RESUMEN
Thyroid dyshormonogenesis (THD) is a heterogeneous group of genetic diseases caused by the total or partial defect in the synthesis or secretion of thyroid hormones. Genetic variants in DUOX2 can cause partial to total iodination organification defects and clinical heterogeneity, from transient to permanent congenital hypothyroidism. The aim of this study was to undertake a molecular characterization and genotype-phenotype correlation in patients with THD and candidate variants in DUOX2. A total of 31 (19.38%) patients from the Catalan Neonatal Screening Program presented with variants in DUOX2 that could explain their phenotype. Fifteen (48.39%) patients were compound heterozygous, 10 (32.26%) heterozygous, and 4 (12.90%) homozygous. In addition, 8 (26.67%) of these patients presented variants in other genes. A total of 35 variants were described, 10 (28.57%) of these variants have not been previously reported in literature. The most frequent variant in our cohort was c.2895_2898del/p.(Phe966SerfsTer29), classified as pathogenic according to reported functional studies. The final diagnosis of this cohort was permanent THD in 21 patients and transient THD in 10, according to reevaluation and/or need for treatment with levothyroxine. A clear genotype-phenotype correlation could not be identified; therefore, functional studies are necessary to confirm the pathogenicity of the variants.
Asunto(s)
Oxidasas Duales , Estudios de Asociación Genética , Humanos , Oxidasas Duales/genética , Oxidasas Duales/metabolismo , Femenino , Masculino , Recién Nacido , Disgenesias Tiroideas/genética , Disgenesias Tiroideas/patología , Fenotipo , Mutación , Genotipo , Hipotiroidismo Congénito/genética , Tamizaje Neonatal , TiroxinaRESUMEN
Congenital hypothyroidism (CHT) is a diverse condition with various genetic etiologies. This study aimed to investigate the utility of next-generation sequencing (NGS) analysis in guiding treatment decisions and predicting prognosis for CHT patients with gland in situ (GIS). A retrospective analysis was conducted on 33 CHT patients with GIS who underwent NGS analysis at a single institution between 2018 and 2023. Patients were classified as having permanent (PCH), transient congenital hypothyroidism, or ambiguous congenital hypothyroidism (ACH) CHT based on their response to levothyroxine discontinuation at 3 years of age. Among the 33 patients, genetic variants were identified in 26, with the most prevalent variants found in DUOX2 (26.92%), TSHR (30.77%), TG (19.35%), and DUOXA2 (19.23%). Patients with high initial thyroid-stimulating hormone levels (>50 mIU/L) and low free thyroxine levels (<0.89 ng/dL) at diagnosis tended to have compound heterozygous or homozygous variants in DUOX2, DUOXA2, and TG, and were more likely to develop PCH. In contrast, patients with heterozygous variants in these genes often exhibited ACH. TSHR variants were associated with diverse clinical manifestations, ranging from PCH to ACH, and were more common in patients with initial thyroid-stimulating hormone levels <50 mIU/L. The study highlights the potential utility of NGS analysis in predicting the clinical course and guiding treatment decisions for CHT patients with GIS. Genetic analysis may aid in determining the appropriate duration of levothyroxine therapy and monitoring strategies, particularly in cases where traditional clinical indicators are inconclusive.
Asunto(s)
Hipotiroidismo Congénito , Oxidasas Duales , Secuenciación de Nucleótidos de Alto Rendimiento , Receptores de Tirotropina , Tiroxina , Humanos , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/tratamiento farmacológico , Hipotiroidismo Congénito/diagnóstico , Hipotiroidismo Congénito/sangre , Femenino , Masculino , Estudios Retrospectivos , Oxidasas Duales/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Tiroxina/uso terapéutico , Receptores de Tirotropina/genética , Preescolar , Lactante , Recién Nacido , Tiroglobulina/genética , Tiroglobulina/sangre , Proteínas de la MembranaRESUMEN
BACKGROUND: Enterobacter cloacae insecticidal proteins have been reported to kill Galleria mellonella larvae through affecting their midgut microbiome. However, the mechanisms involved remain unclear. Here we aim to investigate how the insecticidal proteins act on the midgut Duox-ROS system and microbial community of G. mellonella larvae. METHODS: Reverse transcription qPCR and fluorescence probes were utilized to assess the Duox expression levels and to evaluate quantitative changes of the ROS levels. Sequencing of the 16S rRNA gene sequences of the midgut bacteria of G. mellonella larvae was conducted for further analyses of bacterial diversity, composition, and abundance. RESULTS: After the injection of the insecticidal proteins, the Duox expression levels first increased within 28 h, then dramatically peaked at 36 h, and slowly decreased thereafter. Simultaneously, the ROS levels increased significantly at 36 h, peaked at 48 h, and rapidly declined to the normal level at 60 h. Responsive to the change of the ROS levels, the structure of the midgut microbial community was altered substantially, compared to that of the untreated larvae. The relative abundance of Enterobacteriaceae and other specific pathogenic bacteria increased significantly, whereas that of Lactobacillus decreased sharply. Importantly, notable shifts were observed in the crucial midgut predicted metabolic functions, including membrane transportation, carbohydrate metabolism, and amino acid metabolism. CONCLUSION: Insecticidal proteins of E. cloacae kill G. mellonella larvae mainly through generation of high oxidative stress, alterations of the midgut microbial community and function, and damage to the physiological functions. These findings provide insights into the inhibition mechanism of E. cloacae insecticidal proteins to G. mellonella larvae.
Asunto(s)
Enterobacter cloacae , Microbioma Gastrointestinal , Larva , Mariposas Nocturnas , Especies Reactivas de Oxígeno , Animales , Larva/microbiología , Mariposas Nocturnas/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Insecticidas , Proteínas Bacterianas , ARN Ribosómico 16S , Oxidasas DualesRESUMEN
Hydrogen peroxide, produced by Dual Oxidase (Duox), is essential for thyroid hormone synthesis. Duox activation involves Ca2+ binding to its EF-hand Domain (EFD), which contains two EF-hands (EFs). In this study, we characterized a truncated EFD using spectrometry, calorimetry, electrophoretic mobility, and gel filtration to obtain its Ca2+ binding thermodynamic and kinetics, as well as to assess the associated conformational changes. Our results revealed that its 2nd EF-hand (EF2) exhibits a strong exothermic Ca2+ binding (Ka = 107 M-1) while EF1 shows a weaker binding (Ka = 105 M-1), resulting in the burial of its negatively charged residues. The Ca2+ binding to EFD results in a stable structure with a melting temperature shifting from 67 to 99 °C and induces a structural transition from a dimeric to monomeric form. EF2 appears to play a role in dimer formation in its apo form, while the hydrophobic exposure of Ca2+-bound-EF1 is crucial for dimer formation in its holo form. The result is consistent with structures obtained from Cryo-EM, indicating that a stable structure of EFD with hydrophobic patches upon Ca2+ binding is vital for its Duox's domain-domain interaction for electron transfer.
Asunto(s)
Calcio , Oxidasas Duales , Calcio/metabolismo , Calcio/química , Oxidasas Duales/metabolismo , Oxidasas Duales/química , Motivos EF Hand , Termodinámica , Unión Proteica , Humanos , Cinética , Sitios de Unión , Multimerización de ProteínaRESUMEN
Thyroid dyshormonogenesis (TDH) is responsible for 15%-25% of congenital hypothyroidism (CH) cases. Pathogenetic variants of this common inherited endocrine disorders vary geographically. Unraveling the genetic underpinnings of TDH is essential for genetic counseling and precise therapeutic strategies. This study aims to identify genetic variants associated with TDH in Southern Taiwan using whole exome sequencing (WES). We included CH patients diagnosed through newborn screening at a tertiary medical center from 2011 to 2022. Permanent TDH was determined based on imaging evidence of bilateral thyroid structure and the requirement for continuous medication beyond 3 years of age. Genomic DNA extracted from blood was used for exome library construction, and pathogenic variants were detected using an in-house algorithm. Of the 876 CH patients reviewed, 121 were classified as permanent, with 47 (40%) confirmed as TDH. WES was conducted for 45 patients, and causative variants were identified in 32 patients (71.1%), including DUOX2 (15 cases), TG (8 cases), TSHR (7 cases), TPO (5 cases), and DUOXA2 (1 case). Recurrent variants included DUOX2 c.3329G>A, TSHR c.1349G>A, TG c.1348delT, and TPO c.2268dupT. We identified four novel variants based on genotype, including TSHR c.1135C>T, TSHR c.1349G>C, TG c.2461delA, and TG c.2459T>A. This study underscores the efficacy of WES in providing definitive molecular diagnoses for TDH. Molecular diagnoses are instrumental in genetic counseling, formulating treatment, and developing management strategies. Future research integrating larger population cohorts is vital to further elucidate the genetic landscape of TDH.
Asunto(s)
Hipotiroidismo Congénito , Secuenciación del Exoma , Yoduro Peroxidasa , Receptores de Tirotropina , Humanos , Taiwán , Femenino , Masculino , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/diagnóstico , Recién Nacido , Yoduro Peroxidasa/genética , Receptores de Tirotropina/genética , Oxidasas Duales/genética , Tiroglobulina/genética , Proteínas de Unión a Hierro/genética , Preescolar , Variación Genética , Mutación , Disgenesias Tiroideas/genética , Disgenesias Tiroideas/diagnóstico , Lactante , AutoantígenosRESUMEN
BACKGROUND: Oxidative stress is implicated in the pathogenesis of heart failure. Dual oxidase 1 (DUOX1) might be important in heart failure development through its mediating role in oxidative stress. This study was designed to evaluate the potential role of DUOX1 in heart failure. MATERIALS AND METHODS: AC16 cells were treated with 2 µmol/L of doxorubicin (DOX) for 12, 24, and 48 h to construct a heart failure model. DUOX1 overexpression and silencing in AC16 cell were established. DUOX1 expression was detected by Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Pyroptosis and reactive oxygen species (ROS) production were measured by flow cytometry. RESULTS: Increased DUOX1 expression levels were observed after DOX treatment for 24 h in AC16 cells. DUOX1 silencing inhibited DOX-induced pyroptosis and ROS production. The release of IL-1ß, IL-18, and lactate dehydrogenase (LDH), and expression levels of pyroptosis-related proteins were also decreased. DUOX1 overexpression increased pyroptosis, ROS production, IL-1ß, IL-18, and LDH release, and pyroptosis-related protein expression. N-acetyl-cysteine (NAC) significantly reversed DUOX1-induced pyroptosis, ROS, and related factors. CONCLUSION: These results suggest that DUOX1-derived genotoxicity could promote heart failure development. In the process, oxidative stress and pyroptosis may be involved in the regulation of DUOX1 in heart failure.
Asunto(s)
Oxidasas Duales , Insuficiencia Cardíaca , Estrés Oxidativo , Piroptosis , Especies Reactivas de Oxígeno , Humanos , Caspasa 1/metabolismo , Línea Celular , Doxorrubicina/farmacología , Oxidasas Duales/metabolismo , Oxidasas Duales/genética , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia ArribaRESUMEN
Background: DUOX2 is one of the major causative genes of congenital hypothyroidism (CH). Still, the mutation spectrum and clinical outcomes of biallelic DUOX2 variants are not fully understood. This study aimed to elucidate the molecular features and long-term clinical manifestations of CH caused by multiple pathogenic DUOX2 variants. Methods: A total of 255 patients with CH were screened for rare variants of 11 known causative genes. DUOX2 variants were classified according to their protein structure and residual activity. In vitro assays were performed for several variants of unknown functions. Clinical analyses were conducted for patients with multiple pathogenic variants of DUOX2 but not of other genes. Results: We identified 24 pathogenic variants of DUOX2, together with two benign variants and seven variants of uncertain significance, in 63 patients. The pathogenic variants included three missense substitutions and one frameshift variant that have not yet been linked to CH. Twenty-one patients carried multiple pathogenic DUOX2 variants without any other pathogenic gene variants. Three of the 21 patients harbored homozygous variants. Family analysis, long-read amplicon sequencing, and haplotype phasing confirmed compound heterozygosity of the DUOX2 variants in 14 patients, whereas the allelic positions of the variants in the remaining four patients could not be determined. Of the 21 patients, 19 were treated with levothyroxine; their ages at drug withdrawal ranged from 9 months to 21.4 years. Three patients required retreatment after drug-free intervals of 6 months, 8 months, and 10 years. There were no differences in clinical severity among patients with DUOX2 amorphic/amorphic, amorphic/hypomorphic, and hypomorphic/hypomorphic variants. Conclusions: These results broaden the mutational spectrum of DUOX2. Furthermore, our data imply that patients with multiple pathogenic DUOX2 variants typically exhibit transient CH without significant genotype-phenotype correlations. Most importantly, this study demonstrated for the first time that these patients are at risk of developing recurrent hypothyroidism after a long drug-free interval.
Asunto(s)
Hipotiroidismo Congénito , Oxidasas Duales , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Hipotiroidismo Congénito/genética , Oxidasas Duales/genética , Mutación , NADPH Oxidasas/genética , Tiroxina/uso terapéutico , Adulto JovenRESUMEN
The insights into interactions between host genetics and gut microbiome (GM) in colorectal tumor susceptibility (CTS) remains lacking. We used Collaborative Cross mouse population model to identify genetic and microbial determinants of Azoxymethane-induced CTS. We identified 4417 CTS-associated single nucleotide polymorphisms (SNPs) containing 334 genes that were transcriptionally altered in human colorectal cancers (CRCs) and consistently clustered independent human CRC cohorts into two subgroups with different prognosis. We discovered a set of genera in early-life associated with CTS and defined a 16-genus signature that accurately predicted CTS, the majority of which were correlated with human CRCs. We identified 547 SNPs associated with abundances of these genera. Mediation analysis revealed GM as mediators partially exerting the effect of SNP UNC3869242 within Duox2 on CTS. Intestine cell-specific depletion of Duox2 altered GM composition and contribution of Duox2 depletion to CTS was significantly influenced by GM. Our findings provide potential novel targets for personalized CRC prevention and treatment.
Asunto(s)
Azoximetano , Ratones de Colaboración Cruzada , Neoplasias Colorrectales , Microbioma Gastrointestinal , Polimorfismo de Nucleótido Simple , Animales , Neoplasias Colorrectales/microbiología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inducido químicamente , Humanos , Ratones , Ratones de Colaboración Cruzada/genética , Oxidasas Duales/genética , Oxidasas Duales/metabolismo , Predisposición Genética a la Enfermedad , Masculino , Bacterias/genética , Bacterias/clasificación , Bacterias/metabolismo , Bacterias/aislamiento & purificación , Modelos Animales de Enfermedad , FemeninoRESUMEN
The gut must perform a dual role of protecting the host against toxins and pathogens while harboring mutualistic microbiota. Previous studies suggested that the NADPH oxidase Duox contributes to intestinal homeostasis in Drosophila by producing reactive oxygen species (ROS) in the gut that stimulate epithelial renewal. We find instead that the ROS generated by Duox in the Malpighian tubules leads to the production of Upd3, which enters the gut and stimulates stem cell proliferation. We describe in Drosophila the existence of a countercurrent flow system, which pushes tubule-derived Upd3 to the anterior part of the gut and stimulates epithelial renewal at a distance. Thus, our paper clarifies the role of Duox in gut homeostasis and describes the existence of retrograde fluid flow in the gut, collectively revealing a fascinating example of inter-organ communication.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Mucosa Intestinal , Túbulos de Malpighi , Especies Reactivas de Oxígeno , Animales , Túbulos de Malpighi/metabolismo , Proteínas de Drosophila/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Mucosa Intestinal/metabolismo , Drosophila melanogaster/metabolismo , NADPH Oxidasas/metabolismo , Oxidasas Duales/metabolismo , Oxidasas Duales/genética , Proliferación Celular , Homeostasis , Drosophila/metabolismoRESUMEN
We identified and validated a collection of circular RNAs (circRNAs) in Drosophila melanogaster. We show that depletion of the pro-viral circRNA circATP8B(2), but not its linear siblings, compromises viral infection both in cultured Drosophila cells and in vivo. In addition, circATP8B(2) is enriched in the fly gut, and gut-specific depletion of circATP8B(2) attenuates viral replication in an oral infection model. Furthermore, circATP8B(2) depletion results in increased levels of reactive oxygen species (ROS) and enhanced expression of dual oxidase (Duox), which produces ROS. Genetic and pharmacological manipulations of circATP8B(2)-depleted flies that reduce ROS levels rescue the viral replication defects elicited by circATP8B(2) depletion. Mechanistically, circATP8B(2) associates with Duox, and circATP8B(2)-Duox interaction is crucial for circATP8B(2)-mediated modulation of Duox activity. In addition, Gαq, a G protein subunit required for optimal Duox activity, acts downstream of circATP8B(2). We conclude that circATP8B(2) regulates antiviral defense by modulating Duox expression and Duox-dependent ROS production.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , ARN Circular , Especies Reactivas de Oxígeno , Animales , Especies Reactivas de Oxígeno/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Drosophila melanogaster/inmunología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Replicación Viral , ARN/metabolismo , ARN/genética , NADPH Oxidasas/metabolismo , NADPH Oxidasas/genética , Oxidasas Duales/metabolismo , Oxidasas Duales/genéticaRESUMEN
PURPOSE: Crohn's disease is a chronic gastrointestinal inflammatory disease with possible extraintestinal symptoms. There are predisposing genetic factors and even monogenic variants of the disorder. One of the possible genetic factors are variants of the DUOX2 gene. The protein product of the DUOX2 gene is a dual oxidase enzyme producing H2O2 in the bowel. Reduced H2O2 levels impact mucosal homeostasis and contribute to the development of inflammatory bowel disease. Thus far, only 19 patients with IBD with the DUOX2 variants have been described. METHODS: Here we present a case report of an adolescent female diagnosed at eleven years of age with IBD that was subsequently reclassified as Crohn's disease. She was treated with immunosuppressants and biological therapy but experienced additional complications. Her peripheral blood lymphocyte DNA was studied using massive parallel sequencing. Detected variants were functionally studied. RESULTS: Whole exome sequencing found two novel DUOX2 gene variants: a de novo variant c.3646C>T; p.R1216W and a maternally inherited variant c.3391G>A; p.A1131T which were initially classified as variants of unknown significance. However, follow-up functional studies demonstrated that both DUOX2 variants led to impaired H2O2 generation, which led to their reclassification to the likely pathogenic class according to the ACMG.net. Therefore, we conclude that these variants are causative for the disease. CONCLUSIONS: Identifying novel variants in patients with Crohn's disease and their families is important for precision medicine approaches and understanding of the pathogenesis of likely "monogenic" rare forms of inflammatory bowel disease.
Asunto(s)
Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Humanos , Adolescente , Femenino , Enfermedad de Crohn/genética , Oxidasas Duales/genética , Peróxido de Hidrógeno , Enfermedades Inflamatorias del Intestino/genéticaRESUMEN
Background: Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype-phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. Methods: In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results: Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. Conclusions: We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.
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Hipotiroidismo Congénito , Humanos , China , Hipotiroidismo Congénito/diagnóstico , Hipotiroidismo Congénito/genética , AMP Cíclico , Oxidasas Duales/genética , Mutación , Fenotipo , Receptores de Tirotropina/genética , TirotropinaRESUMEN
Stimulation of human keratinocytes with particulate matter 2.5 (PM2.5) elicits complex signaling events, including a rise in the generation of reactive oxygen species (ROS). However, the mechanisms underlying PM2.5-induced ROS production remain unknown. Here, we show that PM2.5-induced ROS production in human keratinocytes is mediated via the NADPH oxidase (NOXs) system and the Ca2+ signaling pathway. PM2.5 treatment increased the expression of NOX1, NOX4, and a calcium-sensitive NOX, dual oxidase 1 (DUOX1), in human epidermal keratinocyte cell line. PM2.5 bound to aryl hydrocarbon receptor (AhR), and this complex bound to promoter regions of NOX1 and DUOX1, suggesting that AhR acted as a transcription factor of NOX1 and DUOX1. PM2.5 increased the transcription of DUOX1 via epigenetic modification. Moreover, a link between DNA demethylase and histone methyltransferase with the promoter regions of DUOX1 led to an elevation in the expression of DUOX1 mRNA. Interestingly, PM2.5 increased NOX4 expression and promoted the interaction of NOX4 and Ca2+ channels within the cytoplasmic membrane or endoplasmic reticulum, leading to Ca2+ release. The increase in intracellular Ca2+ concentration activated DUOX1, responsible for ROS production. Our findings provide evidence for a PM2.5-mediated ROS-generating system network, in which increased NOX1, NOX4, and DUOX1 expression serves as a ROS signal through AhR and Ca2+ activation.
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NADPH Oxidasas , Receptores de Hidrocarburo de Aril , Humanos , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Oxidasas Duales/genética , Oxidasas Duales/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Material Particulado/toxicidad , Epigénesis GenéticaRESUMEN
NADPH oxidase enzymes (NOX) are involved in all stages of carcinogenesis, but their expression levels and prognostic value in breast cancer (BC) remain unclear. Thus, we aimed to assess the expression and prognostic value of NOX enzymes in BC samples using online databases. For this, mRNA expression from 290 normal breast tissue samples and 1904 BC samples obtained from studies on cBioPortal, Kaplan-Meier Plotter, and The Human Protein Atlas were analyzed. We found higher levels of NOX2, NOX4, and Dual oxidase 1 (DUOX1) in normal breast tissue. NOX1, NOX2, and NOX4 exhibited higher expression in BC, except for the basal subtype, where NOX4 expression was lower. DUOX1 mRNA levels were lower in all BC subtypes. NOX2, NOX4, and NOX5 mRNA levels increased with tumor progression stages, while NOX1 and DUOX1 expression decreased in more advanced stages. Moreover, patients with low expression of NOX1, NOX4, and DUOX1 had lower survival rates than those with high expression of these enzymes. In conclusion, our data suggest an overexpression of NOX enzymes in breast cancer, with certain isoforms showing a positive correlation with tumor progression.
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Neoplasias de la Mama , NADPH Oxidasas , Humanos , Femenino , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Oxidasas Duales/genética , Neoplasias de la Mama/genética , Pronóstico , Especies Reactivas de Oxígeno/metabolismo , ARN Mensajero/genética , Expresión Génica , NADPH Oxidasa 4/genética , NADPH Oxidasa 1/genéticaRESUMEN
OBJECTIVE: To explore the value of whole exome sequencing for the inferential analysis of recessive genetic disease carrier status for couples with a child died of Primary immunodeficiency (PID). METHODS: Clinical data was collected from four couples with a childbearing history of PID who had sought genetic counseling and undergone genetic testing at Henan Provincial People's Hospital from February 2017 to December 2021. Whole exome sequencing (WES) was performed on both partners of each couple, and candidate variants were validated by Sanger sequencing and fluorescent quantitative PCR. Prenatal diagnosis was conducted on fetuses of these couples after confirming the variants. RESULTS: A total of six variants were detected in four genes including IL2RG, BTK, CYBB, and DUOX2. Among these, the c.1265G>A and c.3329G>A variants of the DUOX2 gene and the c.676C>T variant of the IL2RG gene were previously known as pathogenic variants. On the other hand, the Exon5_8del variant of the IL2RG gene, the c.184_185delAC variant of the BTK gene, and the c.472A>T variant of the CYBB gene were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics, the IL2RG: Exon5_8del, BTK: c.184_185delAC and CYBB: c.472A>T variants were classified as likely pathogenic (PVS1+PM2_Supporting+PP4).Prenatal diagnosis was conducted for three couples during their subsequent pregnancies, and the results revealed that the fetuses had the wild-type genotypes at the c.184_185 position of the BTK gene, the c.472 position of the CYBB gene, and the c.676 position of the IL2RG gene. Follow-up examinations one year after birth has found no abnormality in the infants. CONCLUSION: WES is an important tool to infer and analyze the carrier status for couples who had given births to children died of PID and improve the positive detection rate.
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Pruebas Genéticas , Diagnóstico Prenatal , Lactante , Embarazo , Niño , Femenino , Humanos , Secuenciación del Exoma , Oxidasas Duales , Genotipo , MutaciónRESUMEN
In clinical practice, the diagnosis of ulcerative colitis (UC) mainly relies on a comprehensive analysis of a series of signs and symptoms of patients. The current biomarkers for diagnosis of UC and prognostic prediction of anti-TNF-α therapy are inaccurate. The present study aimed to perform an integrative analysis of gene expression profiles in patients with UC. A total of seven datasets from the GEO database that met our strict inclusion criteria were included. After identifying differentially expressed genes (DEGs) between UC patients and healthy individuals, the diagnostic and prognostic utility of the DEGs were then analyzed via least absolute shrinkage and selection operator and support-vector machine recursive feature elimination. Subgroup analyses of the treated and untreated groups, as well as the treatment-response group and non-response group, were also performed. Furthermore, the relationship between the expressions of UC-related genes and infiltration of immune cells in the course of treatment was also investigated. Immunohistochemical (IHC) assay was used to verify the gene expression in inflamed UC tissues. When considering all the applied methods, DUOX2, PI3, S100P, MMP7, and S100A8 had priority to be defined as the characteristic genes among DEGs. The area under curve (AUC) of the five genes, which were all consistently over-expressed, based on an external validation dataset, were all above 0.94 for UC diagnosis. Four of the five genes (DUOX2, PI3, MMP7, and S100A8) were down-regulated between treatment-responsive and nonresponsive patients. A significant difference was also observed concerning the infiltration of immune cells, including macrophage and neutrophil, between the two groups (treatment responsive and nonresponsive). The changes in the expression of DUOX2 and MMP7 based on the IHC assay were highly consistent with the results obtained in the current study. This confirmed the mild to moderate diagnostic and predictive value of DUOX2 and MMP7 in patients with UC. The conducted analyses showed that the expression profile of the five identified biomarkers accurately detects UC, whereas four of the five genes evidently predicted the response to anti-TNF-α therapy.
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Colitis Ulcerosa , Factor de Necrosis Tumoral alfa , Humanos , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/genética , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/patología , Factor de Necrosis Tumoral alfa/genética , Perfilación de la Expresión Génica , Biomarcadores/metabolismo , Pronóstico , Metaloproteinasa 7 de la Matriz/genética , Transcriptoma , Oxidasas Duales/genética , Oxidasas Duales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Femenino , Estudios de Casos y ControlesRESUMEN
Background: Congenital hypothyroidism (CH) is the most common neonatal metabolic disorder. In patients with CH in China, thyroid dyshormonogenesis is more common than thyroid dysgenesis; however, the genetic causes of CH due to thyroid dyshormonogenesis remain largely unknown. Therefore, we aimed at identifying novel candidate causative genes for CH. Methods: To identify novel CH candidate genes, a total of 599 patients with CH were enrolled and next-generation sequencing was performed. The functions of the identified variants were confirmed using HEK293T and FTC-133 cell lines in vitro and in a mouse model organism in vivo. Results: Three pathogenic contactin 6 (CNTN6) variants were identified in two patients with CH. Pedigree analysis showed that CH caused by CNTN6 variants was inherited in an autosomal recessive pattern. The CNTN6 gene was highly expressed in the thyroid in humans and mice. Cntn6 knockout mice presented with thyroid dyshormonogenesis and CH due to the decreased expression of crucial genes for thyroid hormone biosynthesis (Slc5a5, Tpo, and Duox2). All three CNTN6 variants resulted in the blocking of the release of the Notch intracellular domain, which could not translocate into the nucleus, impaired NOTCH1 transcriptional activity, and decreased expression of SLC5A5, TPO, and DUOX2. Further, we found that DTX1 was required for CNTN6 to promote thyroid hormone biosynthesis through Notch signaling. Conclusions: This study demonstrated that CNTN6 is a novel causative gene for CH through the mediation of thyroid hormone biosynthesis via Notch signaling, which provides new insights into the genetic background and mechanisms involved in CH and thyroid dyshormonogenesis.
Asunto(s)
Hipotiroidismo Congénito , Humanos , Animales , Ratones , Hipotiroidismo Congénito/genética , Oxidasas Duales/genética , Células HEK293 , Mutación , Yoduro Peroxidasa/genética , Hormonas Tiroideas , Contactinas/genéticaRESUMEN
BACKGROUND: Ulcerative colitis (UC) presents diagnostic and therapeutic difficulties. The primary objective of this study is to identify efficacious biomarkers for diagnosis and treatment, as well as acquire a deeper understanding of the immuneological characteristics associated with the disease. METHODS: Datasets relating to UC were obtained from GEO database. Among these, three datasets were merged to create a metadata for bioinformatics analysis and machine learning. Additionally, one dataset specifically utilized for external validation. Least absolute shrinkage and selection operator (LASSO) and random forest (RF) were employed to screen signature genes. The artificial neural network (ANN) model and receiver operating characteristic (ROC) curve were used to assess the diagnostic performance of signature genes. The single sample gene set enrichment analysis (ssGSEA) was applied to reveal the immune landscape. Finally, the relationship between the signature genes, immune infiltration, and clinical characteristics was investigated through correlation analysis. RESULT: By intersecting the result of LASSO, RF and WGCNA, 8 signature genes were identified, including S100A8, IL-1B, CXCL1, TCN1, MMP10, GREM1, DUOX2 and SLC6A14. The biological progress of this gene mostly encompasses acute inflammatory response, aggregation and chemotaxis of leukocyte, and response to lipopolysaccharide by mediating IL-17 signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway. Immune infiltration analysis shows 25 immune cells are significantly elevated in UC samples. Moreover, these signature genes exhibit a strong correlation with various immune cells and a mild to moderate correlation with the Mayo score. CONCLUSION: S100A8, IL-1B, CXCL1, TCN1, MMP10, GREM1, DUOX2 and SLC6A14 have been identified as credible potential biomarkers for the diagnosis and therapy of UC. The immune response mediated by these signature biomarkers plays a crucial role in the occurrence and advancement of UC by means of the reciprocal interaction between the signature biomarkers and immune-infiltrated cells.
Asunto(s)
Colitis Ulcerosa , Humanos , Colitis Ulcerosa/genética , Oxidasas Duales , Metaloproteinasa 10 de la Matriz , Aprendizaje Automático , Biomarcadores , Biología ComputacionalRESUMEN
Dual oxidase (Duox) a member of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) family can induce the production of reactive oxygen species (ROS). In vertebrates, the duox gene was indicated to be associated with the mucosal immunity. The roles of the duox gene in invertebrates were mainly studied in insects for the function of maintaining intestinal flora balance. In recent years, some studies have reported that Duox is involved in regulating the production of ROS and plays an important role in defending against the intestinal pathogen infection. However, the molecular mechanism has not been fully illuminated. In this study, a duox 2 involved in the production of H2O2 was identified for the first time in P. clarkii. Mature Pc-Duox 2 is a 7-transmembrane protein molecule that includes PHD, FAD, and NAD domains. Pc-duox 2 was mainly expressed in hemocytes and intestinal tissue. Its expression levels were obviously upregulated after intramuscular or oral infection with V. harveyi. In the RNAi assay, the upregulated trends of H2O2 and total ROS levels in crayfish intestine were significantly suppressed when Pc-duox 2 was knocked down. Compared with the slightly affected SOD activity, the upregulated CAT activity was suppressed more obviously in the crayfish intestine. Furthermore, Pc-duox 2 had an important effect on the maintenance of the structural stability of crayfish the intestine. Further research revealed that the knockdown of Pc-duox 2 could cause an obvious suppression in the upregulated levels of Toll signalling pathway-related genes, including Pc-toll 1, Pc-toll 3, Pc-dorsal, Pc-ALF 5, Pc-crustin 1, and Pc-lysozyme. Ultimately, these changes triggered the accelerated death of crayfish. Overall, we speculated that Pc-duox 2 played an important role in antibacterial innate immunity in the crayfish intestine by regulating the total ROS level.
Asunto(s)
Astacoidea , Peróxido de Hidrógeno , Animales , Oxidasas Duales/genética , Oxidasas Duales/metabolismo , Secuencia de Aminoácidos , Especies Reactivas de Oxígeno/metabolismo , Peróxido de Hidrógeno/metabolismo , Inmunidad Innata/genética , Intestinos , Antibacterianos/metabolismoRESUMEN
Chlorogenic acid (CGA), a polyphenol found mainly in coffee and tea, exerts antioxidant, anti-inflammatory and anti-apoptotic effects at the gastrointestinal level. However, although CGA is known to cross the blood-brain barrier (BBB), its effects on the CNS are still unknown. Oligodendrocytes (OLs), the myelin-forming cells in the CNS, are the main target in demyelinating neuroinflammatory diseases such as multiple sclerosis (MS). We evaluated the antioxidant, anti-inflammatory and anti-apoptotic roles of CGA in M03-13, an immortalized human OL cell line. We found that CGA reduces intracellular superoxide ions, mitochondrial reactive oxygen species (ROS) and NADPH oxidases (NOXs) /dual oxidase 2 (DUOX2) protein levels. The stimulation of M03-13 cells with TNFα activates the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) pathway, leading to an increase in superoxide ion, NOXs/DUOX2 and phosphorylated extracellular regulated protein kinase (pERK) levels. In addition, tumor necrosis factor alpha (TNF-α) stimulation induces caspase 8 activation and the cleavage of poly-ADP-ribose polymerase (PARP). All these TNFα-induced effects are reversed by CGA. Furthermore, CGA induces a blockade of proliferation, driving cells to differentiation, resulting in increased mRNA levels of myelin basic protein (MBP) and proteolipid protein (PLP), which are major markers of mature OLs. Overall, these data suggest that dietary supplementation with this polyphenol could play an important beneficial role in autoimmune neuroinflammatory diseases such as MS.