并使用患者来源的 TINF2 突变细胞系进行互补测试，与 TIN2S 相比，我们还将使用新型免疫沉淀方案和质谱测序方法来确定 TIN2L 是否与 TRF2 之外的其他蛋白质存在差异相互作用。筛查端粒非常短的未遗传特征患者的特定情况 在 SA2 中，我们将通过一系列体外和体内分析确定 TIN2L 磷酸化对 TRF2 结合的影响并阐明其与 DC 簇的关系，从而阐明 TIN2L 磷酸化的功能。 TIN2L 和 TRF2 TRFH 结构域相互作用蛋白之间的关系，并确定它们在 TINF2 突变细胞系中如何改变，这项工作不仅有望揭示 DC 发病机制，而且会产生新的范例。端粒生物学的变化。