Human telomere disease due to disruption of the CCAAT box of the TERC promoter

Abstract

Mutations in the coding region of telomerase complex genes can result in accelerated telomere attrition and human disease. Manifestations of telomere disease include the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia, acute myeloid leukemia, liver cirrhosis, and pulmonary fibrosis. Here, we describe a mutation in the CCAAT box (GCAAT) of the TERC gene promoter in a family in which multiple members had typical features of telomeropathy. The genetic alteration in this critical regulatory sequence resulted in reduced reporter gene activity and absent binding of transcription factor NF-Y, likely responsible for reduced TERC levels, decreased telomerase activity, and short telomeres. This is the first description of a pathogenic mutation in the highly conserved CCAAT box and the first instance of a mutation in the promoter region of TERC producing a telomeropathy. We propose that current mutation-screening strategies should include gene promoter regions for the diagnosis of telomere diseases. This clinical trial was registered at www.clinicaltrials.gov as #NCT00071045.

Figure 1

Pedigree, telomere length, and BM histology. (A) Pedigree of proband (III-3). Individuals II-2 and II-3 are suspected mutation carriers. Slashed symbols indicate deceased (d.) individuals. Neither the proband, her sister, or affected nephew showed abnormal pigmentation of the skin, nail dystrophy, or oral leukoplakia, nor was there evidence of pulmonary or immunologic problems, growth retardation, developmental delay, or microcephaly. During cholecystectomy, an enlarged liver was noticed in III-2; however, this finding was not further evaluated after surgery. (B) Blood leukocyte telomere length (in kilobases) as a function of age. Mutation carriers have very short telomeres in peripheral blood leukocytes. The curve marks the 50th percentile of telomere length for control subjects derived from 298 healthy National Institutes of Health blood bank donors. A sample's telomere length was expressed as a telomere to single-copy gene (T/S) ratio, which was converted to kilobases. BM biopsy showed profound hypocellularity in the proband (III-3; C) and hypocellular BM and eosinophilic ground substance in her sister (III-2; D).

Figure 2

Functional analysis of wild-type and mutant CCAAT boxes of the TERC promoter. (A) Gel shift and supershift assays. Gel shift assay was performed with HeLa nuclear extract with wild-type probe (wt, 5′-Bio/cttggccaatccgtgcggtcgg-3′), a mutant probe (mt1, 5′-Bio/cttgggcaatccgtgcggtcgg-3′), and additional mutant control probes (mt2, 5′-Bio/cttggagtctccgtgcggtcgg-3′; mt3, 5′-Bio/cttggccattccgtgcggtcgg-3′); bold and underlined letters indicate mutated nucleotides. Anti-NF-YA antibody was used for supershift assay. Arrows show bands that were shifted and supershifted with the wt probe but not with the mt1 probe (−58C>G) or additional mutant control probes (mt2 and mt3). (B) A 200-fold molar excess of unlabeled mutant competitor (mt1, mt2, or mt3) did not compete with wild-type binding, whereas wild-type competitor did (indicated with an arrow). Mutant promoter activity in HEK293T (C) or HeLa cells (D) was reduced compared with wild-type in reporter gene assays. Depicted on the x-axis is the 5′ position from the transcriptional start site of TERC for each TERC promoter-luciferase construct; each construct ended at nucleotide +69. Luciferase activity is reported as relative fold increase compared with the empty vector pGL4.18[luc2P/Neo] (given an arbitrary value of 1), normalized to protein concentration. Results shown are means of 3 (HEK293T) or 1 (HeLa) independent experiment performed in duplicate. Error bars indicate SEM. Detailed methods that include sequences of primers and probes used in the present study will be provided on request.