Background Mepolizumab is effective for a subset of severe asthma patients in reducing exacerbation frequency. Identification of an early marker associated with long-term beneficial clinical response would facilitate better targeting of treatment. We set out to determine the impact of mepolizumab from a clinical, blood inflammatory cell and serum proteomics perspective and identify early changes in patients designated responder/non-responder at one year. Methods We enrolled asthma patients that met criteria to be prescribed mepolizumab. Patients had clinical and blood cell measurements at baseline, 12 and 26 weeks. Serum proteomics (Alamar NULISA, 250 panel) was completed at baseline and 12 weeks. Results 37 patients completed the study (24 and 13 responder and non-responder respectively at 1 year). Both Asthma Control Questionnaire (ACQ)-6 and Severe Asthma Questionnaire (SAQ) improved at 12 and 26 weeks and were driven by the responder group. A reduction in blood eosinophils and basophils was observed. There was a significant elevation of only 2/248 serum proteins, IL5 and CCL24 after 12 weeks mepolizumab treatment, with elevation being driven by the responder and non-responder groups respectively. A 12-week improvement in SAQ was associated with higher odds of response at one year (OR 6.75, 95% CI 1.42–24.30; p=0.015). Conclusions We identified early changes in the clinical and protein profile in patients that go onto have a clinically relevant response to mepolizumab providing opportunities for a more personalised asthma management.

Rationale:Idiopathic pulmonary fibrosis (IPF) is a rare, chronic, progressive lung disease with high mortality and few treatment options. Using an additive genetic model, genome-wide association studies (GWAS) have identified multiple risk loci highlighting new genes and pathways of interest. Since IPF risk could also be influenced by non-additive effects, we hypothesised that association analyses using alternative genetic models may provide additional mechanistic insight. Objectives:To perform GWAS of IPF susceptibility to detect associations where the underlying effects are consistent with recessive or dominant genetic models. Methods:We performed GWAS of IPF susceptibility, with logistic regression assuming dominant or recessive genetic models, including 5,159 IPF cases, from clinically-curated sources, and 27,459 controls. We functionally annotated independent signals and performed variant-to-gene mapping, applying fine-mapping to define potentially causal variants and genes. We assessed differential expression levels of genes of interest in publicly available single cell RNAseq data and in primary cells derived from IPF donors and controls. Main Results:We identified five genome-wide significant signals, under a recessive model, that had not been reported previously. These included exonic variants in the cell-cycle gene Polyamine-Modulated Factor 1 (PMF1) and in Epsin 3 (EPN3) genes. We also observed evidence of increased PMF1 expression in airway basal cells of IPF patients compared to controls. Conclusions:Using alternative genetic models in IPF susceptibility GWAS identified new signals and genes, providing new insights into IPF pathogenesis and potential future therapies.

Although breathing seems like a simple action, many individuals encounter difficulties under ever-changing and complex circumstances, which can become increasingly problematic as the individuals age. In this Review, we comprehensively assess the ageing respiratory system. We begin with the physiological structural changes and their effect on lung function associated with ageing, before exploring the genetic and molecular factors that contribute to the loss of respiratory function with age. Defining how biological ageing manifests in the respiratory system and understanding disease-driven hallmarks of accelerated ageing conditions will enable the development of interventions to improve health across age groups, which will be crucial to address population health challenges over the coming decades.

Background:Idiopathic pulmonary fibrosis (IPF) is a rare, incurable lung disease with a median survival of 3-5 years after diagnosis. Treatment options are limited. Genetic association studies can identify new genes involved in disease that might represent potential new drug targets, and it has been shown that drug targets with support from genetic studies are more likely to be successful in clinical development. Previous genome-wide association studies (GWAS) of IPF susceptibility have identified more than 20 signals implicating genes involved in multiple mechanisms, including telomere dysfunction, cell-cell adhesion, host defence immunity, various signalling pathways and, more recently, mitotic spindle assembly complex. Aim:To leverage new datasets and genotype imputation to discover further genes involved in development of IPF that could yield new pathobiological avenues for exploration and to guide future drug target discovery. Methods:We conducted a GWAS of IPF susceptibility including seven IPF case-control studies comprising 5,159 IPF cases and 27,459 controls of European ancestry, where IPF diagnosis was made by a respiratory clinician according to international guidelines. Genotypes were obtained from Whole Genome Sequencing (WGS) or from array-based imputation to the TOPMed WGS reference panel. New signals were replicated in independent biobanks with IPF defined using Electronic Healthcare Records. Bayesian fine-mapping was performed to identify the most likely causal variant(s) and bioinformatic investigation undertaken to map associated variants to putative causal genes. Results:We identified three novel genetic signals of association with IPF susceptibility. Genes prioritised by functional evidence at these signals included MUC1, which encodes a large transmembrane glycoprotein and known biomarker of lung fibrosis, and NTN4 encoding Netrin-4 whose known roles include angiogenesis. The third signal may map to SLC6A6, a taurine and beta-alanine transporter gene, previously implicated in retinal, cardiac and kidney dysfunction. Conclusion:Our study has identified new associations not previously identified by previous large biobank-based studies thereby highlighting the value of utilising clinically-curated IPF case-control studies, and new genotype imputation. We present new evidence for disease-driving roles of MUC1 and of endothelial cell and vascular changes in IPF.

1 Abstract 1.1 Background Many longitudinal omics studies contain only a small number of repeated measurements collected before, during, or after an intervention. Existing approaches, including mixed-effects models and generalized additive models, estimate temporal effects but do not generally provide a discrete representation of trajectory topology that can be queried directly across experimental groups. 1.2 Methods We developed LongOmicsTraj, an open-source R package for topology-based representation and querying of short longitudinal omics trajectories. The framework encodes the direction of change between adjacent visits as up , down , or flat , with the ordered sequence defining an Ordinal Trajectory State (OTS). LongOmicsTraj operates downstream of trajectory estimation and can therefore be applied to empirical summaries or model-derived visit-level estimates, including those from linear mixed-effects models, generalized additive models, and polynomial regression, following a maSigPro-style time-course formulation [1]. OTS labels provide a common representation for topology-based querying, cross-group comparison, and evaluation of higher-level representations such as trajectory clusters. We evaluated the framework using controlled simulations and bronchial biopsy transcriptomic data from the GLUCOLD corticosteroid intervention study (GEO accession GSE36221 ), measured at baseline, 6 months, and 30 months. The biological analysis compared continued inhaled corticosteroid (ICS) treatment, ICS withdrawal after 6 months, and placebo. 1.3 Results In simulations, LongOmicsTraj recovered predefined stable, monotonic, transient, rebound, and oscillatory trajectories with high accuracy when longitudinal signal was sufficiently clear, with performance declining under high-noise conditions and depending partly on the upstream estimator. In GLUCOLD, comparator-aware topology queries reduced 20,358 measured transcripts to 168 genes showing a corticosteroid response that was maintained during continued treatment, reversed following withdrawal, and was not reproduced under placebo. The selected genes included established corticosteroid-response genes and were enriched for immune-cell migration, chemotaxis, cell adhesion, and extracellular-matrix organisation. Topology-aware evaluation of FlexMix trajectory clusters additionally revealed substantial within-cluster temporal heterogeneity, with topology purities of approximately 46–60%. 1.4 Conclusions LongOmicsTraj provides a compact, directly queryable representation of temporal direction and order in short longitudinal omics studies. It complements existing longitudinal estimation and clustering methods by making trajectory structure explicit, enabling structured cross-group queries and quantification of temporal heterogeneity within trajectory clusters.

Abstract Rationale Chronic obstructive pulmonary disease (COPD) represents a leading cause of global morbidity and mortality. Genome-wide association studies (GWAS) have implicated numerous genetic variants in lung function impairment, yet confidently identifying the underlying genes and pathways, and translating these findings into mechanistic insight, remains a significant challenge. Objectives To leverage the genetic amenability and high-throughput screening capability of Drosophila melanogaster to determine the role of candidate causal genes in epithelial cell homeostasis. Methods We performed a loss-of-function analysis of 60 prioritised lung function candidate causal genes implicated from GWAS in two distinct epithelia: the dorsal thorax and trachea. Results We identified 57/60 tested candidate genes that alter at least one aspect of epithelial morphology and behaviour upon knockdown. With a focus on junctional integrity, cell delamination and tissue growth, we identified 11 genes for further study: Sec6, RpS26, pAbp, Arf102f, Riok1, Sra-1, Inpp5e, CG31759, ssh, eIF6 and Rtf1 . Further characterisation found a significant reduction in junctional E-Cadherin levels following Arf102F , Rtf1 , RioK1 and Sra-1 knockdown. Following a secondary screen in the Drosophila tracheal system for priority candidates, Sec6 and RpS26 were associated with significant airway defects and a reduction in larval body size. 8/11 priority genes exhibited differential lung gene expression between controls and patients with COPD. Conclusions These data demonstrate the amenability of Drosophila melanogaster to perform in vivo functional analyses of candidate causal genes at scale. Initial findings implicate several genes in epithelial homeostasis and integrity, providing new mechanistic understanding and potential therapeutic targets for COPD. Graphical Abstract

Background:In ∼10% of asthma patients, symptoms remain uncontrolled despite maximal treatment, representing an unmet clinical need. The causal variants, genes and pathways underlying genetic risk factors have not been fully elucidated, and it is unclear whether there are unique genetic risk factors for this asthma subtype. Methods:We used electronic healthcare records linked to UK Biobank to identify asthma patients with high treatment burden and/or worse outcomes. We performed a genome-wide association study (GWAS) with this case population and healthy controls. We sought replication for associated (p≤5×10-6) signals in four independent studies (12 152 cases and 32 316 controls). Replicated signals were fine-mapped and linked to genes and pathways. Results:In total, 7681 participants met our case definition and showed enrichment for adult-onset asthma, female gender and higher body mass index compared to asthma individuals not meeting case criteria. GWAS with 7681 cases and 38 405 controls revealed 21 reproducible association signals that had previously been associated with asthma, but had a larger effect size in our study. Variant-to-gene mapping highlighted 85 candidate genes, five of which were considered high confidence (BACH2, D2HGDH, IL1RL1, RPS26, SMAD3). Conclusion:We present the first use of electronic healthcare records in UK Biobank to identify a subtype of asthma enriched for patients with high treatment burden and/or worse outcomes. Our findings support the role of known asthma genes, highlighting genetic risk variants with stronger effect in these groups of patients. The prioritised genes provide potential therapeutic opportunities for this difficult-to-treat patient population.

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; differing environmental exposures due to historically sex-biased occupations and diagnostic bias are possible explanations. To date, over 20 independent genetic association signals have been reported for IPF susceptibility, but these have been discovered when combining males and females. The objectives of the present study were to assess whether there is a need to consider sex-specific effects when evaluating genetic risk in clinical prediction models for IPF and to test for sex-specific associations with IPF susceptibility. Methods:We performed a genome-wide single nucleotide polymorphism (SNP)-by-sex interaction study meta-analysis of IPF risk in six independent case-control studies comprising 4561 cases (1280 females, 3281 males) and 22 888 controls (8360 females, 14 528 males) of European genetic ancestry. We used polygenic risk scores (PRSs) comprising common (minor allele frequency >1%) autosomal variants to assess differences in genetic risk prediction between males and females. Results:The predictive accuracy of the PRSs were similar between males and females, regardless of whether using combined or sex-specific association results. Three new independent genetic association signals were identified (p<1×10-6). Conclusions:The predictive accuracy of common autosomal SNP-based PRSs did not vary significantly between males and females. We prioritised three genetic variants whose effect on IPF risk may be modified by sex. These findings would not account for the differences in prevalence between males and females. Future studies should ensure adequate representation of both sexes.

RATIONALE: Impaired lung function predicts mortality and is a diagnostic criterion for chronic obstructive pulmonary disease (COPD). Proteins are often the target of pharmacological interventions, therefore identifying causal links between proteins and lung function could inform understanding of COPD pathophysiology and suggest therapeutic targets. We aim to infer the potential impact of circulating protein levels on lung function, using strictly defined cis protein quantitative trait loci (cis-pQTLs) as genetic instrumental variables for Mendelian randomisation (MR). METHODS: We applied two-sample MR by integrating protein GWAS data (2,923 proteins, 48,195 UK Biobank European participants) with lung function GWAS data (four lung function traits, 149,166 European participants from 36 non-UK Biobank cohorts). We selected strictly defined cis-pQTLs, within 100 kilobase pairs of a transcription start site and strongly associated (P≤5×10-9) with protein levels, and applied single-cis-MR analysis (Wald ratio method). Sensitivity analyses included colocalization analysis (to distinguish causal effects from genomic confounding by linkage disequilibrium), and bidirectional MR to explore possible reverse causation. Replication analysis was conducted where possible. We used the Drug-Gene Interaction Database and phenome-wide association studies (PheWAS) to inform biological and clinical interpretation of identified proteins. RESULTS: We curated 1,841 proteins with a suitable cis-pQTL instrument, and evaluated evidence for causal effects of these proteins on four lung function traits. The single-cis MR analysis implicated 18 proteins for lung function at a Bonferroni-corrected threshold (Wald ratio estimator P<2.72×10-5). Of 10 proteins previously implicated by reported lung function signals, surfactant protein D (SFTPD) has been highlighted in previous respiratory MR analyses and variants in SFTPD have been previously reported to be associated with emphysema; our PheWAS suggested that this variant has a relatively specific effect on lung function as it was associated with no non-respiratory traits at a FDR<1%. In contrast to previous expression QTL evidence, our study suggested that ITGAV inhibition could reduce FEV1/FVC; we note that reduced lung function was also seen in a recent trial of an ITGAV inhibitor (NCT01371305). Our MR analysis implicated 8 novel proteins not implicated by previous GWAS (CCND2, DTD1, PILRA, PTPRK, TDRKH, GRHPR, NUDT5, SLITRK6); in our PheWAS the variants instrumenting these protein levels were associated with a wide range of traits. CONCLUSIONS: Our protein-based approach identified proteins that may be causally related for lung function variability. We highlight known protein drug targets, and identify several new proteins which are potentially therapeutic targets but warrant further follow up for potential utility and safety.

RATIONALE Lung function predicts mortality and is a diagnostic criterion for COPD. Identification of causal genes and the variants and pathways that impact gene function and regulation can inform therapeutic interventions for COPD. Genome-wide association studies (GWAS) of imputed genotypes discovered 1,020 mostly common genetic variants associated with lung function. Whole-exome sequencing (WES) is better suited to study rarer protein-coding variants that may not be well imputed. We analysed the UK Biobank WES data to identify putative causal genes for lung function, not yet detected by GWAS, and to fine map the architecture of causal variants within genes. METHODS We included 343,104 European UK Biobank samples with WES data and four quantitative lung function phenotypes: FEV1, FVC, FEV1/FVC and peak expiratory flow (PEF). We performed single variant tests of 6.7 million variants with minor allele frequency (MAF) <1% and gene-based collapsing tests to enhance power to detect rare variant effects in aggregate. For gene-based tests models were run using MAF filters of <1%, <0.1%, <0.01%, <0.001% and singletons, and 2 variant function criteria: (i) predicted loss-of-function (pLoF); (ii) pLoF + deleterious missense. Qualifying variants were tested in aggregate for 18,468 genes using burden testing and methods accommodating opposing effect directions. RESULTSHMCN1 (Hemicentin-1), previously implicated by a common (MAF 24%) intronic variant, harboured a novel rare (MAF 0.03%) missense variant associated with FEV1/FVC (P=4.78×10-9) and was highly significant in gene-based testing (P=3.27×10-51) with a burden of over 900 pLoF and missense variants of MAF<0.01% contributing. This was notable amongst the other 17 genes with a significant gene-based result (P<2.69×10-6; Bonferroni correction for genes tested), with the next most significant result for LRP1 (P=6.73×10-15) driven by only 2 variants. In total rare variant testing implicated 28 genes, 8 of which have not been previously reported in GWAS. CONCLUSION We discover novel genes associated with lung function and highlight novel variants at known genes. In particular, we show allelic series for lung function – that is, statistically independent genetic variants that each implicate the same gene and show dose-response effects on lung function, such as the common, rare and very rare variants independently implicating HMCN1. Allelic series provide strong evidence to inform functional genomic studies and drug discovery.

ABSTRACT Background Asthma is a heterogeneous disease characterized by overlapping clinical and inflammatory features. Objective This study aimed to provide insight into the systemic inflammatory profile in asthma, greater understanding of asthma endotypes and the contribution of genetic risk factors to both. Methods 4205 patients with asthma aged 16–60 were recruited from UK centers; serum cytokines were quantified from 708, including cytokines associated with Type 1, 2 and 17 inflammation. 3037 patients were genotyped for 25 single nucleotide polymorphisms associated with moderate‐severe asthma. Results Serum cytokines associated with Th2 inflammation showed high coordinated expression for example, IL‐4/IL‐5 (R2 = 0.513). The upper quartile of the serum cytokine data identified 43.7% of patients had high levels for multiple Th2 cytokines. However, the groups defined by serum cytokine profile were not clinically different. Childhood‐onset asthma was characterized by elevated total IgE, allergic rhinitis and dermatitis. Exacerbation prone patients had a higher BMI, smoking pack‐years, asthma control questionnaire score and reduced lung function. Patients with blood eosinophils of > 300 cells/µL had elevated total IgE and lower smoking pack‐years. None of these groups had a differential serum cytokine profile. Asthma risk alleles for; rs61816764 (FLG) and rs9303277 (IKFZ3) were associated with childhood onset disease (p = 2.67 × 10−4 and 2.20 × 10−7; retrospectively). No genetic variant was associated with cytokine levels. Conclusion Systemic inflammation in asthma is complex. Patients had multiple overlapping inflammatory profiles suggesting several disease mechanisms. Genetic risk factors for moderate‐severe asthma confirmed previous associations with childhood onset of asthma.

Rationale Genome-wide association studies (GWAS) identify new genomic signals that drive respiratory disease development. Drug targets supported by genetic studies, including GWAS, are twice as likely to be successful in clinical development [PMID:26121088]. However, a major challenge is prioritisation of GWAS signals and candidate causal genes for pre-clinical experimental follow-up. We identified 1,020 GWAS signals for lung function traits, implicating hundreds of genes and pathways [PMID:36914875]. We describe our prioritisation of non-coding GWAS signals using bioinformatics and CRISPR-based manipulation in a disease-relevant organoid model to identify candidate causal genes with confidence for further functional investigation. Methods Evidence from gene and protein expression datasets, functional annotation, rare variants and nearby genes implicated by Mendelian disease or mouse knockout phenotypes, were combined to map each of the 1020 signals to genes (Variant-to-Gene mapping, V2G). We integrated i) association data for additional respiratory traits (including asthma, IPF, COPD, P<1×10-3), ii) overlap with open chromatin (ATAC-seq, DNA footprinting) and enhancer RNA (eRNA), iii) functionally-informed fine-mapping and iv) significance of lung function association, to identify a long-list of signals for further characterisation. A subset of signals with the strongest V2G evidence (≥3 sources) were first evaluated for their effects on expression of their predicted target gene using CRISPR-interference (CRISPRi). Regions containing prioritised genetic variants were silenced in human alveolar type 2 cell organoids using CRISPRi with dCas9-KRAB and gRNA lentiviral constructs [PMID:34612202], followed by qRT-PCR to assess transcription. Results Of 1020 lung function GWAS signals, 135 had strong V2G mapping (≥3 sources of evidence), 270 were associated with additional respiratory traits, 96 overlapped with DNA footprints or eRNA and 1009 overlapped with open chromatin markers in lung cells. Forty-two signals were prioritised for further investigation based on one or more of those criteria. Of 12 signals prioritised by strong V2G only, CRISPRi-mediated silencing revealed that 9 affected the expression of the predicted target. For example, silencing rs34933034, resulted in an approximately 80% reduction of CSK expression compared to controls (CTRL=1.009±0.149, n=6; KD=0.198±0.226, n=8). In contrast, silencing rs12522114 increased MOCS2-DT expression sevenfold (CTRL=1.011±0.164, n=6; KD=7.271±8.641, n=9). Conclusions We prioritised a viable number of GWAS signals for CRISPRi and validated in silico V2G mapping in a disease-relevant organoid model. Our next tranche of CRISPRi experiments (ongoing) will use a newer dCas9-KRAB-MECP2 CRISPRi tool in single cells for Perturb-seq to evaluate global impacts on gene expression of GWAS signals with high functional priority but low V2G confidence.

Rationale Mepolizumab is effective for a subset of severe asthma patients in reducing exacerbation frequency. Discovery of a predictive/early marker accurately identifying patients that will have a long-term beneficial clinical response would enable targeting of treatment. Objectives We aimed to characterise the nasal methylome and transcriptome post Mepolizumab and identify signatures related to responder/non-responder status. Methods Nasal brushes were taken at baseline (pre-drug) and following 3 months of treatment with Mepolizumab from patients with severe asthma. Both DNA and RNA were extracted. Gene expression was investigated using poly-A RNA sequencing (25M reads) and DNA methylation analysed using the EPIC Array. Measurements and Main Results 27 paired samples were included, 17 patients were clinical responders and 10 were non-responders at one year. Differential gene expression and DNA methylation analyses identified 6719 genes and 53 CpG sites respectively that changed in response to Mepolizumab. There were 1784 genes which were unique to responders and 893 genes unique to non-responders. Pathway analyses revealed unique gene expression signatures. Respiratory disease associations and regulators of ongoing T2 inflammation pathway were still active in non-responders, and there was an inhibition of neutrophil activation pathways in responders. Conclusions There was a significant change in both the transcriptome and methylome in the nasal epithelium in patients three months post-Mepolizumab therapy suggesting broad effects on the airway epithelium in severe asthma. Responder and non-responder group analyses indicate there is a responder-specific gene expression profile that may aid in predicting response at one year.