Dyskeratosis Congenita (DC) is a marrow failure syndrome characterized by skin and nail abnormalities, oral leukoplakia and very short telomeres in circulating leukocytes. Heritable defects in telomere maintenance have been directly implicated in DC by the discovery of mutations in genes encoding components of the telomerase complex: DKC1, TERT, and TERC as well as mutations in the gene encoding the telomere binding protein TINF2. Defective telomeres in DC result in impaired hematopoiesis and predispose to myeloproliferative disorders. Heritable mutations in TERT and TERC have also been implicated in patients presenting with aplastic anemia (AA) and idiopathic pulmonary fibrosis (IPF) without clinical signs of DC. Because short telomeres appear to be associated with increased risks for various human cancers, including head and neck cancer, we sequenced TERT and TERC in two patients with oral carcinoma and anemia.

The first patient presented at age 47 with invasive squamous cell carcinoma (SCC) of the tongue. The patient had a male sibling said to be also suffering from SCC which was not available for analysis and his mother died at age 37 from lymphoma. The patient displayed mild macrocytic anemia and oral leukoplakia. The telomere lengths of peripheral blood cells from the patient, determined by flow-FISH, were found to be below the first percentile expected for his age. In contrast, the leukocyte telomere lengths for the patient’s father and a female sibling were within the normal range. Bi-directional sequence analysis of TERT and TERC was conducted on DNA isolated from whole blood for the three family members. A novel mutation in exon 9 of TERT, C842T, situated within the reverse transcriptase domain of the telomerase enzyme catalytic component was identified in the patient but not in the 2 unaffected relatives. This suggested inheritance of a TERT mutation from the mother. The function of TERT C842T was compared to wildtype (WT) TERT by transfecting WT and mutant TERT cDNA into clonal Jurkat T cells and measuring telomere elongation by flow-FISH following 4 weeks of culture. TERT C842T showed 30% of the elongation obtained with WT TERT (p=0.0034).

The second patient is a 60 yr old male with SCC of the tongue and refractory anemia with ring sideroblasts. The leukocyte telomere length was around the 1st percentile expected for his age. TERT sequencing revealed a three nucleotide deletion resulting in loss of 441E while retaining frame that is expected to impair telomerase activity.

Our data support the concept that mutations in TERT can cause defective telomere maintenance and thereby compromise the proliferation of hematopoietic as well as epithelial cells. The resulting loss of normal cells selects for cells with defective DNA damage checkpoints that are triggered by chromosome ends without telomere repeats. Such cells are at high risk of becoming malignant because their proliferation will be stimulated by the loss of normal cells and their genome is very unstable as telomere function is compromised. Together these factors facilitate and enable clonal evolution of abnormal cells by DNA repair defects and cycles of chromosome fusions/bridge/breakage. Hematological and pathological findings consistent with Dyskeratosis Congenita together with peripheral blood telomere length measurements appear useful parameters to screen for telomere defects in patients and facilitate the discovery of mutations in “telomere maintenance” genes. The TERT mutations in patients with oral carcinomas illustrate that disease manifestations of telomere dysfunction in humans can be very diverse and range from DC, to defective hematopoiesis, pulmonary fibrosis and cancer predisposition.

Disclosures: Lansdorp:Repeat Diagnostics: founder, company specializing in telomere length measurements.

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