Adult-onset pulmonary fibrosis caused by mutations in telomerase

Abstract

Idiopathic pulmonary fibrosis (IPF) is an adult-onset, lethal, scarring lung disease of unknown etiology. Some individuals with IPF have a familial disorder that segregates as a dominant trait with incomplete penetrance. Here we used linkage to map the disease gene in two families to chromosome 5. Sequencing a candidate gene within the interval, TERT, revealed a missense mutation and a frameshift mutation that cosegregated with pulmonary disease in the two families. TERT encodes telomerase reverse transcriptase, which together with the RNA component of telomerase (TERC), is required to maintain telomere integrity. Sequencing the probands of 44 additional unrelated families and 44 sporadic cases of interstitial lung disease revealed five other mutations in TERT. A heterozygous mutation in TERC also was found in one family. Heterozygous carriers of all of the mutations in TERT or TERC had shorter telomeres than age-matched family members without the mutations. Thus, mutations in TERT or TERC that result in telomere shortening over time confer a dramatic increase in susceptibility to adult-onset IPF.

Fig. 1.

Abridged pedigrees of seven families with familial idiopathic interstitial lung disease and TERT (A) or TERC (B) mutations. Individuals with pulmonary fibrosis or an unclassified pulmonary disease due to a lack of medical studies or records are indicated by red and light blue symbols, respectively. Individuals with IPF are distinguished by the asterisks. The presence or absence of a mutation in either gene is indicated by plus or minus signs, respectively. When the mutation was inferred based on the pattern of inheritance, the plus sign is placed in parentheses. The current age or the age at death is listed to the right of each symbol. The family number and the amino acid change in telomerase reverse transcriptase (A) or the mutation in TERC (B) are listed above each family. Mutations in the DNA and protein sequence are abbreviated by convention (33). Amino acids are listed as single letters.

Fig. 2.

Histology of lung from thorascopic lung biopsies from individuals II.6 (A and B), III.4 (C and D), III.12 (E and F), and IV.2 (G and H) from family F11 pictured at ×1.25 magnification (A, C, E, and G) and ×10 magnification (B, D, F, and H). For individuals II.6, III.12, and IV.2, features of usual interstitial pneumonia are seen with a patchy, heterogeneous pattern of normal lung and densely fibrotic lung tissue with architectural distortion, subpleural and paraseptal fibrosis, honeycombing, thickened alveolar septa, and scattered foci of proliferating fibroblasts. The lung biopsy of III.4 shows the obliteration of most alveoli and their replacement by fibrous tissue, prominent fibroblast proliferation, and an absence of normal pulmonary tissue. (Scale bars: A, C, E, and G, 1 mm; B, D, F, and H, 100 μm.)

Fig. 3.

Schematic representation of the functional domains of telomerase reverse transcriptase (A) and telomerase RNA (B) with the position of the mutations in IPF patients relative to the domains. (A) For the telomerase reverse transcriptase, N-terminal region domains (dark green), reverse-transcriptase motifs (blue), and C-terminal region domains (yellow) are shown. Numbers indicate amino acids. Missense mutations are indicated above the diagram with short arrows; deletions causing frameshifts are indicated by the long arrows. (B) Highly conserved domains of telomerase RNA and helices are indicated for telomerase RNA. The r.37a>g mutation disrupts the terminal residue of helix P1b adjacent to the pseudoknot domain. (C) Alignment of the telomerase reverse transcriptase sequences of human, Macaca mulatta (monkey), Canis familiaris (dog), Bos taurus (cow), Mus musculus (mouse), Rattus norvegicus (rat), Gallus gallus (chicken), Xenopus laevis (frog), Saccharomyces cerevisiae (yeast), and Arabidopsis thaliana (plant).

Fig. 4.

Telomerase activity of TERT mutants as measured by the TRAP assay. Telomerase activity of in vitro coexpressed mutant or wild type (wt) telomerase reverse transcriptase (TERT) proteins with mutant or wild-type telomerase RNA (TERC) (A and B) were determined by TRAP. Plasmid constructs encoding TERT and TERC were combined as indicated, in vitro transcribed and translated together, and serially diluted 1:5 before measuring TRAP activity. The positive control (+) is 250 cell equivalents of H1299, a human cancer cell line known to be positive for telomerase activity, as evidenced by the 6-bp incremental TRAP ladder. An aliquot of the highest concentration of the in vitro expressed wild-type telomerase was heat-inactivated at 85°C for 10 min before measuring TRAP activity as a negative control (−). (C) Relative amounts of telomerase activity (percent of wild-type TRAP activity) for seven different TERT mutants and one mutation in telomerase RNA were calculated as a ratio of the intensity of the sample's telomerase products to that of the internal control band as described (30) and normalized to wild-type activity in one representative experiment. Error bars represent SD. Parallel TnT reactions were run by using [³⁵S]methionine and run on an SDS/PAGE gel to confirm equal expression of the TERT wild-type and mutant proteins (data not shown). (D) Plasmid constructs encoding wt TERT or the V747fs TERT mutant were combined at the indicated ratios, in vitro transcribed and translated with TERC, and telomerase activity was measured by TRAP. Parallel TnT reactions were run by using [³⁵S]methionine and run on a SDS/PAGE as shown.

Fig. 5.

Telomere length determined by Southern blotting of chromosomal TRFs of genomic DNA isolated from leukocytes of individuals from families F8 (A), F40 (B), all families (C), and family F11 (D). Abridged pedigrees, the age of each individual, and the presence (+) or absence (−) of a TERT mutation is indicated above each Southern blot in A and B. Open symbols represent normal individuals, blue symbols indicate individuals heterozygous for a mutation in TERT or TERC without IPF, and pink symbols represent individuals heterozygous for a mutation with IPF. (C) Average TRFs of each individual in all families is plotted against age. (D) The percentage of short TRFs (or the relative intensity of TRFs between 1.9 and 4.3 kb over the intensity of TRFs >1.9 kb in length) was plotted against age for all individuals in family F11. Linear regression was used to draw a best fit line through the normal samples.