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Uterine leiomyomas (ULM) are a common cause of solid pelvic tumors in women. Their etiopathogenesis remains unclear. Interleukins (ILs) and their receptors can influence tumor biology of ULM. The aim of this study was to evaluate single nucleotide polymorphisms (SNPs) exhibited in the genes IL4 (rs2070874), IL4R (rs1801275), IL12RB1 (rs11575934), IL12B (rs6887695), IL13 (rs20541) and IL23R (rs7517847) as risk factors for ULM in Slovenian women and to identify associations between corresponding clinical parameters and the analyzed SNPs. In addition, solitary and multiple ULM were compared to identify clinical and/or genetic parameters influencing their occurrence. We conducted a case-control study that included 181 women with leiomyomas and 133 control subjects. Genotyping of selected SNPs was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and high resolution melting (HRM) techniques. The TT genotype of rs20541 (IL13) was significantly associated with decreased risk of ULM compared to both the CC and CT genotypes [p = 0.018; odds ratio (OR) = 0.184; 95% confidence interval (95% CI) = 0.048-0.7121. Using genetic and clinical data to develop a predictive model with logistic regression, we found that adenomyosis, higher age at diagnosis, family history of ULM occurrence, earlier menarche, lower number of pregnancies and lower age at first sexual intercourse, the G allele and genotypes AG and GG of rs1801275 (IL4R) were associated with an increased risk of multiple ULM occurrence. We also found an association between rs20541 (IL13) and 17ß-estradiol serum levels in patients with multiple ULM (p 0.003). Our study showed, for the first time, that rs20541 (IL13) may contribute to susceptibility of ULM development and that rs1801275 (IL4R) can predispose patients to develop multiple ULM.

The aim of this study was to evaluate changes in prognostic risk profiles of women with endometrial cancer by comparing the clinical risk assessment with the integrated molecular risk assessment profiling.

Colorectal cancer (CRC) represents one of the most common malignancies worldwide. Research has indicated that functional gene changes such as single nucleotide polymorphism (SNP) influence carcinogenesis and metastasis and might have an influence on disease relapse. The aim of our study was to evaluate the role of SNPs in selected genes as prognostic markers in resectable CRC.

Curated online interaction databases and gene ontology tools have streamlined the analysis of highly complex gene/protein networks. However, understanding of disease pathogenesis has gradually shifted from a protein-based core to complex interactive networks where non-coding RNA (ncRNA) is thought to play an essential role. As current gene ontology is based predominantly on protein-level information, there is a growing need to analyze networks with ncRNA. In this study, we propose a gene ontology workflow integrating ncRNA using the NPInter V5.0 database. To validate the proposed workflow, we analyzed our previously published curated biomarker datasets for hidden disease susceptibility processes and pharmacogenomics. Our results show a novel involvement of melanogenesis in psoriasis response to biological drugs in general. Hyperpigmentation has been previously observed in psoriasis following treatment with currently indicated biological drugs, thus calling attention to melanogenesis research as a response biomarker in psoriasis. Moreover, our proposed workflow highlights the need to critically evaluate computed ncRNA interactions within databases and a demand for gene ontology analysis of large miRNA blocks.

Background: Breast cancer (BC) comprises multiple subtypes with distinct molecular features, which differ in their interplay with host immunity, prognosis, and treatment. Non-invasive blood analyses can provide valuable insights into systemic immunity during cancer. The aim of this study was to analyze the expression of transcriptional isoforms in peripheral blood mononuclear cells (PBMCs) from BC patients and healthy women to identify potential BC immune biomarkers. Methods: RNA sequencing and isoform-level bioinformatics were performed on PBMCs from 12 triple-negative and 13 luminal A patients. Isoform expression validation by qRT-PCR and clinicopathological correlations were performed in a larger cohort (156 BC patients and 32 healthy women). Results: Transcriptional analyses showed a significant ( p < 0.001) decrease in the ENST00000374459 RASGEF1A isoform in PBMCs of BC compared to healthy subjects, indicating disease-related expression changes. The decrease was associated with higher ctDNA and Ki-67 values. Conclusions: The levels of the RASGEF1A transcriptional isoform ENST00000374459 may have the potential to distinguish between BC and healthy subjects. The downregulation of ENST00000374459 in breast cancer is associated with higher proliferation and ctDNA shedding. Specialized bioinformatics analyses such as isoform analyses hold significant promise in the detection of biomarkers, since standard RNA sequencing analyses may overlook specific transcriptional changes that may be disease-associated and biologically important.

Background: The lack of reliable biomarkers in response to anti-TNFα biologicals hinders personalized therapy for Crohn's disease (CD) patients. The motivation behind our study is to shift the paradigm of anti-TNFα biomarker discovery toward specific immune cell sub-populations using single-cell RNA sequencing and an innovative approach designed to uncover PBMCs gene expression signals, which may be masked due to the treatment or ongoing inflammation; Methods: The single-cell RNA sequencing was performed on PBMC samples from CD patients either naïve to biological therapy, in remission while on adalimumab, or while on ustekinumab but previously non-responsive to adalimumab. Sieves for stringent downstream gene selection consisted of gene ontology and independent cohort genomic profiling. Replication and meta-analyses were performed using publicly available raw RNA sequencing files of sorted immune cells and an association analysis summary. Machine learning, Mendelian randomization, and oligogenic risk score methods were deployed to validate DEGs highly relevant to anti-TNFα therapy response; Results: This study found PLCB1 in CD4 + T cells and CRTAM in double-negative T cells, which met the stringent statistical thresholds throughout the analyses. An additional assessment proved causal inference of both genes in response to anti-TNFα therapy; Conclusions: This study, jointly with an innovative design, uncovered novel candidate genes in the anti-TNFα response landscape of CD, potentially obscured by therapy or inflammation.

Psoriasis is a chronic, immune-mediated and inflammatory skin disease. Although various biological drugs are available for psoriasis treatment, some patients have poor responses or do not respond to treatment. The aim of the present study was to highlight the molecular mechanism of responsiveness to current biological drugs for psoriasis treatment. To this end, we reviewed previously published articles that reported genes associated with treatment response to biological drugs in psoriasis, and gene ontology analysis was subsequently performed using the Cytoscape platform. Herein, we revealed a statistically significant association between NF-kappaB signaling ( p value = 3.37 × 10 -9 ), regulation of granulocyte macrophage colony-stimulating factor production ( p value = 6.20 × 10 -6 ), glial cell proliferation ( p value = 2.41 × 10 -5 ) and treatment response in psoriatic patients. To the best of our knowledge, we are the first to directly associate glial cells with treatment response. Taken together, our study revealed gene ontology (GO) terms, some of which were previously shown to be implicated in the molecular pathway of psoriasis, as novel GO terms involved in responsiveness in psoriatic disease patients., Competing Interests: The authors declare no conflicts of interest.

Multiple sclerosis is a common immune-mediated inflammatory and demyelinating disease. Lower cholecalciferol levels are an established environmental risk factor in multiple sclerosis. Although cholecalciferol supplementation in multiple sclerosis is widely accepted, optimal serum levels are still debated. Moreover, how cholecalciferol affects pathogenic disease mechanisms is still unclear. In the present study, we enrolled 65 relapsing-remitting multiple sclerosis patients who were double-blindly divided into two groups with low and high cholecalciferol supplementation, respectively. In addition to clinical and environmental parameters, we obtained peripheral blood mononuclear cells to analyze DNA, RNA, and miRNA molecules. Importantly, we investigated miRNA-155-5p, a previously published pro-inflammatory miRNA in multiple sclerosis known to be correlated to cholecalciferol levels. Our results show a decrease in miR-155-5p expression after cholecalciferol supplementation in both dosage groups, consistent with previous observations. Subsequent genotyping, gene expression, and eQTL analyses reveal correlations between miR-155-5p and the SARAF gene, which plays a role in the regulation of calcium release-activated channels. As such, the present study is the first to explore and suggest that the SARAF miR-155-5p axis hypothesis might be another mechanism by which cholecalciferol supplementation might decrease miR-155 expression. This association highlights the importance of cholecalciferol supplementation in multiple sclerosis and encourages further investigation and functional cell studies.

Epigenetic studies on the role of DNA-modifying enzymes in HNSCC tumorigenesis have focused on a single enzyme or a group of enzymes. To acquire a more comprehensive insight into the expression profile of methyltransferases and demethylases, in the present study, we examined the mRNA expression of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B, the DNA demethylases TET1, TET2, TET3, and TDG, and the RNA methyltransferase TRDMT1 by RT-qPCR in paired tumor-normal tissue samples from HNSCC patients. We characterized their expression patterns in relation to regional lymph node metastasis, invasion, HPV16 infection, and CpG73 methylation. Here, we show that tumors with regional lymph node metastases (pN+) exhibited decreased expression of DNMT1, 3A and 3B, and TET1 and 3 compared to non-metastatic tumors (pN0), suggesting that metastasis requires a distinct expression profile of DNA methyltransferases/demethylases in solid tumors. Furthermore, we identified the effect of perivascular invasion and HPV16 on DNMT3B expression in HNSCC. Finally, the expression of TET2 and TDG was inversely correlated with the hypermethylation of CpG73, which has previously been associated with poorer survival in HNSCC. Our study further confirms the importance of DNA methyltransferases and demethylases as potential prognostic biomarkers as well as molecular therapeutic targets for HNSCC., Competing Interests: The authors declare no conflicts of interest.

Endometrial cancer is the most common gynecologic malignancy in the developed world. Risk stratification and treatment approaches are changing due to better understanding of tumor biology. Upregulated Wnt signaling plays an important role in cancer initiation and progression with promising potential for development of specific Wnt inhibitor therapy. One of the ways in which Wnt signaling contributes to progression of cancer, is by activating epithelial-to-mesenchymal transition (EMT) in tumor cells, causing the expression of mesenchymal markers, and enabling tumor cells to dissociate and migrate. This study analyzed the expression of Wnt signaling and EMT markers in endometrial cancer. Wnt signaling and EMT markers were significantly correlated with hormone receptors status in EC, but not with other clinico-pathological characteristics. Expression of Wnt antagonist, Dkk1 was significantly different between the ESGO-ESTRO-ESP patient risk assessment categories using integrated molecular risk assessment., 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., (Copyright © 2023 Ledinek, Sobočan, Sisinger, Hojnik, Büdefeld, Potočnik and Knez.)

Asthma is the most prevalent pediatric chronic disease. Bronchodilator drug response (BDR) and fractional exhaled nitric oxide (FeNO) are clinical biomarkers of asthma. Although DNA methylation (DNAm) contributes to asthma pathogenesis, the influence of DNAm on BDR and FeNO is scarcely investigated. This study aims to identify DNAm markers in whole blood associated either with BDR or FeNO in pediatric asthma. We analyzed 121 samples from children with moderate-to-severe asthma. The association of genome-wide DNAm with BDR and FeNO has been assessed using regression models, adjusting for age, sex, ancestry, and tissue heterogeneity. Cross-tissue validation was assessed in 50 nasal samples. Differentially methylated regions (DMRs) and enrichment in traits and biological pathways were assessed. A false discovery rate (FDR) < 0.1 and a genome-wide significance threshold of p < 9 × 10 -8 were used to control for false-positive results. The CpG cg12835256 ( PLA2G12A ) was genome-wide associated with FeNO in blood samples (coefficient= -0.015, p = 2.53 × 10 -9 ) and nominally associated in nasal samples (coefficient = -0.015, p = 0.045). Additionally, three CpGs were suggestively associated with BDR (FDR < 0.1). We identified 12 and four DMRs associated with FeNO and BDR (FDR < 0.05), respectively. An enrichment in allergic and inflammatory processes, smoking, and aging was observed. We reported novel associations of DNAm markers associated with BDR and FeNO enriched in asthma-related processes.

Crohn's disease (CD), rheumatoid arthritis, psoriatic arthritis and other inflammatory diseases comprise a group of chronic diseases with immune-mediated pathogenesis which share common pathological pathways, as well as treatment strategies including anti-TNF biologic therapy. However, the response rate to anti-TNF therapy among those diseases varies, and approximately one third of patients do not respond. Since pharmacogenetic studies for anti-TNF therapy have been more frequent for other related diseases and are rare in CD, the aim of our study was to further explore markers associated with anti-TNF response in other inflammatory diseases in Slovenian CD patients treated with the anti-TNF drug adalimumab (ADA). We enrolled 102 CD patients on ADA, for which the response was defined after 4, 12, 20 and 30 weeks of treatment, using an IBDQ questionnaire and blood CRP value. We genotyped 41 SNPs significantly associated with response to anti-TNF treatment in other diseases. We found novel pharmacogenetic association between SNP rs755622 in the gene MIF (macrophage migration inhibitory factor) and SNP rs3740691 in the gene ARFGAP2 in CD patients treated with ADA. The strongest and most consistent association with treatment response was found for the variant rs2275913 in gene IL17A ( p = 9.73 × 10 -3 ).

Molecular diagnostic tests help clinicians understand the underlying biological mechanisms of their patients' breast cancer (BC) and facilitate clinical management. Several tissue-based mRNA tests are used routinely in clinical practice, particularly for assessing the BC recurrence risk, which can guide treatment decisions. However, blood-based mRNA assays have only recently started to emerge. This review explores the commercially available blood mRNA diagnostic assays for BC. These tests enable differentiation of BC from non-BC subjects (Syantra DX, BCtect), detection of small tumours <10 mm (early BC detection) (Syantra DX), detection of different cancers (including BC) from a single blood sample (multi-cancer blood test Aristotle), detection of BC in premenopausal and postmenopausal women and those with high breast density (Syantra DX), and improvement of diagnostic outcomes of DNA testing (variant interpretation) (+RNAinsight). The review also evaluates ongoing transcriptomic research on exciting possibilities for future assays, including blood transcriptome analyses aimed at differentiating lymph node positive and negative BC, distinguishing BC and benign breast disease, detecting ductal carcinoma in situ, and improving early detection further (expression changes can be detected in blood up to eight years before diagnosing BC using conventional approaches, while future metastatic and non-metastatic BC can be distinguished two years before BC diagnosis).

Background: Uncontrolled pediatric asthma has a large impact on patients and their caregivers. More insight into determinants of uncontrolled asthma is needed. We aim to compare treatment regimens, inhaler techniques, medication adherence and other characteristics of children with controlled and uncontrolled asthma in the: Systems Pharmacology approach to uncontrolled Paediatric Asthma (SysPharmPediA) study., Material and Methods: 145 children with moderate to severe doctor-diagnosed asthma (91 uncontrolled and 54 controlled) aged 6-17 years were enrolled in this multicountry, (Germany, Slovenia, Spain, and the Netherlands) observational, case-control study. The definition of uncontrolled asthma was based on asthma symptoms and/or exacerbations in the past year. Patient-reported adherence and clinician-reported medication use were assessed, as well as lung function and inhalation technique. A logistic regression model was fitted to assess determinants of uncontrolled pediatric asthma., Results: Children in higher asthma treatment steps had a higher risk of uncontrolled asthma (OR (95%CI): 3.30 (1.56-7.19)). The risk of uncontrolled asthma was associated with a larger change in FEV 1 % predicted post and pre-salbutamol (OR (95%CI): 1.08 (1.02-1.15)). Adherence and inhaler techniques were not associated with risk of uncontrolled asthma in this population., Conclusion: This study showed that children with uncontrolled moderate-to-severe asthma were treated in higher treatment steps compared to their controlled peers, but still showed a higher reversibility response to salbutamol. Self-reported adherence and inhaler technique scores did not differ between controlled and uncontrolled asthmatic children. Other determinants, such as environmental factors and differences in biological profiles, may influence the risk of uncontrolled asthma in this moderate to severe asthmatic population., Competing Interests: Declaration of Competing Interest AHAB, EMAS, LDB, SB, PC, SiH, AMH, JK, ADK, OSP, SJHV, and CW have no conflicts of interest to disclose. MG has received the SysPharmPedia and PERMEABLE grants, co-financed by the Ministry of Education, Science and Sport Slovenia (MIZS), and he was also funded by Slovenian Research Agency. SuH has received honoraria from Nutricia. MK has received the ERACoSysMed grant. He was funded by the European Union, the German ministry of education and research, the German research foundation, and Infectopharm and received consulting fees from Bionorica, Sanofi, Novartis, and Bencard. He also received honoraria from ERS, EAACI, ATS, Novartis, Glaxo, Chiesi, Sanofi, Nutricia, Hipp, and Allergopharma. GHK has received grants for his institution outside this study from the Lung Foundation of the Netherlands, The Netherlands Organization of Scientific Research, GSK, TEVA, Vertex, and Ubbo Emmius Foundation. He also received financial support for his institute as membership of scientific advisory boards to GSK and to PURE IMS. AHN has received the ZonMW grant. MWP has received research grants for her institute from Netherlands Lung Foundation and Netherlands Organisation for Health Research and Development and received consulting fees as an advisory board from Sanofi Genzyme. She also received speaker fees from AbbVie and Novartis. She is also chair of the pediatric assembly European Respiratory Society. MPY was funded by ISCIII through AES and EC within the AAL framework, and the SysPharmPedia grant from the ERACoSysMed 1st Joint Transnational Call from the European Union under the Horizon 2020. She was funded by the Ramón y Cajal Program by the Spanish Ministry of Science and Innovation and by the European Social Fund “ESF Investing in your future”. She was funded by the Spanish Ministry of Science and Innovation and the European Regional Development Fund “ERDF A way of making Europe” by the European Union grant [SAF2017–83417R], and by MCIN/AEI/10.13039/501,100,011,033 [grant PID2020–116274RB-I00] (Outside of the submitted work). She was also funded by the Allergopharma Award 2021, Instituto de Salud Carlos III (ISCIII) [CB06/06/1088], and received support from GlaxoSmithKline S.A. (Outside of the submitted work). UP has received the SysPharmPedia grant, co-financed by the Ministry of Education, Science and Sport Slovenia (MIZS), and funded by the Slovenian Research Agency and the Ministry of Education, Science and Sport of the Republic of Slovenia for the PERMEABLE grant. MIA was funded by a full PhD scholarship from the Ministry of Higher Education of the Arab Republic of Egypt during the conduct of the study and was funded by a grant from Stichting Asthma Bestrijding. AHM has received ERANET Systems Medicine and ZonMW grant [project number: 9,003,035,001], SysPharmPediA grant from the ERACoSysMed 1st Joint Transnational Call from the European Union under the Horizon 2020 (AC15/00,015) and she is the PI of P4O2 (Precision Medicine for more Oxygen) public-private partnership sponsored by Health Holland involving many private partners who contribute in cash and/or in kind (Boehringer Ingelheim, Breathomix, Fluidda, Ortec Logiqcare, Philips, Quantib-U, Smartfish, SODAQ, Thirona, TopMD, and Novartis) and received Unrestricted research grant Boehringer Ingelheim and Vertex Innovation Award paid to her institution. She also received consulting fees paid to her institution from Astra Zeneca and Boehringer Ingelheim and received honoraria for lectures paid to her institution from GSK. She is chair of the DSMB SOS BPD study, advisory board member of the CHAMP study, president federation of innovative drug research in the Netherlands (FIGON), and the president European association of systems medicine (EASYM)., (Copyright © 2022. Published by Elsevier B.V.)

The prevention and treatment of skin diseases remains a major challenge in medicine. The search for natural active ingredients that can be used to prevent the development of the disease and complement treatment is on the rise. Natural extracts of ginger and hemp offer a wide range of bioactive compounds with potential health benefits. This study evaluates the effectiveness of hemp and ginger extract as a supportive treatment for skin diseases. It reports a synergistic effect of hemp and ginger extract. The contents of cannabinoids and components of ginger are determined, with the highest being CBD (587.17 ± 8.32 µg/g) and 6-gingerol (60.07 ± 0.40 µg/g). The minimum inhibitory concentration for Staphylococcus aureus (156.5 µg/mL), Escherichia coli (625.2 µg/mL) and Candida albicans (78.3 µg/mL) was also analyzed. Analysis of WM-266-4 cells revealed the greatest decrease in metabolic activity in cells exposed to the extract at a concentration of 1.00 µg/mL. Regarding the expression of genes associated with cellular processes, melanoma aggressiveness, resistance and cell survival, a significant difference was found in the expression of ABCB5 , CAV1 and S100A9 compared with the control (cells not exposed to the extract).

Background: Acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) represent a group of hematological malignancies characterized by the pathogenic clonal expansion of leukemic myeloid cells. The diagnosis and clinical outcome of AML and CML are complicated by genetic heterogeneity of disease; therefore, the identification of novel molecular biomarkers and pharmacological targets is of paramount importance., Methods: RNA-seq-based transcriptome data from a total of five studies were extracted from NCBI GEO repository and subjected to an in-depth bioinformatics analysis to identify differentially expressed genes (DEGs) between AML and CML. A systemic literature survey and functional gene ontology (GO) enrichment analysis were performed for the top 100 DEGs to identify novel candidate genes and biological processes associated with AML and CML., Results: LINC01554, PTMAP12, LOC644936, RPS27AP20 and FAM133CP were identified as novel risk genes for AML and CML. GO enrichment analysis showed that DEGs were significantly associated with pre-RNA splicing, reactive oxygen species and glycoprotein metabolism, the cellular endomembrane system, neutrophil migration and antimicrobial immune response., Conclusions: Our study revealed novel biomarkers and specific biological processes associated with AML and CML. Further studies are required to evaluate their value as molecular targets for managing and treating the myeloid malignancies.

Crohn's disease is a consequence of dysregulated inflammatory response to the host's microbiota. Although anti-TNF treatment improves the quality of the patient's life, a large proportion of patients lose response to the treatment. The past decade of research has led to a continuum of studies showcasing the heterogeneity of anti-TNF response; thus, the aim of the present study was to dissect transcriptome-wide findings to transcript isoform specific levels and combine the analyses with refined information of immune cell landscapes in colon tissue, and subsequently select promising candidates using gene ontology and genomic integration. We enrolled Slovenian Crohn's disease patients who were naïve with respect to adalimumab treatment. We performed colon tissue RNA sequencing and peripheral blood mononuclear cell DNA genotyping with a subsequent contemporary integrative approach to combine immune cell deconvoluted isoform transcript specific transcriptome analysis, gene ontology layering and genomic data. We identified nine genes ( MACF1 , CTSE , HDLBP , HSPA9 , HLA-DMB , TAP2 , LGMN , ANAPC11 , ACP5 ) with 15 transcripts and 16 variants involved in the adalimumab response. Our study identified loci, some of which were previously shown to contribute to inflammatory bowel disease susceptibility, as novel loci involved in adalimumab response in Crohn's disease patients.

Anti-TNF therapy has significantly improved disease control in rheumatoid arthritis, but a fraction of rheumatoid arthritis patients do not respond to anti-TNF therapy or lose response over time. Moreover, the mechanisms underlying non-response to anti-TNF therapy remain largely unknown. To date, many single biomarkers of response to anti-TNF therapy have been published but they have not yet been analyzed as a system of interacting nodes. The aim of our study is to systematically elucidate the biological processes underlying non-response to anti-TNF therapy in rheumatoid arthritis using the gene ontologies of previously published predictive biomarkers. Gene networks were constructed based on published biomarkers and then enriched gene ontology terms were elucidated in subgroups using gene ontology software tools. Our results highlight the novel role of proteasome-mediated protein catabolic processes ( p = 2.91 × 10 -15 ) and plasma lipoproteins ( p = 4.55 × 10 -11 ) in anti-TNF therapy response. The results of our gene ontology analysis help elucidate the biological processes underlying non-response to anti-TNF therapy in rheumatoid arthritis and encourage further study of the highlighted processes., Competing Interests: The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Metformin and 2-deoxy-D-glucose (2DG) exhibit multiple metabolic and immunomodulatory anti-cancer effects, such as suppressed proliferation or PD-L1 expression. Their combination or 2DG alone induce triple-negative breast cancer (TNBC) cell detachment, but their effects on mitochondria, crucial for anchorage-independent growth and metastasis formation, have not yet been evaluated. In the present study, we explored the effects of metformin, 2DG and their combination (metformin + 2DG) on TNBC cell mitochondria in vitro. Metformin + 2DG increased mitochondrial mass in TNBC cells. This was associated with an increased size but not number of morphologically normal mitochondria and driven by the induction of mitochondrial biogenesis rather than suppressed mitophagy. 2DG and metformin + 2DG strongly induced the unfolded protein response by inhibiting protein N-glycosylation. Together with adequate energy stress, this was one of the possible triggers of mitochondrial enlargement. Suppressed N-glycosylation by 2DG or metformin + 2DG also caused PD-L1 deglycosylation and reduced surface expression in MDA-MB-231 cells. PD-L1 was increased in low glucose and normalized by both drugs. 2DG and metformin + 2DG reduced PD-1 expression in Jurkat cells beyond the effects on activation, while cytokine secretion was mostly preserved. Despite increasing mitochondrial mass in TNBC cells, metformin and 2DG could therefore potentially be used as an adjunct therapy to improve anti-tumor immunity in TNBC.

Objectives: Combining of genomic data of different pathologies as a single phenotype has emerged as a useful strategy to identify genetic risk loci shared among immune-mediated diseases. Our study aimed to increase our knowledge of the genetic contribution to Kawasaki disease (KD) and IgA vasculitis (IgAV) by performing the first comprehensive large-scale analysis on the genetic overlap between them., Methods: A total of 1190 vasculitis patients and 11 302 healthy controls were analysed. First, in the discovery phase, genome-wide data of 405 KD patients and 6252 controls and 215 IgAV patients and 1324 controls, all of European origin, were combined using an inverse variance meta-analysis. Second, the top associated polymorphisms were selected for replication in additional independent cohorts (570 cases and 3726 controls). Polymorphisms with P-values ≤5 × 10-8 in the global IgAV-KD meta-analysis were considered as shared genetic risk loci., Results: A genetic variant, rs3743841, located in an intron of the NAGPA gene, reached genome-wide significance in the cross-disease meta-analysis (P = 8.06 × 10-10). Additionally, when IgAV was individually analysed, a strong association between rs3743841 and this vasculitis was also evident [P = 1.25 × 10-7; odds ratio = 1.47 (95% CI 1.27, 1.69)]. In silico functional annotation showed that this polymorphism acts as a regulatory variant modulating the expression levels of the NAGPA and SEC14L5 genes., Conclusion: We identified a new risk locus with pleiotropic effects on the two childhood vasculitides analysed. This locus represents the strongest non-HLA signal described for IgAV to date., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Transcriptome studies of peripheral blood cells can advance our understanding of the systemic immune response to the presence of cancer and the mechanisms underlying cancer onset and progression. This enables the identification of novel minimally invasive immune biomarkers for early cancer detection and personalized cancer management and may bring forward new immunotherapy options. Recent blood gene expression analyses in breast cancer (BC) identified distinct patient subtypes that differed in the immune reaction to cancer and were distinct from the clinical BC subtypes, which are categorized based on expression of specific receptors on tumor cells. Introducing new BC subtypes based on peripheral blood gene expression profiles may be appropriate, since it may assist in BC prognosis, the identification of patients likely to benefit from immunotherapy, and treatment efficacy monitoring. Triple-negative breast cancer (TNBC) is an aggressive, heterogeneous, and difficult-to-treat disease, and identification of novel biomarkers for this BC is crucial for clinical decision-making. A few studies have reported TNBC-enriched blood transcriptional signatures, mostly related to strong inflammation and augmentation of altered immune signaling, that can differentiate TNBC from other classical BC subtypes and facilitate diagnosis. Future research is geared toward transitioning from expression signatures in unfractionated blood cells to those in immune cell subpopulations.

Coordination among multiple signaling pathways ensures an appropriate immune response, where a signaling pathway may impair or augment another signaling pathway. Here, we report a negative feedback regulation of signaling through the key innate immune mediator MyD88 by inflammasome-activated caspase-1. NLRP3 inflammasome activation impaired agonist- or infection-induced TLR signaling and cytokine production through the proteolytic cleavage of MyD88 by caspase-1. Site-specific mutagenesis was used to identify caspase-1 cleavage site within MyD88 intermediary segment. Different cleavage site location within MyD88 defined the functional consequences of MyD88 cleavage between mouse and human cells. LPS/monosodium urate-induced mouse inflammation model corroborated the physiological role of this mechanism of regulation, that could be reversed by chemical inhibition of NLRP3. While Toll/interleukin-1 receptor (TIR) domain released by MyD88 cleavage additionally contributed to the inhibition of signaling, Waldenström's macroglobulinemia associated MyD88 L265P mutation is able to evade the caspase-1-mediated inhibition of MyD88 signaling through the ability of its TIR L265P domain to recruit full length MyD88 and facilitate signaling. The characterization of this mechanism reveals an additional layer of innate immunity regulation., 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., (Copyright © 2022 Avbelj, Hafner-Bratkovič, Lainšček, Manček-Keber, Peternelj, Panter, Treon, Gole, Potočnik and Jerala.)

Background: Women with uterine adenomyosis seeking assisted reproduction have been associated with compromised endometrial receptivity to embryo implantation. To understand the mechanisms involved in this process, we aimed to compare endometrial transcriptome profiles during the window of implantation (WOI) between women with and without adenomyosis., Methods: We obtained endometrial biopsies LH-timed to the WOI from women with sonographic features of adenomyosis (n=10) and controls (n=10). Isolated RNA samples were subjected to RNA sequencing (RNA-seq) by the Illumina NovaSeq 6000 platform and endometrial receptivity classification with a molecular tool for menstrual cycle phase dating (beREADY®, CCHT). The program language R and Bioconductor packages were applied to analyse RNA-seq data in the setting of the result of accurate endometrial dating. To suggest robust candidate pathways, the identified differentially expressed genes (DEGs) associated with the adenomyosis group in the receptive phase were further integrated with 151, 173 and 42 extracted genes from published studies that were related to endometrial receptivity in healthy uterus, endometriosis and adenomyosis, respectively. Enrichment analyses were performed using Cytoscape ClueGO and CluePedia apps., Results: Out of 20 endometrial samples, 2 were dated to the early receptive phase, 13 to the receptive phase and 5 to the late receptive phase. Comparison of the transcriptomics data from all 20 samples provided 909 DEGs (p<0.05; nonsignificant after adjusted p value) in the adenomyosis group but only 4 enriched pathways (Bonferroni p value < 0.05). The analysis of 13 samples only dated to the receptive phase provided suggestive 382 DEGs (p<0.05; nonsignificant after adjusted p value) in the adenomyosis group, leading to 33 enriched pathways (Bonferroni p value < 0.05). These included pathways were already associated with endometrial biology, such as "Expression of interferon (IFN)-induced genes" and "Response to IFN-alpha". Data integration revealed pathways indicating a unique effect of adenomyosis on endometrial molecular organization (e.g., "Expression of IFN-induced genes") and its interference with endometrial receptivity establishment (e.g., "Extracellular matrix organization" and "Tumour necrosis factor production")., Conclusions: Accurate endometrial dating and RNA-seq analysis resulted in the identification of altered response to IFN signalling as the most promising candidate of impaired uterine receptivity in adenomyosis., (© 2021. The Author(s).)

Triple-negative breast cancer (TNBC) is a breast cancer (BC) subtype that accounts for approximately 15-20% of all BC cases. Cancer cell lines (CLs) provide an efficient way to model the disease. We have recently isolated a patient-derived triple-negative BC CL MFUM-BrTNBC-1 and performed a detailed morphological and molecular characterisation and a comprehensive comparison with three commercial BC CLs (MCF-7, MDA-MB-231, MDA-MB-453). Light and fluorescence microscopy were used for morphological studies; immunocytochemical staining for hormone receptor, p53 and Ki67 status; RNA sequencing, qRT-PCR and STR analysis for molecular characterisation; and biomedical image analysis for comparative phenotypical analysis. The patient tissue-derived MFUM-BrTNBC-1 maintained the primary triple-negative receptor status. STR analysis showed a stable and unique STR profile up to the 6th passage. MFUM-BrTNBC-1 expressed EMT transition markers and displayed changes in several cancer-related pathways (MAPK, Wnt and PI3K signalling; nucleotide excision repair; and SWI/SNF chromatin remodelling). Morphologically, MFUM-BrTNBC-1 differed from the commercial TNBC CL MDA-MB-231. The advantages of MFUM-BrTNBC-1 are its isolation from a primary tumour, rather than a metastatic site; good growth characteristics; phenotype identical to primary tissue; complete records of origin; a unique identifier; complete, unique STR profile; quantifiable morphological properties; and genetic stability up to (at least) the 6th passage.

Familial Progressive Hyper- and Hypopigmentation is a pigmentary disorder characterized by a mix of hypo- and hyperpigmented lesions, café-au-lait spots and hypopigmented ash-leaf macules. The disorder was previously linked to KITLG and various mutations have been reported to segregate in different families. Furthermore, association between KITLG mutations and malignancies was also suggested. Exome and SANGER sequencing were performed for identification of KITLG mutations. Functional in silico analyses were additionally performed to assess the findings. We identified a de novo mutation in exon 4 of KITLG gene causing NM&#95;000899.4:c.[329A>T] (chr12:88912508A>T) leading to NP&#95;000890.1:p.(Asp110Val) substitution in the 3rd alpha helix. It was predicted as pathogenic, located in a conserved region and causing an increase in hydrophobicity in the KITLG protein. Our findings clearly confirm an additional hot spot of KITLG mutations in the 3rd alpha helix, which very likely increases the risk of malignancies. To our knowledge the present study provides the strongest evidence of association of the KITLG mutation with both Familial Progressive Hyper- and Hypopigmentation and malignancy due to its' location on somatic cancer mutation locus. Additionally we also address difficulties with classification of the unique phenotype and propose a subtype within broader diagnosis., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Anchorage-independent growth of cancer cells in vitro is correlated to metastasis formation in vivo. Metformin use is associated with decreased breast cancer incidence and currently evaluated in cancer clinical trials. The combined treatment with metformin and 2-deoxy-D-glucose (2DG) in vitro induces detachment of viable MDA-MB-231 breast cancer cells that retain their proliferation capacity. This might be important for cell detachment from primary tumors, but the metabolic changes involved are unknown. We performed LC/MS metabolic profiling on separated attached and detached MDA-MB-231 cells treated with metformin and/or 2DG. High 2DG and metformin plus 2DG altered the metabolic profile similarly to metformin, inferring that metabolic changes are necessary but not sufficient while the specific effects of 2DG are crucial for detachment. Detached cells had higher NADPH levels and lower fatty acids and glutamine levels compared to attached cells, supporting the role of AMPK activation and reductive carboxylation in supporting anchorage-independent survival. Surprisingly, the metabolic profile of detached cells was closer to untreated control cells than attached treated cells, suggesting detachment might help cells adapt to energy stress. Metformin treated cells had higher fatty and amino acid levels with lower purine nucleotide levels, which is relevant for understanding the anticancer mechanisms of metformin., (© 2021. The Author(s).)

Introduction: Severe asthma is a rare disease in children, for which three biologicals, anti-immunoglobulin E, anti-interleukin-5 and anti-IL4RA antibodies, are available in European countries. While global guidelines exist on who should receive biologicals, knowledge is lacking on how those guidelines are implemented in real life and which unmet needs exist in the field. In this survey, we aimed to investigate the status quo and identify open questions in biological therapy of childhood asthma across Europe., Methods: Structured interviews regarding experience with biologicals, regulations on access to the different treatment options, drug selection, therapy success and discontinuation of therapy were performed. Content analysis was used to analyse data., Results: We interviewed 37 experts from 25 European countries and Turkey and found a considerable range in the number of children treated with biologicals per centre. All participating countries provide public access to at least one biological. Most countries allow different medical disciplines to prescribe biologicals to children with asthma, and only a few restrict therapy to specialised centres. We observed significant variation in the time point at which treatment success is assessed, in therapy duration and in the success rate of discontinuation. Most participating centres intend to apply a personalised medicine approach in the future to match patients a priori to available biologicals., Conclusion: Substantial differences exist in the management of childhood severe asthma across Europe, and the need for further studies on biomarkers supporting selection of biologicals, on criteria to assess therapy response and on how/when to end therapy in stable patients is evident., Competing Interests: Conflict of interest: E. Santos-Valente has nothing to disclose. Conflict of interest: H. Buntrock-Döpke has nothing to disclose. Conflict of interest: R. Abou Taam has nothing to disclose. Conflict of interest: S. Arasi has nothing to disclose. Conflict of interest: A. Bakirtas has nothing to disclose. Conflict of interest: J. Lozano Blasco reports personal fees from Novartis and GSK outside the submitted work. Conflict of interest: K. Bønnelykke has nothing to disclose. Conflict of interest: M. Craiu reports a speaker fee for a scientific meeting regarding 5 years of Xolair in Romania (9 May 2018) from Novartis Romania outside the submitted work. Conflict of interest: R. Cutrera has nothing to disclose. Conflict of interest: A. Deschildre reports personal fees and other support from Novartis, GSK and Sanofi outside the submitted work. Conflict of interest: B. Elnazir has nothing to disclose. Conflict of interest: L. Fleming reports grants from Asthma UK, and speakers fees or fees for expert consultation from Teva, Astra Zeneca, Sanofi, Respiri, Novartis; all fees paid direct to her institution and outside the submitted work. Conflict of interest: U. Frey has nothing to disclose. Conflict of interest: M. Gappa reports personal fees from Boehringer Ingelheim, GSK, Novartis and Sanofi outside the submitted work. Conflict of interest: A. Nieto García reports grants for clinical studies and advisory boards membership, as well as lecture fees from Novartis, GSK and MSD. Conflict of interest: K. Skamstrup Hansen has nothing to disclose. Conflict of interest: L. Hanssens has nothing to disclose. Conflict of interest: K. Jahnz-Rozyk received lecture and advisory board fees from AstraZeneca, Novartis, GSK, and Sanofi Aventis, outside the submitted work. Conflict of interest: M. Jesenak has nothing to disclose. Conflict of interest: S. Kerzel reports a speaker fee from Novartis for a lecture that has no topical overlap at all with the current manuscript. Conflict of interest: M. V. Kopp reports personal fees from ALK-Abello GmbH, Chiesi GmbH, Infectopharm GmbH, Novartis GmbH, Sanofi Aventis HmbH and Vertex GmbH, and grants and personal fees from Allergopharma GmbH, outside the submitted work. Conflict of interest: G.H. Koppelman reports grants from Lung Foundation Netherlands, TETRI Foudation, Ubbo Emmius Foundation, Vertex, Teva the Netherlands, GSK, European Union, outside the submitted work; and he participated in advisory board meetings to GSK and Pure IMS (money to institution). Conflict of interest: U. Krivec has nothing to disclose. Conflict of interest: K.A. MacLeod has nothing to disclose. Conflict of interest: M. Mäkelä has nothing to disclose. Conflict of interest: E. Melén reports personal fees from AstraZeneca, Chiesi, Novartis and Sanofi (advisory board fees), outside the submitted work. Conflict of interest: G. Mezei reports a travel grant from LOFARMA outside the submitted work. Conflict of interest: A. Moeller has nothing to disclose. Conflict of interest: A. Moreira has nothing to disclose. Conflict of interest: P. Pohunek reports personal fees for advisory board membership from Novartis and GlaxoSmithKline, and for consultation from Chiesi and AstraZeneca, outside the submitted work. Conflict of interest: P. Minić has nothing to disclose. Conflict of interest: N.W.P. Rutjes reports personal fees from GSK (advisory board on mepolizumab) and Sanofi (advisory board on dupilumab) outside the submitted work. Conflict of interest: P. Sammut has nothing to disclose. Conflict of interest: N. Schwerk reports lecture and advisory board fees from Novartis, Sanofi and Allergopharma; advisory board fees from Boehringer Ingelheim; and lecture fees from Abvie and Infectopharm, all outside the submitted work. Conflict of interest: Z. Szépfalusi has nothing to disclose. Conflict of interest: M. Turkalj has nothing to disclose. Conflict of interest: I. Tzotcheva has nothing to disclose. Conflict of interest: A. Ulmeanu has nothing to disclose. Conflict of interest: S. Verhulst reports grants from GSK during the conduct of the study. Conflict of interest: P. Xepapadaki reports personal fees for advisory services from Uriach, Novartis, Nestle and Nutricia outside the submitted work. Conflict of interest: J. Niggel has nothing to disclose. Conflict of interest: S. Vijverberg reports grants from ZonMW during the conduct of the study. Conflict of interest: A-H. Maitland van der Zee has received research grants outside the submitted work from GSK, Boehringer Ingelheim and Vertex; she is the principal investigator of a P4O2 (Precision Medicine for more Oxygen) public–private partnership sponsored by Health Holland involving many private partners that contribute in cash and/or in kind (Boehringer Ingelheim, Breathomix, Fluidda, Ortec Logiqcare, Philips, Quantib-U, Smartfish, SODAQ, Thirona, TopMD and Novartis); and she has served in advisory boards for AstraZeneca, GSK and Boehringer Ingelheim, with money paid to her institution. Conflict of interest: U. Potocnik was funded by the Ministry of Education, Science and Sport of the Republic of Slovenia, grant PERMEABLE (contract number C3330-19-252012), during the conduct of the study. Conflict of interest: S. M. Reinartz has nothing to disclose. Conflict of interest: C. M. van Drunen has nothing to disclose. Conflict of interest: M. Kabesch reports grants to his institution from the European Union, the German Ministry of Education and Research, and the German Research Foundation, during the conduct of the study; and consultancy fees from Bionorica, Sanofi, Novartis and Bencard, payments for lectures form the ERS, EAACI, ATS, Novartis, Glaxo, Nutricia, Hipp and Allergopharma, and a patent for a method for testing a subject thought to have or be disposed to asthma (European patent application 5 EP070301 135.5), outside the submitted work., (Copyright ©The authors 2021.)

Uterine leiomyomas are tumors, which are hormone driven and originate from the smooth muscle layer of the uterine wall. In addition to known genes in leiomyoma pathogenesis, recent approaches also highlight epigenetic malfunctions as an important mechanism of gene dysregulation. RNA sequencing raw data from pair-matched normal myometrium and fibroid tumors from two independent studies were used as discovery and validation sets and reanalyzed. RNA extracted from normal myometrium and fibroid tumors from 58 Slovenian patients was used as independent confirmation of most significant differentially expressed genes. Subsequently, GWA data from leiomyoma patients were used in order to identify genetic variants at epigenetic marks. Gene Ontology analysis of the overlap of two independent RNA-seq analyses showed that NPTX1 , NPTX2 , CHRM2 , DRD2 and CACNA1A were listed as significant for several enriched GO terms. All five genes were subsequently confirmed in the independent Slovenian cohort. Additional integration and functional analysis showed that genetic variants in these five gene regions are listed at a chromatin structure and state, predicting promoters, enhancers, DNase hypersensitivity and altered transcription factor binding sites. We identified a unique subgroup of dysregulated synaptic signaling genes involved in the biology and pathogenesis of leiomyomas, adding to the complexity of tumor biology.

Inhaled corticosteroids (ICS) are the most common asthma controller medication. An important contribution of genetic factors in ICS response has been evidenced. Here, we aimed to identify novel genetic markers involved in ICS response in asthma. A genome-wide association study (GWAS) of the change in lung function after 6 weeks of ICS treatment was performed in 166 asthma patients from the SLOVENIA study. Patients with an improvement in lung function ≥8% were considered as ICS responders. Suggestively associated variants ( p -value ≤ 5 × 10 -6 ) were evaluated in an independent study ( n = 175). Validation of the association with asthma exacerbations despite ICS use was attempted in European ( n = 2681) and admixed ( n = 1347) populations. Variants previously associated with ICS response were also assessed for replication. As a result, the SNP rs1166980 from the ROBO2 gene was suggestively associated with the change in lung function (OR for G allele: 7.01, 95% CI: 3.29-14.93, p = 4.61 × 10 -7 ), although this was not validated in CAMP. ROBO2 showed gene-level evidence of replication with asthma exacerbations despite ICS use in Europeans (minimum p -value = 1.44 × 10 -5 ), but not in admixed individuals. The association of PDE10A-T with ICS response described by a previous study was validated. This study suggests that ROBO2 could be a potential novel locus for ICS response in Europeans.

The ten most statistically significant estimated glomerular filtration rate (eGFRcrea)-associated loci from genome-wide association studies (GWAs) are tested for associations with chronic kidney disease (CKD) in 208 patients, including dialysis-independent CKD and dialysis-dependent end-stage renal disease (kidney failure). The allele A of intergenic SNP rs2453533 (near GATM ) is more frequent in dialysis-independent CKD patients ( n = 135, adjusted p = 0.020) but not dialysis-dependent kidney failure patients ( n = 73) compared to healthy controls ( n = 309). The allele C of intronic SNP rs4293393 ( UMOD ) is more frequent in healthy controls (adjusted p = 0.042) than in CKD patients. The Allele T of intronic SNP rs9895661 ( BCAS3 ) is associated with decreased eGFRcys (adjusted p = 0.001) and eGFRcrea (adjusted p = 0.017). Our results provide further evidence of a genetic difference between dialysis-dialysis-independent CKD and dialysis-dependent kidney failure, and add the GATM gene locus to the list of loci associated only with dialysis-independent CKD. GATM risk allele carriers in the dialysis-independent group may have a genetic susceptibility to higher creatinine production rather than increased serum creatinine due to kidney malfunction, and therefore, do not progress to dialysis-dependent kidney failure. When using eGFRcrea for CKD diagnosis, physicians might benefit from information about creatinine-increasing loci.

There is a clinical need to identify children with poor asthma control as early as possible, to optimize treatment and/or to find therapeutic alternatives. Here, we present the "Systems Pharmacology Approach to Uncontrolled Pediatric Asthma" (SysPharmPediA) study, which aims to establish a pediatric cohort of moderate-to-severe uncontrolled and controlled patients with asthma, to investigate pathophysiological mechanisms underlying uncontrolled moderate-to-severe asthma in children on maintenance treatment, using a multi-omics systems medicine approach. In this multicenter observational case-control study, moderate-to-severe asthmatic children (age; 6-17 years) were included from four European countries (Netherlands, Germany, Spain, and Slovenia). Subjects were classified based on asthma control and number of exacerbations. Demographics, current and past patient/family history, and clinical characteristics were collected. In addition, systems-wide omics layers, including epi(genomics), transcriptomics, microbiome, proteomics, and metabolomics were evaluated from multiple samples. In all, 145 children were included in this cohort, 91 with uncontrolled (median age = 12 years, 43% females) and 54 with controlled asthma (median age = 11.7 years, 37% females). The two groups did not show statistically significant differences in age, sex, and body mass index z-score distribution. Comprehensive information and diverse noninvasive biosampling procedures for various omics analyses will provide the opportunity to delineate underlying pathophysiological mechanisms of moderate-to-severe uncontrolled pediatric asthma. This eventually might reveal novel biomarkers, which could potentially be used for noninvasive personalized diagnostics and/or treatment.

Rationale: Substantial variability in response to asthma treatment with inhaled corticosteroids (ICS) has been described among individuals and populations, suggesting the contribution of genetic factors. Nonetheless, only a few genes have been identified to date. We aimed to identify genetic variants associated with asthma exacerbations despite ICS use in European children and young adults and to validate the findings in non-Europeans. Moreover, we explored whether a gene-set enrichment analysis could suggest potential novel asthma therapies., Methods: A genome-wide association study (GWAS) of asthma exacerbations was tested in 2681 children of European descent treated with ICS from eight studies. Suggestive association signals were followed up for replication in 538 European asthma patients. Further evaluation was performed in 1773 non-Europeans. Variants revealed by published GWAS were assessed for replication. Additionally, gene-set enrichment analysis focused on drugs was performed., Results: 10 independent variants were associated with asthma exacerbations despite ICS treatment in the discovery phase (p≤5×10 -6 ). Of those, one variant at the CACNA2D3-WNT5A locus was nominally replicated in Europeans (rs67026078; p=0.010), but this was not validated in non-European populations. Five other genes associated with ICS response in previous studies were replicated. Additionally, an enrichment of associations in genes regulated by trichostatin A treatment was found., Conclusions: The intergenic region of CACNA2D3 and WNT5A was revealed as a novel locus for asthma exacerbations despite ICS treatment in European populations. Genes associated were related to trichostatin A, suggesting that this drug could regulate the molecular mechanisms involved in treatment response., Competing Interests: Conflict of interest: N. Hernandez-Pacheco reports grants from Instituto de Salud Carlos III (ISCIII, FI16/00136) and co-funded by the European Social Funds from the European Union (ESF) “ESF invests in your future”, during the conduct of the study. Conflict of interest: S.J. Vijverberg has nothing to disclose. Conflict of interest: E. Herrera-Luis reports grants from the Spanish Ministry of Science, Innovation, and Universities (PRE2018-083837), during the conduct of the study. Conflict of interest: J. Li has nothing to disclose. Conflict of interest: Y.Y. Sio has nothing to disclose. Conflict of interest: R. Granell has nothing to disclose. Conflict of interest: A. Corrales has nothing to disclose. Conflict of interest: C. Maroteau has nothing to disclose. Conflict of interest: R. Lethem has nothing to disclose. Conflict of interest: J. Perez-Garcia has nothing to disclose. Conflict of interest: N. Farzan has nothing to disclose. Conflict of interest: K. Repnik has nothing to disclose. Conflict of interest: M. Gorenjak has nothing to disclose. Conflict of interest: P. Soares has nothing to disclose. Conflict of interest: L. Karimi has nothing to disclose. Conflict of interest: M. Schieck has nothing to disclose. Conflict of interest: L. Pérez-Méndez has nothing to disclose. Conflict of interest: V. Berce has nothing to disclose. Conflict of interest: R. Tavendale has nothing to disclose. Conflict of interest: C. Eng has nothing to disclose. Conflict of interest: O. Sardon has nothing to disclose. Conflict of interest: I. Kull has nothing to disclose. Conflict of interest: S. Mukhopadhyay reports grants from The Gannochy Trust, Perth and Kinross City Council and Scottish Enterprises Tayside, during the conduct of the study. Conflict of interest: M. Pirmohamed reports grants from UK Department of Health and UK Medical Research Council, during the conduct of the study; grants from MRC Clinical Pharmacology Training Scheme (joint funding by MRC and Roche, UCB, Eli Lilly and Novartis), Joint PhD studentship funded by EPSRC and Astra Zeneca and grants from Bristol Myers Squibb, outside the submitted work. Conflict of interest: K.M.C. Verhamme reports grants from ZonMw, during the conduct of the study; and works for a department who in the past received unconditional research grants from Yamanouchi, Pfizer/Boehringer Ingelheim, Novartis and GSK. Conflict of interest: E.G. Burchard reports grants from the National Heart, Lung, and Blood Institute (NHLBI) of the US National Institutes of Health (NIH) (X01HL134589, X01HL134589,R01HL128439, R01HL135156, R01HL141992 and R01HL141845), the National Institute of Environmental Health Sciences (NIEHS) (R01ES015794 and R21ES24844), the National Institute on Minority Health and Health Disparities (NIMHD) (P60MD006902, R01MD010443 and R56MD013312), the National Institute of General Medical Sciences (NIGMS) (RL5GM118984), the Tobacco-Related Disease Research Program (award numbers 24RT-0025 and 27IR-0030), the National Human Genome Research Institute (NHGRI) (U01HG009080), the Sandler Family Foundation, the American Asthma Foundation, the Amos Medical Faculty Development Program from the Robert Wood Johnson Foundation, the Harry Wm. and Diana V. Hind Distinguished Professorship in Pharmaceutical Sciences II, during the conduct of the study. Conflict of interest: M. Kabesch reports grants from European Union, German Ministry of Education and Research, German Research Foundation, during the conduct of the study; personal fees for consultancy from Bionorica, Sanofi, Novartis and Bencard, personal fees for lectures from ERS, EAACI, ATS, Novartis, Glaxo, Nutricia, Hipp and Allergopharma, outside the submitted work. Conflict of interest: D.B. Hawcutt has nothing to disclose. Conflict of interest: E. Melén has nothing to disclose. Conflict of interest: U. Potočnik reports grants from Slovenian Research Agency (P3-0067) and Ministry of Education, Science and Sport Slovenia (MIZS) (SysPharmPediA grant C3330-16-500106), during the conduct of the study. Conflict of interest: F.T. Chew reports grants from Singapore Ministry of Education Academic Research Fund, Singapore Immunology Network, National Medical Research Council (NMRC) (Singapore), Biomedical Research Council (BMRC) (Singapore), and the Agency for Science Technology and Research (A*STAR) (Singapore), during the conduct of the study; and consulting fees from Sime Darby Technology Centre, First Resources Ltd, Genting Plantation and Olam International, outside the submitted work. Conflict of interest: K.G. Tantisira reports grants from U.S. National Institutes of Health, during the conduct of the study. Conflict of interest: S. Turner has nothing to disclose. Conflict of interest: C.M. Palmer has nothing to disclose. Conflict of interest: C. Flores has nothing to disclose. Conflict of interest: M. Pino-Yanes reports grants from Spanish Ministry of Economy, Industry and Competitiveness (funded by the Ramón y Cajal Program, RYC-2015-17205), and Instituto de Salud Carlos III (ISCIII) (funded by ISCIII through AES and EC within AAL framework, and the SysPharmPedia grant from the ERACoSysMed 1st Joint Transnational Call from the European Union under the Horizon 2020, AC15/00015), during the conduct of the study. Conflict of interest: A.H. Maitland-van der Zee reports grants from GSK, during the conduct of the study; grants from Boehringer Ingelheim, personal fees for advisory board work from AstraZeneca and Boehringer Ingelheim, outside the submitted work., (Copyright ©ERS 2021.)