A human somatic cell model for Dyskeratosis Congenita

描述（由申请人提供）： 本基金的具体目的是了解人KARP-1（Ku 86自身抗原相关蛋白-1）的功能，因为它与其作为DC（先天性角化不良）候选基因的作用有关。 DC是一种罕见的遗传性疾病，其特征在于三个皮肤标志：异常皮肤色素沉着，指甲营养不良和粘膜白斑。 该疾病表现出显著的临床异质性，患者还可能患有牙齿、胃肠道、神经、眼科、肺部和/或骨骼异常。 特别是需要持续更新的组织（例如，表皮、粘膜和骨髓谱系）在DC患者中受影响最大，并且与DC相关的所有发病率的70%是由于骨髓衰竭。 来源于DC患者的细胞的特征在于具有i）短端粒，ii）hTR（人端粒RNA）表达水平降低，iii）端粒酶活性降低，iv）基因组不稳定性，和v）增殖性功能障碍。 已经鉴定了两种基因，hTR和DKC 1，当突变时，产生DC。 hTR或DKC 1突变占所有DC患者的约50%，而其他50%的患者具有尚未鉴定的基因突变。 有趣的是，hTR和DKC 1产生的基因产物在细胞定位中是核仁的。 在公开的报告和本申请提供的初步数据中，我们证明了人细胞中Ku 86：KARP-1基因座的单个等位基因的丢失导致与在DC患者中观察到的表型完全相同的表型：i）短端粒，ii）hTR表达水平降低，iii）端粒酶活性降低，iv）基因组不稳定性，和v）增殖性功能障碍。 此外，我们还证明了KARP-1是核仁的。 因此，总的来说，KARP-1是DC的候选基因的证据是令人信服的。 在此，我们描述了将确定KARP-1是否在DC患者中突变并且将阐明KARP-1在人类细胞中端粒维持中的作用的实验。 这些研究的最终目标是利用KARP-1表达改变的人类体细胞系来了解人类患者端粒功能障碍的分子机制。 我们的具体目标是：1。 在DC患者中KARP-1基因突变和/或KARP-1基因表达异常吗？ 2. KARP-1在人体细胞中是必需的吗？ 3. KARP-1及其相互作用物的生化和分子表征。

DESCRIPTION (provided by applicant): The Specific Aims of this grant are directed towards understanding the function of human KARP-1 (Ku86 autoantigen related protein-1) as it relates to its role as a candidate gene for DC (dyskeratosis congenita). DC is a rare inherited disorder characterized by a triad of cutaneous hallmarks: abnormal skin pigmentation, nail dystrophy and mucosal leucoplakia. The disease presents with significant clinical heterogeneity and patients may also be afflicted with dental, gastrointestinal, neurological, ophthalmic, pulmonary and/or skeletal abnormalities. In particular, tissues that need continual renewal (e.g., epidermal, mucosal and bone marrow lineages) are most affected in DC patients and 70% of all the morbidity associated with DC is due to bone marrow failure. Cells derived from DC patients are characterized by possessing i) short telomeres, ii) reduced expression levels of hTR (human telomeric RNA), iii) reduced activity of telomerase, iv) genomic instability, and v) proliferative dysfunction. Two genes, hTR and DKC1, have been identified, that, when mutated, give rise to DC. Mutation of either hTR or DKC1 accounts for about 50% of all DC patients while the other 50% have mutations in genes that have yet to be identified. Interestingly, hTR and DKC1 produce gene products that are nucleolar in cellular localization. In published reports and in preliminary data provided with this application, we demonstrate that the loss of a single allele of the Ku86:KARP-1 locus in human cells results in precisely the same phenotypes that are observed in DC patients: i) short telomeres, ii) reduced expression levels of hTR, iii) reduced activity of telomerase, iv) genomic instability, and v) proliferative dysfunction. In addition, we also demonstrate that KARP-1 is nucleolar. Thus, in toto, the evidence is compelling that KARP-1 is a candidate gene for DC. Herein, we describe experiments that will determine whether KARP-1 is mutated in DC patients and that will elucidate the role(s) of KARP-1 in telomere maintenance in human cells. The ultimate goal of these studies is to use human somatic cell lines altered in their expression of KARP-1 to understand the molecular mechanisms of telomere dysfunction in human patients. Our Specific Aims/Goals are: 1. Is the KARP-1 gene mutated and/or is KARP-1 gene expression aberrant in DC patients? 2. Is KARP-1 essential in human somatic cells? 3. Biochemical and molecular characterization of KARP-1 and its interactors.