Brenda Juan-Guardela

Genetic variants associated with idiopathic pulmonary fibrosis susceptibility and mortality: a genome-wide association study.

BACKGROUND Idiopathic pulmonary fibrosis (IPF) is a devastating disease that probably involves several genetic loci. Several rare genetic variants and one common single nucleotide polymorphism (SNP) of MUC5B have been associated with the disease. Our aim was to identify additional common variants associated with susceptibility and ultimately mortality in IPF. METHODS First, we did a three-stage genome-wide association study (GWAS): stage one was a discovery GWAS; and stages two and three were independent case-control studies. DNA samples from European-American patients with IPF meeting standard criteria were obtained from several US centres for each stage. Data for European-American control individuals for stage one were gathered from the database of genotypes and phenotypes; additional control individuals were recruited at the University of Pittsburgh to increase the number. For controls in stages two and three, we gathered data for additional sex-matched European-American control individuals who had been recruited in another study. DNA samples from patients and from control individuals were genotyped to identify SNPs associated with IPF. SNPs identified in stage one were carried forward to stage two, and those that achieved genome-wide significance (p<5 × 10(-8)) in a meta-analysis were carried forward to stage three. Three case series with follow-up data were selected from stages one and two of the GWAS using samples with follow-up data. Mortality analyses were done in these case series to assess the SNPs associated with IPF that had achieved genome-wide significance in the meta-analysis of stages one and two. Finally, we obtained gene-expression profiling data for lungs of patients with IPF from the Lung Genomics Research Consortium and analysed correlation with SNP genotypes. FINDINGS In stage one of the GWAS (542 patients with IPF, 542 control individuals matched one-by-one to cases by genetic ancestry estimates), we identified 20 loci. Six SNPs reached genome-wide significance in stage two (544 patients, 687 control individuals): three TOLLIP SNPs (rs111521887, rs5743894, rs5743890) and one MUC5B SNP (rs35705950) at 11p15.5; one MDGA2 SNP (rs7144383) at 14q21.3; and one SPPL2C SNP (rs17690703) at 17q21.31. Stage three (324 patients, 702 control individuals) confirmed the associations for all these SNPs, except for rs7144383. Linkage disequilibrium between the MUC5B SNP (rs35705950) and TOLLIP SNPs (rs111521887 [r(2)=0·07], rs5743894 [r(2)=0·16], and rs5743890 [r(2)=0·01]) was low. 683 patients from the GWAS were included in the mortality analysis. Individuals who developed IPF despite having the protective TOLLIP minor allele of rs5743890 carried an increased mortality risk (meta-analysis with fixed-effect model: hazard ratio 1·72 [95% CI 1·24-2·38]; p=0·0012). TOLLIP expression was decreased by 20% in individuals carrying the minor allele of rs5743890 (p=0·097), 40% in those with the minor allele of rs111521887 (p=3·0 × 10(-4)), and 50% in those with the minor allele of rs5743894 (p=2·93 × 10(-5)) compared with homozygous carriers of common alleles for these SNPs. INTERPRETATION Novel variants in TOLLIP and SPPL2C are associated with IPF susceptibility. One novel variant of TOLLIP, rs5743890, is also associated with mortality. These associations and the reduced expression of TOLLIP in patients with IPF who carry TOLLIP SNPs emphasise the importance of this gene in the disease. FUNDING National Institutes of Health; National Heart, Lung, and Blood Institute; Pulmonary Fibrosis Foundation; Coalition for Pulmonary Fibrosis; and Instituto de Salud Carlos III.

Peripheral Blood Mononuclear Cell Gene Expression Profiles Predict Poor Outcome in Idiopathic Pulmonary Fibrosis

Genome-scale transcriptomic profiling of peripheral blood mononuclear cells from patients with idiopathic pulmonary fibrosis reveals that decreased expression of CD28, ICOS, LCK, and ITK predicts mortality. Gene Signature Predicts Mortality Idiopathic pulmonary fibrosis (IPF) is a fatal disease that progresses at different rates. Although no therapies exist, giving patients a more accurate prognosis is highly desirable. To this end, Herazo-Maya and colleagues searched the genomes of cells circulating in the blood of IPF patients and found that four genes may be indicators of poor outcome. Patients were recruited into discovery or replication cohorts from two different medical centers in the United States and followed until death or completion of the study. In both groups, genetic material was isolated from the patients’ peripheral blood mononuclear cells (PBMCs) and analyzed for increased or decreased expression. These gene expression profiles were then correlated with transplant-free survival (TFS). In the discovery cohort, Herazo-Maya et al. found that underexpression of the genes CD28, ICOS, LCK, and ITK was associated with decreased TFS. These findings were confirmed in the replication cohort. This “genomic model” incorporating the four genes was combined with the clinical outputs age, gender, and forced vital capacity to create an even stronger predictor of poor outcome. The authors suggest that the decreased expression of these genes might be linked to lower percentages of CD4+CD28+ T cells in the PBMC population, which could contribute to a mechanistic understanding of why some IPF patients progress differently than others. The findings of this study have the potential to affect the care of patients with IPF as well as the understanding of disease mechanism. However, the combined genomic and clinical predictor will need to be validated in additional independent cohorts before translation. We aimed to identify peripheral blood mononuclear cell (PBMC) gene expression profiles predictive of poor outcomes in idiopathic pulmonary fibrosis (IPF) by performing microarray experiments of PBMCs in discovery and replication cohorts of IPF patients. Microarray analyses identified 52 genes associated with transplant-free survival (TFS) in the discovery cohort. Clustering the microarray samples of the replication cohort using the 52-gene outcome-predictive signature distinguished two patient groups with significant differences in TFS. We studied the pathways associated with TFS in each independent microarray cohort and identified decreased expression of “The costimulatory signal during T cell activation” Biocarta pathway and, in particular, the genes CD28, ICOS, LCK, and ITK, results confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A proportional hazards model, including the qRT-PCR expression of CD28, ICOS, LCK, and ITK along with patient’s age, gender, and percent predicted forced vital capacity (FVC%), demonstrated an area under the receiver operating characteristic curve of 78.5% at 2.4 months for death and lung transplant prediction in the replication cohort. To evaluate the potential cellular source of CD28, ICOS, LCK, and ITK expression, we analyzed and found significant correlation of these genes with the PBMC percentage of CD4+CD28+ T cells in the replication cohort. Our results suggest that CD28, ICOS, LCK, and ITK are potential outcome biomarkers in IPF and should be further evaluated for patient prioritization for lung transplantation and stratification in drug studies.

FK506-Binding Protein 10, a Potential Novel Drug Target for Idiopathic Pulmonary Fibrosis

RATIONALE Increased abundance and stiffness of the extracellular matrix, in particular collagens, is a hallmark of idiopathic pulmonary fibrosis (IPF). FK506-binding protein 10 (FKBP10) is a collagen chaperone, mutations of which have been indicated in the reduction of extracellular matrix stiffness (e.g., in osteogenesis imperfecta). OBJECTIVES To assess the expression and function of FKBP10 in IPF. METHODS We assessed FKBP10 expression in bleomycin-induced lung fibrosis (using quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunofluorescence), analyzed microarray data from 99 patients with IPF and 43 control subjects from a U.S. cohort, and performed Western blot analysis from 6 patients with IPF and 5 control subjects from a German cohort. Subcellular localization of FKBP10 was assessed by immunofluorescent stainings. The expression and function of FKBP10, as well as its regulation by endoplasmic reticulum stress or transforming growth factor-β1, was analyzed by small interfering RNA-mediated loss-of-function experiments, quantitative reverse transcriptase-polymerase chain reaction, Western blot, and quantification of secreted collagens in the lung and in primary human lung fibroblasts (phLF). Effects on collagen secretion were compared with those of the drugs nintedanib and pirfenidone, recently approved for IPF. MEASUREMENTS AND MAIN RESULTS FKBP10 expression was up-regulated in bleomycin-induced lung fibrosis and IPF. Immunofluorescent stainings demonstrated localization to interstitial (myo)fibroblasts and CD68(+) macrophages. Transforming growth factor-β1, but not endoplasmic reticulum stress, induced FKBP10 expression in phLF. The small interfering RNA-mediated knockdown of FKBP10 attenuated expression of profibrotic mediators and effectors, including collagens I and V and α-smooth muscle actin, on the transcript and protein level. Importantly, loss of FKBP10 expression significantly suppressed collagen secretion by phLF. CONCLUSIONS FKBP10 might be a novel drug target for IPF.

Cytokine-Like Factor 1 Gene Expression Is Enriched in Idiopathic Pulmonary Fibrosis and Drives the Accumulation of CD4+ T Cells in Murine Lungs: Evidence for an Antifibrotic Role in Bleomycin Injury

Idiopathic pulmonary fibrosis (IPF) is a progressive and typically fatal lung disease. To gain insight into the pathogenesis of IPF, we reanalyzed our previously published gene expression data profiling IPF lungs. Cytokine receptor-like factor 1 (CRLF1) was among the most highly up-regulated genes in IPF lungs, compared with normal controls. The protein product (CLF-1) and its partner, cardiotrophin-like cytokine (CLC), function as members of the interleukin 6 (IL-6) family of cytokines. Because of earlier work implicating IL-6 family members in IPF pathogenesis, we tested whether CLF-1 expression contributes to inflammation in experimental pulmonary fibrosis. In IPF, we detected CLF-1 expression in both type II alveolar epithelial cells and macrophages. We found that the receptor for CLF-1/CLC signaling, ciliary neurotrophic factor receptor (CNTFR), was expressed only in type II alveolar epithelial cells. Administration of CLF-1/CLC to both uninjured and bleomycin-injured mice led to the pulmonary accumulation of CD4(+) T cells. We also found that CLF-1/CLC administration increased inflammation but decreased pulmonary fibrosis. CLF-1/CLC leads to significantly enriched expression of T-cell-derived chemokines and cytokines, including the antifibrotic cytokine interferon-γ. We propose that, in IPF, CLF-1 is a selective stimulus of type II alveolar epithelial cells and may potentially drive an antifibrotic response by augmenting both T-helper-1-driven and T-regulatory-cell-driven inflammatory responses in the lung.

Integrative phenotyping framework (iPF): integrative clustering of multiple omics data identifies novel lung disease subphenotypes

BackgroundThe increased multi-omics information on carefully phenotyped patients in studies of complex diseases requires novel methods for data integration. Unlike continuous intensity measurements from most omics data sets, phenome data contain clinical variables that are binary, ordinal and categorical.ResultsIn this paper we introduce an integrative phenotyping framework (iPF) for disease subtype discovery. A feature topology plot was developed for effective dimension reduction and visualization of multi-omics data. The approach is free of model assumption and robust to data noises or missingness. We developed a workflow to integrate homogeneous patient clustering from different omics data in an agglomerative manner and then visualized heterogeneous clustering of pairwise omics sources. We applied the framework to two batches of lung samples obtained from patients diagnosed with chronic obstructive lung disease (COPD) or interstitial lung disease (ILD) with well-characterized clinical (phenomic) data, mRNA and microRNA expression profiles. Application of iPF to the first training batch identified clusters of patients consisting of homogenous disease phenotypes as well as clusters with intermediate disease characteristics. Analysis of the second batch revealed a similar data structure, confirming the presence of intermediate clusters. Genes in the intermediate clusters were enriched with inflammatory and immune functional annotations, suggesting that they represent mechanistically distinct disease subphenotypes that may response to immunomodulatory therapies. The iPF software package and all source codes are publicly available.ConclusionsIdentification of subclusters with distinct clinical and biomolecular characteristics suggests that integration of phenomic and other omics information could lead to identification of novel mechanism-based disease sub-phenotypes.

Validation of a 52-gene risk profile for outcome prediction in patients with idiopathic pulmonary fibrosis: an international, multicentre, cohort study

Background The clinical course of Idiopathic Pulmonary Fibrosis (IPF) is unpredictable. Clinical prediction tools are not accurate enough to predict disease outcomes. Methods All-comers with Idiopathic Pulmonary Fibrosis diagnosis were enrolled in a six-cohort study. Peripheral blood mononuclear cells or whole blood was collected at baseline from 425 participants and during follow up from 98 patients. The 52-gene signature was measured by the nCounter® analysis system in four cohorts and extracted from microarray data in two others. The Scoring Algorithm for Molecular Subphenotypes (SAMS) was used to classify patients into low or high risk groups based on a 52-gene signature. Mortality and transplant-free survival were studied using Competing risk and Cox proportional-hazard models, respectively. Time course data and response to anti-fibrotic drugs were analyzed using linear mixed-effect models. Findings The application of SAMS to the 52-gene signature identified two groups of IPF patients (low and high risk) with significant differences in mortality or transplant-free survival in each of the six cohorts (HR 2·03–4·37). Pooled data revealed similar results for mortality (HR:2·18, 95%CI:1·53–3·09, P<0·0001) or transplant-free survival (HR:2·04, 95%CI: 1·52–2·74, P<0·0001). Adding 52-gene risk profiles to the Gender, Age and Physiology (GAP) index significantly improved its mortality predictive accuracy. Temporal changes in SAMS scores were associated with changes in forced vital capacity (FVC) in two cohorts. Untreated patients did not shift their risk profile over time. A simultaneous increase in up score and decrease in down score was predictive of transplant-free survival (HR:3·18· 95%CI 1·16, 8·76, P=0·025) in the Pittsburgh cohort. A simultaneous decrease in up score and increase in down score after initiation of anti-fibrotic drugs was associated with a significant (P=0·005) improvement in FVC in the Yale cohort. Interpretation The peripheral blood 52-gene expression signature is predictive of outcome in patients with IPF. The potential value of the 52-gene signature in predicting response to therapy should be determined in prospective studies.

Matrix Metalloproteinase-19 Promotes Metastatic Behavior In Vitro and Is Associated with Increased Mortality in Non–Small Cell Lung Cancer

RATIONALE Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Matrix metalloproteinases (MMPs) have been implicated in the development and progression of lung cancer, but their role in the molecular pathogenesis of lung cancer remains unclear. We have found that MMP19, a relatively novel member of the MMP family, is overexpressed in lung tumors when compared with control subjects. OBJECTIVES To test the hypothesis that MMP19 plays a significant role in the development and progression of non-small cell lung cancer (NSCLC). METHODS We have analyzed lung cancer gene expression data, immunostained lung tumors for MMP19, and performed in vitro assays to test the effects of MMP19 in NSCLC cells. MEASUREMENTS AND MAIN RESULTS We found that MMP19 gene and protein expression is increased in lung cancer tumors compared with adjacent and histologically normal lung tissues. In three independent datasets, increased MMP19 gene expression conferred a poorer prognosis in NSCLC. In vitro, we found that overexpression of MMP19 promotes epithelial-mesenchymal transition, migration, and invasiveness in multiple NSCLC cell lines. Overexpression of MMP19 with a mutation at the catalytic site did not impair epithelial-mesenchymal transition or expression of prometastasis genes. We also found that miR-30 isoforms, a microRNA family predicted to target MMP19, is markedly down-regulated in human lung cancer and regulates MMP19 expression. CONCLUSIONS Taken together, these findings suggest that MMP19 is associated with the development and progression of NSCLC and may be a potential biomarker of disease severity and outcome.

Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data

Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However, multiplexing involves a trade-off between increased number of sequenced samples and reduced number of reads per sample (i.e., lower depth of coverage). To assess the effect of different sequencing depths owing to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing one, three, six, nine, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization, replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression >1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the log2 number of reads aligning to microRNA loci (R = 0.96). However, most additional microRNAs detected in samples with greater sequencing depth were in the range of expression which had lower fold change reproducibility. These findings elucidate the trade-off between increasing the number of samples in a multiplex with decreasing sequencing depth and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.

Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Fibrosis in Experimental Lung Injury by Enhanced Expression of CXCL12

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2+ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow–derived matrix-producing cells. We found that Twist1-null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-κB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Transcriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may shape the IPF phenotype and regulate inflammation in fibrotic lung injury.