Telomere Diseases

端粒是位于线性染色体末端的重复的六核苷酸序列，其用于保护它们不被识别为染色体断裂;此外，DNA的不对称复制将不可避免地导致遗传物质的丢失，而端粒酶，一种在有丝分裂时添加端粒序列的酶复合物，用于维持基因组的完整性。端粒酶缺陷表现为短端粒和酶活性的丧失：其后果可以在体外和体内测量。DKC 1和TERC（复合物的RNA模板亚基）中的突变是先天性角化不良（再生障碍性贫血的一种体质形式）的某些病例的病因。TERT（编码端粒酶，复合物的限速酶成分）的突变发生在明显获得性再生障碍性贫血和其他疾病中。由于单倍不足的机制，TERT中的杂合突变导致端粒修复缺陷和短端粒。长期用于治疗再生障碍性贫血的雄性激素通过上调包括淋巴细胞和造血祖细胞中的TERT转录和端粒酶活性来起作用。虽然关键的端粒缩短往往导致细胞衰老或凋亡，偶尔细胞成为非整倍体由于染色体的端到端融合。因此，端粒磨损是肿瘤发生的一种机制。 白细胞的端粒长度现在在我们的CLIA实验室中通过基因扩增进行常规测量，使用Quiagen Quagility和Rotor GeneQ提供的机器人方法;高通量分析对研究和临床都很有用，我们的程序经过了患者数据认证。临床样本的测量是再生障碍性贫血的充分诊断所必需的，并且可以预测免疫抑制治疗后的晚期事件，并且可能在其他临床情况下。 我们现在已经建立了单端粒长度分析（STELA），它依赖于使用染色体特异性亚端粒DNA序列的扩增来检测单个染色体中的关键端粒缩短。最近，我们增加了Flow-FISH，这是一种流式细胞术检测，可以测量悬浮液中单个细胞的端粒长度;我们的Flow-FISH检测证实了q-PCR和STELA的结果。 我们已经完成了从严重再生障碍性贫血发展为骨髓增生异常综合征和/或急性髓细胞性白血病的患者中端粒磨损和体细胞基因突变积累的直接比较。 在13例7号单体患者中，端粒磨损明显加速，大约是稳定型再生障碍性贫血患者和健康个体的8倍。 通过STELA证实端粒磨损。 端粒磨损在免疫抑制治疗后早期最为显著，但此后进行性。 相比之下，先前在MDS/AML患者中确定为异常的基因突变相对罕见。 只有三种情况下，突变可以确定在高可变等位基因频率和其余的，当突变存在，他们不太可能解释大单体7克隆。因此，端粒磨损与再生障碍性贫血向7号单体的进展有关。 然而，这些结果需要在我们的基因组数据的背景下考虑（参见再生障碍性贫血病理生理学和治疗年度报告），其中特定MDS/AML基因的突变可预测无进展生存期和总生存期，并与恶性克隆转化相关。 可能的是，这两种白血病的途径都涉及再生障碍性贫血向白血病的转化。 在临床上，我们现在有超过150例端粒病患者，基于qPCR的端粒含量和/或端粒修复复合物基因突变的鉴定。 对这些患者临床表型的全面分析仍在进行中，但TERT和TERC突变之间存在明显差异。 在我们的转诊人群中，TERT突变更常见;它们导致明显更温和的表型。 严重的肺部疾病，通常是纤维化，与TERC突变有关。 相当大比例的端粒病变患者的遗传学基础上没有积极的家族史。 值得注意的是，尽管数据不足以确定复发或克隆演变的可能性，但具有TERT和TERC突变的患者可以对免疫抑制治疗有反应，血液学改善。 最后，我们的达诺唑在端粒病变患者中的研究即将完成。 这种药物一般耐受性良好。 在进入试验的25名患者中，只有4名患者因副作用而停止了男性激素治疗，总体毒性很小。 值得注意的是，肝毒性（由于端粒病变中的肝脏受累，可能在该人群中预期）不常见或不严重。 在有肺部症状和体征的患者中，没有证据表明肺纤维化改善，在一些病例中有进展。 然而，大多数患者的血液学反应：3个月时18/23例，6个月时16/20例，1年时12/14例，2年时10/10例。 该方案的主要生物学终点是基于组织培养实验预测的端粒延长。 端粒的稳定和延长是一致的。 平均而言，进入方案前的端粒磨损为227 bp/年;雄激素制度后，端粒延长平均为430 bp/年。 因此，雄性激素似乎是第一个被正式证明能够延长人类端粒的药物。

Telomeres are repeated hexanucleotide sequences at the ends of linear chromosomes, which serve to protect them from recognition as chromosomal breaks; furthermore, the asymmetric replication of DNA would lead inevitably to a loss of genetic material, and telomerase, an enzymatic complex that adds telomeric sequences at mitosis, functions to maintain genomic integrity. Telomerase deficiency manifests with short telomeres and loss of both enzymatic activity: its consequences can be measured in vitro and in vivo. Mutations in DKC1 and in TERC (the RNA template subunit of the complex) are etiologic in some cases of dyskeratosis congenita, a constitutional form of aplastic anemia. Mutations in TERT (encoding telomerase, the rate limiting enzymatic component of the complex) occur in apparently acquired aplastic anemia and other diseases. Heterozygous mutations in TERT lead to defective telomere repair and short telomeres due to a mechanism of haploinsufficiency. Male hormones, long used to treat aplastic anemia, act by up regulating TERT transcription and telomerase activity, including in lymphocytes and hematopoietic progenitor cells. While critical telomere shortening often leads to either cell senescence or apoptosis, occasional cells become anneuploid due to end-to-end fusion of chromosomes. Thus, telomere attrition is a mechanism for onco genesis. Telomere length of leukocyte is now measured routinely in our CLIA laboratory by gene amplification using robotic methodology provided by a Quiagen Quiagility and Rotor GeneQ; high throughput analysis is useful both for research and in the clinic, and our procedure is certified for patient data. Measurement of clinical samples is required for the adequate diagnosis of aplastic anemia and is predictive of late events after treatment with immunosuppression, and probably in other clinical circumstances. We now have established single telomere length analysis (STELA), which relies on amplification using chromosome specific sub-telomeric DNA sequence to detect critical telomere shortening in individual chromosomes. Recently, we have added Flow-FISH, a flow cytometry assay that allows measurement of telomere length in individual cells in suspension; our Flow-FISH assay confirms results with q-PCR and STELA. We have completed our direct comparison of telomere attrition and accumulation of somatic gene mutations in patients who evolved from severe aplastic anemia to myelodysplastic syndrome and/or acute myeloid leukemia. In 13 monosomy 7 patients, telomere attrition was markedly accelerated, approximately 8-fold greater than in patients with stable aplastic anemia and in healthy individuals. Telomere attrition was confirmed by STELA. Telomere attrition was most marked early after immunosuppressive therapy but progressive thereafter. In contrast, mutations in genes previously identified as abnormal in patients with MDS/AML were relatively infrequent. In only three cases could mutations be identified at high variable alelle frequency and in the remainder, when mutations were present, they were unlikely to explain the large monosomy 7 clone. Therefore, telomere attrition is implicated in the progression to monosomy 7 in aplastic anemia. However, these results need to be considered in the context of our genomic data (see Aplastic Anemia Pathophysiology and Treatment annual report), in which mutations in specific MDS/AML genes were predictive of both progression free and overall survival and linked to malignant clonal transformation. Likely, both routes to leukemia are entailed in transformation from aplastic anemia to leukemic. In the clinic, we now have over 150 patients with telomeropathies, based on telomere content by qPCR and/or the identification of mutations in genes of the telomere repair complex. A full analysis of the clinical phenotypes of these patients is still underway, but there are clear differences between TERT and TERC mutations. TERT mutations are more frequent in our referral population; they result in apparently more mild phenotypes. Profound pulmonary disease, usually fibrosis, is associated with TERC mutations. A substantial proportion of patients with telomeropathies based on genetics do not have a positive family history. Of note, patients with TERT and TERC mutations can respond to immunosuppressive therapy with hematologic improvement, although data are insufficient to determine likelihood of either relapse or clonal evolution. Finally, our study of danozol in patients with telomeropathies is nearing completion. The drug has generally been well tolerated. Of 25 patients who have entered trial, only four have discontinued male hormone therapy due to side effects, and in general toxicities have been minimal. Of note, hepatotoxity, which might be anticipated in this population due to liver involvement in the telomeropathies, was not frequent or severe. There was no evidence of improvement of pulmonary fibrosis in patients with lung symptoms and signs, and in several cases there was progression. However, the majority of patients responded hematologically: 18/23 at three months, 16/20 at six months, 12/14 at 1 year and 10/10 at 2 years. The main biological end point for the protocol was telomere elongation, predicted based on tissue culture experiments. Telomere stabilization and elongation were consistently observed. On average, telomere attrition prior to entering the protocol was 227bp/year; following androgen institution, telomere elongation averaged 430 bp/year. Thus, male hormones appear to be the first agent to be formally demonstrated capable of elongating telomeres in humans.