Haploinsufficiency of telomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita

Abstract

Dyskeratosis congenita is a rare inherited disorder characterized by abnormal skin manifestations. Morbidity and mortality from this disease is usually due to bone marrow failure, but idiopathic pulmonary fibrosis and an increased cancer predisposition also occur. Families with autosomal dominant dyskeratosis congenita display anticipation and have mutations in the telomerase RNA gene. We identified a three-generation pedigree with autosomal dominant dyskeratosis congenita, anticipation, and telomere shortening. We show that a null mutation in motif D of the reverse transcriptase domain of the protein component of telomerase, hTERT, is associated with this phenotype. This mutation leads to haploinsufficiency of telomerase, and telomere shortening occurs despite the presence of telomerase. This finding emphasizes the importance of telomere maintenance and telomerase dosage for maintaining tissue proliferative capacity and has relevance for understanding mechanisms of age-related changes.

Fig. 1.

Pedigree with clinical features and age of onset. Individuals carrying the K902N mutation are indicated as affected. The pedigree displays anticipation in the onset of gray forelock, liver and lung fibrosis, and aplastic anemia. The onset and severity of symptoms correlates with the percentage of short telomeres in bold (% Stel). Short telomeres were defined as the number of telomeres having telomere fluorescent units <15 (Fig. 2). Quantitative FISH studies were done on primary lymphocytes. *, Telomeres measured in fibroblasts.

Fig. 2.

Telomere length distributions of family members. Quantitative FISH shows the proband with shorter average telomere length than either parent in both primary fibroblasts (A) and lymphocytes (B). (C) The proportion of shortest telomeres (lymphocytes) is increased in an affected individual (II7) compared with both an unaffected sibling of comparable age (II6) and an unaffected parent (I2). The percentage of short telomeres in the proband (B) is increased compared with a less severely affected aunt (C, II7) and correlates with anticipation. Average telomere length (Avg) is indicated with individual identifiers shown in Fig. 1.

Fig. 3.

Point mutation in hTERT.(A) Chromatogram showing the presence of a heterozygous mutation G to C in the proband in exon 11 that results in a lysine to asparagine substitution in residue 902. (B) Schematic of hTERT protein with conserved reverse transcriptase domains shown in color. The K902N mutation lies within Motif D, which is conserved among telomerase proteins and across reverse transcriptase families, including HIV reverse transcriptase.

Fig. 4.

Telomerase activity of TERT mutants. In vitro reconstituted telomerase with TERT mutants K902A, K902N, and K902Q were assayed for telomerase activity by using TRAP. Diluted (1/5×, 1/25×, or 1/125×) telomerases were subjected to TRAP assay. A negative control with RNase A-inactivated telomerase was included for the wild-type and mutant enzymes. The labeled product seen in lanes 6-11 represents a very low level of telomerase activity detected by this sensitive PCR-based assay. The activity in these lanes is <1/125 of that of the wild-type enzyme or <0.8%.

Fig. 5.

Effect of mutant TERT K902N on wild-type telomerase activity. Telomerase reconstituted with different ratios (2:1, 1:1, and 1:2) of wild-type and K902N mutant TERT was analyzed by direct telomerase activity assay to determine whether the mutant TERT would inhibit TERT function. The relative activity of each reaction was measured by the intensity of the +4 band. Levels of TERT proteins in each reaction were measured by Western blotting probed with anti-HA antibody. Repeat addition processivity was calculated by comparing the normalized intensity of the major product in the first and second repeats.