Telomere lengths, pulmonary fibrosis and telomerase (TERT) mutations

Abstract

Background: Telomerase is an enzyme that catalyzes the addition of nucleotides on the ends of chromosomes. Rare loss of function mutations in the gene that encodes the protein component of telomerase (TERT) have been described in patients with idiopathic pulmonary fibrosis (IPF). Here we examine the telomere lengths and pulmonary fibrosis phenotype seen in multiple kindreds with heterozygous TERT mutations.

Methods and findings: We have identified 134 individuals with heterozygous TERT mutations from 21 unrelated families. Available medical records, surgical lung biopsies and radiographs were evaluated retrospectively. Genomic DNA isolated from circulating leukocytes has been used to measure telomere lengths with a quantitative PCR assay. We find that telomere lengths of TERT mutation carriers decrease in an age-dependent manner and show progressive shortening with successive generations of mutation inheritance. Family members without TERT mutations have a shorter mean telomere length than normal, demonstrating epigenetic inheritance of shortened telomere lengths in the absence of an inherited TERT mutation. Pulmonary fibrosis is an age-dependent phenotype not seen in mutation carriers less than 40 years of age but found in 60% of men 60 years or older; its development is associated with environmental exposures including cigarette smoking. A radiographic CT pattern of usual interstitial pneumonia (UIP), which is consistent with a diagnosis of IPF, is seen in 74% of cases and a pathologic pattern of UIP is seen in 86% of surgical lung biopsies. Pulmonary fibrosis associated with TERT mutations is progressive and lethal with a mean survival of 3 years after diagnosis. Overall, TERT mutation carriers demonstrate reduced life expectancy, with a mean age of death of 58 and 67 years for males and females, respectively.

Conclusions: A subset of pulmonary fibrosis, like dyskeratosis congenita, bone marrow failure, and liver disease, represents a "telomeropathy" caused by germline mutations in telomerase and characterized by short telomere lengths. Family members within kindreds who do not inherit the TERT mutation have shorter telomere lengths than controls, demonstrating epigenetic inheritance of a shortened parental telomere length set-point.

Figure 1. Abridged pedigrees of kindreds with heterozygous mutations in the gene encoding the protein component of telomerase (TERT).

The arrow indicates the index case. Circles represent females; squares represent males. Symbols with a slash indicate deceased subjects. Individuals with pulmonary fibrosis, blood dyscrasias and liver cirrhosis are indicated by red, green, and yellow symbols, respectively. Roman numerals indicate the generation. Numbers in parentheses indicate individuals for whom no DNA sample was available. The age at the time of consent or the age of death is indicated to the upper right of each symbol. The predicted amino acid changes that result from the TERT mutations are listed above each family. Some of the pedigrees have been modified to hide identifying features.

Figure 2. Telomere lengths of family members from TERT kindreds.

Mean telomere lengths as measured by a quantitative PCR assay for (A) normal subjects and (B) subjects with TERT mutations are plotted against age. (A) The telomere lengths of spouses (open symbols) and family members without TERT mutations (orange filled symbols) are shown relative to the 50^th percentile (center line) and the 10^th to 90^th percentiles for 195 unrelated healthy individuals from 19–89 years of age (blue shaded region). (B) TERT mutation carriers without any clinical disease (open circles), with pulmonary fibrosis (red symbols), blood dyscrasias (green symbols) and liver cirrhosis (yellow symbols) are plotted against the same reference range. (C) Mean observed minus expected age-adjusted telomere length for the indicated groups. The minimum number of successive generations the TERT mutation has segregated in the kindred is indicated; G3 indicates subjects in the third successive generation with TERT mutations, i.e., the children with TERT mutations whose parents and grandparents also had a TERT mutation. G2+1 indicates subjects who do not have a telomerase mutation and are the offspring of individuals that represent the second successive generation with a TERT mutation. Bars show the mean value. (D) Mean observed minus expected age-adjusted telomere lengths for offspring of TERT mutation carriers with (+) and without (−) the mutation. Mean telomere lengths are shorter for offspring of fathers who carry a TERT mutation. P-values of 2.4×10⁻³⁸ (*),1.01×10⁻⁵ (**), 0.01 (***) and linear trend test P-value of 0.04 (****).

Figure 3. Radiographic and pathologic findings of heterozygous TERT mutation carriers with pulmonary fibrosis.

Computed tomography (CT) scans of three different subjects with pulmonary fibrosis. Representative cases are shown with a pattern typical of Usual Interstitial Pneumonia (UIP) with peripheral, basal-predominant fibrosis and moderate to severe honeycombing (A,B), a pattern consistent with UIP with peripheral, basal-predominant fibrosis in the absence of honeycombing (C,D), and a pattern atypical for UIP with fibrosis predominantly affecting the upper lobes and along the bronchi (E, F). Scans are shown at the level of the carina (A, C, E) and the lung base (B, D, F). The majority (25 cases or 86%) of TERT mutation carriers with lung specimens available for review had diagnostic histologic features of UIP. In this low magnification view of UIP (G), typical variegated honeycomb areas (top right) are seen alternating with normal areas (left) and scarred lung (bottom). The case shown in (H) shows increased inflammation and a small, loosely aggregated non-necrotizing granuloma (arrows) that is characterized by a cluster of epitheloid histiocytes and multinucleated giant cells surrounded by chronic inflammation in the interstitium. Panels G and H are shown at 40 and 100-fold magnification, respectively.

Figure 4. Pulmonary fibrosis is a lethal, age-associated phenotype of TERT mutation carriers.

(A) Penetrance of pulmonary fibrosis is shown for men (yellow) and women (blue bars) of different ages. No one less than 40 years of age exhibited pulmonary fibrosis. Penetrance of pulmonary fibrosis for men vs. women 40–49, 50–59 and ≥60 years of age is 14% vs. 2%, 38% vs. 14%, and 60% vs. 50%, respectively. (B) Kaplan-Meier survival curve of 47 different TERT mutation carriers with pulmonary fibrosis demonstrate a mean survival of 3 years after diagnosis.