Telomeres and Ribosomes in Dyskeratosis Congenita

描述(由申请人提供)： 先天性角化不良(DC)是一种罕见的遗传性骨髓衰竭(BMF)综合征，以皮肤色素异常、指甲营养不良和粘膜白斑三联征为特征。DC患者有发生多种癌症的倾向，尤其是造血系统和上消化道的癌症。我们研究的总体目的是了解DC的病理生理和发病机制，以及导致BMF和该疾病患者癌症易感性的分子途径。DC具有常染色体显性、常染色体隐性和X连锁形式的遗传异质性。不同的遗传模式表明，不同基因的突变可能导致相同的表型，这些基因产物可能以共同的途径发挥作用。患有X连锁DC的患者有dyskerin(DKC1)基因突变。Dyskerin是一种核仁蛋白，它与小核仁RNA结合，催化18S和28S核糖体RNA中特定的尿苷残基转化为假尿苷。Dyskerin也是端粒酶复合体的一个组成部分，负责维持端粒，端粒是覆盖在染色体末端的核蛋白结构。在拟议的研究中，我们将研究dyskerin突变的生化和分子后果。我们假设dyskerin在核糖体RNA加工和活性端粒酶复合体的组装中是必不可少的，并且dyskerin导致DC的突变可能会对核糖体RNA加工、端粒酶复合体或两者都产生不同程度的影响，这取决于它们在dyskerin中的位置。为了测试这一点，我们将研究dyskerin零突变对假尿嘧啶、核糖体RNA加工、核糖体生物发生、端粒酶活性和端粒酶复合体完整性的影响。重组酶Cre的表达可以导致dyskerin基因的缺失，目前已经构建了小鼠细胞系和胚胎干细胞。实验将在小鼠组织(肝细胞、骨髓细胞)和ES细胞中进行，从而使我们能够研究Dyskerin基因改变对高水平表达Dyskerin和端粒酶的正常哺乳动物细胞的影响。接下来，我们将把一系列dyskerin突变引入到dyskerin缺失ES细胞的基因组中，并表征这些突变对dyskerin双重功能的影响程度。最后，我们将测试Dyskerin或端粒酶的过度表达是否可以纠正DC突变引起的细胞表型。这些发现可能会为dyskerin和dyskerin突变的功能作用提供重要的见解，这将是更好地理解该病的发病机制和生物学所必需的。更好地了解这种疾病将为设计新的、更有效的DC患者治疗方法提供框架。对核糖体生物发生和端粒维持在人类疾病中的重要性的新见解也可能为其他骨髓衰竭综合征的发病机制提供帮助，在这些综合征中，端粒维持和核糖体生物发生受到损害。

DESCRIPTION (provided by applicant): Dyskeratosis congenita (DC) is a rare inherited bone marrow failure (BMF) syndrome, characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia. Patients with DC have a propensity to develop a variety of cancer, in particular of the hematopoietic system and of the upper gastrointestinal tract. The overall aim of our research is to understand the pathophysiology and pathogenesis of DC and the molecular pathways that lead to BMF and cancer susceptibility in patients with this disease. DC is genetically heterogeneous with autosomal dominant, autosomal recessive, and X-linked forms of the disease. The different patterns of inheritance suggest that mutations in different genes may lead to the same phenotype, and that these gene products might function in a common pathway. Patients with the X-linked form of DC have mutations in the dyskerin (DKC1) gene. Dyskerin is a nucleolar protein that associates with small nucleolar RNAs and catalyzes the conversion of specific uridine residues to pseudouridine in 18S and 28S ribosomal RNA. Dyskerin is also a component of the telomerase complex responsible for the maintenance of telomeres, nucleoprotein structures that cap the ends of chromosomes. In the proposed studies we will investigate the biochemical and molecular consequences of dyskerin mutations. We hypothesize that dyskerin is essential in ribosomal RNA processing and in the assembly of an active telomerase complex, and that mutations in dyskerin causing DC may affect ribosomal RNA processing, the telomerase complex, or both, to a variable extent depending on their location in dyskerin. To test this we will study the consequences of a dyskerin null mutation on pseudouridylation, ribosomal RNA processing, ribosome biogenesis, telomerase activity, and the integrity of the telomerase complex. Mouse lines and embryonic stem (ES) cells have been constructed in which a deletion in the dyskerin gene can be induced by the expression of the recombinase Cre. Experiments will be performed in mouse tissues (liver cells, bone marrow cells), and ES cells thus enabling us to study the effects of genetic alteration of dyskerin in normal mammalian cells with a high level of dyskerin and telomerase expression. Next, we will introduce a series of dyskerin mutations into the genome of dyskerin null ES cells and characterize the extent to which these mutations affect the dual function of dyskerin. Finally, we will test whether over-expression of dyskerin, or of telomerase may correct the cellular phenotype caused by DC mutations. Findings are likely to provide important insights into the functional role of dyskerin and dyskerin mutations which will be essential for a better understanding of the pathogenesis and biology of the disease. A better understanding of the disease will provide the framework for the design of novel and more efficient treatment for patients with DC. New insights gained into the importance of ribosome biogenesis and telomere maintenance in human disease might also shed light on the pathogenesis of other bone marrow failure syndromes in which telomere maintenance and ribosome biogenesis is impaired.