Rationale: Mutations in telomere-related genes have been implicated as the most frequent monogenic cause of Familial Pulmonary Fibrosis (FPF) and are also identified in a subset of patients with sporadic cases of Idiopathic Pulmonary Fibrosis (IPF). The objective of this study was to determine how the clinical features and outcomes of FPF and IPF patients with telomerase mutations differed from those with other genetic risk mechanisms. Methods: DNA was extracted from whole blood collected from 1001 patients with familial IPF and 423 patients with sporadic IPF. They were evaluated for telomere restriction fragment length by Southern blot and for presence of protein altering rare variants (RVs) in telomerase genes by whole exome or whole genome sequencing. The age at diagnosis and longitudinal values of DLCO and FVC (in percent of predicted) were obtained from the medical record and were calculated as an average annualized change. Measurements and Main Results: Eighty-eight of 551 FPF families (16.0%) were found to carry likely/pathogenic RVs in TERT (n=84 individuals), RTEL1 (n=68 individuals), PARN (n=20 individuals) and TERC (n=3). 38% of subjects within the FPF cohort had a peripheral blood mononuclear cell (PBMC) telomere length <10th percentile. Compared to sporadic IPF patients without telomerase mutations (n=357), FPF patients with telomerase mutations were younger at diagnosis, with the average age of diagnosis for familial and sporadic cohorts being 61.5 and 65.9, respectively (P=0.00002). FPF patients were also more likely female and less likely to have used tobacco. Affected subjects in telomere mutation families had also shorter telomere length compared to subjects without telomere-related gene mutations. FPF subjects with likely/pathogenic RVs had accelerated FVC decline compared to subjects without telomere gene RVs (FPF+IPF (-7.7 and -2.4 percentage per year, respectively p=0.03)). There were no differences noted between change in DLCO% between patients with RVs vs non-RV variants when assessed as a group. However, nonsmoking individuals with likely/pathogenic telomere gene rare variants had a faster decline in DLCO when in compared to sporadic IPF patients without telomerase RVs with an average annualized change in DLCO% of –10.2 and –4.0, respectively, (p=0.03). Conclusion: FPF patients with telomerase mutations have a distinct clinical phenotype and molecular genotype, thus supporting the utility of telomere length measurements and genetic testing in patients with FPF.

Rationale: Approximately 1 in 5 cases of idiopathic pulmonary fibrosis (IPF) are familial, and families with highly penetrant PF have increased likelihood of a monogenic disease. We recently described a large multiplex family with >8 affected members who shared a rare variant in the gene encoding for the orphan G-protein coupled receptor GPR87. In these studies, we sought to investigate the mechanism through which GPR87 contributes to the pathogenesis of PF. Methods: Variant allele status was determined by whole-exome and/or Sanger sequencing. Human single-cell RNA-sequencing data from GSE227136 were reanalyzed. GPR87 localization in human tissue-was performed using RNA-ISH (RNAScope). Gpr87 mutant mice were generated using CRISPR-Cas9 and crossed with Scgb1a1-CreERTtDtomato or Krt5-CreERTtDtomato reporter mice for single-dose or repetitive IT bleomycin studies. RNA-ISH and immunofluorescence data were quantified by automated image analysis (HALO, Indica Labs). GPR87 null HBEC3KT cells were generated using CRISPR-Cas9 and WT/mutant rescue studies performed by lentiviral transduction. Results: GPR87 expressed was increased in basal and KRT5-/KRT17+ aberrant basaloid cells in IPF lungs compared to controls and localized to remodeled airways in areas of active collagen production. Gpr87 mutant mice had increased lung fibrosis following single-dose IT bleomycin. Lineage-tracing studies revealed Gpr87+ cells arise from Scgb1a1-labeled airway progenitors (not Krt5+ airway basal cells or AT2 cells) following repetitive bleomycin, with mutant Gpr87 promoting an Krt5+ basal-like fate and restraining alveolar differentiation of distal-airway progenitors. HBEC3-KT cells demonstrated GPR87-dependent adoption of “aberrant basaloid” features upon LPA stimulation. Conclusion: This FPF-associated GPR87 mutation promotes lung fibrosis through regulation of distal airway progenitor cell fate at least in part via acting as an epithelial LPA receptor. GPR87 represents a novel potential epithelial antifibrotic target for IPF.

"Peripheral Blood Telomere Attrition in Persons at Risk for Familial Pulmonary Fibrosis." American Journal of Respiratory and Critical Care Medicine, 0(ja), pp. –

Rationale and Objectives: Up to 20% of idiopathic interstitial lung disease is familial, referred to as familial pulmonary fibrosis (FPF). An integrated analysis of FPF genetic risk was performed by comprehensively evaluating for genetic rare variants (RVs) in a large cohort of FPF kindreds. Methods: Whole-exome sequencing and/or candidate gene sequencing from affected individuals in 569 FPF kindreds was performed, followed by cosegregation analysis in large kindreds, gene burden analysis, gene-based risk scoring, cell-type enrichment analysis, and coexpression network construction. Measurements and Main Results: It was found that 14.9-23.4% of genetic risk in kindreds could be explained by RVs in genes previously linked to FPF, predominantly telomererelated genes. New candidate genes were identified in a small number of families-including SYDE1, SERPINB8, GPR87, and NETO1-and tools were developed for evaluation and prioritization of RV-containing genes across kindreds. Several pathways were enriched for RV-containing genes in FPF, including focal adhesion and mitochondrial complex I assembly. By combining single-cell transcriptomics with prioritized candidate genes, expression of RV-containing genes was discovered to be enriched in smooth muscle cells, type II alveolar epithelial cells, and endothelial cells. Conclusions: In the most comprehensive FPF genetic study to date, the prevalence of RVs in known FPF-related genes was defined, and new candidate genes and pathways relevant to FPF were identified. However, new RV-containing genes shared across multiple kindreds were not identified, thereby suggesting that heterogeneous genetic variants involving a variety of genes and pathways mediate genetic risk in most FPF kindreds.

Rationale: The preclinical natural history of progressive lung fibrosis is poorly understood. Objectives: Our goals were to identify risk factors for interstitial lung abnormalities (ILA) on high-resolution computed tomography (HRCT) scans and to determine progression toward clinical interstitial lung disease (ILD) among subjects in a longitudinal cohort of self-reported unaffected first-degree relatives of patients with familial interstitial pneumonia. Methods: Enrollment evaluation included a health history and exposure questionnaire and HRCT scans, which were categorized by visual assessment as no ILA, early/mild ILA, or extensive ILA. The study endpoint was met when ILA were extensive or when ILD was diagnosed clinically. Among subjects with adequate study time to complete 5-year follow-up HRCT, the proportion with ILD events (endpoint met or radiographic ILA progression) was calculated. Measurements and Main Results: Among 336 subjects, the mean age was 53.1 (SD, 9.9) years. Those with ILA (early/mild [n = 74] or extensive [n = 3]) were older, were more likely to be ever smokers, had shorter peripheral blood mononuclear cell telomeres, and were more likely to carry the MUC5B risk allele. Self-reported occupational or environmental exposures, including aluminum smelting, lead, birds, and mold, were independently associated with ILA. Among 129 subjects with sufficient study time, 25 (19.4%) had an ILD event by 5 years after enrollment; of these, 12 met the study endpoint and another 13 had radiologic progression of ILA. ILD events were more common among those with early/mild ILA at enrollment (63.3% vs. 6.1%; P < 0.0001). Conclusions: Rare and common environmental exposures are independent risk factors for radiologic abnormalities. In 5 years, progression of ILA occurred in most individuals with early ILA detected at enrollment.

Genetic Evaluation and Testing of Patients and Familieswith Idiopathic Pulmonary Fibrosis Jonathan A. Kropski, Lisa R. Young, Joy D. Cogan, Daphne B. Mitchell, Lisa H. Lancaster, John A. Worrell, Cheryl Markin, Na Liu, Wendi R. Mason, Tasha E. Fingerlin, David A. Schwartz, William E. Lawson, Timothy S. Blackwell, John A. Phillips III, and James E. Loyd Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Division of Pulmonary Medicine and Division of Medical Genetics and Genomic Medicine, Department of Pediatrics, and Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Biomedical Research, National Jewish Health, Denver, Colorado; Department of Medicine, University of Colorado, Denver, Colorado; Department of Veterans Affairs Medical Center, Nashville, Tennessee; and Department of Cancer Biology, Department of Cell and Developmental Biology, and Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee

Rationale: Asymptomatic relatives of patients with familial interstitial pneumonia (FIP), the inherited form of idiopathic interstitial pneumonia, carry increased risk for developing interstitial lung disease.Objectives: Studying these at-risk individuals provides a unique opportunity to investigate early stages of FIP pathogenesis and develop predictive models of disease onset.Methods: Seventy-five asymptomatic first-degree relatives of FIP patients (mean age, 50.8 yr) underwent blood sampling and high-resolution chest computed tomography (HRCT) scanning in an ongoing cohort study; 72 consented to bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial biopsies. Twenty-seven healthy individuals were used as control subjects.Measurements and Main Results: Eleven of 75 at-risk subjects (14%) had evidence of interstitial changes by HRCT, whereas 35.2% had abnormalities on transbronchial biopsies. No differences were noted in inflammatory cells in BAL between at-risk individuals and control subjects. At-risk subjects had increased herpesvirus DNA in cell-free BAL and evidence of herpesvirus antigen expression in alveolar epithelial cells (AECs), which correlated with expression of endoplasmic reticulum stress markers in AECs. Peripheral blood mononuclear cell and AEC telomere length were shorter in at-risk individuals than healthy control subjects. The minor allele frequency of the Muc5B rs35705950 promoter polymorphism was increased in at-risk subjects. Levels of several plasma biomarkers differed between at-risk subjects and control subjects, and correlated with abnormal HRCT scans.Conclusions: Evidence of lung parenchymal remodeling and epithelial dysfunction was identified in asymptomatic individuals at risk for FIP. Together, these findings offer new insights into the early pathogenesis of idiopathic interstitial pneumonia and provide an ongoing opportunity to characterize presymptomatic abnormalities that predict progression to clinical disease.

Background Activation of coagulation by expression of tissue factor (TF) in the airspace is a hallmark of acute lung injury (ALI) but the timing of TF activation in relationship to increases in lung permeability and inflammation are unknown. Methods To test the hypothesis that TF is upregulated early in the course of acute bleomycin lung injury and precedes increased permeability and inflammation we studied the early course of bleomycin-induced ALI in mice. Mice were treated with 0.04U intratracheal bleomycin or vehicle control and bronchoalveolar lavage (BAL) and lung tissue were collected daily for 7 days. Whole lung TF mRNA was determined by QT-PCR. TF protein was assessed by ELISA and immunostaining. BAL procoagulant activity was measured by BAL clot time and thrombin-antithrombin complexes. Inflammation was assessed by BAL cell count, differentials and CXCL1/KC concentration. Lung permeability was assessed by BAL protein and lung wet to dry weight ratio. Results Expression of CXCL1 occurred by day 1. BAL protein and lung wet-to-dry weight ratio increased significantly by day 3. TF mRNA and BAL procoagulant activity peaked on day 4 while whole lung TF protein peaked on day 6. Changes in permeability and procoagulant activity preceded inflammatory cell influx which was maximal at day 6 while whole lung TF protein peaked along with inflammation. Conclusion These data demonstrate that cytokine upregulation is the earliest response to bleomycin administration, followed by increased lung permeability, upregulation of TF, and recruitment of inflammatory cells.

Rationale: Up to 20% of cases of idiopathic interstitial pneumonia cluster in families, comprising the syndrome of familial interstitial pneumonia (FIP); however, the genetic basis of FIP remains uncertain in most families.Objectives: To determine if new disease-causing rare genetic variants could be identified using whole-exome sequencing of affected members from FIP families, providing additional insights into disease pathogenesis.Methods: Affected subjects from 25 kindreds were selected from an ongoing FIP registry for whole-exome sequencing from genomic DNA. Candidate rare variants were confirmed by Sanger sequencing, and cosegregation analysis was performed in families, followed by additional sequencing of affected individuals from another 163 kindreds.Measurements and Main Results: We identified a potentially damaging rare variant in the gene encoding for regulator of telomere elongation helicase 1 (RTEL1) that segregated with disease and was associated with very short telomeres in peripheral blood mononuclear cells in 1 of 25 families in our original whole-exome sequencing cohort. Evaluation of affected individuals in 163 additional kindreds revealed another eight families (4.7%) with heterozygous rare variants in RTEL1 that segregated with clinical FIP. Probands and unaffected carriers of these rare variants had short telomeres (<10% for age) in peripheral blood mononuclear cells and increased T-circle formation, suggesting impaired RTEL1 function.Conclusions: Rare loss-of-function variants in RTEL1 represent a newly defined genetic predisposition for FIP, supporting the importance of telomere-related pathways in pulmonary fibrosis.

BACKGROUND:Fibrosing mediastinitis (FM) is an idiosyncratic reaction to infection with Histoplasma capsulatum with a prevalence of 3:100,000 people infected. The rarity of post-histoplasmosis fibrosing mediastinitis (PHFM) in areas where H. capsulatum is endemic suggests that an abnormal immunological host response may be responsible for the development of fibrosis. Our group previously reported an association between subjects with PHFM and human leukocyte antigen (HLA)-A*02. We sought to confirm or extend those findings with application of high resolution HLA typing in a cohort of subjects with PHFM.METHODS:High-resolution HLA typing was performed on DNA samples from a new cohort 34 patients with PHFM. Control cohorts included 707 subjects from the "European American" subset of the National Marrow Donor Program(®) (NMDP) and 700 subjects from Dialysis Clinic, Inc. (DCI). The carriage frequencies of the HLA alleles identified in the PHFM, NMDP, and DCI cohorts were calculated and then all were compared.RESULTS:We found an increase in the carriage frequency of HLA-DQB1*04:02 in PHFM subjects relative to the controls (0.15 versus 0.07 in DCI and 0.05 in NMDP; p = 0.08 and 0.03). Multiple logistic regression showed that DQB1*04:02 was statistically significant (p = 0.04), while DQB1*03:02 and C*03:04 had point estimates of OR > 1, though they did not reach statistical significance. The HLA-A*02 association was not replicated.CONCLUSIONS:HLA-DQB1*04:02 is associated with PHFM, which supports the premise that an aberrant host immune response contributes to the development of PHFM.

Glycosylphophatidylinositol (GPO-anchored proteins play important roles in many biological processes, and mutations affecting proteins involved in the synthesis of the GPI anchor are reported to cause a wide spectrum of intellectual disabilities (IDs) with characteristic additional phenotypic features. Here, we describe a total of five individuals (from three unrelated families) in whom we identified mutations in PGAP3, encoding a protein that is involved in GPI-anchor maturation. Three siblings in a consanguineous Pakistani family presented with profound developmental delay, severe ID, no speech, psychomotor delay, and postnatal microcephaly. A combination of autozygosity mapping and exome sequencing identified a 13.8 Mb region harboring a homozygous c.275G>A (p.Gly92Asp) variant in PGAP3 region 17q11.2-q21.32. Subsequent testing showed elevated serum alkaline phosphatase (ALP), a GPI-anchored enzyme, in all three affected children. In two unrelated individuals in a cohort with developmental delay, ID, and elevated ALP, we identified compound-heterozygous variants c.439dupC (p.Leu147Profs*16) and c.914A>G (p.Asp305Gly) and homozygous variant c.314C>G (p.Pro105Arg). The 1 bp duplication causes a frameshift and nonsense-mediated decay. Further evidence supporting pathogenicity of the missense mutations c.275G>A, c.314C>G, and c.914A>G was provided by the absence of the variants from ethnically matched controls, phylogenetic conservation, and functional studies on Chinese hamster ovary cell lines. Taken together with recent data on PGAP2, these results confirm the importance of the later GPI-anchor remodelling steps for normal neuronal development. Impairment of PGAP3 causes a subtype of hyperphosphatasia with ID, a congenital disorder of glycosylation that is also referred to as Mabry syndrome.

Short telomeres are frequently identified in patients with idiopathic pulmonary fibrosis (IPF) and its inherited form, familial interstitial pneumonia (FIP). We identified a kindred with FIP with short telomeres who did not carry a mutation in known FIP genes TERT or hTR . We performed targeted sequencing of other telomere-related genes to identify the genetic basis of FIP in this kindred. The proband was a 69 year-old man with dyspnea, restrictive pulmonary function test results, and reticular changes on high-resolution CT scan. An older male sibling had died from IPF. The proband had markedly shortened telomeres in peripheral blood and undetectably short telomeres in alveolar epithelial cells. Polymerase chain reaction-based sequencing of NOP10 , TINF2 , NHP2 , and DKC1 revealed that both affected siblings shared a novel A to G 1213 transition in DKC1 near the hTR binding domain that is predicted to encode a Thr405Ala amino acid substitution. hTR levels were decreased out of proportion to DKC1 expression in the T405A DKC1 proband, suggesting this mutation destabilizes hTR and impairs telomerase function. This DKC1 variant represents the third telomere-related gene identified as a genetic cause of FIP. Further investigation into the mechanism by which dyskerin contributes to the development of lung fibrosis is warranted.

The immunomodulatory role of arachidonic acid metabolites in allergic sensitization is undefined. Prostaglandin E-2 (PGE(2)), a product of arachidonic acid metabolism through the cyclooxygenase pathway, has been reported to favor Type 2-like cytokine secretion profiles in murine and human CD4(+) T cells by inhibiting the production of Type 1-associated cytokines. On the basis of these in vitro data, we hypothesized that indomethacin, a nonselective cyclooxygenase inhibitor, would diminish allergen-induced production of Type 2 cytokines in mice, and protect against airway hyperresponsiveness (AHR) to methacholine. We found that ovalbumin-sensitized mice that were treated with indomethacin (OVA-indomethacin mice) had significantly greater AHR (p < 0.05) and higher levels of IL-5 (176 +/- 52 versus 66 +/- 4 pg/ml) and IL-13 (1,226 +/- 279 versus 475 +/- 65 pg/ml) in lung supernatants than mice sensitized with ovalbumin alone (OVA mice), while levels of IL-4 and serum IgE were not different. Lung mRNA expression of the C-C chemokine MCP-1 was increased in OVA-indomethacin mice, while there was no difference between the two groups in lung mRNA expression of eotaxin, MIP-1 alpha, MIP-1 beta, or MIP-2. Histologic examination revealed greater pulmonary interstitial eosinophilia in OVA-indomethacin mice as well. Contrary to our expectations, we conclude that in the BALB/c mouse, cyclooxygenase inhibition during allergen sensitization increases AHR, production of IL-5 and IL-13, and interstitial eosinophilia.

Idiopathic pulmonary fibrosis (IPF) is characterized by interstitial lung infiltrates, dyspnea, and progressive respiratory failure. Reports linking telomerase mutations to familial interstitial pneumonia (FIP) suggest that telomerase activity and telomere length maintenance are important in disease pathogenesis. To investigate the role of telomerase in lung fibrotic remodeling, intratracheal bleomycin was administered to mice deficient in telomerase reverse transcriptase (TERT) or telomerase RNA component (TERC) and to wild-type controls. TERT-deficient and TERC-deficient mice were interbred to the F6 and F4 generation, respectively, when they developed skin manifestations and infertility. Fibrosis was scored using a semiquantitative scale and total lung collagen was measured using a hydroxyprolinemicroplate assay. Telomere lengths were measured in peripheral blood leukocytes and isolated type II alveolar epithelial cells (AECs). Telomerase activity in type II AECs was measured using a real-time polymerase chain reaction (PCR)-based system. Following bleomycin, TERT-deficient and TERC-deficient mice developed an equivalent inflammatory response and similar lung fibrosis (by scoring of lung sections and total lung collagen content) compared to controls, a pattern seen in both early (F1) and later (F6 TERT and F4 TERC) generations. Telomere lengths were reduced in peripheral blood leukocytes and isolated type II AECs from F6 TERT-deficient and F4 TERC-deficient mice compared to controls. Telomerase deficiency in a murine model leads to telomere shortening, but does not predispose to enhanced bleomycin-induced lung fibrosis. Additional genetic or environmental factors may be necessary for development of fibrosis in the presence of telomerase deficiency.