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© 2016 The Author(s).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 importanc

Rationale: Idiopathic interstitial pneumonia (IIP) and its familial variants are progressive and largely untreatable disorders with poorly understood molecular mechanisms. Both the genetics and the histologic type of IIP play a role in the etiology and pathogenesis of interstitial lung disease, but transcriptional signatures of these subtypes are unknown.Objectives: To evaluate gene expression in the lung tissue of patients with usual interstitial pneumonia or nonspecific interstitial pneumonia that was either familial or nonfamilial in origin, and to compare it with gene expression in normal lung parenchyma.Methods: We profiled RNA from the lungs of 16 patients with sporadic IIP, 10 with familial IIP, and 9 normal control subjects on a whole human genome oligonucleotide microarray.Results: Significant transcriptional differences exist in familial and sporadic IIPs. The genes distinguishing the genetic subtypes belong to the same functional categories as transcripts that distinguish IIP from normal samples. Relevant categories include chemokines and growth factors and their receptors, complement components, genes associated with cell proliferation and death, and genes in the Wnt pathway. The role of the chemokine CXCL12 in disease pathogenesis was confirmed in the murine bleomycin model of lung injury, with C57BL/6(CXCR4+/-) mice demonstrating significantly less collagen deposition than C57BL/6(CXCR4+/+) mice. Whereas substantial differences exist between familial and sporadic IIPs, we identified only minor gene expression changes between usual interstitial pneumonia and nonspecific interstitial pneumonia.Conclusions: Taken together, our findings indicate that differences in gene expression profiles between familial and sporadic IIPs may provide clues to the etiology and pathogenesis of IIP. [ABSTRACT FROM AUTHOR]

Background/objectives: Prenatal exposure to ambient air pollution is associated with adverse cardiometabolic outcomes in childhood. We previously observed that prenatal black carbon (BC) was inversely associated with adiponectin, a hormone secreted by adipocytes, in early childhood. Changes to DNA methylation have been proposed as a potential mediator linking in utero exposures to lasting health impacts., Methods: Among 532 mother-child pairs enrolled in the Colorado-based Healthy Start study, we performed an epigenome-wide association study of the relationship between prenatal exposure to a component of air pollution, BC, and DNA methylation in cord blood. Average pregnancy ambient BC was estimated at the mother's residence using a spatiotemporal prediction model. DNA methylation was measured using the Illumina 450K array. We used multiple linear regression to estimate associations between prenatal ambient BC and 429,246 cysteine-phosphate-guanine sites (CpGs), adjusting for potential confounders. We identified differentially methylated regions (DMRs) using DMRff and ENmix-combp. In a subset of participants (n = 243), we investigated DNA methylation as a potential mediator of the association between prenatal ambient BC and lower adiponectin in childhood., Results: We identified 44 CpGs associated with average prenatal ambient BC after correcting for multiple testing. Several genes annotated to the top CpGs had reported functions in the immune system. There were 24 DMRs identified by both DMRff and ENmix-combp. One CpG (cg01123250), located on chromosome 2 and annotated to the UNC80 gene, was found to mediate approximately 20% of the effect of prenatal BC on childhood adiponectin, though the confidence interval was wide (95% CI: 3, 84)., Conclusions: Prenatal BC was associated with DNA methylation in cord blood at several sites and regions in the genome. DNA methylation may partially mediate associations between prenatal BC and childhood cardiometabolic outcomes., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Background: Sarcoidosis is a heterogeneous granulomatous disease with no accurate biomarkers of disease progression. Therefore, we profiled and integrated the DNA methylome, mRNAs, and microRNAs to identify molecular changes associated with sarcoidosis and disease progression that might illuminate underlying mechanisms of disease and potential biomarkers., Methods: Bronchoalveolar lavage cells from 64 sarcoidosis subjects and 16 healthy controls were used. DNA methylation was profiled on Illumina HumanMethylationEPIC arrays, mRNA by RNA-sequencing, and miRNAs by small RNA-sequencing. Linear models were fit to test for effect of sarcoidosis diagnosis and progression phenotype, adjusting for age, sex, smoking, and principal components of the data. We built a supervised multi-omics model using a subset of features from each dataset., Results: We identified 1,459 CpGs, 64 mRNAs, and five miRNAs associated with sarcoidosis versus controls and four mRNAs associated with disease progression. Our integrated model emphasized the prominence of the PI3K/AKT1 pathway, which is important in T cell and mTOR function. Novel immune related genes and miRNAs including LYST, RGS14, SLFN12L, and hsa-miR-199b-5p, distinguished sarcoidosis from controls. Our integrated model also demonstrated differential expression/methylation of IL20RB, ABCC11, SFSWAP, AGBL4, miR-146a-3p, and miR-378b between non-progressive and progressive sarcoidosis., Conclusions: Leveraging the DNA methylome, transcriptome, and miRNA-sequencing in sarcoidosis BAL cells, we detected widespread molecular changes associated with disease, many which are involved in immune response. These molecules may serve as diagnostic/prognostic biomarkers and/or drug targets, although future testing is required for confirmation., (© 2024. The Author(s).)

Background: Type 1 diabetes (T1D) is preceded by a heterogenous pre-clinical phase, islet autoimmunity (IA). We aimed to identify pre vs. post-IA seroconversion (SV) changes in DNAm that differed across three IA progression phenotypes, those who lose autoantibodies (reverters), progress to clinical T1D (progressors), or maintain autoantibody levels (maintainers)., Methods: This epigenome-wide association study (EWAS) included longitudinal DNAm measurements in blood (Illumina 450K and EPIC) from participants in Diabetes Autoimmunity Study in the Young (DAISY) who developed IA, one or more islet autoantibodies on at least two consecutive visits. We compared reverters - individuals who sero-reverted, negative for all autoantibodies on at least two consecutive visits and did not develop T1D (n=41); maintainers - continued to test positive for autoantibodies but did not develop T1D (n=60); progressors - developed clinical T1D (n=42). DNAm data were measured before (pre-SV visit) and after IA (post-SV visit). Linear mixed models were used to test for differences in pre- vs post-SV changes in DNAm across the three groups. Linear mixed models were also used to test for group differences in average DNAm. Cell proportions, age, and sex were adjusted for in all models. Median follow-up across all participants was 15.5 yrs. (interquartile range (IQR): 10.8-18.7)., Results: The median age at the pre-SV visit was 2.2 yrs. (IQR: 0.8-5.3) in progressors, compared to 6.0 yrs. (IQR: 1.3-8.4) in reverters, and 5.7 yrs. (IQR: 1.4-9.7) in maintainers. Median time between the visits was similar in reverters 1.4 yrs. (IQR: 1-1.9), maintainers 1.3 yrs. (IQR: 1.0-2.0), and progressors 1.8 yrs. (IQR: 1.0-2.0). Changes in DNAm, pre- vs post-SV, differed across the groups at one site (cg16066195) and 11 regions. Average DNAm (mean of pre- and post-SV) differed across 22 regions., Conclusion: Differentially changing DNAm regions were located in genomic areas related to beta cell function, immune cell differentiation, and immune cell function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Carry, Vanderlinden, Johnson, Buckner, Steck, Kechris, Yang, Fingerlin, Fiehn, Rewers and Norris.)

Asthma has striking disparities across ancestral groups, but the molecular underpinning of these differences is poorly understood and minimally studied. A goal of the Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) is to understand multi-omic signatures of asthma focusing on populations of African ancestry. RNASeq and DNA methylation data are generated from nasal epithelium including cases (current asthma, N = 253) and controls (never-asthma, N = 283) from 7 different geographic sites to identify differentially expressed genes (DEGs) and gene networks. We identify 389 DEGs; the top DEG, FN1, was downregulated in cases (q = 3.26 × 10 -9 ) and encodes fibronectin which plays a role in wound healing. The top three gene expression modules implicate networks related to immune response (CEACAM5; p = 9.62 × 10 -16 and CPA3; p = 2.39 × 10 -14 ) and wound healing (FN1; p = 7.63 × 10 -9 ). Multi-omic analysis identifies FKBP5, a co-chaperone of glucocorticoid receptor signaling known to be involved in drug response in asthma, where the association between nasal epithelium gene expression is likely regulated by methylation and is associated with increased use of inhaled corticosteroids. This work reveals molecular dysregulation on three axes - increased Th2 inflammation, decreased capacity for wound healing, and impaired drug response - that may play a critical role in asthma within the African Diaspora., (© 2024. The Author(s).)

Genome-wide association studies (GWAS) have become well-powered to detect loci associated with telomere length. However, no prior work has validated genes nominated by GWAS to examine their role in telomere length regulation. We conducted a multi-ancestry meta-analysis of 211,369 individuals and identified five novel association signals. Enrichment analyses of chromatin state and cell-type heritability suggested that blood/immune cells are the most relevant cell type to examine telomere length association signals. We validated specific GWAS associations by overexpressing KBTBD6 or POP5 and demonstrated that both lengthened telomeres. CRISPR/Cas9 deletion of the predicted causal regions in K562 blood cells reduced expression of these genes, demonstrating that these loci are related to transcriptional regulation of KBTBD6 and POP5. Our results demonstrate the utility of telomere length GWAS in the identification of telomere length regulation mechanisms and validate KBTBD6 and POP5 as genes affecting telomere length regulation., (© 2024. The Author(s).)

Maternal educational attainment (MEA) shapes offspring health through multiple potential pathways. Differential DNA methylation may provide a mechanistic understanding of these long-term associations. We aimed to quantify the associations of MEA with offspring DNA methylation levels at birth, in childhood and in adolescence. Using 37 studies from high-income countries, we performed meta-analysis of epigenome-wide association studies (EWAS) to quantify the associations of completed years of MEA at the time of pregnancy with offspring DNA methylation levels at birth (n = 9 881), in childhood (n = 2 017), and adolescence (n = 2 740), adjusting for relevant covariates. MEA was found to be associated with DNA methylation at 473 cytosine-phosphate-guanine sites at birth, one in childhood, and four in adolescence. We observed enrichment for findings from previous EWAS on maternal folate, vitamin-B 12 concentrations, maternal smoking, and pre-pregnancy BMI. The associations were directionally consistent with MEA being inversely associated with behaviours including smoking and BMI. Our findings form a bridge between socio-economic factors and biology and highlight potential pathways underlying effects of maternal education. The results broaden our understanding of bio-social associations linked to differential DNA methylation in multiple early stages of life. The data generated also offers an important resource to help a more precise understanding of the social determinants of health., (© 2023. The Author(s).)

Aim: Assess if cord blood differentially methylated regions (DMRs) representing human metastable epialleles (MEs) associate with offspring adiposity in 588 maternal-infant dyads from the Colorado Health Start Study. Materials & methods: DNA methylation was assessed via the Illumina 450K array (~439,500 CpG sites). Offspring adiposity was obtained via air displacement plethysmography. Linear regression modeled the association of DMRs potentially representing MEs with adiposity. Results & conclusion: We identified two potential MEs, ZFP57 , which associated with infant adiposity change and B4GALNT4 , which associated with infancy and childhood adiposity change. Nine DMRs annotating to genes that annotated to MEs associated with change in offspring adiposity (false discovery rate <0.05). Methylation of approximately 80% of DMRs identified associated with decreased change in adiposity.

Objective: Fetal exposures may impact offspring epigenetic signatures and adiposity. The authors hypothesized that maternal metabolic traits associate with cord blood DNA methylation, which, in turn, associates with child adiposity., Methods: Fasting serum was obtained in 588 pregnant women (27-34 weeks' gestation), and insulin, glucose, high-density lipoprotein cholesterol, triglycerides, and free fatty acids were measured. Cord blood DNA methylation and child adiposity were measured at birth, 4-6 months, and 4-6 years. The association of maternal metabolic traits with DNA methylation (429,246 CpGs) for differentially methylated probes (DMPs) and regions (DMRs) was tested. The association of the first principal component of each DMR with child adiposity was tested, and mediation analysis was performed., Results: Maternal triglycerides were associated with the most DMPs and DMRs of all traits tested (261 and 198, respectively, false discovery rate < 0.05). DMRs were near genes involved in immune function and lipid metabolism. Triglyceride-associated CpGs were associated with child adiposity at 4-6 months (32 CpGs) and 4-6 years (2 CpGs). One, near CD226, was observed at both timepoints, mediating 10% and 22% of the relationship between maternal triglycerides and child adiposity at 4-6 months and 4-6 years, respectively., Conclusions: DNA methylation may play a role in the association of maternal triglycerides and child adiposity., (© 2023 The Authors. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society.)

The placenta undergoes many changes throughout gestation to support the evolving needs of the foetus. There is also a growing appreciation that male and female foetuses develop differently in utero , with unique epigenetic changes in placental tissue. Here, we report meta-analysed sex-specific associations between gestational age and placental DNA methylation from four cohorts in the National Institutes of Health (NIH) Environmental influences on Child Health Outcomes (ECHO) Programme (355 females/419 males, gestational ages 23-42 weeks). We identified 407 cytosine-guanine dinucleotides (CpGs) in females and 794 in males where placental methylation levels were associated with gestational age. After cell-type adjustment, 55 CpGs in females and 826 in males were significant. These were enriched for biological processes critical to the immune system in females and transmembrane transport in males. Our findings are distinct between the sexes: in females, associations with gestational age are largely explained by differences in placental cellular composition, whereas in males, gestational age is directly associated with numerous alterations in methylation levels.

Background: Idiopathic pulmonary fibrosis (IPF) is a heterogeneous disease that is pathologically characterized by areas of normal-appearing lung parenchyma, active fibrosis (transition zones including fibroblastic foci) and dense fibrosis. Defining transcriptional differences between these pathologically heterogeneous regions of the IPF lung is critical to understanding the distribution and extent of fibrotic lung disease and identifying potential therapeutic targets. Application of a spatial transcriptomics platform would provide more detailed spatial resolution of transcriptional signals compared to previous single cell or bulk RNA-Seq studies., Methods: We performed spatial transcriptomics using GeoMx Nanostring Digital Spatial Profiling on formalin-fixed paraffin-embedded (FFPE) tissue from 32 IPF and 12 control subjects and identified 231 regions of interest (ROIs). We compared normal-appearing lung parenchyma and airways between IPF and controls with histologically normal lung tissue, as well as histologically distinct regions within IPF (normal-appearing lung parenchyma, transition zones containing fibroblastic foci, areas of dense fibrosis, and honeycomb epithelium metaplasia)., Results: We identified 254 differentially expressed genes (DEGs) between IPF and controls in histologically normal-appearing regions of lung parenchyma; pathway analysis identified disease processes such as EIF2 signaling (important for cap-dependent mRNA translation), epithelial adherens junction signaling, HIF1α signaling, and integrin signaling. Within IPF, we identified 173 DEGs between transition and normal-appearing lung parenchyma and 198 DEGs between dense fibrosis and normal lung parenchyma; pathways dysregulated in both transition and dense fibrotic areas include EIF2 signaling pathway activation (upstream of endoplasmic reticulum (ER) stress proteins ATF4 and CHOP) and wound healing signaling pathway deactivation. Through cell deconvolution of transcriptome data and immunofluorescence staining, we confirmed loss of alveolar parenchymal signals (AGER, SFTPB, SFTPC), gain of secretory cell markers (SCGB3A2, MUC5B) as well as dysregulation of the upstream regulator ATF4, in histologically normal-appearing tissue in IPF., Conclusions: Our findings demonstrate that histologically normal-appearing regions from the IPF lung are transcriptionally distinct when compared to similar lung tissue from controls with histologically normal lung tissue, and that transition zones and areas of dense fibrosis within the IPF lung demonstrate activation of ER stress and deactivation of wound healing pathways., (© 2023. The Author(s).)

The study provides insights into proteins that may be relevant in BeS and CBD. It provides a framework to investigate the global changes in lung compartment-specific inflammatory cells to better understand the potential interplay of proteins in CBD. https://bit.ly/3PLNTXC., Competing Interests: Conflict of interest: L. Li received support for the present manuscript from NIH HHS/USA grants R01ES023826, R01ES033678, R01ES025722, R01ES034767 and K01ES020857. Conflict of interest: I.V. Yang received support for the present manuscript from NIH HHS/USA grants R01ES023826, R01ES033678 and R01ES025722; consulting fees were received from Eleven P15, outside the submitted work; and she is an unpaid Chair for the American Thoracic Society Section on Genetics and Genomics, outside the submitted work. Conflict of interest: L.A. Maier received support for the present manuscript from NIH HHS/USA grants R01ES023826, R01ES033678 and R01ES025722. Conflict of interest: M. Bhargava received support for the present manuscript from NIH HHS/USA grant R01ES025722; and grants or contracts from R01HL153613 (Comprehensive Proteomic Classifier for the Molecular Characterization of Pulmonary Sarcoidosis; PI M. Bhargava, MPI Maier), KIN-1902-2001 (A Randomized, Double-blind, Placebo-controlled Phase 2 Study with Open Label Extension to Assess the Efficacy and Safety of Namilumab in Subjects with Chronic Pulmonary Sarcoidosis; Site PI M. Bhargava); FSR Pilot Grant (Comprehensive Assessment Of Signal Transduction Pathways in Sarcoidosis; PI M. Bhargava) ATYR1923-C-004 (A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study to Evaluate the Efficacy and Safety of Intravenous Efzofitimod in Patients with Pulmonary Sarcoidosis; Site PI M. Bhargava), Chest Foundation (Inflammatory Protein Panel for Sarcoidosis Diagnosis and Prognosis; PI M. Bhargava), outside the submitted work; and has patents planned, issued or pending (Ingbar D, Rich T, Schumacher R, et al. (2022). Composition and Methods for Treating Pulmonary Edema or Lung Inflammation. American Inventors), outside the submitted work. Conflict of interest: The remaining authors have nothing to disclose., (Copyright ©The authors 2023.)

Objective: Recent advances in epigenetic studies continue to reveal novel mechanisms of gene regulation and control, however little is known on the role of epigenetics in sensorineural hearing loss (SNHL) in humans. We aimed to investigate the methylation patterns of two regions, one in RB1 and another in GJB2 in Filipino patients with SNHL compared to hearing control individuals., Methods: We investigated an RB1 promoter region that was previously identified as differentially methylated in children with SNHL and lead exposure. Additionally, we investigated a sequence in an enhancer-like region within GJB2 that contains four CpGs in close proximity. Bisulfite conversion was performed on salivary DNA samples from 15 children with SNHL and 45 unrelated ethnically-matched individuals. We then performed methylation-specific real-time PCR analysis (qMSP) using TaqMan ® probes to determine percentage methylation of the two regions., Results: Using qMSP, both our cases and controls had zero methylation at the targeted GJB2 and RB1 regions., Conclusion: Our study showed no changes in methylation at the selected CpG regions in RB1 and GJB2 in the two comparison groups with or without SNHL. This may be due to a lack of environmental exposures to these target regions. Other epigenetic marks may be present around these regions as well as those of other HL-associated genes., Competing Interests: Author Disclosure All authors declared no conflicts of interest.

Background: Seasonal variations in environmental exposures at birth or during gestation are associated with numerous adult traits and health outcomes later in life. Whether DNA methylation (DNAm) plays a role in the molecular mechanisms underlying the associations between birth season and lifelong phenotypes remains unclear., Methods: We carried out epigenome-wide meta-analyses within the Pregnancy And Childhood Epigenetic Consortium to identify associations of DNAm with birth season, both at differentially methylated probes (DMPs) and regions (DMRs). Associations were examined at two time points: at birth (21 cohorts, N = 9358) and in children aged 1-11 years (12 cohorts, N = 3610). We conducted meta-analyses to assess the impact of latitude on birth season-specific associations at both time points., Results: We identified associations between birth season and DNAm (False Discovery Rate-adjusted p values < 0.05) at two CpGs at birth (winter-born) and four in the childhood (summer-born) analyses when compared to children born in autumn. Furthermore, we identified twenty-six differentially methylated regions (DMR) at birth (winter-born: 8, spring-born: 15, summer-born: 3) and thirty-two in childhood (winter-born: 12, spring and summer: 10 each) meta-analyses with few overlapping DMRs between the birth seasons or the two time points. The DMRs were associated with genes of known functions in tumorigenesis, psychiatric/neurological disorders, inflammation, or immunity, amongst others. Latitude-stratified meta-analyses [higher (≥ 50°N), lower (< 50°N, northern hemisphere only)] revealed differences in associations between birth season and DNAm by birth latitude. DMR analysis implicated genes with previously reported links to schizophrenia (LAX1), skin disorders (PSORS1C, LTB4R), and airway inflammation including asthma (LTB4R), present only at birth in the higher latitudes (≥ 50°N)., Conclusions: In this large epigenome-wide meta-analysis study, we provide evidence for (i) associations between DNAm and season of birth that are unique for the seasons of the year (temporal effect) and (ii) latitude-dependent variations in the seasonal associations (spatial effect). DNAm could play a role in the molecular mechanisms underlying the effect of birth season on adult health outcomes., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Per- and polyfluoroalkyl substances (PFAS) are ubiquitous, environmentally persistent chemicals, and prenatal exposures have been associated with adverse child health outcomes. Prenatal PFAS exposure may lead to epigenetic age acceleration (EAA), defined as the discrepancy between an individual's chronologic and epigenetic or biological age., Objectives: We estimated associations of maternal serum PFAS concentrations with EAA in umbilical cord blood DNA methylation using linear regression, and a multivariable exposure-response function of the PFAS mixture using Bayesian kernel machine regression., Methods: Five PFAS were quantified in maternal serum (median: 27 weeks of gestation) among 577 mother-infant dyads from a prospective cohort. Cord blood DNA methylation data were assessed with the Illumina HumanMethylation450 array. EAA was calculated as the residuals from regressing gestational age on epigenetic age, calculated using a cord-blood specific epigenetic clock. Linear regression tested for associations between each maternal PFAS concentration with EAA. Bayesian kernel machine regression with hierarchical selection estimated an exposure-response function for the PFAS mixture., Results: In single pollutant models we observed an inverse relationship between perfluorodecanoate (PFDA) and EAA (-0.148 weeks per log-unit increase, 95% CI: -0.283, -0.013). Mixture analysis with hierarchical selection between perfluoroalkyl carboxylates and sulfonates indicated the carboxylates had the highest group posterior inclusion probability (PIP), or relative importance. Within this group, PFDA had the highest conditional PIP. Univariate predictor-response functions indicated PFDA and perfluorononanoate were inversely associated with EAA, while perfluorohexane sulfonate had a positive association with EAA., Conclusions: Maternal mid-pregnancy serum concentrations of PFDA were negatively associated with EAA in cord blood, suggesting a pathway by which prenatal PFAS exposures may affect infant development. No significant associations were observed with other PFAS. Mixture models suggested opposite directions of association between perfluoroalkyl sulfonates and carboxylates. Future studies are needed to determine the importance of neonatal EAA for later child health outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD) are clinically and molecularly heterogeneous diseases. We utilized clustering and integrative network analyses to elucidate roles for microRNAs (miRNAs) and miRNA isoforms (isomiRs) in COPD and ILD pathogenesis. Short RNA sequencing was performed on 351 lung tissue samples of COPD (n = 145), ILD (n = 144) and controls (n = 64). Five distinct subclusters of samples were identified including 1 COPD-predominant cluster and 2 ILD-predominant clusters which associated with different clinical measurements of disease severity. Utilizing 262 samples with gene expression and SNP microarrays, we built disease-specific genetic and expression networks to predict key miRNA regulators of gene expression. Members of miR-449/34 family, known to promote airway differentiation by repressing the Notch pathway, were among the top connected miRNAs in both COPD and ILD networks. Genes associated with miR-449/34 members in the disease networks were enriched among genes that increase in expression with airway differentiation at an air-liquid interface. A highly expressed isomiR containing a novel seed sequence was identified at the miR-34c-5p locus. 47% of the anticorrelated predicted targets for this isomiR were distinct from the canonical seed sequence for miR-34c-5p. Overexpression of the canonical miR-34c-5p and the miR-34c-5p isomiR with an alternative seed sequence down-regulated NOTCH1 and NOTCH4. However, only overexpression of the isomiR down-regulated genes involved in Ras signaling such as CRKL and GRB2. Overall, these findings elucidate molecular heterogeneity inherent across COPD and ILD patients and further suggest roles for miR-34c in regulating disease-associated gene-expression., (© 2023. Springer Nature Limited.)

Objective: Fat content of adipocytes derived from infant umbilical cord mesenchymal stem cells (MSCs) predicts adiposity in children through 4 to 6 years of age. This study tested the hypothesis that MSCs from infants born to mothers with obesity (Ob-MSCs) exhibit adipocyte hypertrophy and perturbations in genes regulating adipogenesis compared with MSCs from infants of mothers with normal weight (NW-MSCs)., Methods: Adipogenesis was induced in MSCs embedded in three-dimensional hydrogel structures, and cell size and number were measured by three-dimensional imaging. Proliferation and protein markers of proliferation and adipogenesis in undifferentiated and adipocyte differentiating cells were measured. RNA sequencing was performed to determine pathways linked to adipogenesis phenotype., Results: In undifferentiated MSCs, greater zinc finger protein (Zfp)423 protein content was observed in Ob- versus NW-MSCs. Adipocytes from Ob-MSCs were larger but fewer than adipocytes from NW-MSCs. RNA sequencing analysis showed that Zfp423 protein correlated with mRNA expression of genes enriched for cell cycle, MSC lineage specification, inflammation, and metabolism pathways. MSC proliferation was not different before differentiation but declined faster in Ob-MSCs upon adipogenic induction., Conclusions: Ob-MSCs have an intrinsic propensity for adipocyte hypertrophy and reduced hyperplasia during adipogenesis in vitro, perhaps linked to greater Zfp423 content and changes in cell cycle pathway gene expression., (© 2023 The Authors. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society.)

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., Competing Interests: Competing interests: ART, AJY and WAF are employees and shareholders of GlaxoSmithKline. LVW reports recent and current research grant funding from GlaxoSmithKline and Orion and consultancy fees from Galapagos. PLM reports recent and current research grant funding from AstraZeneca, consulting fees from Hoffman-La Roche, Boehringer Ingelheim and AstraZeneca and speaker fees from Boehringer Ingelheim and Hoffman-La Roche. TM reports consulting fees from Boehringer Ingelheim, Roche/Genentech, AstraZeneca, Bayer, Blade Therapeutics, Bristol-Myers Squibb, Galapagos, Galecto, GlaxoSmithKline, IQVIA, Pliant, Respivant, Theravance and Veracyte and speaker fees from Boehringer Ingelheim and Roche/Genentech. IVY reports consulting fees from Eleven P15 and is co-chair for the ATS Section of Genetics and Genomics. DAS is a consultant for Vertex Pharmaceuticals and is the founder and chief scientific officer of Eleven P15, a company focused on the early diagnosis and treatment of pulmonary fibrosis. IN reports consulting fees from Boerhinger Ingelheim, Genentech and Sanofi Aventis and participation on the Yale Data Safety Monitoring Board. GJ reports research grant funding from AstraZeneca, Biogen, Galecto, GlaxoSmithKline, RedX and Pliant, consulting fees from Bristol Myers Squibb, Daewoong, Veracyte, Resolution Therapeutics and Pliant, speaker fees from Chiesi, Roche, PatientMPower and AstraZeneca, participation on Boehringer Ingelheim, Galapagos and Vicore data advisory boards and is a trustee for Action for Pulmonary Fibrosis., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Rationale: Idiopathic pulmonary fibrosis (IPF) is a rare, irreversible, and progressive disease of the lungs. Common genetic variants, in addition to nongenetic factors, have been consistently associated with IPF. Rare variants identified by candidate gene, family-based, and exome studies have also been reported to associate with IPF. However, the extent to which rare variants, genome-wide, may contribute to the risk of IPF remains unknown. Objectives: We used whole-genome sequencing to investigate the role of rare variants, genome-wide, on IPF risk. Methods: As part of the Trans-Omics for Precision Medicine Program, we sequenced 2,180 cases of IPF. Association testing focused on the aggregated effect of rare variants (minor allele frequency ⩽0.01) within genes or regions. We also identified individual rare variants that are influential within genes and estimated the heritability of IPF on the basis of rare and common variants. Measurements and Main Results: Rare variants in both TERT and RTEL1 were significantly associated with IPF. A single rare variant in each of the TERT and RTEL1 genes was found to consistently influence the aggregated test statistics. There was no significant evidence of association with other previously reported rare variants. The SNP heritability of IPF was estimated to be 32% (SE = 3%). Conclusions: Rare variants within the TERT and RTEL1 genes and well-established common variants have the largest contribution to IPF risk overall. Efforts in risk profiling or the development of therapies for IPF that focus on TERT , RTEL1 , common variants, and environmental risk factors are likely to have the largest impact on this complex disease.

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.

The gain-of-function minor allele of the MUC5B (mucin 5B, oligomeric mucus/gel-forming) promoter (rs35705950) is the strongest risk factor for idiopathic pulmonary fibrosis (IPF), a devastating fibrotic lung disease that leads to progressive respiratory failure in adults. We have previously demonstrated that Muc5b overexpression in mice worsens lung fibrosis after bleomycin exposure and have hypothesized that excess Muc5b promotes endoplasmic reticulum (ER) stress and apoptosis, stimulating fibrotic lung injury. Here, we report that ER stress pathway members ATF4 (activating transcription factor 4) and ATF6 coexpress with MUC5B in epithelia of the distal IPF airway and honeycomb cyst and that this is more pronounced in carriers of the gain-of-function MUC5B promoter variant. Similarly, in mice exposed to bleomycin, Muc5b expression is temporally associated with markers of ER stress. Using bulk and single-cell RNA sequencing in bleomycin-exposed mice, we found that pathologic ER stress-associated transcripts Atf4 and Ddit3 (DNA damage inducible transcript 3) were elevated in alveolar epithelia of SFTPC -Muc5b transgenic ( SFTPC -Muc5b Tg ) mice relative to wild-type (WT) mice. Activation of the ER stress response inhibits protein translation for most genes by phosphorylation of Eif2α (eukaryotic translation initiation factor 2 alpha), which prevents guanine exchange by Eif2B and facilitates translation of Atf4 . The integrated stress response inhibitor (ISRIB) facilitates interaction of phosphorylated Eif2α with Eif2B, overcoming translation inhibition associated with ER stress and reducing Atf4 . We found that a single dose of ISRIB diminished Atf4 translation in SFTPC -Muc5b Tg mice after bleomycin injury. Moreover, ISRIB resolved the exaggerated fibrotic response of SFTPC -Muc5b Tg mice to bleomycin. In summary, we demonstrate that MUC5B and Muc5b expression is associated with pathologic ER stress and that restoration of normal translation with a single dose of ISRIB promotes lung repair in bleomycin-injured Muc5b-overexpressing mice.

Given the differential risk of type 1 diabetes (T1D) in offspring of affected fathers versus affected mothers and our observation that T1D cases have differential DNA methylation near the imprinted DLGAP2 gene compared to controls, we examined whether methylation near DLGAP2 mediates the association between T1D family history and T1D risk. In a nested case-control study of 87 T1D cases and 87 controls from the Diabetes Autoimmunity Study in the Young, we conducted causal mediation analyses at 12 DLGAP2 region CpGs to decompose the effect of family history on T1D risk into indirect and direct effects. These effects were estimated from two regression models adjusted for the human leukocyte antigen DR3/4 genotype: a linear regression of family history on methylation (mediator model) and a logistic regression of family history and methylation on T1D (outcome model). For 8 of the 12 CpGs, we identified a significant interaction between T1D family history and methylation on T1D risk. Accounting for this interaction, we found that the increased risk of T1D for children with affected mothers compared to those with no family history was mediated through differences in methylation at two CpGs (cg27351978, cg00565786) in the DLGAP2 region, as demonstrated by a significant pure natural indirect effect (odds ratio (OR) = 1.98, 95% confidence interval (CI): 1.06-3.71) and nonsignificant total natural direct effect (OR = 1.65, 95% CI: 0.16-16.62) (for cg00565786). In contrast, the increased risk of T1D for children with an affected father or sibling was not explained by DNA methylation changes at these CpGs. Results were similar for cg27351978 and robust in sensitivity analyses. Lastly, we found that DNA methylation in the DLGAP2 region was associated ( P <0:05) with gene expression of nearby protein-coding genes DLGAP2 , ARHGEF10 , ZNF596 , and ERICH1 . Results indicate that the maternal protective effect conferred through exposure to T1D in utero may operate through changes to DNA methylation that have functional downstream consequences., Competing Interests: Conflicts of Interest The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work.

Chronic beryllium disease (CBD) is a Th1 granulomatous lung disease preceded by sensitization to beryllium (BeS). We profiled the methylome, transcriptome, and selected proteins in the lung to identify molecular signatures and networks associated with BeS and CBD. BAL cell DNA and RNA were profiled using microarrays from CBD ( n  = 30), BeS ( n  = 30), and control subjects ( n  = 12). BAL fluid proteins were measured using Olink Immune Response Panel proteins from CBD ( n  = 22) and BeS ( n  = 22) subjects. Linear models identified features associated with CBD, adjusting for covariation and batch effects. Multiomic integration methods identified correlated features between datasets. We identified 1,546 differentially expressed genes in CBD versus control subjects and 204 in CBD versus BeS. Of the 101 shared transcripts, 24 have significant cis relationships between gene expression and DNA methylation, assessed using expression quantitative trait methylation analysis, including genes not previously identified in CBD. A multiomic model of top DNA methylation and gene expression features demonstrated that the first component separated CBD from other samples and the second component separated control subjects from remaining samples. The top features on component one were enriched for T-lymphocyte function, and the top features on component two were enriched for innate immune signaling. We identified six differentially abundant proteins in CBD versus BeS, with two (SIT1 and SH2D1A) selected as important RNA features in the multiomic model. Our integrated analysis of DNA methylation, gene expression, and proteins in the lung identified multiomic signatures of CBD that differentiated it from BeS and control subjects.

Rationale: Common genetic variants have been associated with idiopathic pulmonary fibrosis (IPF). Objectives: To determine functional relevance of the 10 IPF-associated common genetic variants we previously identified. Methods: We performed expression quantitative trait loci (eQTL) and methylation quantitative trait loci (mQTL) mapping, followed by co-localization of eQTL and mQTL with genetic association signals and functional validation by luciferase reporter assays. Illumina multi-ethnic genotyping arrays, mRNA sequencing, and Illumina 850k methylation arrays were performed on lung tissue of participants with IPF (234 RNA and 345 DNA samples) and non-diseased controls (188 RNA and 202 DNA samples). Measurements and Main Results: Focusing on genetic variants within 10 IPF-associated genetic loci, we identified 27 eQTLs in controls and 24 eQTLs in cases (false-discovery-rate-adjusted P  < 0.05). Among these signals, we identified associations of lead variants rs35705950 with expression of MUC5B and rs2076295 with expression of DSP in both cases and controls. mQTL analysis identified CpGs in gene bodies of MUC5B (cg17589883) and DSP (cg08964675) associated with the lead variants in these two loci. We also demonstrated strong co-localization of eQTL/mQTL and genetic signal in MUC5B (rs35705950) and DSP (rs2076295). Functional validation of the mQTL in MUC5B using luciferase reporter assays demonstrates that the CpG resides within a putative internal repressor element. Conclusions: We have established a relationship of the common IPF genetic risk variants rs35705950 and rs2076295 with respective changes in MUC5B and DSP expression and methylation. These results provide additional evidence that both MUC5B and DSP are involved in the etiology of IPF.

Background: Prenatal exposure to ambient air pollution has been associated with adverse offspring health outcomes. Childhood health effects of prenatal exposures may be mediated through changes to DNA methylation detectable at birth., Methods: Among 429 non-smoking women in a cohort study of mother-infant pairs in Colorado, USA, we estimated associations between prenatal exposure to ambient fine particulate matter (PM 2.5 ) and ozone (O 3 ), and epigenome-wide DNA methylation of umbilical cord blood cells at delivery (2010-2014). We calculated average PM 2.5 and O 3 in each trimester of pregnancy and the full pregnancy using inverse-distance-weighted interpolation. We fit linear regression models adjusted for potential confounders and cell proportions to estimate associations between air pollutants and methylation at each of 432,943 CpGs. Differentially methylated regions (DMRs) were identified using comb-p. Previously in this cohort, we reported positive associations between 3rd trimester O 3 exposure and infant adiposity at 5 months of age. Here, we quantified the potential for mediation of that association by changes in DNA methylation in cord blood., Results: We identified several DMRs for each pollutant and period of pregnancy. The greatest number of significant DMRs were associated with third trimester PM 2.5 (21 DMRs). No single CpGs were associated with air pollutants at a false discovery rate <0.05. We found that up to 8% of the effect of 3rd trimester O 3 on 5-month adiposity may be mediated by locus-specific methylation changes, but mediation estimates were not statistically significant., Conclusions: Differentially methylated regions in cord blood were identified in association with maternal exposure to PM 2.5 and O 3 . Genes annotated to the significant sites played roles in cardiometabolic disease, immune function and inflammation, and neurologic disorders. We found limited evidence of mediation by DNA methylation of associations between third trimester O 3 exposure and 5-month infant adiposity., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Common genetic variants explain less variation in complex phenotypes than inferred from family-based studies, and there is a debate on the source of this 'missing heritability'. We investigated the contribution of rare genetic variants to tobacco use with whole-genome sequences from up to 26,257 unrelated individuals of European ancestries and 11,743 individuals of African ancestries. Across four smoking traits, single-nucleotide-polymorphism-based heritability ([Formula: see text]) was estimated from 0.13 to 0.28 (s.e., 0.10-0.13) in European ancestries, with 35-74% of it attributable to rare variants with minor allele frequencies between 0.01% and 1%. These heritability estimates are 1.5-4 times higher than past estimates based on common variants alone and accounted for 60% to 100% of our pedigree-based estimates of narrow-sense heritability ([Formula: see text], 0.18-0.34). In the African ancestry samples, [Formula: see text] was estimated from 0.03 to 0.33 (s.e., 0.09-0.14) across the four smoking traits. These results suggest that rare variants are important contributors to the heritability of smoking., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of older adults characterized by fibrotic replacement of functional gas exchange units in the lung. The strongest risk factor for IPF is a genetic variantin the promoter region of the gel-forming mucin, MUC5B . To better understand how the MUC5B variant influences development of fibrosis, we used the NicheNet R package and leveraged publicly available single-cell RNA sequencing data to identify and evaluate how epithelia participating in gas exchange are influenced by ligands expressed in control, MUC5B variant, and fibrotic environments. We observed that loss of type-I alveolar epithelia (AECI) characterizes the single-cell RNA transcriptome in fibrotic lung and validated the pattern of AECI loss using single nuclear RNA sequencing. Examining AECI transcriptomes, we found enrichment of transcriptional signatures for IL6 and AREG, which we have previously shown to mediate aberrant epithelial fluidization in IPF and murine bleomycin models. Moreover, we found that the protease ADAM17, which is upstream of IL6 trans-signaling, was enriched in control MUC5B variant donors. We used immunofluorescence to validate a role for enhanced expression of ADAM17 among MUC5B variants, suggesting involvement in IPF pathogenesis and maintenance.

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-β1 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.

Asthma is a common complex respiratory disease characterized by chronic airway inflammation and partially reversible airflow obstruction resulting from genetic and environmental determinants. Because epigenetic marks influence gene expression and can be modified by both environmental exposures and genetic variation, they are increasingly recognized as relevant to the pathogenesis of asthma and may be a key link between environmental exposures and asthma susceptibility. Unlike changes to DNA sequence, epigenetic signatures are dynamic and reversible, creating an opportunity for not only therapeutic targets but may serve as biomarkers to follow disease course and identify molecular subtypes in heterogeneous diseases such as asthma. In this review, we will examine the relationship between asthma and 3 key epigenetic processes that modify gene expression: DNA methylation, modification of histone tails, and noncoding RNAs. In addition to presenting a comprehensive assessment of the existing epigenetic studies focusing on immune regulation in asthma, we will discuss future directions for epigenetic investigation in allergic airway disease., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

We previously identified a novel molecular subtype of idiopathic pulmonary fibrosis (IPF) defined by increased expression of cilium-associated genes, airway mucin gene MUC5B , and KRT5 marker of basal cell airway progenitors. Here we show the association of MUC5B and cilia gene expression in human IPF airway epithelial cells, providing further rationale for examining the role of cilium genes in the pathogenesis of IPF. We demonstrate increased multiciliogenesis and changes in motile cilia structure of multiciliated cells both in IPF and bleomycin lung fibrosis models. Importantly, conditional deletion of a cilium gene, Ift88 (intraflagellar transport 88), in Krt5 basal cells reduces Krt5 pod formation and lung fibrosis, whereas no changes are observed in Ift88 conditional deletion in club cell progenitors. Our findings indicate that aberrant injury-activated primary ciliogenesis and Hedgehog signaling may play a causative role in Krt5 pod formation, which leads to aberrant multiciliogenesis and lung fibrosis. This implies that modulating cilium gene expression in Krt5 cell progenitors is a potential therapeutic target for IPF.