A rare missense p.C125Y mutation in the TNFRSF1A gene identified in a Chinese family with tumor necrosis factor receptor-associated periodic fever syndrome.

Background: Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is a rare autosomal dominant disorder with a low incidence in Asia. The most frequent clinical manifestations include fever, rash, myalgia, joint pain and abdominal pain. Misdiagnosis rates are high because of the clinical and genetic variability of the disease. The pathogenesis of TRAPS is complex and yet to be fully defined. Early genetic diagnosis is the key to precise treatment. Methods: In this study, a Chinese family with suspected TRAPS were analyzed by genome-wide SNP genotyping, linkage analysis and targeted sequencing for identification of mutations in causative genes. To study the pathogenicity of the identified gene mutation, we performed a conservation analysis of the mutation site and protein structure analysis. Flow cytometry was used to detect TNFRSF1A shedding and quantitative real-time PCR were used to assess the activation of unfolded protein response (UPR) in the mutation carriers and healthy individuals. Results: A typical TRAPS family history, with a pattern of autosomal dominant inheritance, led to the identification of a rare mutation in the TNFRSF1A gene (c.G374A [p.Cys125Tyr]) with unknown significance. The patient responded well to corticosteroids, and long-term therapy with colchicine effectively reduced the inflammatory attacks. No amyloid complications occurred during the 6-year follow-up. In silico protein analysis showed that the mutation site is highly conversed and the mutation prevents the formation of intrachain disulfide bonds in the protein. Despite a normal shedding of the TNFRSF1A protein from stimulated monocytes in the TRAPS patients with p.C125Y mutation, the expression of CHOP and the splicing of XBP1 was significantly higher than healthy controls, suggesting the presence of an activation UPR. Conclusion: This is the first report of a Chinese family with the rare p.C125Y mutation in TNFRSF1A. The p.C125Y mutation does not result in aberrant receptor shedding, but instead is associated with an activated UPR in these TRAPS patients, which may provide new insights into the pathogenesis of this rare mutation in TRAPS.

A Novel Homozygous RHOH Variant Associated with T Cell Dysfunction and Recurrent Opportunistic Infections.

RHOH, an atypical small GTPase predominantly expressed in hematopoietic cells, plays a vital role in immune function. A deficiency in RHOH has been linked to epidermodysplasia verruciformis, lung disease, Burkitt lymphoma and T cell defects. Here, we report a novel germline homozygous RHOH c.245G > A (p.Cys82Tyr) variant in a 21-year-old male suffering from recurrent, invasive, opportunistic infections affecting the lungs, eyes, and brain. His sister also succumbed to a lung infection during early adulthood. The patient exhibited a persistent decrease in CD4+ T, B, and NK cell counts, and hypoimmunoglobulinemia. The patient's T cell showed impaired activation upon in vitro TCR stimulation. In Jurkat T cells transduced with RHOHC82Y, a similar reduction in activation marker CD69 up-regulation was observed. Furthermore, the C82Y variant showed reduced RHOH protein expression and impaired interaction with the TCR signaling molecule ZAP70. Together, these data suggest that the newly identified autosomal-recessive RHOH variant is associated with T cell dysfunction and recurrent opportunistic infections, functioning as a hypomorph by disrupting ZAP70-mediated TCR signaling.

Utility of clinical metagenomics in diagnosing malignancies in a cohort of patients with Epstein-Barr virus positivity.

Backgrounds: Differentiation between benign and malignant diseases in EBV-positive patients poses a significant challenge due to the lack of efficient diagnostic tools. Metagenomic Next-Generation Sequencing (mNGS) is commonly used to identify pathogens of patients with fevers of unknown-origin (FUO). Recent studies have extended the application of Next-Generation Sequencing (NGS) in identifying tumors in body fluids and cerebrospinal fluids. In light of these, we conducted this study to develop and apply metagenomic methods to validate their role in identifying EBV-associated malignant disease. Methods: We enrolled 29 patients with positive EBV results in the cohort of FUO in the Department of Infectious Diseases of Huashan Hospital affiliated with Fudan University from 2018 to 2019. Upon enrollment, these patients were grouped for benign diseases, CAEBV, and malignant diseases according to their final diagnosis, and CNV analysis was retrospectively performed in 2022 using samples from 2018 to 2019. Results: Among the 29 patients. 16 of them were diagnosed with benign diseases, 3 patients were diagnosed with CAEBV and 10 patients were with malignant diseases. 29 blood samples from 29 patients were tested for mNGS. Among all 10 patients with malignant diagnosis, CNV analysis suggested neoplasms in 9 patients. Of all 19 patients with benign or CAEBV diagnosis, 2 patients showed abnormal CNV results. The sensitivity and specificity of CNV analysis for the identification for tumors were 90% and 89.5%, separately. Conclusions: The application of mNGS could assist in the identification of microbial infection and malignancies in EBV-related diseases. Our results demonstrate that CNV detection through mNGS is faster compared to conventional oncology tests. Moreover, the convenient collection of peripheral blood samples adds to the advantages of this approach.

Biomarker screening and validation for the differentiation of bloodstream infection from adult-onset Still's disease: A prospective cohort study.

OBJECTIVES: Distinguishing between bloodstream infection (BSI) and adult-onset Still's disease (AOSD) is challenging in practice due to similarities in their clinical and laboratory characteristics. We aimed to identify biomarkers in a prospective cohort of patients with BSI and AOSD for differential diagnosis and prognosis prediction. METHODS: Sixty-four individuals were enrolled in the training set (37 with BSI, 17 with AOSD, and 10 healthy controls). Furthermore, 86 individuals were enrolled in the validation cohort (67 with BSI and 19 with AOSD). Clinical and laboratory data were collected. Blood samples were stimulated using bacteria-specific antigens and levels of several cytokines were detected in the supernatant via Luminex or enzyme-linked immunosorbent assay. RESULTS: Escherichia coli and Klebsiella pneumoniae were the pathogens most frequently responsible for BSI. In the training cohort, the incidence of rash, arthralgia, myalgia, sore throat, lymphadenopathy, leukocytosis, and hyperferritinemia was higher in patients with AOSD than in those with BSI. Procalcitonin was significantly higher in patients with BSI than that in those with AOSD. Interleukin (IL)-6, IL-17A, C-X3-C motif chemokine ligand (CX3CL)-1, and C-X-C motif chemokine ligand 10 (CXCL10) levels were higher in patients with BSI than in those with AOSD. IL-18 was higher among patients with AOSD than in those with BSI. A decision tree analysis showed that a combination of plasma IL-18 and ferritin levels can be used to distinguish BSI from AOSD (diagnostic accuracy: 97.67%, sensitivity: 96.15%, specificity: 100%). Plasma IL-18 levels were positively correlated with ferritin, and were decreased after treatment in both BSI and ASOD groups. CONCLUSIONS: Plasma IL-18 and ferritin levels can be used to differentiate BSI from AOSD. IL-18 may be a potential biomarker for prognosis prediction in BSI and AOSD.

Clinical application of Epstein-Barr virus DNA loads in Epstein-Barr virus-associated diseases: A cohort study.

OBJECTIVES: This study evaluated the diagnostic value of Epstein-Barr virus (EBV) DNA load in blood samples of patients with EBV-associated diseases, and proposed a strategy for the interpretation of positive EBV DNA results. METHODS: Derivation and validation cohorts were established to evaluate the clinical significance of EBV DNA loads in the peripheral blood mononuclear cells (PBMCs) and plasma from EBV-infected patients. EBV DNA loads were compared and receiver operating characteristic curves were employed to assess the optimal cutoff values of EBV DNA for identification of EBV-associated diseases. RESULTS: The derivation and validation cohorts comprised 135 and 71 subjects, respectively. EBV DNA loads in the PBMCs of the EBV-associated diseases group was significantly higher than that of the EBV non-associated diseases group (5.8 × 104 vs 7.8 × 103 copies/106 cells, P<0.0001). The diagnostic cut-off value of viral load in PBMCs for EBV-associated diseases was determined to be 1.6 × 104 copies/106 cells. The combined EBV DNA load cutoff in PBMCs and positive EBV DNA qualitative detection in plasma (>500 copies/mL) allowed for the differentiation of EBV-associated and non-associated diseases; the sensitivity and specificity were 80.6 and 96.8%, respectively. CONCLUSIONS: The strategy of combining EBV DNA loads in PBMCs and plasma will potentially help identify EBV-associated diseases.

Cytokine profiles in cerebrospinal fluid of patients with meningitis at a tertiary general hospital in China.

BACKGROUND: There has been a great deal of evidence indicating that cytokines participate in meningeal inflammation. Different cytokine profiles may be presented in central nervous system (CNS) infection due to different pathogens. We have attempted to investigate cytokine profiles in cerebrospinal fluid (CSF) of patients with CNS infection. METHODS: Forty-three patients with CNS infection including tuberculous meningitis, purulent meningitis and cryptococcal meningitis were enrolled and 11 patients with normal CSF were enrolled as control group. The concentrations of Th1-, Th2- and Th17-type cytokines in CSF were detected using multiplex cytokine assay. Furthermore, the correlation between CSF cytokines and CSF parameters in CNS infection was analyzed. RESULTS: The CSF levels of IL-1ß, IL-4, IL-6, IL-10, IL-17, IL-23, IL-33, IFN-γ, TNF-α and sCD40L among the patients with CNS infection were all higher than control group (all P < 0.05). A remarkable elevation of CSF IL-6 in the patients with CNS infection was observed with the least overlap of the CSF concentrations compared to controls. Moreover, CSF IL-6 levels were strongly negatively correlated with CSF glucose and the CSF/blood glucose ratio (r = -0.4375, P = 0.0042; r = -0.4991, P = 0.0009). CONCLUSIONS: The excessive activation of immune response characterized by elevated levels of CSF Th1-, Th2- and Th17-type cytokines has been observed during CNS infection. Furthermore, we observed negative correlations between CSF IL-6 levels and CSF glucose and CSF/blood glucose ratio in CNS infection. And we suggested that combined CSF IL-6 levels with CSF glucose may serve as a novel biomarker pool for the differential of CNS infection.