Molecular and Clinical Features of Congenital Hypothyroidism Due to Multiple DUOX2 Variants.

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.

Upregulated dual oxidase 1-induced oxidative stress and caspase-1-dependent pyroptosis reflect the etiologies of heart failure.

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.

Duox activation in Drosophila Malpighian tubules stimulates intestinal epithelial renewal through a countercurrent flow.

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.

Genetic and microbial determinants of azoxymethane-induced colorectal tumor susceptibility in Collaborative Cross mice and their implication in human cancer.

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.

Particulate matter stimulates the NADPH oxidase system via AhR-mediated epigenetic modifications.

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.

Functional studies associate novel DUOX2 gene variants detected in heterozygosity to Crohn's disease.

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.

Gene Expression and Prognostic Value of NADPH Oxidase Enzymes in Breast Cancer.

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.

TSHR Variant Screening and Phenotype Analysis in 367 Chinese Patients With Congenital Hypothyroidism.

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.

The circular RNA circATP8B(2) regulates ROS production and antiviral immunity in Drosophila.

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.

[Application of whole exome sequencing for the inferential analysis of recessive genetic disease carrier status for couples with a child died of Primary immunodeficiency].

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.

Screening and experimental validation of diagnostic gene in ulcerative colitis with anti-TNF-α therapy.

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.

Contactin 6, A Novel Causative Gene for Congenital Hypothyroidism, Mediates Thyroid Hormone Biosynthesis Through Notch Signaling.

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.

Biomarkers prediction and immune landscape in ulcerative colitis: Findings based on bioinformatics and machine learning.

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.

Dual oxidase 2 (duox 2) participates in the intestinal antibacterial innate immune responses of Procambarus clarkii by regulating ROS levels.

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.

Antioxidant, Anti-Inflammatory and Pro-Differentiative Effects of Chlorogenic Acid on M03-13 Human Oligodendrocyte-like Cells.

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.

Results of neonatal screening for congenital hypothyroidism and hyperphenylalaninemia in Zhejiang province from 1999 to 2022. / 浙江省1200ä¸‡ä¾‹æ–°ç”Ÿå„¿å ˆå¤©æ€§ç”²çŠ¶è ºåŠŸèƒ½å‡é€€ç—‡ã€é«˜è‹¯ä¸™æ°¨é ¸è¡€ç—‡ç­›æŸ¥ç»“æžœåˆ†æž.

OBJECTIVES: To analyze the results of neonatal screening for congenital hypothyroidism (CH) and hyperphenylalaninemia (HPA) in Zhejiang province from 1999 to 2022. METHODS: A total of 11 922 318 newborns were screened from September 1999 and December 2022 in Zhejiang province. The blood thyroid stimulating hormone (TSH) levels were measured by a fluorescence method and blood phenylalanine (Phe) levels were measured by fluorescence method or tandem mass spectrometry. TSH≥9 µIU/mL was considered positive for CH, while Phe>120 µmol/L and/or Phe/Tyr ratio>2.0 were considered positive for HPA. The positive newborns in screening were recalled, and the gene variations were detected by high-throughput sequencing and MassARRAY tests. RESULTS: The overall neonatal screening rate during 1999-2022 was 89.41% (11 922 318/13 333 929) and the screening rate was increased from 6.46% in 1999 to 100.0% in 2022. A total of 8924 cases of CH were diagnosed among screened newborns with an incidence rate of 1/1336. A total of 563 cases of HPA were diagnosed, including 508 cases of classic phenylketonuria (cPKU) and 55 cases of tetrahydrobiopterin deficiency (BH4D), with an incidence rate of 1/21 176. Ninety-seven out of 8924 cases of CH underwent genetic analysis. Gene mutations were detected in 9 CH related genes, the highest frequency mutations were found in DUOX2 gene (69.0%) with c.3329G>A (p.R1110Q) (18.2%) and c.1588A>T (p.K530X) (17.3%) as the hotspot mutations. There were 81 PAH gene variants detected in a total of 250 cases of cPKU, and c728G>A (p.R243Q) (24.4%), c.721C>T (p.R241C) (15.0%) were the hotspot mutations. Meanwhile 7 novel variants in PAH gene were detected: c.107C>A (p.S36*), c.137G>T (p.G46V), c.148A>G(p.K50E), c.285C>T (p.I95I), c.843-10delTTCC, exon4-7del and c.1066-2A>G. There were 12 PTS gene variants detected in 36 cases of BH4D, and c.259C>T (p.P87S) (31.9%) was the hotspot mutation. CONCLUSIONS: The incident of CH has increased from 1999 to 2022 in Zhejiang province, and it is higher than that of national and global levels; while the incidence of HPA is similar to the national average. DUOX2 gene variation is the most common in CH patients; c.728G>A (p.R243Q) is the hotspot mutation in cPKU patients, while c.259C>T (p.P87S) is the hotspot mutation in BH4D patients.

Placental cartography of NADPH oxidase (NOX) family proteins: Involvement in the pathophysiology of preeclampsia.

The placenta is an essential organ for fetal development. During the first trimester, it undergoes dramatic changes as it develops in an environment poor in oxygen (around 2-3%). From about 10 gestational weeks, oxygen levels increase to 8% in the intervillous chamber. These changes are accompanied by modulation of the activity of NADPH oxidase, a major source of production of reactive oxygen species in the first trimester of pregnancy. The NOX complex is composed of seven different proteins (NOX1-5 and DUOX1-2) whose placental involvements during physiological and pathological pregnancies are largely unknown. The aim of the study was to produce a cartography of NOX family proteins, in terms of RNA, protein expression, and localization during physiological pregnancy and in the case of preeclampsia (PE), in a cohort of early-onset PE (n = 11) and late-onset PE (n = 7) cases. NOX family proteins were mainly expressed in trophoblastic cells (NOX4-5, DUOX1) and modulated during physiological pregnancy. NOX4 underwent an unexpected and hitherto unreported nuclear translocation at term. In the case of PE, two groups stood out: NOX1-3, superoxide producers, were down-regulated (p < 0.05) while NOX4-DUOX1, hydrogen peroxide producers, were up-regulated (p < 0.05), compared to the control group. Mapping of placental NOX will constitute a reference and guide for future investigations concerning its involvement in the pathophysiology of PE.

[Analysis of a child with Very early onset inflammatory bowel disease due to compound heterozygous variants of IL10RA and DUOX2 genes].

OBJECTIVE: To explore the genetic basis of a child with Very early onset inflammatory bowel disease (VEOIBD). METHODS: A female child who had presented at the Children's Hospital of Fudan University on May 23, 2018 due to occurrence of diarrhea and fever 6 days after birth was selected as the study subject. Clinical data of the child was collected. Family-based whole-exome sequencing (WES) was carried out. Candidate variant was verified by Sanger sequencing and PCR of the patient and her parents. RESULTS: The child had developed the symptoms 6 days after birth, with main manifestations including diarrhea, fever, failure to thrive, rectovestibular fistula and hypothyroidism. An enterostomy was performed at the age of 3.5 months due to severe intestinal adhesion and obstruction. Based on her clinical manifestations, colonoscopic finding, and results of biopsies, she was diagnosed with VEOIBD in conjunct with congenital hypothyroidism. Replacement treatment of levothyroxine was given since one month of age. Family-based WES revealed that the child has harbored compound heterozygous variants of the DUOX2 gene, namely c.2654G>T (p.R885L) and c.505C>T (p.R169W), in addition with a heterozygous c.301C>T (p.R101W) variant of the IL10RA gene. Re-analysis of the WES data revealed that the patient also had a 333 bp deletion spanning exon 1 of the IL10RA gene (Chr11: 117857034_117857366). CONCLUSION: For patients with VEOIBD, genetic testing is recommended. Presence of additional DUOX2 gene variants might have exacerbated the clinical symptoms in this patient. Above finding has facilitated genetic counseling and prenatal diagnosis for this family, and raised clinicians' awareness of this rare disease.

[Key Prediction Genes of Nasopharyngeal Carcinoma:Screening Based on Systematic Bioinformatics and Validation by Cell Experiments].

Objective To screen out the potential prediction genes for nasopharyngeal carcinoma(NPC)from the gene microarray data of NPC samples and then verify the genes by cell experiments.Methods The NPC dataset was downloaded from Gene Expression Omnibus,and limma package was employed to screen out the differentially expressed genes.Weighted correlation network analysis package was used for weighted gene co-expression network analysis,and Venn diagram was drawn to find the common genes.The gene ontology annotation and Kyoto encyclopedia of genes and genomes pathway enrichment were then performed for the common genes.The biomarkers for NPC were further explored by protein-protein interaction network,LASSO regression,and non-parametric tests.Real-time quantitative PCR and Western blotting were employed to determine the mRNA and protein levels of key predictors of NPC,so as to verify the screening results.Results There were 622 up-regulated genes and 351 down-regulated genes in the GSE12452 dataset.A total of 116 common genes were obtained by limma analysis and weighted gene co-expression network analysis.The common genes were mainly involved in the biological processes of cell proliferation and regulation and regulation of intercellular adhesion.They were mainly enriched in Rap1,Ras,and tumor necrosis factor signaling pathways.Six key genes were screened out,encoding angiopoietin-2(ANGPT2),dual oxidase 2(DUOX2),coagulation factor Ⅲ(F3),interleukin-15(IL-15),lipocalin-2,and retinoic acid receptor-related orphan receptor B(RORB).Real-time quantitative PCR and Western blotting showed that the NPC cells had up-regulated mRNA and protein levels of ANGPT2 and IL-15 and down-regulated mRNA and protein levels of DUOX2,F3,and RORB,which was consistent with the results predicted by bioinformatics.Conclusion ANGPT2,DUOX2,F3,IL-15 and RORB are potential predictive molecular markers and therapeutic targets for NPC,which may be involved in Rap1,Ras,tumor necrosis factor and other signaling pathways.

Analysis and identification of ferroptosis-related genes in ulcerative colitis.

BACKGROUND: Previous studies have shown that ferroptosis is associated with the pathogenesis of ulcerative colitis (UC). Therefore, this study aimed to identify key ferroptosis-related genes (FRGs) associated with the diagnosis of UC. METHODS: UC-related expression datasets were downloaded from the Gene Expression Omnibus (GEO) database. First, Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify UC-related genes (UCRGs). Differentially expressed genes (DEGs) between normal and UC groups were screened in GSE87466, and DEGs were subjected to an intersection analysis with FRGs and UCRGs to obtain ferroptosis-related DEGs (FR DEGs). Then a protein-protein interaction (PPI) network was constructed for FR DEGs. The hub genes were extracted based on the degree, Maximum Neighborhood Component (MNC), closeness, and Maximal Clique Centrality (MCC). Biomarkers with diagnostic values were screened by support vector machine (SVM) and the least absolute shrinkage and selection operator (LASSO) algorithms. Next, the infiltration of immune cells was compared between UC and normal groups, and the correlation between different immune cells and diagnostic genes was analyzed. The biological functions, classical pathways, and intermolecular interaction networks of diagnostic genes were characterized utilizing ingenuity pathway analysis (IPA). Finally, a TF-mRNA network was constructed and potential small-molecule compounds were screened. RESULTS: Thirty-six FR DEGs were obtained, and these were enriched in biological processes such as positive regulation of cytokine production, cytokine-mediated signalling pathway, long-chain fatty acid-CoA ligase activity, etc. Among 18 hub genes, five genes (ALOX5, TIMP1, TNFAIP3, SOCS1, DUOX2) were captured with diagnostic values for UC, and they displayed significant differences between UC and normal groups. Sixteen immune cell infiltrates were significantly different between UC and normal groups, such as activated dendritic cells and resting dendritic cells. TNFAIP3 and ALOX5 were positively correlated with neutrophils, and TIMP1, SOCS1, ALOX5, and DUOX2 were negatively correlated with M2 macrophages. IPA showed that diagnostic genes were related to 43 function modules and activated 17 pathways. The constructed TF-mRNA regulatory network comprised three diagnostic genes and 17 differentially expressed TFs. Potential small-molecule compounds including helveticoside and cymarin were identified. CONCLUSION: Our findings yielded several promising FRGs for UC, providing a scientific reference for further studies on the pathogenesis of UC.