Susan E. Stanley

Telomere dysfunction causes alveolar stem cell failure

Significance Idiopathic pulmonary fibrosis and emphysema are leading causes of mortality, but there are no effective therapies. Mutations in telomerase are the most common identifiable risk factor for idiopathic pulmonary fibrosis. They also predispose to severe emphysema in smokers, occurring at a frequency similar to α-1 antitrypsin deficiency. The work shown here points to alveolar stem cell senescence as a driver of these pathologies. Epithelial stem cell failure was associated with secondary inflammatory recruitment and exquisite susceptibility to injury from “second hits.” The findings suggest that efforts to reverse the stem cell failure state directly, rather than its secondary consequences, may be an effective therapy approach in telomere-mediated lung disease. Telomere syndromes have their most common manifestation in lung disease that is recognized as idiopathic pulmonary fibrosis and emphysema. In both conditions, there is loss of alveolar integrity, but the underlying mechanisms are not known. We tested the capacity of alveolar epithelial and stromal cells from mice with short telomeres to support alveolar organoid colony formation and found that type 2 alveolar epithelial cells (AEC2s), the stem cell-containing population, were limiting. When telomere dysfunction was induced in adult AEC2s by conditional deletion of the shelterin component telomeric repeat-binding factor 2, cells survived but remained dormant and showed all the hallmarks of cellular senescence. Telomere dysfunction in AEC2s triggered an immune response, and this was associated with AEC2-derived up-regulation of cytokine signaling pathways that are known to provoke inflammation in the lung. Mice uniformly died after challenge with bleomycin, underscoring an essential role for telomere function in AEC2s for alveolar repair. Our data show that alveoloar progenitor senescence is sufficient to recapitulate the regenerative defects, inflammatory responses, and susceptibility to injury that are characteristic of telomere-mediated lung disease. They suggest alveolar stem cell failure is a driver of telomere-mediated lung disease and that efforts to reverse it may be clinically beneficial.

Altered hepatic triglyceride content after partial hepatectomy without impaired liver regeneration in multiple murine genetic models

Liver regeneration is impaired following partial hepatectomy (PH) in mice with genetic obesity and hepatic steatosis and also in wild‐type mice fed a high‐fat diet. These findings contrast with other data showing that liver regeneration is impaired in mice in which hepatic lipid accumulation is suppressed by either pharmacologic leptin administration or by disrupted glucocorticoid signaling. These latter findings suggest that hepatic steatosis may actually be required for normal liver regeneration. We have reexamined this relationship using several murine models of altered hepatic lipid metabolism. Liver fatty acid (FA) binding protein knockout mice manifested reduced hepatic triglyceride (TG) content compared to controls, with no effect on liver regeneration or hepatocyte proliferation. Examination of early adipogenic messenger RNAs revealed comparable induction in liver from both genotypes despite reduced hepatic steatosis. Following PH, hepatic TG was reduced in intestine‐specific microsomal TG transfer protein deleter mice, which fail to absorb dietary fat, increased in peroxisome proliferator activated receptor alpha knockout mice, which exhibit defective FA oxidation, and unchanged (from wild‐type mice) in liver‐specific FA synthase knockout mice in which endogenous hepatic FA synthesis is impaired. Hepatic TG increased in the regenerating liver in all models, even in animals in which lipid accumulation is genetically constrained. However, in no model—and over a >90‐fold range of hepatic TG content—was liver regeneration significantly impaired following PH. Conclusion: Although hepatic TG content is widely variable and increases during liver regeneration, alterations in neither exogenous or endogenous lipid metabolic pathways, demonstrated to promote or diminish hepatic steatosis, influence hepatocyte proliferation. (HEPATOLOGY 2008.)

Telomerase mutations in smokers with severe emphysema

Mutations in the essential telomerase genes TERT and TR cause familial pulmonary fibrosis; however, in telomerase-null mice, short telomeres predispose to emphysema after chronic cigarette smoke exposure. Here, we tested whether telomerase mutations are a risk factor for human emphysema by examining their frequency in smokers with chronic obstructive pulmonary disease (COPD). Across two independent cohorts, we found 3 of 292 severe COPD cases carried deleterious mutations in TERT (1%). This prevalence is comparable to the frequency of alpha-1 antitrypsin deficiency documented in this population. The TERT mutations compromised telomerase catalytic activity, and mutation carriers had short telomeres. Telomerase mutation carriers with emphysema were predominantly female and had an increased incidence of pneumothorax. In families, emphysema showed an autosomal dominant inheritance pattern, along with pulmonary fibrosis and other telomere syndrome features, but manifested only in smokers. Our findings identify germline mutations in telomerase as a Mendelian risk factor for COPD susceptibility that clusters in autosomal dominant families with telomere-mediated disease including pulmonary fibrosis.

The short and long telomere syndromes: paired paradigms for molecular medicine

Recent advances have defined a role for abnormally short telomeres in a broad spectrum of genetic disorders. They include rare conditions such as dyskeratosis congenita as well pulmonary fibrosis and emphysema. Now, there is new evidence that some familial cancers, such as melanoma, are caused by mutations that lengthen telomeres. Here, we examine the significance of these short and long telomere length extremes for understanding the molecular basis of age-related disease and cancer.

Exome Sequencing Identifies Mutant TINF2 in a Family With Pulmonary Fibrosis

BACKGROUND Short telomeres are a common defect in idiopathic pulmonary fibrosis, yet mutations in the telomerase genes account for only a subset of these cases. METHODS We identified a family with pulmonary fibrosis, idiopathic infertility, and short telomeres. RESULTS Exome sequencing of blood-derived DNA revealed two mutations in the telomere-binding protein TINF2. The first was a 15-base-pair deletion encompassing the exon 6 splice acceptor site, and the second was a missense mutation, Thr284Arg. Haplotype analysis indicated both variants fell on the same allele. However, lung-derived DNA showed predominantly the Thr284Arg allele, indicating that the deletion seen in the blood was acquired and may have a protective advantage because it diminished expression of the missense mutation. This mosaicism may represent functional reversion in telomere syndromes similar to that described for Fanconi anemia. No mutations were identified in over 40 uncharacterized pulmonary fibrosis probands suggesting that mutant TINF2 accounts for a small subset of familial cases. However, similar to affected individuals in this family, we identified a history of male and female infertility preceding the onset of pulmonary fibrosis in 11% of TERT and TR mutation carriers (five of 45). CONCLUSIONS Our findings identify TINF2 as a mutant telomere gene in familial pulmonary fibrosis and suggest that infertility may precede the presentation of pulmonary fibrosis in a small subset of adults with telomere syndromes.

Loss-of-function mutations in the RNA biogenesis factor NAF1 predispose to pulmonary fibrosis–emphysema

Mutations in the RNA biogenesis factor NAF1 cause short telomeres and, consequently, age-related lung disease in people. Tidy telomeres make for healthier lungs Telomeres are the protective caps that prevent the ends of chromosomes from unraveling. People carrying mutations in the protein or RNA component of telomerase, the enzyme that makes telomeres have short telomeres and a serious and often fatal lung disease—pulmonary fibrosis. Now, Stanley et al. find in several patients that other mutations, specifically those that interfere with RNA biogenesis, can also cause both short telomeres and lung disease. This work expands our understanding of how telomeres are maintained and their role in human disease. Chronic obstructive pulmonary disease and pulmonary fibrosis have been hypothesized to represent premature aging phenotypes. At times, they cluster in families, but the genetic basis is not understood. We identified rare, frameshift mutations in the gene for nuclear assembly factor 1, NAF1, a box H/ACA RNA biogenesis factor, in pulmonary fibrosis–emphysema patients. The mutations segregated with short telomere length, low telomerase RNA levels, and extrapulmonary manifestations including myelodysplastic syndrome and liver disease. A truncated NAF1 was detected in cells derived from patients, and, in cells in which the frameshift mutation was introduced by genome editing, telomerase RNA levels were reduced. The mutant NAF1 lacked a conserved carboxyl-terminal motif, which we show is required for nuclear localization. To understand the disease mechanism, we used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) to generate Naf1+/− mice and found that they had half the levels of telomerase RNA. Other box H/ACA RNA levels were also decreased, but rRNA pseudouridylation, which is guided by snoRNAs, was intact. Moreover, first-generation Naf1+/− mice showed no evidence of ribosomal pathology. Our data indicate that disease in NAF1 mutation carriers is telomere-mediated; they show that NAF1 haploinsufficiency selectively disturbs telomere length homeostasis by decreasing the levels of telomerase RNA while sparing rRNA pseudouridylation.

Hepatopulmonary syndrome is a frequent cause of dyspnea in the short telomere disorders.

BACKGROUND Telomere syndromes have their most common manifestation in idiopathic pulmonary fibrosis and emphysema. The short telomere defect in these patients may manifest systemically as bone marrow failure and liver disease. We sought to understand the causes of dyspnea in telomerase and telomere gene mutation carriers who have no parenchymal lung disease. METHODS Clinical and pathologic data were reviewed as part of a Johns Hopkins-based natural history study of short telomere syndromes including dyskeratosis congenita. RESULTS Hepatopulmonary syndrome (HPS) was diagnosed in nine of 42 cases (21%). Their age at presentation was significantly younger than that of cases initially presenting with pulmonary fibrosis and emphysema (median, 25 years vs 55 years; P < .001). Cases had evidence of intra- and extrapulmonary arteriovascular malformations that caused shunt physiology. Nodular regenerative hyperplasia was the most frequent histopathologic abnormality, and it was seen in the absence of cirrhosis. Dyspnea and portal hypertension were progressive, and the median time to death or liver transplantation was 6 years (range, 4-10 years; n = 6). In cases that underwent liver transplantation, dyspnea and hypoxia improved, but pulmonary fibrosis subsequently developed. CONCLUSIONS This report identifies HPS as a frequent cause of dyspnea in telomerase and telomere gene mutation carriers. While it usually precedes the development of parenchymal lung disease, HPS may also co-occur with pulmonary fibrosis and emphysema. Recognizing this genetic diagnosis is critical for management, especially in the lung and liver transplantation setting.

Radiation Sensitivity and Radiation Necrosis in the Short Telomere Syndromes

We report radiation necrosis as the first manifestation of a germline mutation in telomerase. A 60-year-old woman presented with defective wound healing after multiple surgical explorations of the chest wall to exclude breast cancer recurrence. At age 42, she received a diagnosis of stage I estrogen receptor–positive left breast infiltrating ductal carcinoma on routine mammography. Lumpectomy with axillary node dissection revealed T1N0 disease. Her adjuvant radiation therapy consisted of 50.4 Gy in 28 fractions followed by a 10-Gy boost in 5 fractions to the tumor bed. She did well for 12 years, when a left rib fracture was incidentally noted on imaging. This prompted multiple needle biopsies which showed atypia but no malignancy. Open surgical biopsy revealed the anterior ribs within the radiation field were replaced with soft tissue, but no malignancy was found. The postoperative course was complicated by multiple wound infections requiring surgical debridement and failed healing of skin grafts left her with an open chest wall wound (Fig. 1A). Fig. 1 Clinical features and cell survival of cells with short telomeres after irradiation. (A) Nonhealing open, left anterior chest wall wound (arrow) after open biopsy and failed grafts. (B) Basilar honeycombing typical of idiopathic pulmonary fibrosis (arrows). ... She was evaluated at our center for a second opinion. Computed tomography (CT) imaging showed fractured left ribs and chest wall abnormalities consistent with radiation necrosis. In addition, in the contralateral lung, there was basilar honeycombing characteristic of idiopathic pulmonary fibrosis (IPF) (Fig. 1A and B). Family history revealed 2 siblings who died from IPF and myelodysplastic syndrome, a complex pathognomonic for the diagnosis of a short telomere syndrome (2). Genetic evaluation revealed a deleterious mutation in the telomerase RNA gene, TR, that was also found in her siblings (n.80, T>A). The mutation predicted disruption of the essential pseudoknot domain of TR and was associated with short telomere length, below the age-adjusted 10th percentile (Fig. 1C). Additional chest wall reconstruction was deferred given the known risk of respiratory failure after anesthesia and elective surgery in IPF patients (3). During the subsequent year, her wound improved with conservative care, but she developed worsening respiratory symptoms, and subsequently died cancer-free from end-stage lung disease. In order to test whether short telomeres may predispose to radiation sensitivity, we used a modified cell survival assay that is used in the diagnosis of double-strand break syndromes such as ataxia-telangiectasia (A-T) (1, 4). We examined lymphoblastoid cells derived from 7 other telomerase and telomere gene mutation carriers (Fig. 1C). Four of the carriers were asymptomatic, 2 had IPF, and 1 had bone marrow failure. Cells from these subjects showed significantly compromised survival compared to controls and were similarly sensitive to A-T on average (Fig. 1D and E). In 3 of 7 cases (43%), the surviving fraction at 1 Gy fell at or below 21%, a threshold considered to be diagnostic of A-T in some settings (1). Neither the age nor the extent of the telomere defect predicted the 1-Gy surviving fraction. Radiation sensitivity and radiation necrosis are rare complications of radiation therapy, but they are well documented in patients with defective DNA doublestrand break repair such as A-T (5). The data we show here indicate that patients with short telomere syndromes are similarly sensitive to radiation. To our knowledge, this is the first report of radiation necrosis in a telomerase mutation carrier. Sensitivity to radiation in the setting of telomere length abnormalities was first documented in telomerase null mice with short telomeres (6), and our data highlight the relevance of these observations in clinical settings. Syndromes marked by short telomeres manifest as IPF, emphysema, and aplastic anemia (7). Mutations in telomere and telomerase genes are the most common cause of familial pulmonary fibrosis cases, accounting for one-third of cases (7); they also rival alpha-1 antitrypsin deficiency as a risk factor for emphysema (8). Short telomere syndrome patients most often present in mid-to-late adulthood and have no associated dysmorphic features. A cancer diagnosis is estimated to affect up to 10% of severe cases, such as in dyskeratosis congenita, and predominantly manifests as myelodysplasia-acute myeloid leukemia and, more rarely, squamous cell head and neck carcinomas (9). There are sparse data regarding cancer treatment outcomes in this population. After bone marrow transplantation, pulmonary fibrosis is a nearly uniform complication, and lung shielding has evolved as a standard to minimize this risk (10). Awareness of this genetic diagnosis is important in clinical settings, where ionizing radiation is used such as in assessing the risk of X ray–based imaging as a screening tool in pulmonary fibrosis, as well as in a preference for radiation-sparing regimens in hematopoietic stem cell transplantation. Patients suspected of having telomere syndromes and who have a personal or family history of IPF-emphysema, especially, or myelodysplastic syndrome, should be identifiable by telomere length measurement combined with genetic testing.

Diagnostic utility of telomere length testing in a hospital-based setting

Significance This study defines clinical indications for using telomere length (TL) measurement as a diagnostic tool in a hospital setting. It shows that, in contrast to other methods, TL measurement by flow cytometry and FISH (flowFISH) can be standardized, and has reproducible and definable upper and lower normal boundaries. In telomerase mutation carriers and carriers of other mutant telomere maintenance genes, TL had prognostic value, correlating with the age of onset of short telomere syndrome phenotypes, as well as the predominant complication. In a prospective study, TL results were actionable in one-fourth of cases with idiopathic bone marrow failure affecting the stem cell donor choice and/or treatment regimen. The data show that, for targeted clinical indications, and in a hospital setting, TL measurement by flowFISH informs patient care decisions. Telomere length (TL) predicts the onset of cellular senescence in vitro but the diagnostic utility of TL measurement in clinical settings is not fully known. We tested the value of TL measurement by flow cytometry and FISH (flowFISH) in patients with mutations in telomerase and telomere maintenance genes. TL had a discrete and reproducible normal range with definable upper and lower boundaries. While TL above the 50th age-adjusted percentile had a 100% negative predictive value for clinically relevant mutations, the lower threshold in mutation carriers was age-dependent, and adult mutation carriers often overlapped with the lowest decile of controls. The extent of telomere shortening correlated with the age at diagnosis as well as the short telomere syndrome phenotype. Extremely short TL caused bone marrow failure and immunodeficiency in children and young adults, while milder defects manifested as pulmonary fibrosis-emphysema in adults. We prospectively examined whether TL altered treatment decisions for newly diagnosed idiopathic bone marrow failure patients and found abnormally short TL enriched for patients with mutations in some inherited bone marrow failure genes, such as RUNX1, in addition to telomerase and telomere maintenance genes. The result was actionable, altering the choice of treatment regimen and/or hematopoietic stem cell donor in one-fourth of the cases (9 of 38, 24%). We conclude that TL measurement by flowFISH, when used for targeted clinical indications and in limited settings, can influence treatment decisions in ways that improve outcome.

Systematic Computational Identification of Variants That Activate Exonic and Intronic Cryptic Splice Sites

We developed a variant-annotation method that combines sequence-based machine-learning classification with a context-dependent algorithm for selecting splice variants. Our approach is distinctive in that it compares the splice potential of a sequence bearing a variant with the splice potential of the reference sequence. After training, classification accurately identified 168 of 180 (93.3%) canonical splice sites of five genes. The combined method, CryptSplice, identified and correctly predicted the effect of 18 of 21 (86%) known splice-altering variants in CFTR, a well-studied gene whose loss-of-function variants cause cystic fibrosis (CF). Among 1,423 unannotated CFTR disease-associated variants, the method identified 32 potential exonic cryptic splice variants, two of which were experimentally evaluated and confirmed. After complete CFTR sequencing, the method found three cryptic intronic splice variants (one known and two experimentally verified) that completed the molecular diagnosis of CF in 6 of 14 individuals. CryptSplice interrogation of sequence data from six individuals with X-linked dyskeratosis congenita caused by an unknown disease-causing variant in DKC1 identified two splice-altering variants that were experimentally verified. To assess the extent to which disease-associated variants might activate cryptic splicing, we selected 458 pathogenic variants and 348 variants of uncertain significance (VUSs) classified as high confidence from ClinVar. Splice-site activation was predicted for 129 (28%) of the pathogenic variants and 75 (22%) of the VUSs. Our findings suggest that cryptic splice-site activation is more common than previously thought and should be routinely considered for all variants within the transcribed regions of genes.