Rare variants and survival of patients with idiopathic pulmonary fibrosis.

Background: The clinical course of idiopathic pulmonary fibrosis (IPF) is highly variable and unpredictable, with multiple genetic variants influencing IPF outcomes. Notably, rare pathogenic variants in telomere-related genes are associated with poorer clinical outcomes in these patients. Here we assessed whether rare qualifying variants (QVs) in monogenic adult-onset pulmonary fibrosis (PF) genes are associated with IPF survival. Using polygenic risk scores (PRS), we also evaluated the influence of common IPF risk variants in individuals carrying these QVs. Methods: We identified QVs in telomere and non-telomere genes linked to monogenic PF forms using whole-genome sequences (WGS) from 888 Pulmonary Fibrosis Foundation Patient Registry (PFFPR) individuals. We also derived a PRS for IPF (PRS-IPF) from 19 previously published common sentinel IPF variants. Using regression models, we then examined the mutual relationships of QVs and PRS-IPF and their association with survival. Validation of results was sought in WGS from an independent IPF study (PROFILE, n=472), and results from the two cohorts were meta-analyzed. Results: Carriers of QVs in monogenic adult-onset PF genes, representing nearly 1 out of 6 IPF patients, were associated with lower PRS-IPF (Odds Ratio [OR]: 1.79; 95% Confidence Interval [CI]: 1.15-2.81; p=0.010) and shorter survival (Hazard Ratio [HR]: 1.53; 95% CI: 1.12-2.10; p=7.3×10 -3 ). Notably, carriers of pathogenic variants at telomere genes showed the strongest association with survival (HR: 1.76; 95% CI: 1.13-2.76; p=0.013). The meta-analysis of the results showed a consistent direction of effect across both cohorts. Conclusions: We revealed the opposite effects of QVs and PRS-IPF on IPF survival. Thus, a distinct IPF molecular subtype might be defined by QVs in monogenic adult-onset PF genes. Assessing the carrier status for QVs and modelling PRS-IPF promises to further contribute to predicting disease progression among IPF patients.

Molecular Endotypes of Idiopathic Pulmonary Fibrosis: A Latent Class Analysis of Two Multicenter Observational Cohorts.

Rationale: Idiopathic pulmonary fibrosis (IPF) causes irreversible fibrosis of the lung parenchyma. Although antifibrotic therapy can slow IPF progression, treatment response is variable. There exists a critical need to develop a precision medicine approach to IPF. Objectives: To identify and validate biologically driven molecular endotypes of IPF. Methods: Latent class analysis (LCA) was independently performed in prospectively recruited discovery (n = 875) and validation (n = 347) cohorts. Twenty-five plasma biomarkers associated with fibrogenesis served as class-defining variables. The association between molecular endotype and 4-year transplant-free survival was tested using multivariable Cox regression adjusted for baseline confounders. Endotype-dependent differential treatment response to future antifibrotic exposure was then assessed in a pooled cohort of patients naive to antifibrotic therapy at the time of biomarker measurement (n = 555). Measurements and Main Results: LCA independently identified two latent classes in both cohorts (P < 0.0001). WFDC2 (WAP four-disulfide core domain protein 2) was the most important determinant of class membership across cohorts. Membership in class 2 was characterized by higher biomarker concentrations and a higher risk of death or transplant (discovery, hazard ratio [HR], 2.02; 95% confidence interval [CI], 1.64-2.48; P < 0.001; validation, HR, 1.95; 95% CI, 1.34-2.82; P < 0.001). In pooled analysis, significant heterogeneity in treatment effect was observed between endotypes (P = 0.030 for interaction), with a favorable antifibrotic response in class 2 (HR, 0.64; 95% CI, 0.45-0.93; P = 0.018) but not in class 1 (HR, 1.19; 95% CI, 0.77-1.84; P = 0.422). Conclusions: In this multicohort study, we identified two novel molecular endotypes of IPF with divergent clinical outcomes and responses to antifibrotic therapy. Pending further validation, these endotypes could enable a precision medicine approach for future IPF clinical trials.

Biological Age, Chronological Age, and Survival in Pulmonary Fibrosis: A Causal Mediation Analysis.

Rationale: Accelerated biological aging has been implicated in the development of interstitial lung disease (ILD) and other diseases of aging but remains poorly understood. Objectives: To identify plasma proteins that mediate the relationship between chronological age and survival association in patients with ILD. Methods: Causal mediation analysis was performed to identify plasma proteins that mediated the chronological age-survival relationship in an idiopathic pulmonary fibrosis discovery cohort. Proteins mediating this relationship after adjustment for false discovery were advanced for testing in an independent ILD validation cohort and explored in a chronic obstructive pulmonary disease cohort. A proteomic-based measure of biological age was constructed and survival analysis performed, assessing the impact of biological age and peripheral blood telomere length on the chronological age-survival relationship. Measurements and Main Results: Twenty-two proteins mediated the chronological age-survival relationship after adjustment for false discovery in the idiopathic pulmonary fibrosis discovery cohort (n = 874), with 19 remaining significant mediators of this relationship in the ILD validation cohort (n = 983) and one mediating this relationship in the chronic obstructive pulmonary disease cohort. Latent transforming growth factor-ß binding protein 2 and ectodysplasin A2 receptor showed the strongest mediation across cohorts. A proteomic measure of biological age completely attenuated the chronological age-survival association and better discriminated survival than chronological age. Results were robust to adjustment for peripheral blood telomere length, which did not mediate the chronological age-survival relationship. Conclusions: Molecular measures of aging completely mediate the relationship between chronological age and survival, suggesting that chronological age has no direct effect on ILD survival.

A multidimensional classifier to support lung transplant referral in patients with pulmonary fibrosis.

BACKGROUND: Lung transplantation remains the sole curative option for patients with idiopathic pulmonary fibrosis (IPF), but donor organs remain scarce, and many eligible patients die before transplant. Tools to optimize the timing of transplant referrals are urgently needed. METHODS: Least absolute shrinkage and selection operator was applied to clinical and proteomic data generated as part of a prospective cohort study of interstitial lung disease (ILD) to derive clinical, proteomic, and multidimensional logit models of near-term death or lung transplant within 18 months of blood draw. Model-fitted values were dichotomized at the point of maximal sensitivity and specificity, and decision curve analysis was used to select the best-performing classifier. We then applied this classifier to independent IPF and non-IPF ILD cohorts to determine test performance characteristics. Cohorts were restricted to patients aged ≤72 years with body mass index 18 to 32 to increase the likelihood of transplant eligibility. RESULTS: IPF derivation, IPF validation, and non-IPF ILD validation cohorts consisted of 314, 105, and 295 patients, respectively. A multidimensional model comprising 2 clinical variables and 20 proteins outperformed stand-alone clinical and proteomic models. Following dichotomization, the multidimensional classifier predicted near-term outcome with 70% sensitivity and 92% specificity in the IPF validation cohort and 70% sensitivity and 80% specificity in the non-IPF ILD validation cohort. CONCLUSIONS: A multidimensional classifier of near-term outcomes accurately discriminated this end-point with good test performance across independent IPF and non-IPF ILD cohorts. These findings support refinement and prospective validation of this classifier in transplant-eligible individuals.

Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men.

Lung cancer is the third most common cancer with Black/AA men showing higher risk and poorer outcomes than NHW men. Lung cancer disparities are multifactorial, driven by tobacco exposure, inequities in care access, upstream health determinants, and molecular determinants including biological and genetic factors. Elevated expressions of protein arginine methyltransferases (PRMTs) correlating with poorer prognosis have been observed in many cancers. Most importantly, our study shows that PRMT6 displays higher expression in lung cancer tissues of Black/AA men compared to NHW men. In this study, we investigated the underlying mechanism of PRMT6 and its cooperation with PRMT1 to form a heteromer as a driver of lung cancer. Disrupting PRMT1/PRMT6 heteromer by a competitive peptide reduced proliferation in non-small cell lung cancer cell lines and patient-derived organoids, therefore, giving rise to a more strategic approach in the treatment of Black/AA men with lung cancer and to eliminate cancer health disparities.

Association study of human leukocyte antigen variants and idiopathic pulmonary fibrosis.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia marked by progressive lung fibrosis and a poor prognosis. Recent studies have highlighted the potential role of infection in the pathogenesis of IPF, and a prior association of the HLA-DQB1 gene with idiopathic fibrotic interstitial pneumonia (including IPF) has been reported. Owing to the important role that the human leukocyte antigen (HLA) region plays in the immune response, here we evaluated if HLA genetic variation was associated specifically with IPF risk. Methods: We performed a meta-analysis of associations of the HLA region with IPF risk in individuals of European ancestry from seven independent case-control studies of IPF (comprising 5159 cases and 27 459 controls, including a prior study of fibrotic interstitial pneumonia). Single nucleotide polymorphisms, classical HLA alleles and amino acids were analysed and signals meeting a region-wide association threshold of p<4.5×10-4 and a posterior probability of replication >90% were considered significant. We sought to replicate the previously reported HLA-DQB1 association in the subset of studies independent of the original report. Results: The meta-analysis of all seven studies identified four significant independent single nucleotide polymorphisms associated with IPF risk. However, none met the posterior probability for replication criterion. The HLA-DQB1 association was not replicated in the independent IPF studies. Conclusion: Variation in the HLA region was not consistently associated with risk in studies of IPF. However, this does not preclude the possibility that other genomic regions linked to the immune response may be involved in the aetiology of IPF.

Machine Learning of Plasma Proteomics Classifies Diagnosis of Interstitial Lung Disease.

Rationale: Distinguishing connective tissue disease-associated interstitial lung disease (CTD-ILD) from idiopathic pulmonary fibrosis (IPF) can be clinically challenging. Objectives: To identify proteins that separate and classify patients with CTD-ILD and those with IPF. Methods: Four registries with 1,247 patients with IPF and 352 patients with CTD-ILD were included in analyses. Plasma samples were subjected to high-throughput proteomics assays. Protein features were prioritized using recursive feature elimination to construct a proteomic classifier. Multiple machine learning models, including support vector machine, LASSO (least absolute shrinkage and selection operator) regression, random forest, and imbalanced Random Forest, were trained and tested in independent cohorts. The validated models were used to classify each case iteratively in external datasets. Measurements and Main Results: A classifier with 37 proteins (proteomic classifier 37 [PC37]) was enriched in the biological process of bronchiole development and smooth muscle proliferation and immune responses. Four machine learning models used PC37 with sex and age score to generate continuous classification values. Receiver operating characteristic curve analyses of these scores demonstrated consistent areas under the curve of 0.85-0.90 in the test cohort and 0.94-0.96 in the single-sample dataset. Binary classification demonstrated 78.6-80.4% sensitivity and 76-84.4% specificity in the test cohort and 93.5-96.1% sensitivity and 69.5-77.6% specificity in the single-sample classification dataset. Composite analysis of all machine learning models confirmed 78.2% (194 of 248) accuracy in the test cohort and 82.9% (208 of 251) in the single-sample classification dataset. Conclusions: Multiple machine learning models trained with large cohort proteomic datasets consistently distinguished CTD-ILD from IPF. Many of the identified proteins are involved in immune pathways. We further developed a novel approach for single-sample classification, which could facilitate honing the differential diagnosis of ILD in challenging cases and improve clinical decision making.

Genome-wide SNP-sex interaction analysis of susceptibility to idiopathic pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition that is more prevalent in males than females. The reasons for this are not fully understood, with differing environmental exposures due to historically sex-biased occupations, or diagnostic bias, being possible explanations. To date, over 20 independent genetic variants have been identified to be associated with IPF susceptibility, but these have been discovered when combining males and females. Our aim was to test for the presence of sex-specific associations with IPF susceptibility and assess whether there is a need to consider sex-specific effects when evaluating genetic risk in clinical prediction models for IPF. Methods: We performed genome-wide single nucleotide polymorphism (SNP)-by-sex interaction studies of IPF risk in six independent IPF case-control studies and combined them using inverse-variance weighted fixed effect meta-analysis. In total, 4,561 cases (1,280 females and 2,281 males) and 23,500 controls (8,360 females and 14,528 males) of European genetic ancestry were analysed. We used polygenic risk scores (PRS) to assess differences in genetic risk prediction between males and females. Findings: Three independent genetic association signals were identified. All showed a consistent direction of effect across all individual IPF studies and an opposite direction of effect in IPF susceptibility between females and males. None had been previously identified in IPF susceptibility genome-wide association studies (GWAS). The predictive accuracy of the PRSs were similar between males and females, regardless of whether using combined or sex-specific GWAS results. Interpretation: We prioritised three genetic variants whose effect on IPF risk may be modified by sex, however these require further study. We found no evidence that the predictive accuracy of common SNP-based PRSs varies significantly between males and females.

Associations of Plasma Omega-3 Fatty Acids With Progression and Survival in Pulmonary Fibrosis.

BACKGROUND: Preclinical experiments suggest protective effects of omega-3 fatty acids and their metabolites in lung injury and fibrosis. Whether higher intake of omega-3 fatty acids is associated with disease progression and survival in humans with pulmonary fibrosis is unknown. RESEARCH QUESTION: What are the associations of plasma omega-3 fatty acid levels (a validated marker of omega-3 nutritional intake) with disease progression and transplant-free survival in pulmonary fibrosis? STUDY DESIGN AND METHODS: Omega-3 fatty acid levels were measured from plasma samples of patients with clinically diagnosed pulmonary fibrosis from the Pulmonary Fibrosis Foundation Patient Registry (n = 150), University of Virginia (n = 58), and University of Chicago (n = 101) cohorts. The N-3 index (docosahexaenoic acid + eicosapentaenoic acid) was the primary exposure variable of interest. Linear-mixed effects models with random intercept and slope were used to examine associations of plasma omega-3 fatty acid levels with changes in FVC and diffusing capacity for carbon monoxide over a period of 12 months. Cox proportional hazards models were used to examine transplant-free survival. Stratified analyses by telomere length were performed in the University of Chicago cohort. RESULTS: Most of the cohort were patients with idiopathic pulmonary fibrosis (88%) and male patients (74%). One-unit increment in log-transformed N-3 index plasma level was associated with a change in diffusing capacity for carbon monoxide of 1.43 mL/min/mm Hg per 12 months (95% CI, 0.46-2.41) and a hazard ratio for transplant-free survival of 0.44 (95% CI, 0.24-0.83). Cardiovascular disease history, smoking, and antifibrotic usage did not significantly modify associations. Omega-3 fatty acid levels were not significantly associated with changes in FVC. Higher eicosapentaenoic acid plasma levels were associated with longer transplant-free survival among University of Chicago participants with shorter telomere length (P value for interaction = .02). INTERPRETATION: Further research is needed to investigate underlying biological mechanisms and whether omega-3 fatty acids are a potential disease-modifying therapy.

Commensal Oral Microbiota, Disease Severity and Mortality in Fibrotic Lung Disease.

RATIONALE: Oral microbiota associate with diseases of the mouth and serve as a source of lung microbiota. However, the role of oral microbiota in lung disease is unknown. OBJECTIVES: To determine associations between oral microbiota and disease severity and death in idiopathic pulmonary fibrosis. METHODS: We analyzed 16S rRNA gene and shotgun metagenomic sequencing data of buccal swabs from 511 patients with idiopathic pulmonary fibrosis in the multicenter CleanUP-IPF trial. Buccal swabs were collected from usual care, and antimicrobial cohorts. Microbiome data was correlated with measures of disease severity using principal component analysis and linear regression models. Associations between the buccal microbiome and mortality were determined using Cox additive models, Kaplan Meier analysis and Cox proportional hazards models. MEASUREMENTS AND MAIN RESULTS: Greater buccal microbial diversity associated with lower forced vital capacity (FVC) at baseline [mean diff -3.60: 95% CI -5.92 to -1.29 percent predicted FVC per 1 unit increment]. The buccal proportion of Streptococcus correlated positively with FVC [mean diff 0.80: 95% CI 0.16-1.43 percent predicted per 10% increase] (n=490). Greater microbial diversity was associated with an increased risk of death [HR 1.73: 95% CI 1.03-2.90] while a greater proportion of Streptococcus was associated with a reduced risk of death [HR 0.85: 95% CI 0.73 to 0.99]. The Streptococcus genus was mainly comprised of Streptococcus mitis species. CONCLUSIONS: Increasing buccal microbial diversity predicts disease severity and death in IPF. The oral commensal Streptococcus mitis spp associates with preserved lung function and improved survival.

Proteomic Biomarkers of Survival in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) causes progressive lung scarring and high mortality. Reliable and accurate prognostic biomarkers are urgently needed. OBJECTIVE: To identify and validate circulating protein biomarkers of IPF survival. METHODS: High-throughput proteomic data were generated using prospectively collected plasma samples from patients with IPF from the Pulmonary Fibrosis Foundation Patient Registry (discovery cohort) and the Universities of California-Davis, Chicago, and Virginia (validation cohort). Proteins associated with three-year transplant-free survival (TFS) were identified using multivariable Cox proportional hazards regression. Those associated with TFS after adjustment for false discovery in the discovery cohort were advanced for testing in the validation cohort, with proteins maintaining TFS association with consistent effect direction considered validated. After combining cohorts, functional analyses were performed, and machine learning used to derive a proteomic signature of TFS. MAIN RESULTS: Of 2921 proteins tested in the discovery cohort (n=871), 231 were associated with differential TFS. Of these, 140 maintained TFS association with consistent effect direction in the validation cohort (n=355). After combining cohorts, validated proteins with strongest TFS association were latent-transforming growth factor beta-binding protein 2 (HR 2.43, 95% CI 2.09-2.82), collagen alpha-1(XXIV) chain (HR 2.21; 95% CI 1.86-2.39) and keratin 19 (HR 1.60; 95% CI 1.47-1.74). In decision curve analysis, a proteomic signature of TFS outperformed a similarly derived clinical prediction model. CONCLUSIONS: In largest proteomic investigation of IPF outcomes performed to date, we identified and validated 140 protein biomarkers of TFS. These results shed important light on potential drivers of IPF progression.

Telomere length and immunosuppression in non-idiopathic pulmonary fibrosis interstitial lung disease.

BACKGROUND: Studies suggest a harmful pharmacogenomic interaction exists between short leukocyte telomere length (LTL) and immunosuppressants in idiopathic pulmonary fibrosis (IPF). It remains unknown if a similar interaction exists in non-IPF interstitial lung disease (ILD). METHODS: A retrospective, multicentre cohort analysis was performed in fibrotic hypersensitivity pneumonitis (fHP), unclassifiable ILD (uILD) and connective tissue disease (CTD)-ILD patients from five centres. LTL was measured by quantitative PCR for discovery and replication cohorts and expressed as age-adjusted percentiles of normal. Inverse probability of treatment weights based on propensity scores were used to assess the association between mycophenolate or azathioprine exposure and age-adjusted LTL on 2-year transplant-free survival using weighted Cox proportional hazards regression incorporating time-dependent immunosuppressant exposure. RESULTS: The discovery and replication cohorts included 613 and 325 patients, respectively. In total, 40% of patients were exposed to immunosuppression and 22% had LTL <10th percentile of normal. fHP and uILD patients with LTL <10th percentile experienced reduced survival when exposed to either mycophenolate or azathioprine in the discovery cohort (mortality hazard ratio (HR) 4.97, 95% CI 2.26-10.92; p<0.001) and replication cohort (mortality HR 4.90, 95% CI 1.74-13.77; p=0.003). Immunosuppressant exposure was not associated with differential survival in patients with LTL ≥10th percentile. There was a significant interaction between LTL <10th percentile and immunosuppressant exposure (discovery pinteraction=0.013; replication pinteraction=0.011). Low event rate and prevalence of LTL <10th percentile precluded subgroup analyses for CTD-ILD. CONCLUSION: Similar to IPF, fHP and uILD patients with age-adjusted LTL <10th percentile may experience reduced survival when exposed to immunosuppression.

Telomere length associates with chronological age and mortality across racially diverse pulmonary fibrosis cohorts.

Pulmonary fibrosis (PF) is characterized by profound scarring and poor survival. We investigated the association of leukocyte telomere length (LTL) with chronological age and mortality across racially diverse PF cohorts. LTL measurements among participants with PF stratified by race/ethnicity were assessed in relation to age and all-cause mortality, and compared to controls. Generalized linear models were used to evaluate the age-LTL relationship, Cox proportional hazards models were used for hazard ratio estimation, and the Cochran-Armitage test was used to assess quartiles of LTL. Standardized LTL shortened with increasing chronological age; this association in controls was strengthened in PF (R = -0.28; P < 0.0001). In PF, age- and sex-adjusted LTL below the median consistently predicted worse mortality across all racial groups (White, HR = 2.21, 95% CI = 1.79-2.72; Black, HR = 2.22, 95% CI = 1.05-4.66; Hispanic, HR = 3.40, 95% CI = 1.88-6.14; and Asian, HR = 2.11, 95% CI = 0.55-8.23). LTL associates uniformly with chronological age and is a biomarker predictive of mortality in PF across racial groups.

Central lung gene expression associates with myofibroblast features in idiopathic pulmonary fibrosis.

RATIONALE: Contribution of central lung tissues to pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unknown. OBJECTIVE: To ascertain the relationship between cell types of IPF-central and IPF-peripheral lung explants using RNA sequencing (RNA-seq) transcriptome. METHODS: Biopsies of paired IPF-central and IPF-peripheral along with non-IPF lungs were selected by reviewing H&E data. Criteria for differentially expressed genes (DEG) were set at false discovery rate <5% and fold change >2. Computational cell composition deconvolution was performed. Signature scores were computed for each cell type. FINDINGS: Comparison of central IPF versus non-IPF identified 1723 DEG (1522 upregulated and 201 downregulated). Sixty-two per cent (938/1522) of the mutually upregulated genes in central IPF genes were also upregulated in peripheral IPF versus non-IPF. Moreover, 85 IPF central-associated genes (CAG) were upregulated in central IPF versus both peripheral IPF and central non-IPF. IPF single-cell RNA-seq analysis revealed the highest CAG signature score in myofibroblasts and significantly correlated with a previously published activated fibroblasts signature (r=0.88, p=1.6×10-4). CAG signature scores were significantly higher in IPF than in non-IPF myofibroblasts (p=0.013). Network analysis of central-IPF genes identified a module significantly correlated with the deconvoluted proportion of myofibroblasts in central IPF and anti-correlated with inflammation foci trait in peripheral IPF. The module genes were over-represented in idiopathic pulmonary fibrosis signalling pathways. INTERPRETATION: Gene expression in central IPF lung regions demonstrates active myofibroblast features that contributes to disease progression. Further elucidation of pathological transcriptomic state of cells in the central regions of the IPF lung that are relatively spared from morphological rearrangements may provide insights into molecular changes in the IPF progression.

PCSK6 and Survival in Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. A better understanding of the molecular drivers of IPF progression is needed. Objectives: To identify and validate molecular determinants of IPF survival. Methods: A staged genome-wide association study was performed using paired genomic and survival data. Stage I cases were drawn from centers across the United States and Europe and stage II cases from Vanderbilt University. Cox proportional hazards regression was used to identify gene variants associated with differential transplantation-free survival (TFS). Stage I variants with nominal significance (P < 5 × 10-5) were advanced for stage II testing and meta-analyzed to identify those reaching genome-wide significance (P < 5 × 10-8). Downstream analyses were performed for genes and proteins associated with variants reaching genome-wide significance. Measurements and Main Results: After quality controls, 1,481 stage I cases and 397 stage II cases were included in the analysis. After filtering, 9,075,629 variants were tested in stage I, with 158 meeting advancement criteria. Four variants associated with TFS with consistent effect direction were identified in stage II, including one in an intron of PCSK6 (proprotein convertase subtilisin/kexin type 6) reaching genome-wide significance (hazard ratio, 4.11 [95% confidence interval, 2.54-6.67]; P = 9.45 × 10-9). PCSK6 protein was highly expressed in IPF lung parenchyma. PCSK6 lung staining intensity, peripheral blood gene expression, and plasma concentration were associated with reduced TFS. Conclusions: We identified four novel variants associated with IPF survival, including one in PCSK6 that reached genome-wide significance. Downstream analyses suggested that PCSK6 protein plays a potentially important role in IPF progression.

Longitudinal lung function and gas transfer in individuals with idiopathic pulmonary fibrosis: a genome-wide association study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an incurable lung disease characterised by progressive scarring leading to alveolar stiffness, reduced lung capacity, and impeded gas transfer. We aimed to identify genetic variants associated with declining lung capacity or declining gas transfer after diagnosis of IPF. METHODS: We did a genome-wide meta-analysis of longitudinal measures of forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLCO) in individuals diagnosed with IPF. Individuals were recruited to three studies between June, 1996, and August, 2017, from across centres in the US, UK, and Spain. Suggestively significant variants were investigated further in an additional independent study (CleanUP-IPF). All four studies diagnosed cases following American Thoracic Society/European Respiratory Society guidelines. Variants were defined as significantly associated if they had a meta-analysis p<5 × 10-8 when meta-analysing across all discovery and follow-up studies, had consistent direction of effects across all four studies, and were nominally significant (p<0·05) in each study. FINDINGS: 1329 individuals with a total of 5216 measures were included in the FVC analysis. 975 individuals with a total of 3361 measures were included in the DLCO analysis. For the discovery genome-wide analyses, 7 611 174 genetic variants were included in the FVC analysis and 7 536 843 in the DLCO analysis. One variant (rs115982800) located in an antisense RNA gene for protein kinase N2 (PKN2) showed a genome-wide significant association with FVC decline (-140 mL/year per risk allele [95% CI -180 to -100]; p=9·14 × 10-12). INTERPRETATION: Our analysis identifies a genetic variant associated with disease progression, which might highlight a new biological mechanism for IPF. We found that PKN2, a Rho and Rac effector protein, is the most likely gene of interest from this analysis. PKN2 inhibitors are currently in development and signify a potential novel therapeutic approach for IPF. FUNDING: Action for Pulmonary Fibrosis, Medical Research Council, Wellcome Trust, and National Institutes of Health National Heart, Lung, and Blood Institute.

Cluster analysis of transcriptomic datasets to identify endotypes of idiopathic pulmonary fibrosis.

BACKGROUND: Considerable clinical heterogeneity in idiopathic pulmonary fibrosis (IPF) suggests the existence of multiple disease endotypes. Identifying these endotypes would improve our understanding of the pathogenesis of IPF and could allow for a biomarker-driven personalised medicine approach. We aimed to identify clinically distinct groups of patients with IPF that could represent distinct disease endotypes. METHODS: We co-normalised, pooled and clustered three publicly available blood transcriptomic datasets (total 220 IPF cases). We compared clinical traits across clusters and used gene enrichment analysis to identify biological pathways and processes that were over-represented among the genes that were differentially expressed across clusters. A gene-based classifier was developed and validated using three additional independent datasets (total 194 IPF cases). FINDINGS: We identified three clusters of patients with IPF with statistically significant differences in lung function (p=0.009) and mortality (p=0.009) between groups. Gene enrichment analysis implicated mitochondrial homeostasis, apoptosis, cell cycle and innate and adaptive immunity in the pathogenesis underlying these groups. We developed and validated a 13-gene cluster classifier that predicted mortality in IPF (high-risk clusters vs low-risk cluster: HR 4.25, 95% CI 2.14 to 8.46, p=3.7×10-5). INTERPRETATION: We have identified blood gene expression signatures capable of discerning groups of patients with IPF with significant differences in survival. These clusters could be representative of distinct pathophysiological states, which would support the theory of multiple endotypes of IPF. Although more work must be done to confirm the existence of these endotypes, our classifier could be a useful tool in patient stratification and outcome prediction in IPF.