Adult-onset pulmonary fibrosis caused by mutations
Kalliopi D. Tsakiri*, Jennifer T. Cronkhite*, Phillip J. Kuan*, Chao Xing*†, Ganesh Raghu‡, Jonathan C. Weissler§,
Randall L. Rosenblatt§, Jerry W. Shay¶, and Christine Kim Garcia*§?
*McDermott Center for Human Growth and Development,†Center for Clinical Sciences,§Department of Internal Medicine, Division of Pulmonary and
Critical Care Medicine,¶Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390;
and‡Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Washington Medical Center, 1959 NE Pacific,
Seattle, WA 98195-6522
Edited by Michael S. Brown, University of Texas Southwestern Medical Center, Dallas, TX, and approved March 26, 2007 (received for review
February 2, 2007)
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 muta-
tion 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 unre-
lated families and 44 sporadic cases of interstitial lung disease re-
vealed five other mutations in TERT. A heterozygous mutation in
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.
genetics ? idiopathic pulmonary fibrosis ? telomeres ? aging
decade and increases in prevalence with advanced age (1, 2). Mean
survival after diagnosis is 3 years (3). The clinical presentation of
IPF is similar to that of all of the different scarring lung diseases,
fibrosis and symptoms of a chronic cough and shortness of breath.
IPF is distinguished from the other interstitial lung diseases by its
unknown etiology, by characteristic abnormalities on pulmonary
function tests and radiographs, and by biopsy findings, which
include evidence of injury occurring over time with foci of repli-
cating fibroblasts at the interface between normal and scarred lung
Unlike other interstitial lung diseases, IPF does not respond to
immunosuppressive therapies and its clinical course is marked by
proven to prolong life expectancy.
Approximately one of every 50 patients with IPF has an affected
first-degree family member (4). The inheritance pattern is most
consistent with autosomal dominant with incomplete penetrance.
The clinical presentation of familial IPF is indistinguishable from
sporadic IPF except that the age of onset tends to be earlier (55
approach to map the culprit gene in two large families to chromo-
in a candidate gene, TERT, which encodes the catalytic component
of telomerase, and one heterozygous mutation in TERC, the
increased susceptibility to developing IPF.
diopathic pulmonary fibrosis (IPF) is a devastating progressive
fibrotic disease of the lungs that typically presents after the fifth
We have collected 46 families with two or more cases of
idiopathic interstitial lung disease, with many of those affected
meeting the clinical criteria for IPF (3). To localize the gene
nucleotide polymorphism (SNP) linkage scan in two of the
largest Caucasian families in our collection (Fig. 1). Families F11
and F31 include five individuals with IPF, five with pulmonary
fibrosis and six with unclassified pulmonary disease [Table 1 and
supporting information (SI) Table 2]. Thorascopic lung biopsies
were available from four family members of family F11 (Fig. 2);
three biopsy samples had a histologic pattern typical of IPF
whereas the fourth (III.4) had generalized fibrosis. Both families
of odds (LOD) score of 2.8. Among the genes we evaluated was
TERT, which was considered a candidate because a mutation in
this gene was recently reported to cause autosomal dominant
dyskeratosis congenita (DKC), a disorder in which 20% of
affected individuals develop pulmonary fibrosis (6, 7).
The 16 exons and consensus splicing sequences of TERT were
F31 was heterozygous for a deletion of thymidine at position 2240
in the cDNA, which creates a frameshift in the reading frame and
is predicted to result in a truncated protein missing half of the
proband of family F11 was heterozygous for a transition mutation
(CGT 3 CAT) in codon 865 that is predicted to change a highly
conserved arginine to a histidine. This arginine is part of the
reverse transcriptase proteins (8) (Fig. 3). All family members with
IPF or pulmonary fibrosis were heterozygous for these mutations.
For both families, some family members with TERT mutations
exhibited other clinical features of DKC (7), such as osteopo-
rosis/osteopenia, anemia and cancer (Table 1), but none of the
affected individuals had the mucocutaneous lesions typical of
DKC. Some family members who inherited the TERT mutation
had no evidence of pulmonary disease.
We sequenced the coding regions of TERT in the probands of 44
additional families with idiopathic interstitial lung disease. Four
additional sequence variations, including three missense mutations
and one 177-bp deletion, were found (Fig. 3). We also sequenced
Author contributions: J.W.S. and C.K.G. designed research; K.D.T., J.T.C., P.J.K., and C.X.
performed research; G.R., J.C.W., and R.L.R. contributed new reagents/analytic tools; and
J.W.S. and C.K.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
Abbreviations: DKC, dyskeratosis congenita; IPF, idiopathic pulmonary fibrosis; LOD, log-
arithm of odds; TRAP, telomere repeat amplification protocol; TRF, terminal restriction
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
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the coding region of TERT in 44 individuals with sporadic inter-
stitial lung disease and no family history of interstitial lung disease.
arginine at codon 865 found to be mutated in family F11. Thus, a
total of two frameshift deletions and five missense mutations in
TERT were identified in patients with pulmonary fibrosis (Fig. 3).
None of the seven mutations were found in 94 locally collected,
ethnically matched individuals, nor were they detected in 200
patients with aplastic anemia or a multiethnic panel of 528 indi-
viduals sequenced as controls (9). Electropherograms of all of the
sequence mutations are shown in SI Fig. 6, and all common
polymorphisms found by sequencing are listed in SI Table 3.
Telomerase has two essential components: the catalytic protein
encoded by TERT and the telomerase RNA (TERC) that provides
a template for the repeat sequence added in tandem to the ends of
chromosomes. Multiple different mutations in TERC have been
451 bp of genomic DNA that encodes TERC in our 46 unrelated
probands of familial IPF and in all of the cases of sporadic
pulmonary fibrosis. One proband of a Caucasian family (F61) was
heterozygous for an adenosine and guanine at position 37. This
mutation affects the terminal residue of the P1b helix that is known
to function in telomerase template boundary definition (11). This
change was not detected in 94 ethnically matched controls but was
identified previously in a person with severe aplastic anemia who
was a compound heterozygote for variants in TERC (12).
To assess the functional significance of the TERT and TERC
mutations, the activity of in vitro coexpressed recombinant telom-
erase protein and RNA was determined by the telomere repeat
amplification protocol (TRAP) assay. As expected, the mutation
(V747fs, Fig. 4 A and C) missing half the reverse transcriptase
missense mutations in TERT (Fig. 4 B and C) and the mutation in
TERC (Fig. 4 A and C) produced levels of telomerase activity that
ranged from zero to 100% of wild-type activity. Mixing different
amounts of the V747fs TERT construct with the wild-type TERT
construct did not affect the activity of the wild-type protein,
suggesting a mechanism of haploinsufficiency (Fig. 4D).
To determine whether the mutations we identified in TERT and
TERC affected telomere length in vivo, we analyzed the telomere
lengths of genomic DNA isolated from leukocytes. Genomic DNA
containing telomeres and subtelomeric regions is resistant to di-
gestion by restriction enzymes and the terminal restriction frag-
ments (TRFs) can be visualized by using concatamers of the
telomere sequence (TTAGGG) as a probe using a modification of
the Southern blot procedure.
Telomere length is influenced by many different factors, includ-
ing age (13, 14) and the number of generations the mutation is
transmitted (6, 15). When compared with normal family members
of similar age, the mean telomere length was significantly shorter
for those individuals who were heterozygous for a mutation in
TERT or TERC (Fig. 5 A and B). All of the mutations caused
telomere shortening, even those that were not associated with a
detectable decrease in telomerase activity by the in vitro TRAP
The mean telomere length of asymptomatic heterozygous carriers
was similar to those with IPF in the aggregate analysis (Fig. 5C).
When telomere lengths were analyzed within families, the
family members with clinical disease tended to have shorter
TRFs than the asymptomatic carriers. Because the shortest
telomeres, not the average telomere length, limit cell growth,
(16) we analyzed each family separately and estimated the
proportion of short TRFs (arbitrarily set as those between 1.9
and 4.3 kb) for age-similar family members. For family 11 (Fig.
5D), the individuals with the highest proportion of short TRFs
were the two individuals with IPF. Another individual with a
TERT mutation in this family had very short TRFs but did not
have IPF. This individual had cirrhosis of unknown etiology.
Unclassified Pulmonary Disease
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.
Abridged pedigrees of seven families with familial idiopathic interstitial lung disease and TERT (A) or TERC (B) mutations. Individuals with pulmonary
Tsakiri et al.
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This report provides previously undescribed molecular insight into
the pathogenesis of IPF and expands the scope of diseases caused
by mutations in telomerase. The individuals with TERT or TERC
mutations have none of the physical manifestations of DKC and
were all ascertained by the lung fibrosis phenotype, distinguishing
them from previously described patients with mutations in these
genes (6, 9, 10, 17). None had aplastic anemia but several exhibited
a mild to moderate anemia and one individual developed neutro-
penia after lung transplantation. A number of individuals had
evidence of axial osteoporosis; in many cases, this diagnosis pre-
ceded or was determined in the absence of lung disease. All
developed pulmonary fibrosis during their adult years with no
history of pediatric lung disease, in contrast with kindreds associ-
ated with surfactant protein C mutations (18, 19).
of telomerase deficiency. It is not yet been reported whether aged
telomerase-deficient mice also develop similar lung pathology (20).
The pathologic features of IPF suggest that the lung injury is focal,
affecting scattered portions of the lung parenchyma and recurring
pulmonary phenotype is incompletely penetrant and may be influ-
enced not only by the nature of the mutations but also by environ-
mental influences, such as cigarette smoking. Smoking causes
telomere shortening in a dose-dependent manner (22) and is
associated with familial interstitial pneumonia (23). In this study,
the average age of death of the smokers with a mutation in TERT
or TERC (58 years, n ? 13) was 10 years earlier than that of the
nonsmokers (68 years, n ? 7). Telomere length is also influenced
Table 1. Molecular and clinical data of individuals in families with idiopathic interstitial lung disease
Family DNA changeAA changeSubject
Osteopenia Anemia CancerOtherDiagnosis Smoker
c.3346?3522del E1116fsX1127 I.1
Pulmonary function test measurements were obtained prior to lung transplantation. FVC, forced vital capacity; DLco, diffusing capacity for carbon monoxide;
HP, hypersensitivity pneumonitis; ?, unknown; ?, yes; ?, no; TB, tuberculosis.
*TLC (total lung capacity) measurement reported.
†All families are Caucasian, except F8 and F34, who are Hispanic.
‡Diagnosis of osteoporosis/osteopenia made prior to or in the absence of treatment with steroids.
www.pnas.org?cgi?doi?10.1073?pnas.0701009104Tsakiri et al.
is the predominant phenotype and why smoking may exacerbate
this disease. Interestingly, a recent trial with high doses of the
IPF (25). Therapeutics directed toward enhancing telomerase
activity or delaying telomere shortening may lead to novel treat-
ments for IPF in the future.
Because telomerase protein expression is generally restricted
to cells with the capacity to proliferate (26), IPF may result, in
part, from the loss or senescence of a cell population in the lung
able to respond to repetitive injuries over time. Telomerase may
be a marker for identifying resident stem cells that promote
regeneration and prevent premature aging of the lung.
Materials and Methods
Clinical Studies. This study was approved by the University of
Texas Southwestern Institutional Review Board. Written in-
formed consent was obtained from all participants. Each par-
ticipant completed a medical questionnaire and medical records
were obtained when available. All of the families had two or
more cases of idiopathic interstitial lung disease; 34 of the 46
families had individuals with IPF (3). All of the sporadic cases
carried a diagnosis of idiopathic interstitial lung disease; 31
individuals had IPF. Genomic DNA was isolated from leuko-
cytes with an Autopure LS (Qiagen, Valencia, CA). For four
individuals, DNA was isolated and amplified from formalin-
fixed paraffin embedded archival samples as follows. Paraffin
was removed from tissue shavings by serial extraction with 1 ml
each of xylene, a 1:1 mixture of xylene:ethyl ethanol, and ethyl
ethanol. The pellet was dried and the DNA was isolated by using
the tissue protocol of the QIAamp DNA Mini Kit (Qiagen). The
final product was amplified according to the GenomePlex Whole
Genome Amplification Kit (Sigma, St. Louis, MO).
Histology. Photography of hematoxylin and eosin-stained slides
of formalin fixed paraffin embedded lung samples were carried
out on a Leica DM2000 photomicroscope by using an Optronics
DEI-750 analog CCD color camera. Images were captured by
using Image J v1.23 acquisition and analysis software (Scion).
Genotyping. Genomic DNA was genotyped by using the Illumina
Linkage IVb SNP panel of ?6,000 polymorphic SNPs by the
University of Texas Southwestern Microarray Core. Call rates
varied from 99.7–100% for Autopure-purified DNA and between
samples. Individuals with IPF, pulmonary fibrosis, or unclassified
pulmonary disease were considered ‘‘affected,’’ and all others were
assigned an unknown affectation status. We used the software
MERLIN (27) to screen the entire genome by using multipoint
linkage analysis and a model-free method (28) followed by evalu-
ation of the regions with the highest signals by using a model-based
method. Analysis of both families F11 and F31 revealed the highest
peak on chromosome 5p15 with a model-free LOD score of 2.82
(P ? 2.0 ? 10?4) and a model-based LOD score of 2.68 (P ? 2.1 ?
10?3). The 1-LOD drop interval spanned 4.3 Mb and extended
from the end of chromosome 5p to rs959937.
Sequencing and Mutation Analysis. Intronic primers were designed
by using ELXR (29) to sequence both exons and splice sites. After
performed on an ABI 3700 automated sequencer by BigDye
terminator cycle sequencing reagents (Applied Biosystems). All
PCR conditions and primers are listed in SI Table 4. All sequences
were determined in both directions, the mutations were confirmed
comparative alignment were obtained from the NCBI web site at
www.ncbi.nlm.nih.gov. The comparison of telomerase reverse tran-
a ClustalW generated alignment (www.ebi.ac.uk/clustalw) by using
the default settings.
TRAP Assays. Telomerase was reconstituted by expressing human
translation system (Promega, Madison, WI), and its activity was
quantified by the Cy5-fluorescent gel-based TRAP assay as de-
into the parental plasmids pGRN121, pGRN125, and pKT26
(described below) with the use of the QuikChange site-directed
mutagenesis kit (Stratagene). The c.2240delT mutation and its
wild-type control were generated by PCR and directionally sub-
cloned into pCITE-4a (Novagen). The complete coding sequences
for all of the mutants were verified by sequencing. Telomerase
RNA was amplified from genomic DNA by PCR using oligonu-
TGGG-3?, digested with HindIII and BamHI, and subcloned into
pUC18 to generate plasmid pKT26. Linearized TERT constructs
(0.5 ?g) and FspI-linearized telomerase RNA constructs (0.25 ?g)
were used as substrates in 25-?l TnT reactions. The reactions were
serially diluted 1:5 in 1? TRAP buffer starting with 0.3–0.5 ?l of
the TnT reaction. After the extension of the substrate by telomer-
ase, each sample was treated with 1 unit of RNaseH (Invitrogen)
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, honey-
The lung biopsy of III.4 shows the obliteration of most alveoli and their replace-
ment by fibrous tissue, prominent fibroblast proliferation, and an absence of
Tsakiri et al.
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for 45 min at 37°C before PCR analysis of the products. Repeat
experiments showed the same relative trend of TRAP activities. A
lated products were run on a 7% SDS/PAGE gel, fixed in 50%
methanol and 10% glacial acid for 1 h, impregnated with Kodak
B CDE E-IT12 CP E-II E-III E-IVQFP TEN
e l b
a i r
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).
Schematic representation of the functional domains of telomerase reverse transcriptase (A) and telomerase RNA (B) with the position of the mutations
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
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.
Telomerase activity of TERT mutants as measured by the TRAP assay. Telomerase activity of in vitro coexpressed mutant or wild type (wt) telomerase
www.pnas.org?cgi?doi?10.1073?pnas.0701009104Tsakiri et al.
En3hance (PerkinElmer), dried, and exposed to film at ?80°C for Download full-text
TRF Analysis. TRF analysis of genomic DNA isolated from
leukocytes was performed as described (32). The mean TRF was
determined as described except a grid of 200, instead of 30, boxes
was placed over each lane. The percentage of short telomeres
was calculated by dividing the relative signal intensity of each
lane (between 1.9 and 4.3 kb) by the relative signal intensity of
the entire lane (between 1.9 and 19 kb).
We thank the affected individuals and their families for their participa-
tion in this study; Alison Cook, Holly Brookman, and especially Melissa
Nolasco for excellent technical assistance; Robert Barnes for assistance
with the linkage analysis; Russell Turner, Yong Zhao, Nuno Gomes, and
other members of the J.W.S. and Wright laboratory for assistance with
the telomerase assays; Yolanda Mageto, Fernando Torres, and Todd
Hoopman for referral of cases; and Helen Hobbs, Jonathan Cohen, Mike
Brown, and Joe Goldstein for helpful discussions. K.D.T. is a graduate
student from the University of Crete. This work was supported by the
University of Texas Southwestern President’s Research Council Young
Researcher Award and National Institutes of Health Grant K23
RR020632 (to C.K.G.). This work was also supported in part by the
James M. Collins Center for Biomedical Research and the Will Rogers
Institute (to J.C.W.) and the Lung Cancer Specialized Programs of
Research Excellence P50 CA75907 and NASA Specialized Center of
Research NNJ05HD36G (to J.W.S.).
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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
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.
Telomere length determined by Southern blotting of chromosomal TRFs of genomic DNA isolated from leukocytes of individuals from families F8 (A),
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