Telomere Diseases

端粒是线性染色体末端重复的六核苷酸序列，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损失，而端粒酶是一种在有丝分裂时增加端粒序列的酶复合物，其功能是维持基因组的完整性。端粒酶缺乏表现为端粒缩短和酶活性丧失：其后果可以在体外和体内测量。DKC1和TERC（复合体的RNA模板亚基）的突变是一些先天性角化不良病例的病因，先天性角化不良是再生障碍性贫血的一种构成形式。TERT（编码端粒酶，该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。TERT的杂合突变由于单倍体功能不全的机制导致端粒修复缺陷和端粒缩短。长期以来用于治疗再生障碍性贫血的男性激素通过上调TERT转录和端粒酶活性起作用，包括淋巴细胞和造血祖细胞。虽然关键端粒缩短通常导致细胞衰老或凋亡，但偶尔细胞由于染色体的端到端融合而成为整倍体。因此，端粒磨损是肿瘤发生的一种机制。

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.