MOLECULAR STUDIES OF BONE MARROW FAILURE

描述(由申请人提供)：我们研究的总体目标是改善骨髓衰竭(BMF)患者的诊断、护理和治疗。我们的研究重点之一是先天性角化不良(DC)，这是一种罕见的遗传性BMF，与典型的三种粘膜皮肤特征有关，包括异常色素沉着、营养不良的指甲改变和口腔粘膜白斑。已经在DC患者中发现了6个不同基因的突变，这些基因都与端粒的维持有关。在我们的上一次资助期间，我们调查了诊断为BMF的患者及其家人中TERC和端粒酶催化亚单位TERT突变的频率、遗传力、外显率和表达能力。我们的研究确定了由于TERC或TERT基因突变而导致的BMF患者中的少数但独特的人群。疾病的外显性和表现力是高度可变的，这取决于突变的基因、突变的性质和突变遗传的世代数。在BMF患者中，外周血细胞短端粒的测量被发现是识别DC患者的一种敏感但非特异性的方法。我们的研究和其他研究人员的研究表明，具有DC经典临床特征的患者只是由于端粒维护缺陷而患有BMF的个人的“冰山一角”。由于这些发现，人们对短端粒和端粒维持基因突变的临床意义存在困惑和分歧。我们假设，造血干细胞的早衰是DC患者BMF的基础，而早衰的主要原因是端粒功能障碍。在这项拟议的研究中，我们将纵向监测BMF患者的端粒长度和DC相关基因的突变，并确定端粒缩短或端粒长度的比率是否与BMF的严重程度相关。我们将研究新发现的DC相关基因序列改变对端粒末端端粒酶活性的功能影响，并探讨导致患者原代细胞端粒缩短和过早衰老的途径。我们将测试端粒酶活性和/或抗氧化剂的增加是否会延缓突变细胞的衰老。这些原则验证实验可能为DC患者以及可能与BMF和癌症易感性相关的其他相关疾病确定新的治疗选择。 与公共健康相关：骨髓产生血细胞，用于对抗感染、止血和向组织输送氧气。遗传性骨髓衰竭综合征(IBMFS)患者无法产生足够数量的血细胞，导致严重残疾和过早死亡。其中一些患者的关键基因发生了突变，这些基因决定了染色体末端的长度和完整性，也就是所谓的端粒。传统上，这些患者还会出现额外的临床表现，包括皮肤变化、舌头上的白斑和指甲脆，这导致了疾病的名称，先天性角化不良(DC)。我们和其他人的工作表明，这些DC相关基因的突变比最初假设的要频繁得多，大多数患者没有典型的相关表现。然而，在骨髓衰竭(BMF)时，所有DC患者的端粒都较短。有多种机制可以缩短端粒。它们都会导致细胞停止生长并过早衰老。这项拟议的研究将表征导致DC患者端粒缩短的机制，并测试抑制这些机制是否会改善细胞生长，防止或延缓这些细胞的过早衰老。这些实验有望为DC患者找到新的治疗方法，也可能为其他类型的BMF患者找到新的治疗方法。

DESCRIPTION (provided by applicant): The overall goal of our research is to improve the diagnosis, care, and treatment of patients with bone marrow failure (BMF). One focus of our research is dyskeratosis congenita (DC), a rare inherited form of BMF associated with a classic triad of mucocutaneous features including abnormal pigmentation, dystrophic nail changes, and leukoplakia of the oral mucosa. Mutations in 6 different genes, all involved in the maintenance of telomeres, have been identified in patients with DC. During our last funding period we investigated the frequency, heritability, penetrance, and expressivity of mutations in TERC and in the telomerase catalytic subunit TERT in patients diagnosed with BMF and their families. Our studies identified small but distinctive populations of patients with BMF due to TERC or TERT gene mutations. Disease penetrance and expressivity were highly variable depending on the gene mutated, the nature of the mutation, and the number of generations the mutation had been inherited. Among patients with BMF the measurement of short telomeres in peripheral blood cells was found to be a sensitive though nonspecific method for identifying patients with DC. Our studies and those of other investigators have shown that patients with the classic clinical features of DC are only the "tip of the iceberg" of individuals who have BMF due to defective telomere maintenance. Because of these discoveries there is confusion and disagreement about the clinical significance of short telomeres and mutations in telomere maintenance genes. We hypothesize that premature senescence of hematopoietic stem cells is the basis of BMF in patients with DC and that the major cause of premature senescence is dysfunctional telomeres. In the proposed research we will longitudinally monitor telomere length in patients with BMF and mutations in DC associated genes and determine whether the rate of telomere shortening or telomere length correlates with the severity of BMF. We will investigate the functional consequences of newly identified sequence alterations in DC associated genes on telomerase activity at the telomere end and investigate the pathways that lead to short telomeres and premature senescence in primary cells from patients with the disease. We will test whether an increase in telomerase activity and/or antioxidants will delay the onset of senescence in the mutant cells. These proof-of-principle experiments may identify new treatment options for patients with DC and possibly other related conditions associated with BMF and cancer predisposition. PUBLIC HEALTH RELEVANCE: The bone marrow produces blood cells that serve to fight infections, stop bleeding, and transport oxygen to tissues. Patients with inherited bone marrow failure syndromes (IBMFS) are unable to make sufficient numbers of blood cells, resulting in major disability and early death. Some of these patients have mutations in critical genes that determine the length and integrity of the chromosome end, known as the telomere. Classically these patients present with additional clinical manifestations, including alterations of the skin, white spots on the tongue and brittle fingernails, which led to the disease name, dyskeratosis congenita (DC). Our work and that of others has shown that mutations in these DC associated genes are much more frequent than originally presumed and that the majority of patients do not present with the classic associated manifestations. However, at the time of bone marrow failure (BMF) all patients with DC have short telomeres. There are multiple mechanisms that shorten the telomeres. They all cause cells to stop growing and to age prematurely. The proposed research will characterize the mechanisms that lead to short telomeres in patients with DC and test whether the inhibition of these mechanisms will improve cell growth and prevent or delay premature aging of these cells. These experiments will hopefully identify new treatments for patients with DC and possibly also for patients with other types of BMF.