Molecular Genetics of Dyskeratosis Congenita

 描述(申请人提供)：先天性角化不良(DC)是一种由端粒维护缺陷引起的多系统疾病。骨髓衰竭、骨髓增生异常综合征、肺纤维化、实体瘤和肝脏疾病是DC危及生命的表现。我们研究的目标是了解DC的分子遗传学，从而为可能不仅与DC相关而且与其他与端粒缩短相关的疾病的靶向干预开辟道路。编码端粒保护素复合体成分TIN2的TINF2突变是DC的原因。一个突出的问题是，为什么TINF2的杂合和从头突变导致极端的端粒缩短和严重的早发性疾病，而端粒酶成分基因的杂合突变导致更严重的疾病表现和世代更早的疾病发病。迄今为止报道的所有DC相关的TINF2突变，无论是错义还是截断，都聚集在一个34个氨基酸的片段上，位于短(TIN2S1-354)和长(TIN2L1-451)TIN2亚型的中心。我们的研究重点是未研究的TIN2L亚型，它要么在DC簇中含有错义突变，要么在存在截断突变时不表达。我们的初步数据表明，TIN2L和TIN2S不是Shelterin的等同成分，最常见的DC相关错义突变对TIN2L有有害影响，但对TIN2S没有影响。此外，我们发现TIN2L包含一个保守的磷酸化位点，当突变时，对TIN2L的功能影响与DC簇内的突变相同。最后，我们发现TIN2L和TRF2之间的相互作用的增强需要TRF2-F120，TRF2-F120是TRF同源结构域(TRFH)中的一个关键残基，作为各种DNA加工和DNA损伤反应因子的对接位点。我们假设TIN2L调节TRFH结构域相互作用蛋白访问TRF2的能力，TIN2L活性的改变和TIN2L对端粒的募集增加导致患者端粒缩短。在这项建议的特定目的(SA)1中，我们将通过产生一系列等位基因突变细胞系并与患者来源的TINF2突变细胞系进行互补测试，来确定TIN2S和TIN2L对端粒结构和功能的相对影响。我们还将利用一种新的免疫沉淀方案和随后的质谱测序方法来确定TIN2L是否与TIN2S相比，与TRF2以外的其他蛋白质有不同的相互作用。最后，我们将筛查具有非常短端粒的无基因特征的患者的特定基因。 TIN2L基因突变。在SA2中，我们将通过一系列体外和体内分析来确定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。在SA3中，我们将确定TIN2L和TRF2 TRFH结构域相互作用蛋白之间的功能相互作用，并确定它们在TINF2突变细胞系中是如何改变的。这项工作不仅有望阐明DC的发病机制，而且还将在端粒生物学中产生新的范式转变。

 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.