Incidence and Progression of Fibrotic Lung Disease in an At-Risk Cohort.

Rationale: Relatives of patients with familial interstitial pneumonia (FIP) are at increased risk for pulmonary fibrosis and develop preclinical pulmonary fibrosis (PrePF). Objectives: We defined the incidence and progression of new-onset PrePF and its relationship to survival among first-degree relatives of families with FIP. Methods: This is a cohort study of family members with FIP who were initially screened with a health questionnaire and chest high-resolution computed tomography (HRCT) scan, and approximately 4 years later, the evaluation was repeated. A total of 493 asymptomatic first-degree relatives of patients with FIP were evaluated at baseline, and 296 (60%) of the original subjects participated in the subsequent evaluation. Measurements and Main Results: The median interval between HRCTs was 3.9 years (interquartile range, 3.5-4.4 yr). A total of 252 subjects who agreed to repeat evaluation were originally determined not to have PrePF at baseline; 16 developed PrePF. A conservative estimate of the annual incidence of PrePF is 1,023 per 100,000 person-years (95% confidence interval, 511-1,831 per 100,000 person-years). Of 44 subjects with PrePF at baseline, 38.4% subjects had worsening dyspnea compared with 15.4% of those without PrePF (P = 0.002). Usual interstitial pneumonia by HRCT (P < 0.0002) and baseline quantitative fibrosis score (P < 0.001) are also associated with worsening dyspnea. PrePF at the initial screen is associated with decreased survival (P < 0.001). Conclusions: The incidence of PrePF in this at-risk population is at least 100-fold higher than that reported for sporadic idiopathic pulmonary fibrosis (IPF). Although PrePF and IPF represent distinct entities, our study demonstrates that PrePF, like IPF, is progressive and associated with decreased survival.

Idiopathic Pulmonary Fibrosis: A Genetic Disease That Involves Mucociliary Dysfunction of the Peripheral Airways.

Idiopathic pulmonary fibrosis (IPF) is an incurable complex genetic disorder that is associated with sequence changes in 7 genes (MUC5B, TERT, TERC, RTEL1, PARN, SFTPC, and SFTPA2) and with variants in at least 11 novel loci. We have previously found that 1) a common gain-of-function promoter variant in MUC5B rs35705950 is the strongest risk factor (genetic and otherwise), accounting for 30-35% of the risk of developing IPF, a disease that was previously considered idiopathic; 2) the MUC5B promoter variant can potentially be used to identify individuals with preclinical pulmonary fibrosis and is predictive of radiologic progression of preclinical pulmonary fibrosis; and 3) MUC5B may be involved in the pathogenesis of pulmonary fibrosis with MUC5B message and protein expressed in bronchiolo-alveolar epithelia of IPF and the characteristic IPF honeycomb cysts. Based on these considerations, we hypothesize that excessive production of MUC5B either enhances injury due to reduced mucociliary clearance or impedes repair consequent to disruption of normal regenerative mechanisms in the distal lung. In aggregate, these novel considerations should have broad impact, resulting in specific etiologic targets, early detection of disease, and novel biologic pathways for use in the design of future intervention, prevention, and mechanistic studies of IPF.

Muc5b plays a role in the development of inflammation and fibrosis in hypersensitivity pneumonitis induced by Saccharopolyspora rectivirgula.

Previously we have shown that a gain-of-function MUC5B promoter variant (rs35705950) is the strongest risk factor for the development of idiopathic pulmonary fibrosis. We have also found that Muc5b overexpression reduces mucociliary clearance in mice, potentially leading to recurrent injury to the bronchoalveolar epithelia. Hypersensitivity pneumonitis (HP) is induced by inhalation of numerous causative antigens that may be affected by mucociliary clearance. We conducted this study to determine the role of Muc5b in a mouse model of HP induced by Saccharopolyspora rectivirgula (SR) antigen. We used Muc5b-deficient and wild-type (WT) mice to determine whether Muc5b plays a role in inflammation and fibrosis at 3 and 6 wk in an SR model of HP. We measured cell concentrations and MUC5B expression in whole lung lavage (WLL) and quantified fibrosis using hydroxyproline assay and second harmonic generation. Muc5b expression in WLL fluid was significantly increased in SR-exposed WT mice compared with saline controls. WT mice challenged with SR developed more inflammation and lung fibrosis at 6 wk compared with 3 wk postexposure. Moreover, we found that 6 wk following challenge with SR, Muc5b-deficient mice had less lung inflammation and less lung fibrosis than Muc5b WT mice. Furthermore, Muc5b-deficient mice had significantly lower concentrations of TGF-ß1 in the WLL compared with Muc5b WT mice at 6 wk of exposure. Muc5b appears to play a role in fibrosis in the animal model of HP and this may have implications for HP in humans.

Genes, other than Muc5b, play a role in bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an incurable genetic disease that affects 5 million people worldwide. The gain-of-function MUC5B promoter variant rs35705950 is the dominant genetic risk factor for IPF, yet has a low penetrance. This raises the possibility that other genes and transcripts affect the penetrance of MUC5B. Previously, we have shown that the concentration of Muc5b in bronchoalveolar epithelia is directly associated with the extent and persistence of bleomycin-induced lung fibrosis in mice. In this study, we investigated whether bleomycin-induced lung injury is Muc5b dependent in genetically divergent strains of mice. Specifically, mice from the eight Diversity Outbred (DO) founders were phenotyped for Muc5b expression and lung fibrosis 3 wk after intratracheal bleomycin administration. Although we identified strains with low Muc5b expression and minimal lung fibrosis (CAST/EiJ and PWK/PhJ) and strains with high Muc5b expression and extensive lung fibrosis (NZO/H1LtJ and WSB/EiJ), there also were strains that did not demonstrate a clear relationship between Muc5b expression and lung fibrosis (129S1/SvlmJ, NOD/ShiLtJ, and C57BL/6J, A/J). Hierarchical clustering suggests that other factors may work in concert with or potentially independent of Muc5b to promote bleomycin-induced lung injury and fibrosis. This study suggests that these strains and their recombinant inbred crosses may prove helpful in identifying the genes and transcripts that interact with Muc5b and cause lung fibrosis.

Chronic Hypersensitivity Pneumonitis, an Interstitial Lung Disease with Distinct Molecular Signatures.

Rationale: Chronic hypersensitivity pneumonitis (CHP) is caused by an immune response to antigen inhalation and is characterized by variable histopathological and clinical features. A subset of subjects with CHP have usual interstitial pneumonia and appear to be clinically similar to subjects with idiopathic pulmonary fibrosis (IPF).Objectives: To determine the common and unique molecular features of CHP and IPF.Methods: Transcriptome analysis of lung samples from CHP (n = 82), IPF (n = 103), and unaffected controls (n = 103) was conducted. Differential gene expression was determined adjusting for sex, race, age, and smoking history and using false discovery rate to control for multiple comparisons.Measurements and Main Results: When compared with controls, we identified 413 upregulated and 317 downregulated genes in CHP and 861 upregulated and 322 downregulated genes in IPF. Concordantly upregulated or downregulated genes in CHP and IPF were related to collagen catabolic processes and epithelial development, whereas genes specific to CHP (differentially expressed in CHP when compared with control and not differentially expressed in IPF) were related to chemokine-mediated signaling and immune responsiveness. Using weighted gene coexpression network analysis, we found that among subjects with CHP, genes involved in adaptive immunity or epithelial cell development were associated with improved or reduced lung function, respectively, and that MUC5B expression was associated with epithelial cell development. MUC5B expression was also associated with lung fibrosis and honeycombing.Conclusions: Gene expression analysis of CHP and IPF identified signatures common to CHP and IPF, as well as genes uniquely expressed in CHP. Select modules of gene expression are characterized by distinct clinical and pathological features of CHP.

Resequencing Study Confirms That Host Defense and Cell Senescence Gene Variants Contribute to the Risk of Idiopathic Pulmonary Fibrosis.

Rationale: Several common and rare genetic variants have been associated with idiopathic pulmonary fibrosis, a progressive fibrotic condition that is localized to the lung. Objectives: To develop an integrated understanding of the rare and common variants located in multiple loci that have been reported to contribute to the risk of disease. Methods: We performed deep targeted resequencing (3.69 Mb of DNA) in cases (n = 3,624) and control subjects (n = 4,442) across genes and regions previously associated with disease. We tested for associations between disease and 1) individual common variants via logistic regression and 2) groups of rare variants via sequence kernel association tests. Measurements and Main Results: Statistically significant common variant association signals occurred in all 10 of the regions chosen based on genome-wide association studies. The strongest risk variant is the MUC5B promoter variant rs35705950, with an odds ratio of 5.45 (95% confidence interval, 4.91-6.06) for one copy of the risk allele and 18.68 (95% confidence interval, 13.34-26.17) for two copies of the risk allele (P = 9.60 × 10-295). In addition to identifying for the first time that rare variation in FAM13A is associated with disease, we confirmed the role of rare variation in the TERT and RTEL1 gene regions in the risk of IPF, and found that the FAM13A and TERT regions have independent common and rare variant signals. Conclusions: A limited number of common and rare variants contribute to the risk of idiopathic pulmonary fibrosis in each of the resequencing regions, and these genetic variants focus on biological mechanisms of host defense and cell senescence.

MUC5B variant is associated with visually and quantitatively detected preclinical pulmonary fibrosis.

BACKGROUND: Relatives of patients with familial interstitial pneumonia (FIP) are at increased risk for pulmonary fibrosis. We assessed the prevalence and risk factors for preclinical pulmonary fibrosis (PrePF) in first-degree relatives of patients with FIP and determined the utility of deep learning in detecting PrePF on CT. METHODS: First-degree relatives of patients with FIP over 40 years of age who believed themselves to be unaffected by pulmonary fibrosis underwent CT scans of the chest. Images were visually reviewed, and a deep learning algorithm was used to quantify lung fibrosis. Genotyping for common idiopathic pulmonary fibrosis risk variants in MUC5B and TERT was performed. FINDINGS: In 494 relatives of patients with FIP from 263 families of patients with FIP, the prevalence of PrePF on visual CT evaluation was 15.6% (95% CI 12.6 to 19.0). Compared with visual CT evaluation, deep learning quantitative CT analysis had 84% sensitivity (95% CI 0.72 to 0.89) and 86% sensitivity (95% CI 0.83 to 0.89) for discriminating subjects with visual PrePF diagnosis. Subjects with PrePF were older (65.9, SD 10.1 years) than subjects without fibrosis (55.8 SD 8.7 years), more likely to be male (49% vs 37%), more likely to have smoked (44% vs 27%) and more likely to have the MUC5B promoter variant rs35705950 (minor allele frequency 0.29 vs 0.21). MUC5B variant carriers had higher quantitative CT fibrosis scores (mean difference of 0.36%), a difference that remains significant when controlling for age and sex. INTERPRETATION: PrePF is common in relatives of patients with FIP. Its prevalence increases with age and the presence of a common MUC5B promoter variant. Quantitative CT analysis can detect these imaging abnormalities.

The relationship between complement C3 expression and the MUC5B genotype in pulmonary fibrosis.

The common gain-of-function MUC5B promoter variant ( rs35705950 ) is the strongest risk factor for the development of idiopathic pulmonary fibrosis (IPF). While the role of complement in IPF is controversial, both MUC5B and the complement system play a role in lung host defense. The aim of this study was to evaluate the relationship between complement component 3 (C3) and MUC5B in patients with IPF and in bleomycin-induced lung injury in mice. To do this, we evaluated C3 gene expression in whole lung tissue from 300 subjects with IPF and 175 healthy controls. Expression of C3 was higher in IPF than healthy controls {1.40-fold increase [95% confidence interval (CI) 1.31-1.50]; P < 0.0001} and even greater among IPF subjects with the highest-risk IPF MUC5B promoter genotype [TT vs. GG = 1.59-fold (95% CI 1.15-2.20); P < 0.05; TT vs. GT = 1.66-fold (95% CI 1.20-2.30); P < 0.05]. Among subjects with IPF, C3 expression was significantly higher in the lung tissue without microscopic honeycombing than in the lung tissue with microscopic honeycombing [1.40-fold increase (95% CI 1.23- 1.59); P < 0.01]. In mice, while bleomycin exposure increased Muc5b protein expression, C3-deficient mice were protected from bleomycin-induced lung injury. In aggregate, our findings indicate that the MUC5B promoter variant is associated with higher C3 expression and suggest that the complement system may contribute to the pathogenesis of IPF.

Quantitative high-resolution computed tomography fibrosis score: performance characteristics in idiopathic pulmonary fibrosis.

We evaluated performance characteristics and estimated the minimal clinically important difference (MCID) of data-driven texture analysis (DTA), a high-resolution computed tomography (HRCT)-derived measurement of lung fibrosis, in subjects with idiopathic pulmonary fibrosis (IPF).The study population included 141 subjects with IPF from two interventional clinical trials who had both baseline and nominal 54- or 60-week follow-up HRCT. DTA scores were computed and compared with forced vital capacity (FVC), diffusing capacity of the lung for carbon monoxide, distance covered during a 6-min walk test and St George's Respiratory Questionnaire scores to assess the method's reliability, validity and responsiveness. Anchor- and distribution-based methods were used to estimate its MCID.DTA had acceptable reliability in subjects appearing stable according to anchor variables at follow-up. Correlations between the DTA score and other clinical measurements at baseline were moderate to weak and in the hypothesised directions. Acceptable responsiveness was demonstrated by moderate to weak correlations (in the directions hypothesised) between changes in the DTA score and changes in other parameters. Using FVC as an anchor, MCID was estimated to be 3.4%.Quantification of lung fibrosis extent on HRCT using DTA is reliable, valid and responsive, and an increase of ∼3.4% represents a clinically important change.

Idiopathic Pulmonary Fibrosis: Data-driven Textural Analysis of Extent of Fibrosis at Baseline and 15-Month Follow-up.

Purpose To evaluate associations between pulmonary function and both quantitative analysis and visual assessment of thin-section computed tomography (CT) images at baseline and at 15-month follow-up in subjects with idiopathic pulmonary fibrosis (IPF). Materials and Methods This retrospective analysis of preexisting anonymized data, collected prospectively between 2007 and 2013 in a HIPAA-compliant study, was exempt from additional institutional review board approval. The extent of lung fibrosis at baseline inspiratory chest CT in 280 subjects enrolled in the IPF Network was evaluated. Visual analysis was performed by using a semiquantitative scoring system. Computer-based quantitative analysis included CT histogram-based measurements and a data-driven textural analysis (DTA). Follow-up CT images in 72 of these subjects were also analyzed. Univariate comparisons were performed by using Spearman rank correlation. Multivariate and longitudinal analyses were performed by using a linear mixed model approach, in which models were compared by using asymptotic χ2 tests. Results At baseline, all CT-derived measures showed moderate significant correlation (P < .001) with pulmonary function. At follow-up CT, changes in DTA scores showed significant correlation with changes in both forced vital capacity percentage predicted (ρ = -0.41, P < .001) and diffusing capacity for carbon monoxide percentage predicted (ρ = -0.40, P < .001). Asymptotic χ2 tests showed that inclusion of DTA score significantly improved fit of both baseline and longitudinal linear mixed models in the prediction of pulmonary function (P < .001 for both). Conclusion When compared with semiquantitative visual assessment and CT histogram-based measurements, DTA score provides additional information that can be used to predict diminished function. Automatic quantification of lung fibrosis at CT yields an index of severity that correlates with visual assessment and functional change in subjects with IPF. © RSNA, 2017.

Desmoplakin Variants Are Associated with Idiopathic Pulmonary Fibrosis.

RATIONALE: Sequence variation, methylation differences, and transcriptional changes in desmoplakin (DSP) have been observed in patients with idiopathic pulmonary fibrosis (IPF). OBJECTIVES: To identify novel variants in DSP associated with IPF and to characterize the relationship of these IPF sequence variants with DSP gene expression in human lung. METHODS: A chromosome 6 locus (7,370,061-7,606,946) was sequenced in 230 subjects with IPF and 228 control subjects. Validation genotyping of disease-associated variants was conducted in 936 subjects with IPF and 936 control subjects. DSP gene expression was measured in lung tissue from 334 subjects with IPF and 201 control subjects. MEASUREMENTS AND MAIN RESULTS: We identified 23 sequence variants in the chromosome 6 locus associated with IPF. Genotyping of selected variants in our validation cohort revealed that noncoding intron 1 variant rs2744371 (odds ratio = 0.77, 95% confidence interval [CI] = 0.66-0.91, P = 0.002) is protective for IPF, and a previously described IPF-associated intron 5 variant (rs2076295) is associated with increased risk of IPF (odds ratio = 1.36, 95% CI = 1.19-1.56, P < 0.001) after controlling for sex and age. DSP expression is 2.3-fold increased (95% CI = 1.91-2.71) in IPF lung tissue (P < 0.0001). Only the minor allele at rs2076295 is associated with decreased DSP expression (P = 0.001). Staining of fibrotic and normal human lung tissue localized DSP to airway epithelia. CONCLUSIONS: Sequence variants in DSP are associated with IPF, and rs2076295 genotype is associated with differential expression of DSP in the lung. DSP expression is increased in IPF lung and concentrated in the airway epithelia, suggesting a potential role for DSP in the pathogenesis of IPF.

MUC5B promoter polymorphism and interstitial lung abnormalities.

BACKGROUND: A common promoter polymorphism (rs35705950) in MUC5B, the gene encoding mucin 5B, is associated with idiopathic pulmonary fibrosis. It is not known whether this polymorphism is associated with interstitial lung disease in the general population. METHODS: We performed a blinded assessment of interstitial lung abnormalities detected in 2633 participants in the Framingham Heart Study by means of volumetric chest computed tomography (CT). We evaluated the relationship between the abnormalities and the genotype at the rs35705950 locus. RESULTS: Of the 2633 chest CT scans that were evaluated, interstitial lung abnormalities were present in 177 (7%). Participants with such abnormalities were more likely to have shortness of breath and chronic cough and reduced measures of total lung and diffusion capacity, as compared with participants without such abnormalities. After adjustment for covariates, for each copy of the minor rs35705950 allele, the odds of interstitial lung abnormalities were 2.8 times greater (95% confidence interval [CI], 2.0 to 3.9; P<0.001), and the odds of definite CT evidence of pulmonary fibrosis were 6.3 times greater (95% CI, 3.1 to 12.7; P<0.001). Although the evidence of an association between the MUC5B genotype and interstitial lung abnormalities was greater among participants who were older than 50 years of age, a history of cigarette smoking did not appear to influence the association. CONCLUSIONS: The MUC5B promoter polymorphism was found to be associated with interstitial lung disease in the general population. Although this association was more apparent in older persons, it did not appear to be influenced by cigarette smoking. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT00005121.).

Radiologic-pathologic discordance in biopsy-proven usual interstitial pneumonia.

The aim of this study was to compare the clinical, radiological and histological findings in a large population of subjects enrolled during a multicentre study of idiopathic pulmonary fibrosis, with a focus on discordance between imaging and histologic diagnoses of usual interstitial pneumonia (UIP).Two independent radiologists retrospectively reviewed 241 subjects who underwent high-resolution computed tomography (HRCT) and surgical lung biopsies. HRCT findings were classified as UIP, possible UIP and inconsistent with UIP. Histological findings were classified as definite, probable, possible and not UIP.Of the 241 cases, 102 (42.3%) had HRCT findings of UIP, 64 (26.6%) had possible UIP and 75 (31.1%) were inconsistent with UIP. Among those with UIP on HRCT, 99 (97.1%) had histologically definite or probable UIP (concordant group), and 71 (94.7%) of those with "inconsistent" HRCT features had histologically definite or probable UIP (discordant group). Discordant subjects were slightly younger and less likely to be smokers than concordant subjects, but no survival differences were identified.In this population of patients enrolled with a diagnosis of idiopathic pulmonary fibrosis, 94.7% of those with HRCT findings "inconsistent with UIP" demonstrated histological UIP. This suggests that the term "inconsistent with UIP" is misleading.

Genome-wide imputation study identifies novel HLA locus for pulmonary fibrosis and potential role for auto-immunity in fibrotic idiopathic interstitial pneumonia.

BACKGROUND: Fibrotic idiopathic interstitial pneumonias (fIIP) are a group of fatal lung diseases with largely unknown etiology and without definitive treatment other than lung transplant to prolong life. There is strong evidence for the importance of both rare and common genetic risk alleles in familial and sporadic disease. We have previously used genome-wide single nucleotide polymorphism data to identify 10 risk loci for fIIP. Here we extend that work to imputed genome-wide genotypes and conduct new RNA sequencing studies of lung tissue to identify and characterize new fIIP risk loci. RESULTS: We performed genome-wide genotype imputation association analyses in 1616 non-Hispanic white (NHW) cases and 4683 NHW controls followed by validation and replication (878 cases, 2017 controls) genotyping and targeted gene expression in lung tissue. Following meta-analysis of the discovery and replication populations, we identified a novel fIIP locus in the HLA region of chromosome 6 (rs7887 P meta = 3.7 × 10(-09)). Imputation of classic HLA alleles identified two in high linkage disequilibrium that are associated with fIIP (DRB1*15:01 P = 1.3 × 10(-7) and DQB1*06:02 P = 6.1 × 10(-8)). Targeted RNA-sequencing of the HLA locus identified 21 genes differentially expressed between fibrotic and control lung tissue (Q < 0.001), many of which are involved in immune and inflammatory response regulation. In addition, the putative risk alleles, DRB1*15:01 and DQB1*06:02, are associated with expression of the DQB1 gene among fIIP cases (Q < 1 × 10(-16)). CONCLUSIONS: We have identified a genome-wide significant association between the HLA region and fIIP. Two HLA alleles are associated with fIIP and affect expression of HLA genes in lung tissue, indicating that the potential genetic risk due to HLA alleles may involve gene regulation in addition to altered protein structure. These studies reveal the importance of the HLA region for risk of fIIP and a basis for the potential etiologic role of auto-immunity in fIIP.

Rare variants in RTEL1 are associated with familial interstitial pneumonia.

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.

Relationship between gene expression and lung function in Idiopathic Interstitial Pneumonias.

BACKGROUND: Idiopathic interstitial pneumonias (IIPs) are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium. Since lung function is a key determinant of survival, we reasoned that the transcriptional profile in IIP lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to IIPs. RESULTS: Using gene expression profiling of 167 lung tissue specimens with IIP diagnosis and 50 control lungs, we identified genes whose expression is associated with changes in lung function (% predicted FVC and % predicted DLCO) modeled as categorical (severe vs mild disease) or continuous variables while adjusting for smoking status and IIP subtype; false discovery rate (FDR) approach was used to correct for multiple comparisons. This analysis identified 58 transcripts that are associated with mild vs severe disease (categorical analysis), including those with established role in fibrosis (ADAMTS4, ADAMTS9, AGER, HIF-1α, SERPINA3, SERPINE2, and SELE) as well as novel IIP candidate genes such as rhotekin 2 (RTKN2) and peptidase inhibitor 15 (PI15). Protein-protein interactome analysis of 553 genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of IIPs is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. Our findings were validated in an independent cohort of 131 IIPs and 40 controls at the mRNA level and for one gene (RTKN2) at the protein level by immunohistochemistry in a subset of samples. CONCLUSIONS: We identified commonalities and differences in gene expression among different subtypes of IIPs. Disease progression, as characterized by lower measures of FVC and DLCO, results in marked changes in expression of novel and established genes and pathways involved in IIPs. These genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for IIP severity.

Incorporating genetics into the identification and treatment of Idiopathic Pulmonary Fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis, the most common form of idiopathic interstitial pneumonia, is characterized by progressive, irreversible scarring of the lung parenchyma. Idiopathic pulmonary fibrosis has a poor prognosis, and there are no medical therapies available that have been shown to improve survival. It is usually sporadic, but there is evidence of familial clustering of pulmonary fibrosis, suggesting a genetic basis for this disease. More recently, studies have confirmed that specific genetic variants are associated with both familial and sporadic forms of pulmonary fibrosis. DISCUSSION: Although there are common and rare genetic variants that have been associated with the risk of developing pulmonary fibrosis, the genotyping of patients is not a generally accepted strategy. Better understanding of the interplay between genetic risk and environmental exposure is likely needed to inform both treatment and disease prevention. Several identified disease-associated genetic variants have implications for disease progression and survival, but systematic studies of known genetic variants and their influence on therapeutic efficacy are lacking. Future investigations should focus on understanding phenotypic differences between patients carrying different risk alleles, and clinical studies should be designed to control for the influence of different genetic risk variants on patient outcomes. Inherited genetic factors play a significant role in the risk of developing pulmonary fibrosis. Future studies will be needed to characterize patient phenotypes and to understand how these genetic factors will influence clinical decision-making for both diagnosis and treatment of idiopathic pulmonary fibrosis.

Relationship of DNA methylation and gene expression in idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is an untreatable and often fatal lung disease that is increasing in prevalence and is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control gene expression and are likely to regulate the IPF transcriptome. OBJECTIVES: To identify methylation marks that modify gene expression in IPF lung. METHODS: We assessed DNA methylation (comprehensive high-throughput arrays for relative methylation arrays [CHARM]) and gene expression (Agilent gene expression arrays) in 94 patients with IPF and 67 control subjects, and performed integrative genomic analyses to define methylation-gene expression relationships in IPF lung. We validated methylation changes by a targeted analysis (Epityper), and performed functional validation of one of the genes identified by our analysis. MEASUREMENTS AND MAIN RESULTS: We identified 2,130 differentially methylated regions (DMRs; <5% false discovery rate), of which 738 are associated with significant changes in gene expression and enriched for expected inverse relationship between methylation and expression (P < 2.2 × 10(-16)). We validated 13/15 DMRs by targeted analysis of methylation. Methylation-expression quantitative trait loci (methyl-eQTL) identified methylation marks that control cis and trans gene expression, with an enrichment for cis relationships (P < 2.2 × 10(-16)). We found five trans methyl-eQTLs where a methylation change at a single DMR is associated with transcriptional changes in a substantial number of genes; four of these DMRs are near transcription factors (castor zinc finger 1 [CASZ1], FOXC1, MXD4, and ZDHHC4). We studied the in vitro effects of change in CASZ1 expression and validated its role in regulation of target genes in the methyl-eQTL. CONCLUSIONS: These results suggest that DNA methylation may be involved in the pathogenesis of IPF.

Familial and sporadic idiopathic pulmonary fibrosis: making the diagnosis from peripheral blood.

BACKGROUND: Peripheral blood biomarkers might improve diagnostic accuracy for idiopathic pulmonary fibrosis (IPF). RESULTS: Gene expression profiles were obtained from 89 patients with IPF and 26 normal controls. Samples were stratified according to severity of disease based on pulmonary function. The stratified dataset was split into subsets; two-thirds of the samples were selected to comprise the training set, while one-third was reserved for the validation set. Bayesian probit regression was used on the training set to develop a gene expression model for IPF versus normal. The gene expression model was tested by using it on the validation set to perform class prediction. Unsupervised clustering failed to discriminate between samples of different severity. Therefore, samples of all severities were included in the training and validation sets, in equal proportions. A gene signature model was developed from the training set. The model was built in an iterative fashion with the number of gene features selected to minimize the misclassification error in cross validation. The final model was based on the top 108 discriminating genes in the training set. The signature was successfully applied to the validation set, ROC area under the curve = 0.893, p < 0.0001. Using the optimal threshold (0.74) accurate class predictions were made for 77% of the test cases with sensitivity = 0.70, specificity = 1.00. CONCLUSIONS: By using Bayesian probit regression to develop a model, we show that it is entirely possible to make a diagnosis of IPF from the peripheral blood with gene signatures.