A New Registry for Genetic Diagnosis of Inborn Errors of Immunity within the Military Health System

Genotypic definition of monogenic inborn errors of immunity (IEIs) continues to accelerate with broader access to next generation sequencing, underscoring this aggregated group of disorders as a major health burden impacting both civilian and military populations. At an estimated prevalence of 1 in 1200 individuals, IEIs affect ~8,000 patients within the Military Health System (MHS). Despite access to targeted gene/exome panels at military treatment facilities, most affected patients never receive a definitive genetic diagnosis that would significantly improve clinical care. To address this gap, we established the first registry of IEI patients within the MHS with the goal of identifying known and novel pathogenic genetic defects to increase diagnosis rates and enhance clinical care. Using the registry, a research protocol was opened in July 2022. Since July we have enrolled 75 IEI patients encompassing a breadth of phenotypes including severe and recurrent infections, bone marrow failure, autoimmunity/autoinflammation, atopic disease, and malignancy. Enrolled patients provide blood and bone marrow samples for whole genome, ultra-deep targeted panel and comprehensive transcriptome sequencing, plus cryopreservation of peripheral blood mononuclear cells for future functional studies. We are also implementing and developing analytical methods for identifying and interrogating non-coding and structural variants. Suspected pathogenic variants are adjudicated by a clinical molecular geneticist using state-of-the-art analysis pipelines. These analyses subsequently inform in vitro experiments to validate causative mutations using cell reporter systems and primary patient cells. Clinical variant validation and return of genetic results are planned with genetic counseling provided. As a proof of principle, this integrated genetic evaluation pipeline revealed a novel, candidate TLR7 nonsense variant in two adolescent brothers who both endured critical COVID-19 pneumonia, requiring mechanical ventilation and extracorporeal membrane oxygenation. Our protocol is therefore poised to greatly enrich clinical genetics resources available in the MHS for IEI patients, contributing to better diagnosis rates, informed family counseling, and targeted treatments that collectively improve the health and readiness of the military community. Moreover, our efforts should yield new mechanistic insights on immune pathogenesis for a broad variety of known and novel IEIs.Copyright © 2023 Elsevier Inc.

Antidepressant-like effects of geniposide in chronic unpredictable mild stress-induced mice by regulating the circ_0008405/miR-25-3p/Gata2 and Oip5os1/miR-25-3p/Gata2 networks.

Evidence exists suggesting the anti-depressive activities of geniposide (GP), a major compound in Gardenia jasminoides Ellis. Accordingly, the present study attempts to explore the anti-depressive mechanism of GP in chronic unpredictable mild stress (CUMS)-induced depression-like behaviors of mice. CUMS-induced mice were given GP daily and subjected to behavioral tests to observe the effect of GP on the depression-like behaviors. It was noted that GP administration reduced depression-like behaviors in CUMS mice. Transcriptome sequencing was conducted in three control and three CUMS mice. Differentially expressed circRNAs, lncRNAs and mRNAs were then screened by bioinformatics analyses. Intersection analysis of the transcriptome sequencing results with the bioinformatics analysis results was followed to identify the candidate targets. We found that Gata2 alleviated depression-like behaviors via the metabolism- and synapse-related pathways. Gata2 was a target of miR-25-3p, which had binding sites to circ_0008405 and Oip5os1. circ_0008405 and Oip5os1 competitively bound to miR-25-3p to release the expression of Gata2. GP administration ameliorated depression-like behaviors in CUMS mice through regulation of the circ_0008405/miR-25-3p/Gata2 and Oip5os1/miR-25-3p/Gata2 crosstalk networks. Taken together, GP may exert a potential antidepressant-like effect on CUMS mice, which is ascribed to regulation of the circ_0008405/miR-25-3p/Gata2 and Oip5os1/miR-25-3p/Gata2 crosstalk networks.

Full-length transcriptome analysis of two chemotype and functional characterization of genes related to sesquiterpene biosynthesis in Atractylodes lancea.

Atractylodes lancea (Thunb.) DC. is an important medicinal plant mainly distributed in China. A. lancea is rich in volatile oils and has a significant effect on various diseases, including coronavirus disease 2019 (COVID-19). Based on the signature constituents of volatile oils, A. lancea is divided into two chemotypes: the Dabieshan and Maoshan chemotype. Gas chromatography-mass spectrometry (GC-MS) results revealed that the hinesol and ß-eudesmol contents in the Dabieshan chemotype were higher than those in the Maoshan chemotype. Next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing technologies were combined to investigate the molecular mechanisms of sesquiterpenoid biosynthesis in A. lancea. A total of 42 differentially expressed genes (DEGs) for terpenoid biosynthesis were identified in the two chemotype groups, and nine full-length terpene synthase (TPS) genes were identified. Subcellular localization revealed that AlTPS1 and AlTPS2 proteins were localized in the nucleus and endoplasmic reticulum. They use FPP as a substrate to generate sesquiterpenoids. AlTPS1 catalyzes biosynthesis of elemol while AlTPS2 is observed to perform ß-farnesene synthase activity. This study provides information for understanding the differences in the accumulation of terpenoids in two chemotypes of A. lancea and lays a foundation for further elucidation of the molecular mechanism of sesquiterpenoid biosynthesis.

Incidence and immunomic features of apyretic COVID-19 in patients affected by solid tumors: a prospective cohort study.

BACKGROUND AND RATIONALE: Little is known about SARS-CoV-2 seroconversion in asymptomatic patients affected by solid cancer, and whether it is associated with specific transcriptomics changes in peripheral blood mononuclear cells (PBMC). METHODS: Patients affected by solid cancer treated in a top comprehensive cancer center in Italy during the first COVID-19 pandemic wave, and negative for COVID-19-symptoms since the first detection of COVID-19 in Italy, were prospectively evaluated by SARS-CoV-2 serology in the period between April 14th and June 23rd 2020. Follow-up serologies were performed, every 21-28 days, until August 23rd 2020. All SARS-CoV-2 IgM + patients underwent confirmatory nasopharyngeal swab (NPS). PBMCs from a subset of SARS-CoV-2 IgM + patients were collected at baseline, at 2 months, and at 7 months for transcriptome sequencing. RESULTS: SARS-CoV-2 serology was performed on 446 of the 466 recruited patients. A total of 14 patients (3.14%) tested positive for at least one SARS-CoV-2 immunoglobulin in the period between April 14th and August 23rd 2020. Incidence of SARS-CoV-2 IgM decreased from 1.48% in the first month of the accrual to 0% in the last month. Viral RNA could not be detected in any of the NPS. PBMC serial transcriptomic analysis showed progressive downregulation of interleukin 6 upregulated signatures, chemokine-mediated signaling and chemokine-chemokine receptor KEGG pathways. B- and T-cell receptor pathways (p-values = 0.0002 and 0.017 respectively) were progressively upregulated. CONCLUSIONS: SARS-CoV-2 seroconversion rate in asymptomatic patients affected by solid cancer is consistent with that of asymptomatic COVID-19 assessed in the general population through NPS at the peak of the first wave. Transcriptomic features over time in IgM + asymptomatic cases are suggestive of previous viral exposure.

Molecular signature of postmortem lung tissue from COVID-19 patients suggests distinct trajectories driving mortality.

To elucidate the molecular mechanisms that manifest lung abnormalities during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, we performed whole-transcriptome sequencing of lung autopsies from 31 patients with severe COVID-19 and ten uninfected controls. Using metatranscriptomics, we identified the existence of two distinct molecular signatures of lethal COVID-19. The dominant 'classical' signature (n=23) showed upregulation of the unfolded protein response, steroid biosynthesis and complement activation, supported by massive metabolic reprogramming leading to characteristic lung damage. The rarer signature (n=8) that potentially represents 'cytokine release syndrome' (CRS) showed upregulation of cytokines such as IL1 and CCL19, but absence of complement activation. We found that a majority of patients cleared SARS-CoV-2 infection, but they suffered from acute dysbiosis with characteristic enrichment of opportunistic pathogens such as Staphylococcus cohnii in 'classical' patients and Pasteurella multocida in CRS patients. Our results suggest two distinct models of lung pathology in severe COVID-19 patients, which can be identified through complement activation, presence of specific cytokines and characteristic microbiome. These findings can be used to design personalized therapy using in silico identified drug molecules or in mitigating specific secondary infections.

Transcriptomic analysis of the innate immune signatures of a SARS-CoV-2 protein subunit vaccine ZF2001 and an mRNA vaccine RRV.

Analysis of large-scale gene expression post vaccination can provide an overview of immune responses. We used transcriptional approaches to comprehensively analyze the innate immune response signatures elicited by protein subunit (PS) vaccine ZF2001 and an mRNA vaccine named RRV. A fine-grained time-dependent dissection of large-scale gene expression post immunization revealed that ZF001 induced MHC class II-related genes, including cd74 and H2-Aa, more expeditiously than the RRV. Notably, the RRV induced MHC class I-related genes such as Tap1/2, B2m, and H2-D1/K1. At day 21 post immunization, the titres of binding and neutralization antibody (NAb) induced by both vaccines were comparable, which were accordant with the expression level of genes essential to BCR/TCR signalling transduction and B/T cells activation at day 7. However, compared to ZF2001, the early responses of RRV were more robust, including the activation of pattern recognition receptors (PRRs), expression of genes involved in RNA degradation, and transcription inhibition, which are directly related to anti-viral signals. This pattern also coincided with the induction of cytokines by the RRV. Generally, the transcriptomic patterns of two very different vaccines mapped here provide a framework for establishing correlates between the induction of genes and protection, which can be tailored for evoking specific and potent immune responses against SARS-CoV-2.

Severe Adaptive Immune Suppression May Be Why Patients With Severe COVID-19 Cannot Be Discharged From the ICU Even After Negative Viral Tests.

During the COVID-19 pandemic, a phenomenon emerged in which some patients with severe disease were critically ill and could not be discharged from the ICU even though they exhibited negative viral tests. To explore the underlying mechanism, we collected blood samples from these patients and analyzed the gene expression profiles of peripheral immune cells. We found that all enrolled patients, regardless of changes in genes related to different symptoms and inflammatory responses, showed universally and severely decreased expression of adaptive immunity-related genes, especially those related to T/B cell arms and HLA molecules, and that these patients exhibited long-term secondary infections. In addition, no significant change was found in the expression of classic immunosuppression molecules including PD-1, PD-L1, and CTLA-4, suggesting that the adaptive immune suppression may not be due to the change of these genes. According to the published literatures and our data, this adaptive immunosuppression is likely to be caused by the "dysregulated host response" to severe infection, similar to the immunosuppression that exists in other severely infected patients with sepsis.

Host transcriptomic profiling of COVID-19 patients with mild, moderate, and severe clinical outcomes.

Characterizing key molecular and cellular pathways involved in COVID-19 is essential for disease prognosis and management. We perform shotgun transcriptome sequencing of human RNA obtained from nasopharyngeal swabs of patients with COVID-19, and identify a molecular signature associated with disease severity. Specifically, we identify globally dysregulated immune related pathways, such as cytokine-cytokine receptor signaling, complement and coagulation cascades, JAK-STAT, and TGF- ß signaling pathways in all, though to a higher extent in patients with severe symptoms. The excessive release of cytokines and chemokines such as CCL2, CCL22, CXCL9 and CXCL12 and certain interferons and interleukins related genes like IFIH1, IFI44, IFIT1 and IL10 were significantly higher in patients with severe clinical presentation compared to mild and moderate presentations. Differential gene expression analysis identified a small set of regulatory genes that might act as strong predictors of patient outcome. Our data suggest that rapid transcriptome analysis of nasopharyngeal swabs can be a powerful approach to quantify host molecular response and may provide valuable insights into COVID-19 pathophysiology.

Localization of Cell Receptor-Related Genes of SARS-CoV-2 in the Kidney through Single-Cell Transcriptome Analysis.

BACKGROUND: The new coronavirus (SARS-CoV-2), which has been responsible for the recent coronavirus disease 2019 (COVID-19) pandemic, uses the cell receptor angiotensin converting enzyme-2 (ACE2) for entry and the serine protease TMPRSS2 for spike (S) protein priming. Meanwhile, the presence of B0AT1 (SLC6A19) may prevent TMPRSS2 from accessing the cutting position on ACE2. Identifying the expression of these cell receptor-related genes of SARS-CoV-2 is critical for understanding the pathogenesis of SARS-CoV-2 in various tissues, especially in the kidney. METHODS: The single-cell transcription datasets of the human cell landscape (HCL) and other relevant single-cell transcription databases were used to analyze the expression of ACE2, TMPRSS2, and SLC6A19 in various organs and tissues, but mainly in the kidney. RESULTS: ACE2 was significantly expressed in the S1, S2, and S3 segments of proximal tubule (PT) cells. TMPRSS2 was widely expressed in several renal tubule populations extending from the PT cells to the collection system cell type, of which intercalated cells and the distal convoluted tubule cells showed more significant expression than PT cells. Unexpectedly, although expressed on various renal tubule populations, SLC6A19 was mainly enriched in PT cells, in line with ACE2 expression. Although alveolar-type (AT) 2 cells of the lung and intestinal epithelial cells expressed ACE2 as well as PT cells, AT 2 cells significantly expressed TMPRSS2 but not SLC6A19, while all 3 genes were significantly expressed in intestinal epithelial cells. CONCLUSIONS: ACE2 was widely expressed in specific cell subgroups of various human tissues, especially in intestinal epithelial cells, kidney PT cells, and also AT 2 cells of the lung. These 3 types of cells demonstrated significant differences in the distribution of the cell receptor-related genes of SARS-CoV-2, which may indicate the diversity of cell surface structures and differences in the affinity between SARS-CoV-2 and cells.