In this article, we show short telomere lengths of circulating leukocytes in 25% or more of all subjects with familial or sporadic idiopathic interstitial pneumonia. The findings of the inherited mutations in telomerase in families and individuals with IPF initially suggested that telomerase dysfunction is important for the molecular pathogenesis of this disease. Here we find that of 71 unrelated probands, 12 (17%) with heterozygous mutations in TERT or TERC and an additional 14 (20%) probands without telomerase mutations have mean telomere lengths of circulating leukocytes that are shorter than the 10th percentile by the TRFL assay. Therefore, 37% of probands with the familial form of the disease have evidence of short telomeres. Similarly, of 75 unrelated sporadic cases, 2 (3%) have mutations in TERT and 17 have TRFLs less than the 10th percentile; so 25% of sporadic cases have short telomeres. These findings suggest that short telomere lengths are commonly associated with both the familial and sporadic forms of pulmonary fibrosis and can be only partially explained at the molecular level by coding mutations in TERT and TERC. The sequencing of both genes has been limited to the coding regions and their surrounding intronic splice sites. We cannot rule out noncoding mutations or small deletions in either gene. In one expanded family, the short telomere phenotype segregates with pulmonary fibrosis as an autosomal dominant trait but does not cosegregate with haplotypes about TERT and TERC, suggesting that other genetic loci may contribute to this trait (data not shown).
All families collected with familial pulmonary fibrosis have at least one affected member carrying a diagnosis of an idiopathic interstitial pneumonia or unclassifiable interstitial pneumonia; other affected family members have interstitial lung disease. Although IPF is the most common diagnosis among the affecteds, it is not the only diagnosis. In fact, only 65% of case subjects with coding mutations in TERT
meet the clinical definition of IPF. Other mutation carriers within the same family have been diagnosed with nonspecific interstitial pneumonitis, granulomatous lung disease, and coal worker pneumoconiosis. Similarly, the occurrence of pathologic findings of diverse subtypes of non–usual interstitial pneumonitis in the same family has been reported for other cohorts of familial pulmonary fibrosis kindreds (30
). Granulomatous lung disease, as seen in the proband of family F106, has been associated with telomerase mutations earlier; we previously described one of the affected individuals in family F71 with chronic hypersensitivity whose open lung biopsy showed usual interstitial pneumonitis with features of noncaseating granulomas (2
). The mutations in telomerase appear to increase the susceptibility to interstitial lung disease in general and are not associated with one particular clinicopathologic subtype. This raises the possibility that although the diagnosis of a specific interstitial lung disease may differ for individual family members with telomerase mutations due to different environmental or occupational exposures, the identified genetic predisposition leads to a tissue repair response of fibrosis in reaction to injury.
We also found that IPF is the most common, but not the only, pulmonary diagnosis seen in those familial and sporadic case subjects without telomerase mutations and whose telomere lengths are less than the 10th percentile. A diagnosis of IPF was found in 50–85% of these groups; other diagnoses such as nonspecific interstitial pneumonitis and cryptogenic organizing pneumonia were made by open lung biopsy. Patients in this study were collected on the basis of a diagnosis of idiopathic interstitial pneumonia, but we again found that telomere shortening was not associated with one particular clinicopathologic subtype. A collected cohort of patients with idiopathic, familial, or anorexigen-associated pulmonary arterial hypertension did not demonstrate an excess of individuals with short telomere lengths, suggesting some specificity of the association between pulmonary fibrosis and short telomeres. Additional cohorts of patients with different pulmonary phenotypes will be needed to delineate the range of pulmonary diagnoses associated with telomere shortening.
Although most individuals who carry a heterozygous mutation in TERT or TERC have short telomeres, 15% do not fall below the 10th percentile by the TRFL assay. Analysis of these individuals by the quantitative PCR method also demonstrates that they fall within the normal range (data not shown). In contrast, all of the individuals with diagnoses of pulmonary fibrosis and who carry a heterozygous mutation in TERT or TERC are more than 48 years of age and have telomere lengths below the 10th percentile predicted line. This strongly suggests that the pulmonary fibrosis phenotype is related to older age and short telomere lengths in this molecularly defined group of patients. Leukocyte telomere length may be influenced by other genetic, intrinsic, tissue-specific, or environmental effects. It is currently unknown whether the other mutation carriers have subclinical manifestations of the disease or whether they demonstrate incomplete penetrance. It is also not known whether the nature and degree of telomere shortening seen in circulating leukocytes is representative of the lung cells within these subjects. For eight control subjects and four subjects heterozygous for the TERT P702L mutation, we see a correlation between telomere lengths of DNA isolated from circulating leukocytes and oral buccal epithelial cells (see Figure E4), suggesting that the germline mutations have a global effect on telomere shortening.
Because most of the case subjects in this study have been collected or referred from lung transplantation centers, many are severely affected, having demonstrated progressive worsening of disease despite treatment or withdrawal from presumed culprit exposures. An important question concerns whether mutations in telomerase or telomere length offer any prognostic information regarding the natural history of the disease. Although we have found that more subjects with the telomerase mutation had died or undergone transplantation over the course of this study than those without mutations in TERT or TERC (50 vs. 32%), these results are not statistically significant. Similarly, there is a trend in that more familial and sporadic case subjects without telomerase mutations below the 10th percentile had died or undergone lung transplantation than those with telomere lengths greater than the 10th percentile (43–53% vs. 25–31%), but these trends are not significant in both groups. We did not find a significant correlation between telomere length and diffusion capacity measurements for these individuals. Prospective studies and/or analysis of larger cohorts will be needed to determine whether telomerase mutations or telomere shortening are associated with rate of progression or severity of the pulmonary fibrosis phenotype.
Smoking is a known risk factor for IPF (32
) and for the development of interstitial lung disease in at-risk individuals in kindreds with the familial form of the disease (30
). Those with mutations in TERT
had a lower cumulative amount of cigarette smoking than those without mutations, 12.5 versus 19.2 pack-years. It may be that smoking of any level (even small amounts) may increase the risk of developing pulmonary fibrosis in those with an inherited predisposition. Similar results have been found in studies of carriers of a major lung cancer susceptibility locus identified by linkage analysis (33
). Cigarette smoking and other environmental effects are likely important modifiers for the development of organ-specific disease in subjects with a global risk of telomerase dysfunction.
One of the mutations reported in this study, V694M, was identified in a 60-year-old smoker with no evidence of bone marrow dysfunction and whose 80-year-old mother had a history of pulmonary fibrosis. This same mutation has been reported in a 34-year-old man with moderate aplastic anemia that did not respond to immunosuppression and has short telomere lengths as measured by flow–fluorescence in situ
). The development of pulmonary fibrosis versus bone marrow dysfunction in telomerase mutation carriers may be related to secondary “hits,” such as environmental toxins (cigarette smoking), fibrosis-prone intrinsic host mesenchymal responses to injury (34
), or other susceptibilities. Understanding the influences on the development of lung disease in telomerase mutation carriers will be important to delineate.
In epidemiologic studies, IPF is diagnosed more commonly in males than females (1
). Of the 20 case subjects with pulmonary fibrosis and telomerase mutations for which we had available DNA, 17 are male. The male-to-female ratio of 5.7:1 in this group suggests a lower penetrance of the pulmonary fibrosis phenotype in females with these dominantly inherited telomerase mutations. Women with TERT
mutations and pulmonary fibrosis tend to be, on average, 11.9 years older than their male counterparts (66.0 vs. 54.1 yr for women and men, respectively) and many do not fit the narrow clinical diagnosis of IPF, having apical lung-predominant pulmonary fibrosis. The male-to-female ratio is 1.4:1 and 1.3:1 for the familial and sporadic case subjects without telomerase mutations, respectively. We did observe shorter mean age-adjusted telomere lengths for the men in all groups () and found a significant correlation between telomere length and sex after controlling for age and ethnicity by multiple regression. These findings suggest that some of the observed excess of male cases may be explained by their shorter telomere lengths.
Both assays used in this study are straightforward and can measure telomere lengths of genomic DNA samples. We included only case subjects for whom we have good-quality DNA isolated from circulating leukocytes. The TRFL assay provides an indirect, rather than a direct, measure of telomere length because the undigested lengths of DNA contain telomeres and subtelomeric segments. To minimize the size of subtelomeric sequences, we digested the genomic DNA with six different 4-bp restriction enzymes. We observed a comparable rate of telomere length attrition with age (17 bp/yr) in normal individuals of this age range, as have other investigators who have used this same method (18
). The quantitative PCR measurement of telomere length offers an independent method for estimating telomere length from genomic DNA and we found a good correlation between these two methods. It had been previously shown that a quantitative PCR method for estimating telomere length is fast, inexpensive, and requires significantly less DNA than measurement by Southern blotting (13
). From the TRFL assay, but not the quantitative PCR assay, we can estimate the percentage of short telomeres in each sample which is the most biologically relevant measure (36
Telomere length is known to be a heritable trait with parental effects (37
). Twin studies suggest that telomere size, as measured by the TRFL assay, displays a heritability of 36–78% (22
). The quantitative trait of TRFLs has been mapped to various loci in normal subjects (38
) or in small families with heart disease (19
), but the causative genetic variants within these genomic intervals have not been pinpointed. Understanding the genetic underpinnings of telomere shortening in pulmonary fibrosis may lead to a more complete understanding of how this process contributes to the risk of developing irreversible lung scarring. In addition, such studies may more clearly define the pulmonary as well as other organ phenotypes associated with telomere shortening in aging humans.