Telomerase mutations in smokers with severe emphysema

Abstract

Mutations in the essential telomerase genes TERT and TR cause familial pulmonary fibrosis; however, in telomerase-null mice, short telomeres predispose to emphysema after chronic cigarette smoke exposure. Here, we tested whether telomerase mutations are a risk factor for human emphysema by examining their frequency in smokers with chronic obstructive pulmonary disease (COPD). Across two independent cohorts, we found 3 of 292 severe COPD cases carried deleterious mutations in TERT (1%). This prevalence is comparable to the frequency of alpha-1 antitrypsin deficiency documented in this population. The TERT mutations compromised telomerase catalytic activity, and mutation carriers had short telomeres. Telomerase mutation carriers with emphysema were predominantly female and had an increased incidence of pneumothorax. In families, emphysema showed an autosomal dominant inheritance pattern, along with pulmonary fibrosis and other telomere syndrome features, but manifested only in smokers. Our findings identify germline mutations in telomerase as a Mendelian risk factor for COPD susceptibility that clusters in autosomal dominant families with telomere-mediated disease including pulmonary fibrosis.

Figure 3. Pedigrees of telomere syndrome cases with emphysema.

(A) Pedigrees of emphysema cases with telomere defects and their relatives’ clinical history. The asterisk denotes individuals with DNA sequence data available and/or telomere length measurement performed. Boldface indicates individuals who carried the mutant gene and/or had very short telomeres (shown in B). DC, dyskeratosis congenita, a telomere syndrome defined by mucocutaneous features; CS, positive smoking history; NS, never-smoker; BMF, bone marrow failure; d., age at death from lung disease; ds, disease. (B) Measurement of lymphocyte telomere length by flow cytometry and FISH shows the short telomere defect in affected members relative to age-matched controls. The nomogram was based on data from 400 controls. (C–F) Apical and mid-lung chest CT cuts from two female cases (2.II.1 [C and D] and 5.II.3 [E and F]) show severe apical emphysema with blebs. In addition, 2.II.1 has a right-sided pneumothorax that arose spontaneously.

Figure 2. Radiographic and pulmonary function studies of telomerase mutation carriers with COPD.

(A–D) High-resolution inspiratory CT images from COPDGene subjects with telomerase mutations. The panels are labeled with the subject’s mutation. Images from TERT mutation carriers show apical centrilobular emphysema (A–D). In the subject with TERT Arg599Gln, bronchiectasis and a reticular, subpleural interstitial lung abnormality could also be appreciated (B). (E) Rate of change in forced expiratory volume in 1 second (FEV₁) from baseline across the 5 years of the Lung Health Study in the subject with a TERT His925Gln mutation. The rate of change is graphed relative to the highest and lowest quartiles of the 5,887 study population and based onlongitudinal data available through dbGaP from 163 and 174 participants, respectively. Error bars represent SEM.

Figure 1. Functional consequences of telomerase variants identified by chronic obstructive pulmonary disease subjects.

(A) Chromatograms of PCR-amplified products of variants identified by next-generation sequencing. (B) Gel image of telomere repeat ladder generated from wild-type and mutant telomerases reconstituted in vivo and immunopurified. The decreased intensity of the DNA repeat products reflects impaired enzymatic activity of TERT Arg599Gln, Thr726Met, and His925Gln. TERT Asp868Asn is a negative control, catalytically defective in one of the aspartic acid residues essential for reverse transcription. ³²P end-labeled 18-mer oligonucleotide was included as an internal control for the recovery of DNA products. (C) Northern blot for TR levels from immunopurified telomerases (top). Western blot for TERT expression in cells (bottom) was performed with anti-FLAG and anti-GAPDH antibodies for ectopically expressed FLAG-tagged TERT and endogenous GAPDH, respectively. (D) Mean telomerase activity was derived from 4 activity assays from cell lysates prepared from two separate transfections. (E) Relative telomere length as measured by quantitative PCR in age-matched controls (ages 37–64, n = 8), TERT mutation carriers (ages 46–57, n = 3) from the COPDGene Study and the Lung Health Study (LHS), telomerase mutation carriers with pulmonary fibrosis (ages 45–63, TERT n = 2, TR n = 3), and COPDGene controls: a homozygous SERPINA1 Glu366Lys mutation carrier (formerly coded Glu342Lys, rs28929474, PI*ZZ genotype, age 46) and the control TERT Arg653Cys variant (age 68). T/S ratio, ratio of telomere repeat number to single gene copy number. ^†PI*ZZ mutation carrier. (F) Lymphocyte telomere length by flow cytometry and FISH of a TERT mutation carrier and telomerase mutation carriers with pulmonary fibrosis relative to a nomogram of 400 controls. Error bars represent SEM, and 2-sided P values were calculated using Student’s t test.