Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution.

DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B-induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi.

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.

Phycomyces MADB interacts with MADA to form the primary photoreceptor complex for fungal phototropism.

The fungus Phycomyces blakesleeanus reacts to environmental signals, including light, gravity, touch, and the presence of nearby objects, by changing the speed and direction of growth of its fruiting body (sporangiophore). Phototropism, growth toward light, shares many features in fungi and plants but the molecular mechanisms remain to be fully elucidated. Phycomyces mutants with altered phototropism were isolated approximately 40 years ago and found to have mutations in the mad genes. All of the responses to light in Phycomyces require the products of the madA and madB genes. We showed that madA encodes a protein similar to the Neurospora blue-light photoreceptor, zinc-finger protein WC-1. We show here that madB encodes a protein similar to the Neurospora zinc-finger protein WC-2. MADA and MADB interact to form a complex in yeast 2-hybrid assays and when coexpressed in E. coli, providing evidence that phototropism and other responses to light are mediated by a photoresponsive transcription factor complex. The Phycomyces genome contains 3 genes similar to wc-1, and 4 genes similar to wc-2, many of which are regulated by light in a madA or madB dependent manner. We did not detect any interactions between additional WC proteins in yeast 2-hybrid assays, which suggest that MADA and MADB form the major photoreceptor complex in Phycomyces. However, the presence of multiple wc genes in Phycomyces may enable perception across a broad range of light intensities, and may provide specialized photoreceptors for distinct photoresponses.

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.

Protein-DNA interactions in the promoter region of the Phycomyces carB and carRA genes correlate with the kinetics of their mRNA accumulation in response to light.

Carotene biosynthesis in Phycomyces is photoinducible and carried out by phytoene dehydrogenase (encoded by carB) and a bifunctional enzyme possessing lycopene cyclase and phytoene synthase activities (carRA). A light pulse followed by periods of darkness produced similar biphasic responses in the expression of the carB and carRA genes, indicating their coordinated regulation. Specific binding complexes were formed between the carB-carRA intergenic region and protein extracts from wild type mycelia grown in the dark or 8min after irradiation. These two conditions correspond to the points at which the expression of both genes is minimal, suggesting that these binding complexes are involved in the down-regulation of photocarotenogenesis in Phycomyces. Protein extracts from carotene mutants failed to form the dark retardation complex, suggesting a role of these genes in the regulation of photocarotenogenesis. In contrast, protein extracts from phototropic mutants formed dark retardation complexes identical to that of the wild type.

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.

A new PTGDR promoter polymorphism in a population of children with asthma.

Recently, functional genetic variants of the PTGDR gene have been associated with asthma. The objective of this work was to study polymorphisms of the promoter region of PTGDR and their haplotype and diplotype combinations in a Spanish population of children with asthma. In this study, 200 Caucasian individuals were included. Asthma was specialist-physician diagnosed according to the ATS criteria. The polymorphisms were analyzed by direct sequencing. In the study, the new polymorphism (-613C > T) in the promoter region of PTGDR was analyzed. The CT genotype was more common in controls (17%) than in patients with asthma (1%) (p-value = 0.0003; OR, 0.057; 95% CI, 0.007-0.441). The CCCT CCCC diplotype (promoter positions -613, -549, -441, and -197) was more frequent in the group of patients with asthma [Fisher's p-value = 0.012; OR, 10.24; 95% CI (1.25-83.68)]; this diplotype is unambiguous. To our knowledge, this is the first study of -613C > T PTGDR polymorphism in patients. This analysis provides more complete information on influence of diplotype combinations of PTGDR polymorphisms in asthma.

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.

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.

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)

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.