Molecular Genetics of Dyskeratosis Congenita

 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。骨髓衰竭、骨髓增生异常综合征、肺纤维化、实体瘤和肝病是DC的危及生命的表现。我们研究的目标是了解DC的分子遗传学，从而为有针对性的干预措施开辟途径，这些干预措施不仅可能与DC有关，而且可能与端粒缩短相关的其他疾病有关。编码端粒掩蔽蛋白复合物组分TIN 2的TIN 2突变是DC的原因。一个突出的问题是为什么TINF 2中的杂合和新生突变导致极端端粒缩短和严重的早发性疾病，而端粒酶组分基因中的杂合突变导致疾病的更严重表现和家族中几代人的逐渐早发性疾病。迄今为止报道的所有DC相关的TINF 2突变，无论是错义还是截短，都集中在位于短（TIN 2S 1 -354）和长（TIN 2L 1 -451）TIN 2同种型中心的34个氨基酸片段中。我们的研究集中在未研究的TIN 2L亚型，其在DC簇内含有错义突变或在存在截短突变时不表达。我们的初步数据表明，TIN 2L和TIN 2S不是shelterin的等效组分，并且最常见的DC相关错义突变对TIN 2L而不是TIN 2S具有有害作用。此外，我们发现TIN 2L含有一个保守的磷酸化位点，当突变时，其对TIN 2L的功能作用与DC簇内的突变相同。最后，我们发现TIN 2L和TRF 2之间增强的相互作用需要TRF 2-F120，TRF 2-F120是TRF同源（TRFH）结构域内的关键残基，其作为各种DNA加工和DNA损伤反应因子的对接位点。我们假设TIN 2L调节TRFH结构域相互作用蛋白进入TRF 2的能力，并且改变TIN 2L活性和增强TIN 2L向端粒的募集驱动患者的端粒缩短。在本提案的具体目标（SA）1中，我们将通过产生一系列等位基因突变细胞系并使用患者来源的TINF 2突变细胞系进行互补试验，确定TIN 2S和TIN 2L对端粒结构和功能的相对影响。我们还将确定，如果TIN 2L相比，TIN 2S，差异与其他蛋白质的相互作用，除了TRF 2使用一种新的免疫沉淀协议，然后通过质谱测序方法。最后，我们将筛选基因特征不明的端粒非常短的患者， TIN 2L突变。在SA 2中，我们将通过一系列体外和体内分析，确定TIN 2L磷酸化对TRF 2结合的影响并阐明其与DC簇的关系，从而阐明TIN 2L磷酸化的功能。在SA 3中，我们将确定TIN 2L和TRF 2 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TINF 2突变细胞系中如何改变。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.