Human Telomere Disease Due to Disruption of the CCAAT Box of the TERC Promoter

Abstract 2405

Telomeres, the structures capping the ends of linear chromosomes, consist of hundreds to thousands of TTAGGG repeats. To prevent critical telomere shortening, highly proliferative cells express telomerase (encoded by TERT), a reverse transcriptase that adds TTAGGG repeats to telomeres, using TERC as its RNA template. Mutations in TERT (resulting in amino acid changes and reduced enzymatic activity), in TERC (resulting in changes in RNA structure and impaired binding to telomerase), or in other components of the telomerase complex lead to accelerated telomere attrition. Clinically, the telomeropathies can manifest as the bone marrow (BM) failure syndromes dyskeratosis congenita and aplastic anemia; acute myeloid leukemia; hepatic nodular regenerative hyperplasia and cirrhosis; and familial pulmonary fibrosis. Sequencing strategies for the diagnosis of telomere disease are based on screening exons and their flanking regions of telomerase genes for mutations. Clearly pathogenic mutations in the promoter region of TERT or TERC have not been described. Here, we report a mutation in the CCAAT box of the TERC promoter region in a severely affected pedigree, leading to telomere disease. The index patient, a 39-year old Caucasian female with a family history of BM failure and leukemia, was diagnosed with aplastic anemia. Her peripheral blood telomere length was extremely short in comparison to age-matched healthy controls by quantitative PCR (patient, 5.4 kb; expected for age, 8.2 kb; 2.9 standard deviations below the age-matched average). Telomere lengths of her sister and her affected nephew also were very short, while an unaffected nephew had normal telomere length. No mutations were found in TERT, the coding region of TERC, or exon 6a of TINF2. However, the proband, her sister, her affected nephew, but not the unaffected nephew, carried a heterozygous −58 C>G in the CCAAT box (position −58 to −54) of the TERC promoter region. The mutation was not present in 378 healthy subjects. Gel shift and supershift assays with HeLa nuclear extract and anti-NF-YA antibody displayed binding and supershift for the wild-type but not for the −58C>G mutant biotinylated probe. Reporter gene experiments in HEK293T cells transfected with wild-type or mutant promoter showed a 9-fold decrease in luciferase activity in the mutant promoter as compared to the wild-type. These results indicate that the CCAAT-box disruption leads to reduced TERC levels, lower telomerase activity, and ultimately short telomeres, as observed in the present pedigree. The CCAAT box is an important and highly conserved promoter element that is frequently present in promoter regions of RNA polymerase II transcribed genes. In vitro, the CCAAT box in the TERC promoter has been shown to be critical for maintaining basal transcription of TERC by binding NF-Y. In vivo, mutations have been described in the CCAAT boxes of the ^Aγ and ^Gγ gene promoters, leading to hereditary persistence of fetal haemoglobin, a benign condition that benefits sickle cell anemia and β-thalassemia patients. We here provide the first instance of a mutation in a CCAAT box etiologic in human disease and the first example of a TERC promoter mutation producing a telomeropathy. Pathogenic mutations in telomerase genes may be located in promoter and likely also more distant regulatory sequences.