Telomere shortening in familial and sporadic pulmonary fibrosis

Abstract

Rationale: Heterozygous mutations in the coding regions of the telomerase genes, TERT and TERC, have been found in familial and sporadic cases of idiopathic interstitial pneumonia. All affected patients with mutations have short telomeres.

Objectives: To test whether telomere shortening is a frequent mechanism underlying pulmonary fibrosis, we have characterized telomere lengths in subjects with familial or sporadic disease who do not have coding mutations in TERT or TERC.

Methods: Using a modified Southern blot assay, the telomerase restriction fragment length method, and a quantitative polymerase chain reaction assay we have measured telomere lengths of genomic DNA isolated from circulating leukocytes from normal control subjects and subjects with pulmonary fibrosis.

Measurements and main results: All affected patients with telomerase mutations, including case subjects heterozygous for newly reported mutations in TERT, have short telomere lengths. A significantly higher proportion of probands with familial pulmonary fibrosis (24%) and sporadic case subjects (23%) in which no coding mutation in TERT or TERC was found had telomere lengths less than the 10th percentile when compared with control subjects (P = 2.6 x 10(-8)). Pulmonary fibrosis affectation status was significantly associated with telomerase restriction fragment lengths, even after controlling for age, sex, and ethnicity (P = 6.1 x 10(-11)). Overall, 25% of sporadic cases and 37% of familial cases of pulmonary fibrosis had telomere lengths less than the 10th percentile.

Conclusions: A significant fraction of individuals with pulmonary fibrosis have short telomere lengths that cannot be explained by coding mutations in telomerase. Telomere shortening of circulating leukocytes may be a marker for an increased predisposition toward the development of this age-associated disease.

Figure 1.

(A) Abridged pedigrees of kindreds F55, F80, F106, F107, and F119 with familial pulmonary fibrosis and TERT mutations; (B) schematic representation of the functional domains of TERT with the position of the mutations found in pulmonary fibrosis subjects relative to the domains; (C) alignment of the TERT sequences of human, Macaca mulatta (monkey), Canis familiaris (dog), Bos taurus (cow), Mus musculus (mouse), Rattus norvegicus (rat), Gallus gallus (chicken), Xenopus laevis (frog), Schizosaccharomyces pombe (yeast), and Arabidopsis thaliana (plant); and (D) relative telomerase activity of TERT mutants as measured by the telomere repeat amplification protocol (TRAP) assay. In (A), shaded symbols indicate individuals with pulmonary fibrosis (pink) or lung disease (blue); the presence or absence of the mutation is indicated by plus or minus signs, respectively. The current age or the age at death is listed to the right of each symbol. Mutations in the DNA and protein sequence are abbreviated by convention. Amino acids are listed as single letters. Additional details of the clinical features of individuals are listed in Table E1 in the online supplement. In (B), the N-terminal region domains (green), the reverse transcriptase motifs (blue), and C-terminal region domains (yellow) are shown. New mutations described in this article are indicated in boldface type; mutations previously described in Reference (italics) and Reference (roman) are shown for comparison. In (D), relative amounts of telomerase activity for seven different TERT mutants were calculated as a ratio of the intensity of the sample's telomerase products to that of an internal control band and normalized to wild-type activity in one representative experiment. Error bars represent the SD. Parallel reactions using [³⁵S]methionine were run on a sodium dodecyl sulfate–polyacrylamide gel to confirm equal expression of the TERT wild-type and mutant proteins.

Figure 2.

Mean terminal restriction fragment lengths (TRFLs) for (A) normal control subjects and (B) individuals from families with TERT or TERC mutations plotted against age. Open circles represent unrelated normal control subjects in (A). Symbols in (B) represent those without heterozygous TERT or TERC mutations (yellow circles) and those with TERT or TERC mutations either with (pink circles) or without (solid circles) a diagnosis of pulmonary fibrosis. The blue region delineates the 10th to 90th percentile predicted bands of the mean TRFLs for the normal control subjects. Linear regression was used to draw a best-fit line through the normal samples.

Figure 3.

Telomere length determined by the terminal restriction fragment length (TRFL) assay of (A) subjects in kindreds with familial pulmonary fibrosis and (B) sporadic cases of idiopathic interstitial lung disease. Abridged pedigrees, the age of each individual, and the presence (+) or absence (–) of the TERT mutation are indicated above each Southern blot in (A) and (B). Open symbols represent normal individuals; solid symbols indicate individuals with pulmonary fibrosis.

Figure 4.

Telomere length as determined by the (A–C) terminal restriction fragment length (TRFL) assay and by the (D–F) quantitative polymerase chain reaction (PCR) assay for normal control subjects (A and D), probands of kindreds with familial pulmonary fibrosis (B and E), and sporadic case subjects with idiopathic interstitial lung disease (C and F) plotted against age. Open circles represent normal control subjects, red triangles represent unrelated probands and sporadic case subjects with pulmonary fibrosis, and solid circles represent probands and sporadic case subjects with pulmonary fibrosis and TERT or TERC mutations. The blue region delineates the 10th to 90th percentile predicted bands of the mean TRFLs or ln(relative T/S ratio) for the normal control subjects (T/S ratio, ratio of the copy number of telomere DNA to a single-copy gene). Linear regression analysis of the TRFL data of normal control subjects (A) established a linear relationship between telomere length and age by the following equation: TRFL = 6.87 – 0.0169 × age (P = 6.1 × 10⁻¹²). Linear regression analysis of the normal subjects (D) established a linear relationship between the logarithm of the relative T/S ratio and age by the following equation: ln(relative T/S ratio) = 1.02 – 0.00451 × age (P = 2.67 × 10⁻¹⁰).

Figure 5.

A measure of the percent short telomeres [logit(p)] for (A) normal control subjects, (B) probands of kindreds with familial pulmonary fibrosis, and (C) sporadic cases with idiopathic interstitial lung disease plotted against age. Open circles represent normal control subjects, red triangles represent unrelated probands and sporadic cases with pulmonary fibrosis, solid circles represent probands and sporadic cases with pulmonary fibrosis and TERT or TERC mutations. The blue region delineates the 10th to 90th percentile predicted bands of the logit(p) for the normal control subjects. Linear regression analysis of the logit(p) established a linear relationship for normal subjects between this measure and age by the following equation (where p is the percentage of short telomeres): logit(p) = ln[p/(1 – p)] = −2.56 + 0.0148 × age (P = 3.4 × 10⁻¹²).

Figure 6.

Telomere length as determined by the quantitative polymerase chain reaction assay for patients with pulmonary arterial hypertension and control subjects plotted against age. Red triangles represent unrelated cases of idiopathic, familial, or anorexigen-associated pulmonary arterial hypertension; solid circles represent available spouse control subjects. The mean age of both the case subjects and spouses is 52 years. This cohort of pulmonary hypertension case subjects includes 83% female and 17% male patients of the following ethnicities: white (75%), black (7%), Hispanic (10%), and other (7%).