Interleukin 5 Receptor Subunit Alpha Expression as a Potential Biomarker in Patients with Nasal Polyposis.

Chronic Rhinosinusitis with Nasal Polyposis (CRSwNP) affects the quality of life of patients suffering from it. The search for a suitable biomarker has been conducted over the last decades. Interleukin 5 receptor subunit alpha (IL-5Rα) involves the activation, maintenance, and survival of eosinophils, which are highly tied to chronic inflammatory processes of the airways, like asthma or CRSwNP. In this study, we evaluate the utility of IL5RA as a genetic biomarker in CRSwNP. IL5RA mRNA expression level was analyzed in different groups of patients by performing qPCR assays. A significant increase in IL5RA expression was observed in CRSwNP patients, especially those with asthma and atopy. We found differences in expression levels when comparing groups with or without polyposis or asthma, as well as some atypical cases related to eosinophil levels. That opens a path to future studies to further characterize groups of patients with common features in the context of pharmacogenetics and in an era towards developing a more precise personalized treatment with IL-5Rα as a therapeutic target for CRSwNP.

RNY3 modulates cell proliferation and IL13 mRNA levels in a T lymphocyte model: a possible new epigenetic mechanism of IL-13 regulation

Allergic asthma is the most common type of asthma. It is characterized by TH2 cell–driven inflammation in which interleukin-13 (IL-13) plays a pivotal role. Cytoplasmic RNAs (Y-RNAs), a variety of non-coding RNAs that are dysregulated in many cancer types, are also differentially expressed in patients with allergic asthma. Their function in the development of the disease is still unknown. We investigated the potential role of RNY3 RNA (hY3) in the TH2 cell inflammatory response using the Jurkat cell line as a model. hY3 expression levels were modulated to mimic the upregulation effect in allergic disease. We evaluated the effect of hY3 over cell stimulation and the expression of the TH2 cytokine IL13. Total RNA was isolated and retrotranscribed, and RNA levels were assessed by qPCR. In Jurkat cells, hY3 levels increased upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. When transfecting with high levels of hY3 mimic molecules, cell proliferation rate decreased while IL13 mRNA levels increased upon stimulation compared to stimulated control cells. Our results show the effect of increased hY3 levels on cell proliferation and the levels of IL13 mRNA in Jurkat cells. Also, we showed that hY3 could act over other cells via exosomes. This study opens up new ways to study the potential regulatory function of hY3 over IL-13 production and its implications for asthma development. (AU)

RNY3 modulates cell proliferation and IL13 mRNA levels in a T lymphocyte model: a possible new epigenetic mechanism of IL-13 regulation.

Allergic asthma is the most common type of asthma. It is characterized by TH2 cell-driven inflammation in which interleukin-13 (IL-13) plays a pivotal role. Cytoplasmic RNAs (Y-RNAs), a variety of non-coding RNAs that are dysregulated in many cancer types, are also differentially expressed in patients with allergic asthma. Their function in the development of the disease is still unknown. We investigated the potential role of RNY3 RNA (hY3) in the TH2 cell inflammatory response using the Jurkat cell line as a model. hY3 expression levels were modulated to mimic the upregulation effect in allergic disease. We evaluated the effect of hY3 over cell stimulation and the expression of the TH2 cytokine IL13. Total RNA was isolated and retrotranscribed, and RNA levels were assessed by qPCR. In Jurkat cells, hY3 levels increased upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. When transfecting with high levels of hY3 mimic molecules, cell proliferation rate decreased while IL13 mRNA levels increased upon stimulation compared to stimulated control cells. Our results show the effect of increased hY3 levels on cell proliferation and the levels of IL13 mRNA in Jurkat cells. Also, we showed that hY3 could act over other cells via exosomes. This study opens up new ways to study the potential regulatory function of hY3 over IL-13 production and its implications for asthma development.

Effects of Therapeutic Antibodies on Gene and Protein Signatures in Asthma Patients: A Comparative Systematic Review.

Several biologic therapies that target inflammatory modulators are now used for treating patients with uncontrolled, severe asthma. Knowledge about how this type of treatment modifies the molecular milieu is rapidly increasing. Thus, this systematic review aimed to compile the reported effects of therapeutic antibodies on the transcriptome or proteome of asthma patients. Studies of asthmatic patients under biological treatment describing transcriptomic or proteomic changes upon treatment were included. Preclinical or single gene/protein studies were not considered. PubMed and Scopus search was performed in August and September 2021. Following PRISMA guidelines and GRADE recommendations, we selected 12 studies on gene or protein expression changes in patients treated with the antibodies currently approved by EMA and the FDA. All studies were at low risk of bias as per the RoB2 tool. Different gene clusters have been identified to change upon omalizumab treatment, found a reduction in eosinophil-associated gene signatures after benralizumab treatment, and protein profiles were different in patients treated with mepolizumab and in those treated with benralizumab. The main potential biomarkers proposed by the selected studies are shown. These results may contribute to discovering biomarkers of response and selecting the best therapy for each patient.

Ten Years of Experience Support Pharmacogenetic Testing to Guide Individualized Drug Therapy.

Precision medicine utilizing the genetic information of genes involved in the metabolism and disposition of drugs can not only improve drug efficacy but also prevent or minimize adverse events. Polypharmacy is common among multimorbid patients and is associated with increased adverse events. One of the main objectives in health care is safe and efficacious drug therapy, which is directly correlated to the individual response to treatment. Precision medicine can increase drug safety in many scenarios, including polypharmacy. In this report, we share our experience utilizing precision medicine over the past ten years. Based on our experience using pharmacogenetic (PGx)-informed prescribing, we implemented a five-step precision medicine protocol (5SPM) that includes the assessment of the biological-clinical characteristics of the patient, current and past prescription history, and the patient's PGx test results. To illustrate our approach, we present cases highlighting the clinical relevance of precision medicine with a focus on patients with a complex history and polypharmacy.

PTGDR2 Expression in Peripheral Blood as a Potential Biomarker in Adult Patients with Asthma.

BACKGROUND: Precision medicine is a promising strategy to identify biomarkers, stratify asthmatic patients according to different endotypes, and match them with the appropriate therapy. This proof-of-concept study aimed to investigate whether gene expression in peripheral blood could provide a valuable noninvasive approach for the molecular phenotyping of asthma. METHODS: We performed whole-transcriptome RNA sequencing on peripheral blood of 30 non-atopic non-asthmatic controls and 30 asthmatic patients. A quantitative PCR (qPCR) validation study of PTGDR2 that encodes for CRTH2 receptor, expressed in cells involved in T2 inflammation, was developed in a cohort of 361 independent subjects: 94 non-asthmatic non-atopic controls, 187 asthmatic patients [including 82 with chronic rhinosinusitis with nasal polyposis (CRSwNP) and 24 with aspirin-exacerbated respiratory disease (AERD)], 52 with allergic rhinitis, and 28 with CRSwNP without asthma. RESULTS: PTGDR2 was one of the most differentially overexpressed genes in asthmatic patients' peripheral blood (p-value 2.64 × 106). These results were confirmed by qPCR in the validation study, where PTGDR2 transcripts were significantly upregulated in asthmatic patients (p < 0.001). This upregulation was mainly detected in some subgroups such as allergic asthma, asthma with CRSwNP, AERD, eosinophilic asthma, and severe persistent asthma. PTGDR2 expression was detected in different blood cell types, and its correlation with eosinophil counts showed differences in some groups of asthmatic patients. CONCLUSIONS: We found that PTGDR2 expression levels could identify asthma patients, introduce a minimally invasive biomarker for adult asthma molecular phenotyping, and add additional information to blood eosinophils. Although further studies are required, analyzing PTGDR2 expression levels in peripheral blood of asthmatics might assist in selecting patients for treatment with specific antagonists.

Increased TPSAB1 Copy Number in a Family With Elevated Basal Serum Levels of Tryptase.

Background: Some recent familial studies have described a pattern of autosomal dominant inheritance for increased basal serum tryptase (BST), but no correlation with mRNA expression and gene dose have been reported. Objective: We analyzed TPSAB1 mRNA expression and gene dose in a four-member family with high BST and in two control subjects. Methods: Blood samples were collected from the family and control subjects. Complete morphologic, immunophenotypical, and molecular bone marrow mast cell (MC) studies were performed. mRNA gene expression and gene dose were performed in a LightCycler 480 instrument. Genotype and CNV were performed by quantitative real-time digital PCR (qdPCR). Results: CNV analysis revealed a hereditary copy number gain genotype (3ß2α) present in all the family members studied. The elevated total BST in the family members correlated with a significant increase in tryptase gene expression and dose. Conclusions and Clinical Relevance: We present a family with hereditary α-tryptasemia and elevated BST which correlated with a high expression of tryptase genes and an increased gene dose. The family members presented with atypical MC-mediator release symptoms or were even asymptomatic. Clinicians should be aware that elevated BST does not always mean an MC disorder.

Genetics and Epigenetics of Atopic Dermatitis: An Updated Systematic Review.

BACKGROUND: Atopic dermatitis is a common inflammatory skin disorder that affects up to 15-20% of the population and is characterized by recurrent eczematous lesions with intense itching. As a heterogeneous disease, multiple factors have been suggested to explain the nature of atopic dermatitis (AD), and its high prevalence makes it necessary to periodically compile and update the new information available. In this systematic review, the focus is set at the genetic and epigenetic studies carried out in the last years. METHODS: A systematic literature review was conducted in three scientific publication databases (PubMed, Cochrane Library, and Scopus). The search was restricted to publications indexed from July 2016 to December 2019, and keywords related to atopic dermatitis genetics and epigenetics were used. RESULTS: A total of 73 original papers met the inclusion criteria established, including 9 epigenetic studies. A total of 62 genes and 5 intergenic regions were described as associated with AD. CONCLUSION: Filaggrin (FLG) polymorphisms are confirmed as key genetic determinants for AD development, but also epigenetic regulation and other genes with functions mainly related to the immune system and extracellular matrix, reinforcing the notion of skin homeostasis breakage in AD.

Molecular Analysis of IL-5 Receptor Subunit Alpha as a Possible Pharmacogenetic Biomarker in Asthma.

Background: Asthma is a heterogeneous syndrome with a broad clinical spectrum and high drug response variability. The inflammatory response in asthma involves multiple effector cells and mediator molecules. Based on asthma immunopathogenesis, precision medicine can be a promising strategy for identifying biomarkers. Biologic therapies acting on the IL-5/IL-5 receptor axis have been developed. IL-5 promotes proliferation, differentiation and activation of eosinophils by binding to the IL-5 receptor, located on the surface of eosinophils and basophils. This study aimed to investigate the expression of IL5RA in patients with several types of asthma and its expression after treatment with benralizumab, a biologic directed against IL-5 receptor subunit alpha. Methods: Sixty peripheral blood samples, 30 from healthy controls and 30 from asthmatic patients, were selected for a transcriptomic RNAseq study. Differential expression analysis was performed by statistical assessment of fold changes and P-values. A validation study of IL5RA expression was developed using qPCR in 100 controls and 187 asthmatic patients. The effect of benralizumab on IL5RA expression was evaluated in five patients by comparing expression levels between pretreatment and after 3 months of treatment. The IL5RA mRNA levels were normalized to GAPDH and TBP expression values for each sample. Calculations were made by the comparative ΔΔCt method. All procedures followed the MIQE guidelines. Results: IL5RA was one of the most differentially overexpressed coding transcripts in the peripheral blood of asthmatic patients (P = 8.63E-08 and fold change of 2.22). In the qPCR validation study, IL5RA expression levels were significantly higher in asthmatic patients than in controls (P < 0.001). Significant expression differences were present in different asthmatic types. In the biological drug study, patients treated with benralizumab showed a significant decrease in IL5RA expression and blood eosinophil counts. A notable improvement in ACT and lung function was also observed in these patients. Conclusions: These results indicate that IL5RA is overexpressed in patients with different types of asthma. It could help identify which asthmatic patients will respond more efficiently to benralizumab, moving toward a more personalized asthma management. Although further studies are required, IL5RA could play a role as a biomarker and pharmacogenetic factor in asthma.

Promoter genotyping and mRNA expression - based analysis of the PTGDR gene in allergy

BACKGROUND: Prostaglandin D2 receptors are acquiring a relevant role as potential therapeutic targets in allergy. PTGDR has been described as a candidate gene in allergic disease, although functional studies on this gene are lacking. OBJECTIVE: The objective of this case-control study was to investigate the potential role of PTGDR in allergy. METHODS: The study population comprised 195 allergic patients and 112 healthy controls. The PTGDR promoter polymorphisms -1289G>A, -1122T>C, -881C>T, -834C>T, -613C>T, -549T>C, -441C>T, -197T>C, and -95G>T were amplified by polymerase chain reaction (PCR) and sequenced. PTGDR expression levels were analyzed using quantitative PCR and normalized to GAPDH and TBP mRNA levels. All procedures were performed following the Minimum Information for Publication of Quantitative Real-Time PCR Experiment guidelines. RESULTS: PTGDR expression levels were significantly higher in allergic patients than in controls (P<.001). Receiver operating characteristic analysis for expression of PTGDR showed a sensitivity of 81.4% compared with 67% for IgE levels. In addition, differences in the genotypic distribution of the polymorphisms -1289G>A and -1122T>C were found in allergic patients (P=.009). CONCLUSIONS: The results indicate that PTGDR overexpression is associated with allergy. The polymorphisms -1289G>A and -1122T>C partly explain the variation in expression we observed. PTGDR expression could have a potential role as a biomarker and pharmacogenetic factor in allergy

ANTECEDENTES: Los receptores de la prostaglandina D2 están adquiriendo un papel relevante como posibles dianas terapéuticas en la alergia. El gen PTGDR ha sido descrito como un gen candidato en una enfermedad alérgica, sin embargo, faltan estudios funcionales sobre este gen. OBJETIVO: El objetivo de este estudio de casos y controles fue analizar el posible papel del gen PTGDR en la alergia. MÉTODOS: Se incluyeron 195 pacientes alérgicos y 112 controles sanos. Un fragmento de la región promotora de PTGDR que comprendía las posiciones polimórficas -1289G> A, -1122T>C, -881C>T, -834C>T, -613C>T, -549T>C, -441C>T, -197T>C y -95G>T fue amplificado mediante la reacción en cadena de la polimerasa y secuenciado. Los niveles de expresión de PTGDR se analizaron mediante q-PCR y se normalizaron a los niveles de ARNm de GAPDH y TBP. Todos los procedimientos se realizaron siguiendo la guía MIQE. RESULTADOS: Los niveles de expresión de PTGDR fueron significativamente superiores en los pacientes alérgicos que en los controles (p < 0,001). El análisis ROC para la expresión de PTGDR mostró una sensibilidad del 81,4% en comparación con el 67% para los niveles de IgE. Además, se encontraron diferencias en la distribución genotípica de los polimorfismos -1289G>A y -1122T>C en pacientes alérgicos (p = 0,009). CONCLUSIONES: Los resultados indican que la sobreexpresión de PTGDR se asocia con la alergia. Además, los polimorfismos -1289G>A y -1122T>C contribuyen a explicar parte de la variación de expresión observada. La expresión de PTGDR podría tener un papel potencial como biomarcador y factor farmacogenético en la alergia

YRNAs overexpression and potential implications in allergy.

BACKGROUND: Small non-coding RNAs (snRNAs) develop important functions related to epigenetic regulation. YRNAs are snRNAs involved in the initiation of DNA replication and RNA stability that regulate gene expression. They have been related to autoimmune, cancer and inflammatory diseases but never before to allergy. In this work we described for the first time in allergic patients the differential expression profile of YRNAs, their regulatory mechanisms and their potential as new diagnostic and therapeutic targets. METHODS: From a previous whole RNAseq study in B cells of allergic patients, differential expression profiles of coding and non-coding transcripts were obtained. To select the most differentially expressed non coding transcripts, fold change and p-values were analyzed. A validation of the expression differences detected was developed in an independent cohort of 304 individuals, 208 allergic patients and 96 controls by using qPCR. Potential binding and retrotransponibility capacity were characterized by in silico structural analysis. Using a novel bioinformatics approach, RNA targets identification, functional enrichment and network analyses were performed. RESULTS: We found that almost 70% of overexpressed non-coding transcripts in allergic patients corresponded to YRNAs. From the three more differentially overexpressed candidates, increased expression was independently confirmed in the peripheral blood of allergic patients. Structural analysis suggested a protein binding capacity decrease and an increase in retrotransponibility. Studies of RNA targets allowed the identification of sequences related to the immune mechanisms underlying allergy. CONCLUSIONS: Overexpression of YRNAs is observed for the first time in allergic patients. Structural and functional information points to their implication on regulatory mechanisms of the disease.

PTGDR expression is upregulated through retinoic acid receptors (RAR) mechanism in allergy.

Functional studies suggest that promoter polymorphisms of the Prostaglandin D Receptor (PTGDR) gene can be involved in asthma. All-trans Retinoic acid (ATRA) has also been linked to allergic diseases. We have previously described the PTGDR promoter activation mediated by ATRA through response elements (RARE) at position -549T> C. In this study we aimed to analyze the effect of retinoic acid (RA) on the expression of PTGDR, the production of cytokines as well as to evaluate the binding of RA receptors to RA-Response Elements (RARE) sequences. A549 cells were transfected with vectors carrying different PTGDR haplotypes and treated with all-Trans Retinoic Acid (ATRA). PTGDR expression was measured by qPCR. Chromatin Immunoprecipitation assays (ChIP) were performed in ATRA stimulated KU812 cells and in PBMCs of patients carrying CTCT, CCCC or CCCT haplotypes. In addition, a broad panel of cytokines was analyzed by cytometric bead assay in A549 cells. The expression of PTGDR increased in A549 cells transfected with PTGDR-variants. The CCCC haplotype showed a significantly higher expression compared with CTCT. However, we found that RA up-regulated PTGDR expression through RARα mainly in the CTCT variant. Experiments on PBMCs from allergic patients carrying the -549T and -549C variant of the PTGDR promoter after ATRA and RAR antagonist administration confirmed the modulation of PTGDR by ATRA. The cytokine analysis showed that IL4 and IL6 levels were significantly increased in A549 cells transfected with PTGDR. In addition, ATRA treatment decreased the levels of IL4, IL6 and TNFα in A549 cells, whereas it increased IL4 and TNFα levels in PTGDR-transfected cells. We observed genetic differences in the regulation of PTGDR by ATRA that could contribute to the phenotypic differences observed in allergic patients. Our findings showed that RAR modulation by PTGDR might have an impact on Th2 responses, suggesting that RAR could be a potential therapeutic target in allergic inflammation.

Cluster Analysis Identifies 3 Phenotypes within Allergic Asthma.

BACKGROUND: Asthma is a heterogeneous chronic disease with different clinical expressions and responses to treatment. In recent years, several unbiased approaches based on clinical, physiological, and molecular features have described several phenotypes of asthma. Some phenotypes are allergic, but little is known about whether these phenotypes can be further subdivided. OBJECTIVE: We aimed to phenotype patients with allergic asthma using an unbiased approach based on multivariate classification techniques (unsupervised hierarchical cluster analysis). METHODS: From a total of 54 variables of 225 patients with well-characterized allergic asthma diagnosed following American Thoracic Society (ATS) recommendation, positive skin prick test to aeroallergens, and concordant symptoms, we finally selected 19 variables by multiple correspondence analyses. Then a cluster analysis was performed. RESULTS: Three groups were identified. Cluster 1 was constituted by patients with intermittent or mild persistent asthma, without family antecedents of atopy, asthma, or rhinitis. This group showed the lowest total IgE levels. Cluster 2 was constituted by patients with mild asthma with a family history of atopy, asthma, or rhinitis. Total IgE levels were intermediate. Cluster 3 included patients with moderate or severe persistent asthma that needed treatment with corticosteroids and long-acting ß-agonists. This group showed the highest total IgE levels. CONCLUSIONS: We identified 3 phenotypes of allergic asthma in our population. Furthermore, we described 2 phenotypes of mild atopic asthma mainly differentiated by a family history of allergy.

PTGDR gene expression and response to dexamethasone treatment in an in vitro model.

Asthma is a multifactorial pathology influenced by environmental and genetic factors. Glucocorticoid treatment decreases symptoms by regulating genes involved in the inflammatory process through binding to specific DNA sequences. Polymorphisms located in the promoter region of the Prostaglandin D Receptor (PTGDR) gene have been related to asthma. We aimed to analyze the effect of PTGDR promoter haplotypes on gene expression and response to corticosteroid therapy. A549 lung epithelial cells were transfected with vectors carrying four different PTGDR haplotypes (CTCT, CCCC, CCCT and TCCT), and treated with dexamethasone. Different approaches to study the promoter activity (Dual Luciferase Reporter System), gene expression levels (qPCR) and cytokine secretion (Multiplexed Bead-based Flow Cytometric) were used. In addition, in silico analysis was also performed. Cells carrying the TCCT haplotype showed the lowest promoter activity (p-value<0.05) and mRNA expression levels in basal conditions. After dexamethasone treatment, cells carrying the wild-type variant CTCT showed the highest response, and those carrying the TCCT variant the lowest (p-value<0.05) in luciferase assays. Different transcription factor binding patterns were identified in silico. Moreover, differences in cytokine secretion were also found among different promoter haplotypes. Polymorphisms of PTGDR gene influence basal promoter activity and gene expression, as well as the cytokine secretory pattern. Furthermore, an association between these positions and response to corticoid treatment was observed.

Pharmacogenetics and the treatment of asthma.

Heterogeneity defines both the natural history of asthma as well as patient's response to treatment. Pharmacogenomics contribute to understand the genetic basis of drug response and thus to define new therapeutic targets or molecular biomarkers to evaluate treatment effectiveness. This review is initially focused on different genes so far involved in the pharmacological response to asthma treatment. Specific considerations regarding allergic asthma, the pharmacogenetics aspects of polypharmacy and the application of pharmacogenomics in new drugs in asthma will also be addressed. Finally, future perspectives related to epigenetic regulatory elements and the potential impact of systems biology in pharmacogenetics of asthma will be considered.

A Ras GTPase associated protein is involved in the phototropic and circadian photobiology responses in fungi.

Light is an environmental signal perceived by most eukaryotic organisms and that can have major impacts on their growth and development. The MadC protein in the fungus Phycomyces blakesleeanus (Mucoromycotina) has been postulated to form part of the photosensory input for phototropism of the fruiting body sporangiophores, but the madC gene has remained unidentified since the 1960s when madC mutants were first isolated. In this study the madC gene was identified by positional cloning. All madC mutant strains contain loss-of-function point mutations within a gene predicted to encode a GTPase activating protein (GAP) for Ras. The madC gene complements the Saccharomyces cerevisiae Ras-GAP ira1 mutant and the encoded MadC protein interacts with P. blakesleeanus Ras homologs in yeast two-hybrid assays, indicating that MadC is a regulator of Ras signaling. Deletion of the homolog in the filamentous ascomycete Neurospora crassa affects the circadian clock output, yielding a pattern of asexual conidiation similar to a ras-1 mutant that is used in circadian studies in N. crassa. Thus, MadC is unlikely to be a photosensor, yet is a fundamental link in the photoresponses from blue light perceived by the conserved White Collar complex with Ras signaling in two distantly-related filamentous fungal species.