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Original Research Diffuse Lung Disease
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Telomere Length in Interstitial Lung Diseases Reinier Snetselaar, MSc; Coline H. M. van Moorsel, PhD; Karin M. Kazemier, BSc; Joanne J. van der Vis, BSc; Pieter Zanen, MD, PhD; Matthijs F. M. van Oosterhout, MD, PhD; and Jan C. Grutters, MD, PhD
Interstitial lung disease (ILD) is a heterogeneous group of rare diseases that primarily affect the pulmonary interstitium. Studies have implicated a role for telomere length (TL) maintenance in ILD, particularly in idiopathic interstitial pneumonia (IIP). Here, we measure TL in a wide spectrum of sporadic and familial cohorts of ILD and compare TL between patient cohorts and control subjects.
BACKGROUND:
METHODS: A multiplex quantitative polymerase chain reaction method was used to measure TL in 173 healthy subjects and 359 patients with various ILDs, including familial interstitial pneumonia (FIP). The FIP cohort was divided into patients carrying TERT mutations, patients carrying SFTPA2 or SFTPC mutations, and patients without a proven mutation (FIP-no mutation). RESULTS: TL in all cases of ILD was significantly shorter compared with those of control subjects (P range: .038 to , .0001). Furthermore, TL in patients with idiopathic pulmonary fibrosis (IPF) was significantly shorter than in patients with other IIPs (P 5 .002) and in patients with sarcoidosis (P , .0001). Within the FIP cohort, patients in the FIP-telomerase reverse transcriptase (TERT) group had the shortest telomeres (P , .0001), and those in the FIP-no mutation group had TL comparable to that of patients with IPF (P 5 .049). Remarkably, TL of patients with FIP-surfactant protein (SFTP) was significantly longer than in patients with IPF, but similar to that observed in patients with other sporadic IIPs.
The results show telomere shortening across all ILD diagnoses. The difference in TL between the FIP-TERT and FIP-SFTP groups indicates the distinction between acquired and innate telomere shortening. Short TL in the IPF and FIP-no mutation groups is indicative of an innate telomere-biology defect, while a stress-induced, acquired telomere shortening might be the underlying process for the other ILD diagnoses. CHEST 2015; 148(4):1011-1018
CONCLUSIONS:
Manuscript received December 9, 2014; revision accepted April 20, 2015; originally published Online First May 14, 2015. ABBREVIATIONS: COP 5 cryptogenic organizing pneumonia; CTDILD 5 connective tissue disease-associated interstitial lung disease; FIP 5 familial interstitial pneumonia; HP 5 hypersensitivity pneumonitis; IIP 5 idiopathic interstitial pneumonia; ILD 5 interstitial lung disease; iNSIP 5 idiopathic nonspecific interstitial pneumonia; IPF 5 idiopathic pulmonary fibrosis; PCR 5 polymerase chain reaction; SFTP 5 surfactant protein; SR-ILD 5 smoking-related interstitial lung disease; TERT 5 telomerase reverse transcriptase; TL 5 telomere length; T/S 5 telomere/single-copy gene AFFILIATIONS: From the Center of Interstitial Lung Diseases, Department of Pulmonology (Mr Snetselaar; Drs van Moorsel, Zanen, and Grutters; and Mss Kazemier and van der Vis), Department of Clinical Chemistry (Ms van der Vis), and Department of Pathology (Dr van Oosterhout), St. Antonius Hospital, Nieuwegein; and the Division of Heart and Lung (Drs van Moorsel, Zanen, and Grutters and Ms Kazemier), University Medical Center Utrecht, Utrecht, The Netherlands.
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Part of this study was presented at the International World Association for Sarcoidosis and Other Granulomatous Disorders (WASOG) Conference on Diffuse Parenchymal Lung Diseases, June 6-7, 2013, Paris, France, and the Pittsburgh-Munich International Lung Conference, October 23-24, 2014, Pittsburgh, PA. FUNDING/SUPPORT: Funding for this study was received from the St. Antonius Research Fund and the Pender Foundation. CORRESPONDENCE TO: Coline H. M. van Moorsel, PhD, Center of Interstitial Lung Diseases, Department of Pulmonology, St. Antonius Hospital, Koekoekslaan 1, 3435 CM Nieuwegein, The Netherlands; e-mail: [email protected] © 2015 AMERICAN COLLEGE OF CHEST PHYSICIANS. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.14-3078
1011
Interstitial lung diseases (ILDs) are a group of diseases that primarily affect the pulmonary interstitium. Although ILD is a heterogeneous group of diagnoses, they are classified together based on similar clinical, radiologic, physiologic, or pathologic features.1 Four groups are distinguished within ILD: diseases with a known cause, idiopathic interstitial pneumonias (IIPs), granulomatous diseases, and a miscellaneous group.2 The etiology of a number of ILDs is unknown, which presents limitations to classification and, hence, to treatment.3 Therefore, it is important to investigate which features are common and which are unique in ILD. Multiple ILDs have been associated with short telomere length (TL).4-6 Telomeres protect genetic information by acting as a buffer against the chromosomal shortening that is inherent to cell growth. Critical shortening of the telomeres leads to cell-cycle arrest. Maintaining TL is necessary, therefore, for ongoing cell proliferation.7 Loss in TL can be restored by the ribonucleoprotein telomerase. The relevance of telomere biology in ILD was first discovered in IIP. Patients with familial disease were found to carry mutations in the telomere maintenance genes TERT and TERC.8,9 These patients also had distinctly short telomeres in their blood cells. Next, a cohort of patients with IIP who did not carry these mutations, both familial and sporadic, was also shown to have shorter telomeres, compared with control subjects. A significant portion of these patients who did not carry the mutations had TL below that of the 10th and even below the first percentile of control subjects.4,6 Telomere biology-related genetic factors are suggested to underlie telomere shortening in these patients. This suggestion is underlined by discoveries of familial disease causing mutations in other telomere biology-related genes beside TERT and TERC, such as DKC1 in the telomerase complex, TINF2 in the shelterin complex, and RTEL1, which interacts with the shelterin complex.10-12 Associa-
Materials and Methods Patients and Control Subjects A total of 359 patients diagnosed with ILD at the Department of Pulmonology of the St. Antonius Hospital in Nieuwegein were retrospectively included in this study. The patients were diagnosed with IPF, idiopathic nonspecific interstitial pneumonia (iNSIP), cryptogenic organizing pneumonia (COP), smoking-related ILD (SR-ILD), hypersensitivity pneumonitis (HP), sarcoidosis, connective tissue diseaseassociated ILD (CTD-ILD), or FIP. Diagnosis was made in accordance with international guidelines.2,23-25 For IIP cases with coexisting patterns, multidisciplinary discussion determined the clinical significance of the individual patterns.25 FIP was defined as two or more first-degree family members with IIP and was documented in 67 patients in 49 different families, and 18 affected family members. Upon first visit to the out-
1012 Original Research
tions with short TL have also been described in other diseases, such as asthma, COPD,13,14 cardiovascular disease,15 and cancer.16 Therefore, it is important to distinguish between genetically predisposed, innately telomere-related diseases and diseases in which short telomeres are acquired due to increased oxidative stress, inflammation, or accelerated cell turnover.17 It has been suggested, therefore, that the degree of difference in TL between healthy control subjects and patients determines if the short telomeres reflect acquired stress states or innate, telomere-driven, degenerative changes.18 We hypothesized that measuring TL in a broad selection of ILD diagnoses would allow us to identify ILD diagnoses with an innate telomere-related pathobiology. Therefore, we measured TL of peripheral blood cells in healthy control subjects and in seven different ILD diagnoses. Subsequently, we determined the degree of difference between healthy control subjects and ILD. We also assessed differences in TL among ILDs and, in particular, among the different forms of IIP. In familial IIP, also called familial interstitial pneumonia (FIP), it has been found that a diagnosis of idiopathic pulmonary fibrosis (IPF) is most frequent, but all subtypes of IIP can be present.19 In this group, two classes of diseasecausing mutations can be distinguished. Besides the telomerase-related mutations, there are mutations in surfactant proteins (SFTPs) that are known to cause FIP.20,21 The surfactant mutations cause endoplasmic reticulum stress, which can lead to pulmonary fibrosis.22 An effect of these mutations on TL, however, has never been investigated. To further explore TL in ILD, we subdivided the FIP cohort into three groups—those with the SFTP mutation (FIP-SFTP), those with telomerase reverse transcriptase (TERT)-related mutations (FIP-TERT), and those who did not carry a mutation (FIP-no mutation)—and compared these to the nonfamilial ILD data.
patient clinic, patients are asked to fill out a questionnaire regarding familial disease status. In case of a positive anamnesis for familial disease, the possibility of FIP and retrieval of further medical information were discussed by the respective physician. Fourteen of the 49 families have been described by van Moorsel et al21 as “[familial pulmonary fibrosis] FPF 1-10, 15, and 17-19.” A histopathologic pattern of usual interstitial pneumonia was present in these patients, sometimes with coexistence or superimposition of other patterns, as is also known from other FIP reports.8,19 Patients with FIP had been screened for mutations on all exons of the genes TERT, TERC, and SFTPC, and on exon 6 of SFTPA2. Mutation-carrying familial patients were classified as a separate group and subdivided in SFTPC-C or -A2 carriers (FIP-SFTP) and telomerase mutation carriers (FIP-TERT). No mutations were found in TERC. The remaining FIP cohort, therefore, consisted only of patients without an identified mutation (FIP-no mutation). The control subjects
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comprised 173 self-reported healthy employees of St. Antonius Hospital. The medical ethical committee of St. Antonius Hospital in Nieuwegein approved this study (approval no. W14.056 and R05.08A) and subjects gave formal written informed consent. Quantitative Polymerase Chain Reaction Telomere Length
(Bio-Rad Laboratories Inc), and all quantitative PCR TL measurements were performed in triplicate. Along each PCR run, four independent samples were measured for quality control and an average interassay coefficient of variation of , 10% was found. An average intraassay variation of , 5% was found. Statistical Methods
Genomic DNA was extracted from peripheral WBCs using a magnetic beads-based method (chemagic DNA blood 10k kit; Perkin Elmer Inc). TL was measured using the monochrome multiplex, quantitative polymerase chain reaction (PCR) method previously described.26 Briefly, the relative TL was estimated from the ratio of the telomere repeat copy number to a single gene (human b-globin gene) copy number (telomere/single-copy gene [T/S] ratio) for each sample, using standard curves from a serial dilution of a genomic DNA pool. Reactions were performed on a Bio-Rad MyiQ real-time PCR detection system
For statistical analysis, SPSS Statistics, version 22 (IBM Corporation) and GraphPad Prism 5 (GraphPad Software Inc) were used. A general linear model analysis of variance was used to determine differences in TL between the tested cohorts. Age and sex were modeled as covariates. Post hoc analysis was done using Fisher least significant difference. The relation between relative TL and age in control subjects was used to calculate the 10th, fifth, and first percentiles of control subjects’ TL. Fisher exact test was used to compare percentages of cases and control subjects below the 10th, fifth, and first percentiles.
Results
with sarcoidosis had the smallest difference in TL compared with control subjects (P 5 .004).
Relative TL was determined in control subjects and patients diagnosed with the following ILDs: sarcoidosis, HP, CTD-ILD, iNSIP, SR-ILD, COP, IPF, and FIP (Table 1). TL in ILD
First, we determined the effect of an ILD diagnosis on TL using a general linear model with age and sex as covariates. This analysis showed that TL in the study population was significantly determined by an ILD diagnosis (P , .0001) when adjusted for age and sex. Post hoc comparison showed a significant difference in TL between the control group and all tested ILDs separately (Fig 1A). The largest differences in average TL compared with control subjects were found for IPF, FIP-no mutation, and FIP-TERT (P , .001). Patients TABLE 1
Post hoc comparison also revealed a number of notable differences and similarities in TL between separate ILDs (Fig 1A). First, TL in patients with sporadic IPF was significantly shorter than in those with sarcoidosis (P , .001) and in those with the following IIP diagnoses: iNSIP (P 5 .005), and SR-ILD (P 5 .031); a trend toward significance was found for COP (P 5 .113). Second, a significant difference was found between the two granulomatous ILDs: TL in sarcoidosis was longer than in HP (P 5 .009). Finally, no significant difference was found between CTD-ILD and iNSIP (P 5 .276). Patients in the FIP-no mutation group had TL comparable with patients with sporadic IPF. Between familial patients, TERT mutation carriers had the shortest
] ILD Cohorts and Mean Telomere Length in Peripheral Blood Subjects
Cohort Control
No.
(Male/Female)
Age, Median (IQR), y
T/S Ratio, Mean (SD)
173
(105/68)
49 (31-57)
0.975 (0.10)
Sarcoidosis
67
(38/29)
50 (43-64)
0.927 (0.10)
HP
40
(19/21)
60 (50-67)
0.870 (0.10)
CTD-ILD
29
(11/18)
62 (44-66)
0.865 (0.09)
iNSIP
26
(19/7)
68 (60-76)
0.879 (0.09)
SR-ILD
13
(6/7)
46 (41-55)
0.907 (0.12)
8
(4/4)
67 (60-71)
0.883 (0.06)
COP IPF
109
(112/21)
68 (59-72)
0.818 (0.11)
28
(19/13)
59 (53-70)
0.822 (0.09)
FIP-TERT
27
(23/6)
63 (57-67)
0.710 (0.09)
FIP-SFTP
12
(7/8)
33 (30-59)
0.895 (0.10)
FIP-no mutation
COP 5 cryptogenic organizing pneumonia; CTD-ILD 5 connective tissue disease-associated interstitial lung disease; FIP-no mutation 5 familial interstitial pneumonia, nonmutation carrier; FIP-SFTP 5 surfactant mutation carriers; FIP-TERT 5 telomerase mutation carriers; HP 5 hypersensitivity pneumonitis; ILD 5 interstitial lung disease; iNSIP 5 idiopathic nonspecific interstitial pneumonia; IPF 5 idiopathic pulmonary fibrosis; IQR 5 interquartile range; SR-ILD 5 smoking-related interstitial lung disease; T/S 5 telomere/single-copy gene.
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1013
Figure 1 – Telomere length in interstitial lung disease. A, The estimated marginal means of the T/S ratio per cohort, adjusted for age and sex, in peripheral WBCs. Whiskers represent SEs. B-E, T/S ratio per patient. The dotted line represents linear regression for control subjects. Solid lines represent the 95th and fifth predicted percentiles for control subjects. B, Sporadic idiopathic interstitial pneumonias (IIPs): IPF, iNSIP, SR-ILD, and COP. C, FIP-no mutation, FIP-TERT, and FIP-SFTP. D, Granulomatous ILDs: sarcoidosis and HP. E, Patients with CTD-ILD. COP 5 cryptogenic organizing pneumonia; CTD-ILD 5 connective tissue disease-associated interstitial lung disease; FIP-no mutation 5 familial interstitial pneumonia, nonmutation carriers; FIP-SFTP 5 familial interstitial pneumonia, surfactant-mutation carriers; FIP-TERT 5 familial interstitial pneumonia, telomerase-mutation carriers; HP 5 hypersensitivity pneumonitis; iNSIP 5 idiopathic nonspecific interstitial pneumonia; IPF 5 idiopathic pulmonary fibrosis; SR-ILD 5 smoking-related interstitial lung disease; T/S 5 telomere/single-copy gene. 1014 Original Research
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telomeres and differed significantly from patients in the FIP-SFTP or FIP-no mutation groups (P , .001). On the other hand, patients with an SFTP mutation had the longest telomeres, comparable with that found in patients with the following IIPs: iNSIP, SR-ILD, and COP, and significantly longer than TL in patients with IPF (P 5 .049) and in the FIP-no mutation group (P 5 .037).
high (85%) and remained high at the fifth and first percentiles. The FIP-TERT cohort had significantly more patients below the fifth percentile (P 5 .022) than FIPSFTP. Dividing the HP patient cohort in a group with (n 5 20) and without (n 5 20) fibrosis did not show a significant difference in relative TL (data not shown).
Patient Proportions Below the 10th Percentile
This study shows that telomeres of patients with ILD are shorter compared with those of healthy control subjects. However, the degree of difference in TL differs significantly between the different ILD diagnoses and particularly within the IIP subgroup. Within familial disease, TL correlates with the underlying genetic cause. This is a large study on TL and ILD, including eight different ILD diagnoses and 532 subjects, and is the first study, to our knowledge, to separately analyze different subclasses of IIP and FIP.
Per ILD, the degree of difference of TL compared with control subjects was examined by determining the proportion of patients who had TL below that of the 10th, fifth, and first percentiles of control subjects (Table 2). In control subjects, a significant linear relation between relative TL and age (P 5 .0024) was present and relative TL decreased by 0.002 units/y. No such relation between TL and age was seen in any of the ILD cohorts. T/S ratio for each ILD per patient is shown in Figures 1B-E. In all ILDs except COP, the proportion of patients with TL below the 10th percentile differed significantly from control subjects (P , .05) (Table 2). Below the fifth percentile, the proportion of patients with HP, CTD-ILD, SR-ILD, IPF, FIP, and FIP-TERT was still significantly higher than in control subjects (P , .05). Below the first percentile, the proportion of patients with sarcoidosis was significantly higher than control subjects (P 5 .016). Only in IPF, FIP-no mutation, and FIP-TERT groups did a significant proportion of patients have TL below the 10th, fifth, and first percentiles. The proportion of patients in the FIP-TERT group whose TL was below the 10th percentile was extremely TABLE 2
] Percentages of Patients per Cohort With Telomere Length Below 10th, Fifth, and First Percentiles
Cohort Control
10th
5th
8.7
6.9
1st 1.7
Sarcoidosis
19.4a
14.9
9.0a
HP
35.0a
22.5a
7.5
CTD-ILD
41.4a
27.6a
3.4
iNSIP
30.8
7.7
3.8
SR-ILD
46.2a
23.1a
7.7
COP
12.5
IPF
55.0a
45.9a
16.5a
FIP-no mutation
50.0a
39.3a
21.4a
FIP-TERT
85.2a
81.5a
59.3a
FIP-SFTP
33.3
25
a
a
0
See Table 1 legend for expansion of abbreviations. P , .05 compared with control subjects.
a
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0
0
Discussion
Previous studies have shown that a significant proportion of patients with sporadic IIP have reduced TLs.4,6 Two IIP TL studies reported finding no significant difference in TL between the different IIP diagnoses.4,6 It was suggested that insufficient statistical power was present to detect differences. The studies had been performed in mixed IIP cohorts of 73 patients6 and 62 patients,4 and consisted of 63% and 81% patients with IPF, respectively. To analyze differences between IIP cohorts, we included a large number of patients with non-IPF IIP in our study and found that TL is significantly shorter in patients with IPF compared with those with iNSIP or SR-ILD, while a trend toward significance was found for COP (P 5 .113). Shortest mean TL was found in patients with FIP who carried a TERT mutation. In previous studies, patients with FIP, whether carrying a mutation in TERT and TERC, had been shown to have significantly shorter telomeres compared with control subjects.6,8,9 Here, we confirmed these findings both for the FIP and FIP-TERT groups and also determined, for the first time to our knowledge, TL in familial patients with a mutation in surfactant genes SFTPA2 or SFTPC. More importantly, patients in the FIP-SFTP group have significantly longer telomeres than those in the FIP-TERT group. This supports the presence of divergent etiologies for FIP: one based on telomere biology,27-29 which is most profound in patients with FIP-TERT, and one based on local endoplasmic reticulum stress, which is most profound in patients with FIP-SFTP.22 IPF TL has previously been compared with control subjects and patients with HP.30 In that study, the proportions of patients with IPF (5.7%) and those with 1015
HP (5.8%) with TL below that of the 10th percentile of control subjects were comparable to the control population.30 Others have found a proportion of 25% of patients with IPF below the 10th percentile.6 Our results showed a significantly high proportion of 55% and 35% below the 10th percentile for patients with IPF and those with HP, respectively. The difference between these studies could possibly be attributed to a difference in disease severity between the studied cohorts. A limitation of the present study is that all patients were derived from a single center. The ILD outpatient clinic of St. Antonius Hospital is a tertiary referral and transplantation center for ILD in The Netherlands, which might lead to a patient population with more severe ILD cases in advanced stages of disease. TL might not only be dependent on the diagnosis but may also change during disease. In the future, serial TL measurements might be performed to further investigate this possibility. A significant difference in TL between control subjects and patients with sarcoidosis has been previously reported5 and was also found in our study. However, we show that in comparison with other ILDs, patients with sarcoidosis have the smallest loss of TL. This indicates a minimal role of telomere-related pathobiology in sarcoidosis. No difference in TL between control subjects and patients with sarcoidosis has also been reported,31 but was thought to be due to the small sarcoidosis sample size (n 5 22). A notable observation in our sarcoidosis cohort is the significant proportion of patients with TL below that of the first percentile of control subjects. Of these six patients, five were between 38 and 47 years old and male sex, corresponding with previously reported ageand sex-related acceleration of loss of TL in sarcoidosis.5 Multiple factors influence TL. Patients with FIP-SFTP do not suffer from genetically determined telomeropathy22,32 but do have severe interstitial lung fibrosis with clinical and pathologic findings similar to those seen in IPF.21 Although not driven by telomeropathy, we found that TL was significantly shorter in patients in the FIPSFTP group than in control subjects. This degree of telomere shortening in patients with FIP-SFTP most likely represents the acquired decrease of TL due to disease.18,33 A similar degree of TL loss was found in iNSIP, SR-ILD, and COP, suggesting that these diseases are also not driven by telomere-related pathology, but instead have acquired shorter telomeres in peripheral blood cells. TL in patients with HP and those with CTD-ILD was slightly shorter than that found in patients
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with FIP-SFTP and other non-IPF IIP. A significant proportion of patients with HP and patients with CTD-ILD had TL below the fifth percentile and a significant difference in TL between these ILD cases and control subjects has been shown before.31 Decreased TL can be acquired by exogenous factors like increased oxidative stress due to smoking, but also can be due to increased proliferation of immune cells.34,35 Both antigen presentation and chronic viral infection are associated with T-cell proliferation and subsequent telomere attrition.36,37 Multiple ILDs, including sarcoidosis, COP, HP, and CTD–ILD are associated with a significant systemic upregulation of the immune system. Their short TL might be due, therefore, to increased proliferation of immune cells, which has been shown to be associated with decreased TL in peripheral blood cells.38 Interestingly, no difference in TL was found between (sub)acute HP and chronic HP. This suggests a limit to the extent of telomere shortening as a result of prolonged immune responses. The patients with FIP-SFTP had a significantly higher mean TL than those in the FIP-no mutation group and patients with IPF. Within our FIP-no mutation and IPF cohort, some individual patients had a near-normal TL suggestive of nontelomere- or surfactant-related disease etiology. On the other hand, some patients with IPF and patients with FIP have very short telomeres, comparable with those seen in FIP-TERT, suggesting an innate telomere-related pathobiology.18 TL is a heritable trait, and compromised telomerase function leads, over generations, to increasingly shorter telomeres. In IPF, a genetic polymorphism in TERT has been found to confer IPF susceptibility.39 This supports the possibility of a telomere-driven pathology in patients with sporadic IPF. TL in these patients would already be compromised upon encountering exogenous risk factors and subsequent cell-proliferative demands. Telomere pathology has been linked to epithelial cell senescence32 and increased senescence of epithelial cells lining fibroblast foci has been shown in IPF by Minegawa et al.40 They also showed increased senescence of epithelial cells lining fibroblast foci in IPF.40 Telomere-induced senescent cells were shown to produce a senescence-associated secretory phenotype in vitro.41 This phenotype could subsequently lead to lung remodeling.32 Also, additional hits to a senescent epithelium, like cigarette smoke, viral infections, or gastroesophageal reflux, would contribute to the onset of IPF.42,43 Identification of telomere-driven disease in at least a subgroup of IPF could lead to a new classification based
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on presence or absence of telomere aberrations. However, further research is needed on the association of such a group with clinical and molecular differentiating markers. Furthermore, it is possible that therapeutic interventions have different outcomes for telomere pathology-driven IPF compared with IPF based on other etiologies. Because the present study supports an important role for telomere shortening in the pathogenesis of IPF, TL might become an important parameter in the design of future clinical trials. In conclusion, TL in patients with ILD is shorter compared with that of control subjects, and TL in patients
Acknowledgments Author contributions: R. S., C. H. M. v. M., and J. C. G. had full access to all of the data in the study, and take responsibility for the integrity of the data and the accuracy of the data analysis, including and especially any adverse effects. R. S. served as principal author. R. S., C. H. M. v. M., K. M. K., J. J. v. d. V., and J. C. G. contributed to the study concept and design; K. M. K. and J. J. v. d. V. contributed to data collection; R. S., C. H. M. v. M., K. M. K., J. J. v. d. V., P. Z., and J. C. G. contributed to data analysis; R. S., C. H. M. v. M., P. Z., M. F. M. v. O., and J. C. G. contributed to data interpretation; R. S., C. H. M. v. M., M. F. M. v. O., and J. C. G. contributed to the writing of the manuscript; R. S., C. H. M. v. M., K. M. K., J. J. v. d. V., P. Z., M. F. M. v. O., and J. C. G. approved the final manuscript. Conflict of interest: None declared. Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript.
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with ILD differs significantly among different diagnoses. This indicates a varying role of telomere biology in ILD. A remarkable difference is also shown between patients with FIP carrying a mutation in TERT, who have the shortest telomeres, and patients with FIP with an SFTPC or SFTPA2 mutation, who carry the longest telomeres in IIP. The decrease in TL in most ILDs is comparable to that seen in FIP-SFTP and is likely acquired through increased oxidative stress or inflammatory responses. However, the telomeres in patients with IPF and those with other FIP are much shorter, suggesting an important role for an innate, genetically predisposed, telomeredriven disease in many of these patients.
5. Guan JZ, Maeda T, Sugano M, et al. An analysis of telomere length in sarcoidosis. J Gerontol A Biol Sci Med Sci. 2007; 62(11):1199-1203. 6. Cronkhite JT, Xing C, Raghu G, et al. Telomere shortening in familial and sporadic pulmonary fibrosis. Am J Respir Crit Care Med. 2008;178(7):729-737. 7. Young NS. Telomere biology and telomere diseases: implications for practice and research. Hematology Am Soc Hematol Educ Program. 2010;2010:30-35. 8. Armanios MY, Chen JJ, Cogan JD, et al. Telomerase mutations in families with idiopathic pulmonary fibrosis. N Engl J Med. 2007;356(13):1317-1326. 9. Tsakiri KD, Cronkhite JT, Kuan PJ, et al. Adult-onset pulmonary fibrosis caused by mutations in telomerase. Proc Natl Acad Sci U S A. 2007;104(18):7552-7557. 10. Alder JK, Stanley SE, Wagner CL, Hamilton M, Hanumanthu VS, Armanios M. Exome sequencing identifies mutant TINF2 in a family with pulmonary fibrosis. Chest. 2015.147(5):1361-1368. 11. Cogan JD, Kropski JA, Zhao M, et al; University of Washington Center for Mendelian Genomics. Rare variants in RTEL1 are associated with familial interstitial pneumonia. Am J Respir Crit Care Med. 2015;191(6):646-655. 12. Kropski JA, Mitchell DB, Markin C, et al. A novel dyskerin (DKC1) mutation is associated with familial interstitial pneumonia. Chest. 2014;146(1):e1-e7. 13. Albrecht E, Sillanpää E, Karrasch S, et al. Telomere length in circulating leukocytes is associated with lung function and disease. Eur Respir J. 2014;43(4):983-992. 14. Belsky DW, Shalev I, Sears MR, et al. Is chronic asthma associated with shorter leukocyte telomere length at midlife? Am J Respir Crit Care Med. 2014;190(4): 384-391. 15. Haycock PC, Heydon EE, Kaptoge S, Butterworth AS, Thompson A, Willeit P. Leucocyte telomere length and risk of cardiovascular disease: systematic review and meta-analysis. BMJ. 2014;349:g4227. 16. Prescott J, Wentzensen IM, Savage SA, De Vivo I. Epidemiologic evidence for
a role of telomere dysfunction in cancer etiology. Mutat Res. 2012;730(1-2):75-84. 17. Kong CM, Lee XW, Wang X. Telomere shortening in human diseases. FEBS J. 2013;280(14):3180-3193. 18. Armanios M. Telomeres and age-related disease: how telomere biology informs clinical paradigms. J Clin Invest. 2013; 123(3):996-1002. 19. Steele MP, Speer MC, Loyd JE, et al. Clinical and pathologic features of familial interstitial pneumonia. Am J Respir Crit Care Med. 2005;172(9):1146-1152. 20. Wang Y, Kuan PJ, Xing C, et al. Genetic defects in surfactant protein A2 are associated with pulmonary fibrosis and lung cancer. Am J Hum Genet. 2009;84(1):52-59. 21. van Moorsel CH, van Oosterhout MF, Barlo NP, et al. Surfactant protein C mutations are the basis of a significant portion of adult familial pulmonary fibrosis in a Dutch cohort. Am J Respir Crit Care Med. 2010;182(11):1419-1425. 22. Tanjore H, Blackwell TS, Lawson WE. Emerging evidence for endoplasmic reticulum stress in the pathogenesis of idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2012; 302(8):L721-L729. 23. Costabel U, Hunninghake GW; Sarcoidosis Statement Committee. American Thoracic Society. European Respiratory Society. World Association for Sarcoidosis and Other Granulomatous Disorders. ATS/ERS/WASOG statement on sarcoidosis. Eur Respir J. 1999;14(4): 735-737. 24. Raghu G, Collard HR, Egan JJ, et al; ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med. 2011;183(6): 788-824. 25. Travis WD, Costabel U, Hansell DM, et al; ATS/ERS Committee on Idiopathic Interstitial Pneumonias. An official American Thoracic Society/European Respiratory Society statement: update of the international multidisciplinary
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classification of the idiopathic interstitial pneumonias. Am J Respir Crit Care Med. 2013;188(6):733-748.
32. Armanios M, Blackburn EH. The telomere syndromes. Nat Rev Genet. 2012;13(10): 693-704.
38. Andrews NP, Fujii H, Goronzy JJ, Weyand CM. Telomeres and immunological diseases of aging. Gerontology. 2010;56(4):390-403.
26. Cawthon RM. Telomere length measurement by a novel monochrome multiplex quantitative PCR method. Nucleic Acids Res. 2009;37(3):e21.
33. Townsley DM, Dumitriu B, Young NS. Bone marrow failure and the telomeropathies. Blood. 2014;124(18): 2775-2783.
27. Gansner JM, Rosas IO. Telomeres in lung disease. Transl Res. 2013;162(6): 343-352.
34. Valdes AM, Andrew T, Gardner JP, et al. Obesity, cigarette smoking, and telomere length in women. Lancet. 2005;366(9486): 662-664.
39. Mushiroda T, Wattanapokayakit S, Takahashi A, et al; Pirfenidone Clinical Study Group. A genome-wide association study identifies an association of a common variant in TERT with susceptibility to idiopathic pulmonary fibrosis. J Med Genet. 2008;45(10):654-656.
35. Plunkett FJ, Franzese O, Belaramani LL, et al. The impact of telomere erosion on memory CD81 T cells in patients with X-linked lymphoproliferative syndrome. Mech Ageing Dev. 2005;126(8): 855-865.
40. Minagawa S, Araya J, Numata T, et al. Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-b-induced senescence of human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2011;300(3):L391-L401.
30. Liu T, Ullenbruch M, Young Choi Y, et al. Telomerase and telomere length in pulmonary fibrosis. Am J Respir Cell Mol Biol. 2013;49(2):260-268.
36. Rufer N, Brümmendorf TH, Kolvraa S, et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood. J Exp Med. 1999;190(2):157-167.
41. Coppé JP, Desprez PY, Krtolica A, Campisi J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol. 2010;5:99-118.
31. Stuart BD, Lee JS, Kozlitina J, et al. Effect of telomere length on survival in patients with idiopathic pulmonary fibrosis: an observational cohort study with independent validation. Lancet Respir Med. 2014;2(7):557-565.
37. Effros RB, Allsopp R, Chiu CP, et al. Shortened telomeres in the expanded CD28-CD81 cell subset in HIV disease implicate replicative senescence in HIV pathogenesis. AIDS. 1996;10(8): F17-F22.
28. Holohan B, Wright WE, Shay JW. Cell biology of disease: telomeropathies: an emerging spectrum disorder. J Cell Biol. 2014;205(3):289-299. 29. Kropski JA, Lawson WE, Young LR, Blackwell TS. Genetic studies provide clues on the pathogenesis of idiopathic pulmonary fibrosis. Dis Model Mech. 2013;6(1):9-17.
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42. Alder JK, Guo N, Kembou F, et al. Telomere length is a determinant of emphysema susceptibility. Am J Respir Crit Care Med. 2011;184(8):904-912. 43. Wuyts WA, Agostini C, Antoniou KM, et al. The pathogenesis of pulmonary fibrosis: a moving target. Eur Respir J. 2013;41(5):1207-1218.
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Original Research Diffuse Lung Disease
]
Telomere Length in Interstitial Lung Diseases Reinier Snetselaar, MSc; Coline H. M. van Moorsel, PhD; Karin M. Kazemier, BSc; Joanne J. van der Vis, BSc; Pieter Zanen, MD, PhD; Matthijs F. M. van Oosterhout, MD, PhD; and Jan C. Grutters, MD, PhD
Interstitial lung disease (ILD) is a heterogeneous group of rare diseases that primarily affect the pulmonary interstitium. Studies have implicated a role for telomere length (TL) maintenance in ILD, particularly in idiopathic interstitial pneumonia (IIP). Here, we measure TL in a wide spectrum of sporadic and familial cohorts of ILD and compare TL between patient cohorts and control subjects.
BACKGROUND:
METHODS: A multiplex quantitative polymerase chain reaction method was used to measure TL in 173 healthy subjects and 359 patients with various ILDs, including familial interstitial pneumonia (FIP). The FIP cohort was divided into patients carrying TERT mutations, patients carrying SFTPA2 or SFTPC mutations, and patients without a proven mutation (FIP-no mutation). RESULTS: TL in all cases of ILD was significantly shorter compared with those of control subjects (P range: .038 to , .0001). Furthermore, TL in patients with idiopathic pulmonary fibrosis (IPF) was significantly shorter than in patients with other IIPs (P 5 .002) and in patients with sarcoidosis (P , .0001). Within the FIP cohort, patients in the FIP-telomerase reverse transcriptase (TERT) group had the shortest telomeres (P , .0001), and those in the FIP-no mutation group had TL comparable to that of patients with IPF (P 5 .049). Remarkably, TL of patients with FIP-surfactant protein (SFTP) was significantly longer than in patients with IPF, but similar to that observed in patients with other sporadic IIPs.
The results show telomere shortening across all ILD diagnoses. The difference in TL between the FIP-TERT and FIP-SFTP groups indicates the distinction between acquired and innate telomere shortening. Short TL in the IPF and FIP-no mutation groups is indicative of an innate telomere-biology defect, while a stress-induced, acquired telomere shortening might be the underlying process for the other ILD diagnoses. CHEST 2015; 148(4):1011-1018
CONCLUSIONS:
Manuscript received December 9, 2014; revision accepted April 20, 2015; originally published Online First May 14, 2015. ABBREVIATIONS: COP 5 cryptogenic organizing pneumonia; CTDILD 5 connective tissue disease-associated interstitial lung disease; FIP 5 familial interstitial pneumonia; HP 5 hypersensitivity pneumonitis; IIP 5 idiopathic interstitial pneumonia; ILD 5 interstitial lung disease; iNSIP 5 idiopathic nonspecific interstitial pneumonia; IPF 5 idiopathic pulmonary fibrosis; PCR 5 polymerase chain reaction; SFTP 5 surfactant protein; SR-ILD 5 smoking-related interstitial lung disease; TERT 5 telomerase reverse transcriptase; TL 5 telomere length; T/S 5 telomere/single-copy gene AFFILIATIONS: From the Center of Interstitial Lung Diseases, Department of Pulmonology (Mr Snetselaar; Drs van Moorsel, Zanen, and Grutters; and Mss Kazemier and van der Vis), Department of Clinical Chemistry (Ms van der Vis), and Department of Pathology (Dr van Oosterhout), St. Antonius Hospital, Nieuwegein; and the Division of Heart and Lung (Drs van Moorsel, Zanen, and Grutters and Ms Kazemier), University Medical Center Utrecht, Utrecht, The Netherlands.
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Part of this study was presented at the International World Association for Sarcoidosis and Other Granulomatous Disorders (WASOG) Conference on Diffuse Parenchymal Lung Diseases, June 6-7, 2013, Paris, France, and the Pittsburgh-Munich International Lung Conference, October 23-24, 2014, Pittsburgh, PA. FUNDING/SUPPORT: Funding for this study was received from the St. Antonius Research Fund and the Pender Foundation. CORRESPONDENCE TO: Coline H. M. van Moorsel, PhD, Center of Interstitial Lung Diseases, Department of Pulmonology, St. Antonius Hospital, Koekoekslaan 1, 3435 CM Nieuwegein, The Netherlands; e-mail: [email protected] © 2015 AMERICAN COLLEGE OF CHEST PHYSICIANS. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.14-3078
1011
Interstitial lung diseases (ILDs) are a group of diseases that primarily affect the pulmonary interstitium. Although ILD is a heterogeneous group of diagnoses, they are classified together based on similar clinical, radiologic, physiologic, or pathologic features.1 Four groups are distinguished within ILD: diseases with a known cause, idiopathic interstitial pneumonias (IIPs), granulomatous diseases, and a miscellaneous group.2 The etiology of a number of ILDs is unknown, which presents limitations to classification and, hence, to treatment.3 Therefore, it is important to investigate which features are common and which are unique in ILD. Multiple ILDs have been associated with short telomere length (TL).4-6 Telomeres protect genetic information by acting as a buffer against the chromosomal shortening that is inherent to cell growth. Critical shortening of the telomeres leads to cell-cycle arrest. Maintaining TL is necessary, therefore, for ongoing cell proliferation.7 Loss in TL can be restored by the ribonucleoprotein telomerase. The relevance of telomere biology in ILD was first discovered in IIP. Patients with familial disease were found to carry mutations in the telomere maintenance genes TERT and TERC.8,9 These patients also had distinctly short telomeres in their blood cells. Next, a cohort of patients with IIP who did not carry these mutations, both familial and sporadic, was also shown to have shorter telomeres, compared with control subjects. A significant portion of these patients who did not carry the mutations had TL below that of the 10th and even below the first percentile of control subjects.4,6 Telomere biology-related genetic factors are suggested to underlie telomere shortening in these patients. This suggestion is underlined by discoveries of familial disease causing mutations in other telomere biology-related genes beside TERT and TERC, such as DKC1 in the telomerase complex, TINF2 in the shelterin complex, and RTEL1, which interacts with the shelterin complex.10-12 Associa-
Materials and Methods Patients and Control Subjects A total of 359 patients diagnosed with ILD at the Department of Pulmonology of the St. Antonius Hospital in Nieuwegein were retrospectively included in this study. The patients were diagnosed with IPF, idiopathic nonspecific interstitial pneumonia (iNSIP), cryptogenic organizing pneumonia (COP), smoking-related ILD (SR-ILD), hypersensitivity pneumonitis (HP), sarcoidosis, connective tissue diseaseassociated ILD (CTD-ILD), or FIP. Diagnosis was made in accordance with international guidelines.2,23-25 For IIP cases with coexisting patterns, multidisciplinary discussion determined the clinical significance of the individual patterns.25 FIP was defined as two or more first-degree family members with IIP and was documented in 67 patients in 49 different families, and 18 affected family members. Upon first visit to the out-
1012 Original Research
tions with short TL have also been described in other diseases, such as asthma, COPD,13,14 cardiovascular disease,15 and cancer.16 Therefore, it is important to distinguish between genetically predisposed, innately telomere-related diseases and diseases in which short telomeres are acquired due to increased oxidative stress, inflammation, or accelerated cell turnover.17 It has been suggested, therefore, that the degree of difference in TL between healthy control subjects and patients determines if the short telomeres reflect acquired stress states or innate, telomere-driven, degenerative changes.18 We hypothesized that measuring TL in a broad selection of ILD diagnoses would allow us to identify ILD diagnoses with an innate telomere-related pathobiology. Therefore, we measured TL of peripheral blood cells in healthy control subjects and in seven different ILD diagnoses. Subsequently, we determined the degree of difference between healthy control subjects and ILD. We also assessed differences in TL among ILDs and, in particular, among the different forms of IIP. In familial IIP, also called familial interstitial pneumonia (FIP), it has been found that a diagnosis of idiopathic pulmonary fibrosis (IPF) is most frequent, but all subtypes of IIP can be present.19 In this group, two classes of diseasecausing mutations can be distinguished. Besides the telomerase-related mutations, there are mutations in surfactant proteins (SFTPs) that are known to cause FIP.20,21 The surfactant mutations cause endoplasmic reticulum stress, which can lead to pulmonary fibrosis.22 An effect of these mutations on TL, however, has never been investigated. To further explore TL in ILD, we subdivided the FIP cohort into three groups—those with the SFTP mutation (FIP-SFTP), those with telomerase reverse transcriptase (TERT)-related mutations (FIP-TERT), and those who did not carry a mutation (FIP-no mutation)—and compared these to the nonfamilial ILD data.
patient clinic, patients are asked to fill out a questionnaire regarding familial disease status. In case of a positive anamnesis for familial disease, the possibility of FIP and retrieval of further medical information were discussed by the respective physician. Fourteen of the 49 families have been described by van Moorsel et al21 as “[familial pulmonary fibrosis] FPF 1-10, 15, and 17-19.” A histopathologic pattern of usual interstitial pneumonia was present in these patients, sometimes with coexistence or superimposition of other patterns, as is also known from other FIP reports.8,19 Patients with FIP had been screened for mutations on all exons of the genes TERT, TERC, and SFTPC, and on exon 6 of SFTPA2. Mutation-carrying familial patients were classified as a separate group and subdivided in SFTPC-C or -A2 carriers (FIP-SFTP) and telomerase mutation carriers (FIP-TERT). No mutations were found in TERC. The remaining FIP cohort, therefore, consisted only of patients without an identified mutation (FIP-no mutation). The control subjects
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comprised 173 self-reported healthy employees of St. Antonius Hospital. The medical ethical committee of St. Antonius Hospital in Nieuwegein approved this study (approval no. W14.056 and R05.08A) and subjects gave formal written informed consent. Quantitative Polymerase Chain Reaction Telomere Length
(Bio-Rad Laboratories Inc), and all quantitative PCR TL measurements were performed in triplicate. Along each PCR run, four independent samples were measured for quality control and an average interassay coefficient of variation of , 10% was found. An average intraassay variation of , 5% was found. Statistical Methods
Genomic DNA was extracted from peripheral WBCs using a magnetic beads-based method (chemagic DNA blood 10k kit; Perkin Elmer Inc). TL was measured using the monochrome multiplex, quantitative polymerase chain reaction (PCR) method previously described.26 Briefly, the relative TL was estimated from the ratio of the telomere repeat copy number to a single gene (human b-globin gene) copy number (telomere/single-copy gene [T/S] ratio) for each sample, using standard curves from a serial dilution of a genomic DNA pool. Reactions were performed on a Bio-Rad MyiQ real-time PCR detection system
For statistical analysis, SPSS Statistics, version 22 (IBM Corporation) and GraphPad Prism 5 (GraphPad Software Inc) were used. A general linear model analysis of variance was used to determine differences in TL between the tested cohorts. Age and sex were modeled as covariates. Post hoc analysis was done using Fisher least significant difference. The relation between relative TL and age in control subjects was used to calculate the 10th, fifth, and first percentiles of control subjects’ TL. Fisher exact test was used to compare percentages of cases and control subjects below the 10th, fifth, and first percentiles.
Results
with sarcoidosis had the smallest difference in TL compared with control subjects (P 5 .004).
Relative TL was determined in control subjects and patients diagnosed with the following ILDs: sarcoidosis, HP, CTD-ILD, iNSIP, SR-ILD, COP, IPF, and FIP (Table 1). TL in ILD
First, we determined the effect of an ILD diagnosis on TL using a general linear model with age and sex as covariates. This analysis showed that TL in the study population was significantly determined by an ILD diagnosis (P , .0001) when adjusted for age and sex. Post hoc comparison showed a significant difference in TL between the control group and all tested ILDs separately (Fig 1A). The largest differences in average TL compared with control subjects were found for IPF, FIP-no mutation, and FIP-TERT (P , .001). Patients TABLE 1
Post hoc comparison also revealed a number of notable differences and similarities in TL between separate ILDs (Fig 1A). First, TL in patients with sporadic IPF was significantly shorter than in those with sarcoidosis (P , .001) and in those with the following IIP diagnoses: iNSIP (P 5 .005), and SR-ILD (P 5 .031); a trend toward significance was found for COP (P 5 .113). Second, a significant difference was found between the two granulomatous ILDs: TL in sarcoidosis was longer than in HP (P 5 .009). Finally, no significant difference was found between CTD-ILD and iNSIP (P 5 .276). Patients in the FIP-no mutation group had TL comparable with patients with sporadic IPF. Between familial patients, TERT mutation carriers had the shortest
] ILD Cohorts and Mean Telomere Length in Peripheral Blood Subjects
Cohort Control
No.
(Male/Female)
Age, Median (IQR), y
T/S Ratio, Mean (SD)
173
(105/68)
49 (31-57)
0.975 (0.10)
Sarcoidosis
67
(38/29)
50 (43-64)
0.927 (0.10)
HP
40
(19/21)
60 (50-67)
0.870 (0.10)
CTD-ILD
29
(11/18)
62 (44-66)
0.865 (0.09)
iNSIP
26
(19/7)
68 (60-76)
0.879 (0.09)
SR-ILD
13
(6/7)
46 (41-55)
0.907 (0.12)
8
(4/4)
67 (60-71)
0.883 (0.06)
COP IPF
109
(112/21)
68 (59-72)
0.818 (0.11)
28
(19/13)
59 (53-70)
0.822 (0.09)
FIP-TERT
27
(23/6)
63 (57-67)
0.710 (0.09)
FIP-SFTP
12
(7/8)
33 (30-59)
0.895 (0.10)
FIP-no mutation
COP 5 cryptogenic organizing pneumonia; CTD-ILD 5 connective tissue disease-associated interstitial lung disease; FIP-no mutation 5 familial interstitial pneumonia, nonmutation carrier; FIP-SFTP 5 surfactant mutation carriers; FIP-TERT 5 telomerase mutation carriers; HP 5 hypersensitivity pneumonitis; ILD 5 interstitial lung disease; iNSIP 5 idiopathic nonspecific interstitial pneumonia; IPF 5 idiopathic pulmonary fibrosis; IQR 5 interquartile range; SR-ILD 5 smoking-related interstitial lung disease; T/S 5 telomere/single-copy gene.
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1013
Figure 1 – Telomere length in interstitial lung disease. A, The estimated marginal means of the T/S ratio per cohort, adjusted for age and sex, in peripheral WBCs. Whiskers represent SEs. B-E, T/S ratio per patient. The dotted line represents linear regression for control subjects. Solid lines represent the 95th and fifth predicted percentiles for control subjects. B, Sporadic idiopathic interstitial pneumonias (IIPs): IPF, iNSIP, SR-ILD, and COP. C, FIP-no mutation, FIP-TERT, and FIP-SFTP. D, Granulomatous ILDs: sarcoidosis and HP. E, Patients with CTD-ILD. COP 5 cryptogenic organizing pneumonia; CTD-ILD 5 connective tissue disease-associated interstitial lung disease; FIP-no mutation 5 familial interstitial pneumonia, nonmutation carriers; FIP-SFTP 5 familial interstitial pneumonia, surfactant-mutation carriers; FIP-TERT 5 familial interstitial pneumonia, telomerase-mutation carriers; HP 5 hypersensitivity pneumonitis; iNSIP 5 idiopathic nonspecific interstitial pneumonia; IPF 5 idiopathic pulmonary fibrosis; SR-ILD 5 smoking-related interstitial lung disease; T/S 5 telomere/single-copy gene. 1014 Original Research
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telomeres and differed significantly from patients in the FIP-SFTP or FIP-no mutation groups (P , .001). On the other hand, patients with an SFTP mutation had the longest telomeres, comparable with that found in patients with the following IIPs: iNSIP, SR-ILD, and COP, and significantly longer than TL in patients with IPF (P 5 .049) and in the FIP-no mutation group (P 5 .037).
high (85%) and remained high at the fifth and first percentiles. The FIP-TERT cohort had significantly more patients below the fifth percentile (P 5 .022) than FIPSFTP. Dividing the HP patient cohort in a group with (n 5 20) and without (n 5 20) fibrosis did not show a significant difference in relative TL (data not shown).
Patient Proportions Below the 10th Percentile
This study shows that telomeres of patients with ILD are shorter compared with those of healthy control subjects. However, the degree of difference in TL differs significantly between the different ILD diagnoses and particularly within the IIP subgroup. Within familial disease, TL correlates with the underlying genetic cause. This is a large study on TL and ILD, including eight different ILD diagnoses and 532 subjects, and is the first study, to our knowledge, to separately analyze different subclasses of IIP and FIP.
Per ILD, the degree of difference of TL compared with control subjects was examined by determining the proportion of patients who had TL below that of the 10th, fifth, and first percentiles of control subjects (Table 2). In control subjects, a significant linear relation between relative TL and age (P 5 .0024) was present and relative TL decreased by 0.002 units/y. No such relation between TL and age was seen in any of the ILD cohorts. T/S ratio for each ILD per patient is shown in Figures 1B-E. In all ILDs except COP, the proportion of patients with TL below the 10th percentile differed significantly from control subjects (P , .05) (Table 2). Below the fifth percentile, the proportion of patients with HP, CTD-ILD, SR-ILD, IPF, FIP, and FIP-TERT was still significantly higher than in control subjects (P , .05). Below the first percentile, the proportion of patients with sarcoidosis was significantly higher than control subjects (P 5 .016). Only in IPF, FIP-no mutation, and FIP-TERT groups did a significant proportion of patients have TL below the 10th, fifth, and first percentiles. The proportion of patients in the FIP-TERT group whose TL was below the 10th percentile was extremely TABLE 2
] Percentages of Patients per Cohort With Telomere Length Below 10th, Fifth, and First Percentiles
Cohort Control
10th
5th
8.7
6.9
1st 1.7
Sarcoidosis
19.4a
14.9
9.0a
HP
35.0a
22.5a
7.5
CTD-ILD
41.4a
27.6a
3.4
iNSIP
30.8
7.7
3.8
SR-ILD
46.2a
23.1a
7.7
COP
12.5
IPF
55.0a
45.9a
16.5a
FIP-no mutation
50.0a
39.3a
21.4a
FIP-TERT
85.2a
81.5a
59.3a
FIP-SFTP
33.3
25
a
a
0
See Table 1 legend for expansion of abbreviations. P , .05 compared with control subjects.
a
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0
0
Discussion
Previous studies have shown that a significant proportion of patients with sporadic IIP have reduced TLs.4,6 Two IIP TL studies reported finding no significant difference in TL between the different IIP diagnoses.4,6 It was suggested that insufficient statistical power was present to detect differences. The studies had been performed in mixed IIP cohorts of 73 patients6 and 62 patients,4 and consisted of 63% and 81% patients with IPF, respectively. To analyze differences between IIP cohorts, we included a large number of patients with non-IPF IIP in our study and found that TL is significantly shorter in patients with IPF compared with those with iNSIP or SR-ILD, while a trend toward significance was found for COP (P 5 .113). Shortest mean TL was found in patients with FIP who carried a TERT mutation. In previous studies, patients with FIP, whether carrying a mutation in TERT and TERC, had been shown to have significantly shorter telomeres compared with control subjects.6,8,9 Here, we confirmed these findings both for the FIP and FIP-TERT groups and also determined, for the first time to our knowledge, TL in familial patients with a mutation in surfactant genes SFTPA2 or SFTPC. More importantly, patients in the FIP-SFTP group have significantly longer telomeres than those in the FIP-TERT group. This supports the presence of divergent etiologies for FIP: one based on telomere biology,27-29 which is most profound in patients with FIP-TERT, and one based on local endoplasmic reticulum stress, which is most profound in patients with FIP-SFTP.22 IPF TL has previously been compared with control subjects and patients with HP.30 In that study, the proportions of patients with IPF (5.7%) and those with 1015
HP (5.8%) with TL below that of the 10th percentile of control subjects were comparable to the control population.30 Others have found a proportion of 25% of patients with IPF below the 10th percentile.6 Our results showed a significantly high proportion of 55% and 35% below the 10th percentile for patients with IPF and those with HP, respectively. The difference between these studies could possibly be attributed to a difference in disease severity between the studied cohorts. A limitation of the present study is that all patients were derived from a single center. The ILD outpatient clinic of St. Antonius Hospital is a tertiary referral and transplantation center for ILD in The Netherlands, which might lead to a patient population with more severe ILD cases in advanced stages of disease. TL might not only be dependent on the diagnosis but may also change during disease. In the future, serial TL measurements might be performed to further investigate this possibility. A significant difference in TL between control subjects and patients with sarcoidosis has been previously reported5 and was also found in our study. However, we show that in comparison with other ILDs, patients with sarcoidosis have the smallest loss of TL. This indicates a minimal role of telomere-related pathobiology in sarcoidosis. No difference in TL between control subjects and patients with sarcoidosis has also been reported,31 but was thought to be due to the small sarcoidosis sample size (n 5 22). A notable observation in our sarcoidosis cohort is the significant proportion of patients with TL below that of the first percentile of control subjects. Of these six patients, five were between 38 and 47 years old and male sex, corresponding with previously reported ageand sex-related acceleration of loss of TL in sarcoidosis.5 Multiple factors influence TL. Patients with FIP-SFTP do not suffer from genetically determined telomeropathy22,32 but do have severe interstitial lung fibrosis with clinical and pathologic findings similar to those seen in IPF.21 Although not driven by telomeropathy, we found that TL was significantly shorter in patients in the FIPSFTP group than in control subjects. This degree of telomere shortening in patients with FIP-SFTP most likely represents the acquired decrease of TL due to disease.18,33 A similar degree of TL loss was found in iNSIP, SR-ILD, and COP, suggesting that these diseases are also not driven by telomere-related pathology, but instead have acquired shorter telomeres in peripheral blood cells. TL in patients with HP and those with CTD-ILD was slightly shorter than that found in patients
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with FIP-SFTP and other non-IPF IIP. A significant proportion of patients with HP and patients with CTD-ILD had TL below the fifth percentile and a significant difference in TL between these ILD cases and control subjects has been shown before.31 Decreased TL can be acquired by exogenous factors like increased oxidative stress due to smoking, but also can be due to increased proliferation of immune cells.34,35 Both antigen presentation and chronic viral infection are associated with T-cell proliferation and subsequent telomere attrition.36,37 Multiple ILDs, including sarcoidosis, COP, HP, and CTD–ILD are associated with a significant systemic upregulation of the immune system. Their short TL might be due, therefore, to increased proliferation of immune cells, which has been shown to be associated with decreased TL in peripheral blood cells.38 Interestingly, no difference in TL was found between (sub)acute HP and chronic HP. This suggests a limit to the extent of telomere shortening as a result of prolonged immune responses. The patients with FIP-SFTP had a significantly higher mean TL than those in the FIP-no mutation group and patients with IPF. Within our FIP-no mutation and IPF cohort, some individual patients had a near-normal TL suggestive of nontelomere- or surfactant-related disease etiology. On the other hand, some patients with IPF and patients with FIP have very short telomeres, comparable with those seen in FIP-TERT, suggesting an innate telomere-related pathobiology.18 TL is a heritable trait, and compromised telomerase function leads, over generations, to increasingly shorter telomeres. In IPF, a genetic polymorphism in TERT has been found to confer IPF susceptibility.39 This supports the possibility of a telomere-driven pathology in patients with sporadic IPF. TL in these patients would already be compromised upon encountering exogenous risk factors and subsequent cell-proliferative demands. Telomere pathology has been linked to epithelial cell senescence32 and increased senescence of epithelial cells lining fibroblast foci has been shown in IPF by Minegawa et al.40 They also showed increased senescence of epithelial cells lining fibroblast foci in IPF.40 Telomere-induced senescent cells were shown to produce a senescence-associated secretory phenotype in vitro.41 This phenotype could subsequently lead to lung remodeling.32 Also, additional hits to a senescent epithelium, like cigarette smoke, viral infections, or gastroesophageal reflux, would contribute to the onset of IPF.42,43 Identification of telomere-driven disease in at least a subgroup of IPF could lead to a new classification based
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on presence or absence of telomere aberrations. However, further research is needed on the association of such a group with clinical and molecular differentiating markers. Furthermore, it is possible that therapeutic interventions have different outcomes for telomere pathology-driven IPF compared with IPF based on other etiologies. Because the present study supports an important role for telomere shortening in the pathogenesis of IPF, TL might become an important parameter in the design of future clinical trials. In conclusion, TL in patients with ILD is shorter compared with that of control subjects, and TL in patients
Acknowledgments Author contributions: R. S., C. H. M. v. M., and J. C. G. had full access to all of the data in the study, and take responsibility for the integrity of the data and the accuracy of the data analysis, including and especially any adverse effects. R. S. served as principal author. R. S., C. H. M. v. M., K. M. K., J. J. v. d. V., and J. C. G. contributed to the study concept and design; K. M. K. and J. J. v. d. V. contributed to data collection; R. S., C. H. M. v. M., K. M. K., J. J. v. d. V., P. Z., and J. C. G. contributed to data analysis; R. S., C. H. M. v. M., P. Z., M. F. M. v. O., and J. C. G. contributed to data interpretation; R. S., C. H. M. v. M., M. F. M. v. O., and J. C. G. contributed to the writing of the manuscript; R. S., C. H. M. v. M., K. M. K., J. J. v. d. V., P. Z., M. F. M. v. O., and J. C. G. approved the final manuscript. Conflict of interest: None declared. Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript.
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