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A multivariant total subclass analysis has been performed for a control cohort (n=15) and a long COVID patient cohort (n=15) measuring the IgG1, IgG2, IgG3, IgG4 and IgE response to the following 14 variants of SARS-CoV-2: Wuhan, Alpha, Delta, BA.1, BA.2, BA.5, EG.5.1, XBB.1.5, BA.2.75, CH.1.1, BA.2.12.1, BQ.1.1, JN.1, and KP.3. Significant differences (p < 0.05 and p < 0.005) between concentrations of IgG subclasses by variant were found in 24% of variants and in mean-normalised distributions. The medians of the mean-normalised distributions were significantly lower for IgG1 (p < 0.05) in long COVID patients compared with controls, and significantly higher (p < 0.005) for levels of IgG3, IgG4 and IgE for long COVID patients. A preliminary diagnostics classification analysis performed by variation of the mean-normalised upper and lower percentiles symmetrically for IgG3 showed a long COVID diagnostic sensitivity of 80%, and specificity of 80% for the 60th percentile threshold of the control cohort. Three types of long COVID can be identified: patients with at least one variant below the threshold (hypo-immune), patients with at least one variant above the threshold (hyperimmune) and patients with IgG3 levels within the reference range. The multivariant subclass spectrum indicates IgG4 and IgE elevations due to potential chronic antigen exposure from persistent virus or autoimmunity and may indicate potential therapeutic interventions.

BackgroundAn adverse female sex-bias exists across many autoimmune disorders, yet its underlying mechanisms, particularly the role of sex hormones, remains poorly understood. Furthermore, the physiological influence of sex hormones in regulating T cell function remains undefined. We examined for the critical role of estrogen and progesterone, in regulating CD4+ T cell responses, specifically with respect to inflammation and their bone erosion potential in RA. MethodsInflammatory markers, circulating antibodies, sex hormone receptors, ER and PR levels were investigated in both RA patients and controls. Further, RA CD4+ T cells were stimulated in varying concentrations of estradiol and progesterone and assessed for modulation in cytokines, transcription factors, RANKL, and FasL expression. Subsequent ex-vivo studies were performed to examine the role of sex hormones in modulating T cell responses. ResultsRA patients displayed systemic inflammation and high circulating antibodies, with significantly higher expression in synovial fluid. Higher expression of ER and PR was evinced on RA CD4+ T cells. Upon hormone stimulation, two cohorts of patients namely responders and non-responders were observed with respect to modulation in cytokines, transcription factors, RANKL, and FasL expression. Our ex-vivo Th1 and Th17 cells demonstrated that sex hormones play a physiological role in modulating inflammation and have bone erosion potential. ConclusionOur findings demonstrate the pivotal significance of sex hormones in modulating TCR responses, thereby regulating inflammation and bone erosion in RA pathology. Further dissection of TCR signaling pathways with respect to sex hormone stimulation may provide promising targets for therapeutic implications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/25342530v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@b74525org.highwire.dtl.DTLVardef@1cc01aorg.highwire.dtl.DTLVardef@1881e4forg.highwire.dtl.DTLVardef@17de2a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionAdverse drug reactions (ADRs) remain a major public health issue, and genetic factors contribute importantly to interindividual variability in drug response. Pharmacogenetic testing helps reduce ADR risk by optimizing drug selection and dosage, particularly in monogenic disorders. Material and MethodsWhole-exome sequencing of 6,739 samples from the Russian population was performed using the MGIEasy Universal DNA Library Prep Set on the DNBSEQ-G400 platform (MGI). Variants in 48 genes were examined, focusing on inherited arrhythmias (Long QT syndrome, Short QT syndrome, Timothy syndrome, Andersen-Tawil syndrome, Brugada syndrome, Atrial fibrillation, Catecholaminergic polymorphic ventricular tachycardia), enzyme deficiencies (Glucose-6-Phosphate Dehydrogenase Deficiency [G6PDD], Porphyrias), Dravet Syndrome (DS) and Malignant Hyperthermia (MH). All identified variants had been reported at least once as pathogenic (P) or likely pathogenic (LP) in ClinVar, along with those occasionally classified as variants of uncertain significance (VUS). Each variant was manually re-evaluated according to ACMG criteria. ResultsA total of 75 unique variants in 18 genes were observed in 119 individuals (1.77%), including 21 carriers and 13 women with a G6PD mutation. Of these, 46 variants were classified as P, 21 as LP, and 8 as VUS. Missense variants accounted for the largest proportion (73.33%). The most affected genes were KCNQ1 (24/119), which exhibited the highest number of unique variants (18), G6PD (20/119), SCN1A (15/119), and RYR1 (14/119). Regarding associated conditions, mutations linked to arrhythmias were found in 51 individuals, MH in 27, G6PDD in 20, DS in 15, and Porphyrias in 6. ConclusionsIncorporating genetic information on both common and rare clinically actionable variants into therapeutic decision-making has the potential to improve medication safety, reduce preventable ADRs, and enhance the effectiveness of personalized pharmacotherapy.

BackgroundCervical cancer is one of the most common malignant tumors of the female reproductive system. Existing treatments provide limited benefit for patients with advanced, recurrent or metastatic disease, and reliable prognostic markers are lacking. In this study we integrated multi-omic data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database. Protein-coding genes meeting the criteria of an adjusted P value < 0.05 and |log2 fold-change| > 5 were screened; 693 genes were identified. We further focused on three genes related to the tumor microenvironment--interleukin 21 (IL21), C-X-C motif chemokine ligand 9 (CXCL9) and cluster of differentiation 1A (CD1A)--and performed differential expression analysis, survival analysis, clinical stage analysis and immune infiltration correlation analysis to clarify their prognostic value and potential mechanisms in cervical cancer. Results(1) CXCL9 and CD1A were highly expressed in cervical cancer tissues, and all three genes showed high expression across different pathological stages without stage-dependent differences; (2) high expression of IL21, CXCL9 and CD1A improved patient prognosis and was positively associated with overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI); (3) expression of IL21, CXCL9 and CD1A was closely correlated with infiltration of multiple immune cells: IL21 correlated with total T cells, helper T cells and B cells, CXCL9 correlated with T cells and activated dendritic cells, and CD1A correlated with immature dendritic cells. ConclusionIL21, CXCL9 and CD1A are potential prognostic biomarkers and key immunomodulatory factors in cervical cancer. This study provides a new direction for immunotherapy and individualized precision treatment of cervical cancer.

PURPOSEBEST1-associated inherited retinal disease constitutes one of the largest inherited retinal disease patient populations across the world. Innovative therapies are currently in development to address this significant unmet need. To better understand the scale of unmet need, and the distribution of phenotypes and genotypes, we conducted a meta-analysis of BEST1 patients reported in the literature to provide up-to-date patient number estimates. METHODSWe utilized the GeneScape(R) IRD Patient Atlas, an expertly curated database of [~]73K retinal disease patients undergoing extensive genetic testing, in combination with a dedicated literature search to estimate the proportion of IRD patients attributed to BEST1 using fixed effects weighting. This was also translated to patient number estimates for each country. Further extrapolation of patient subtypes was estimated based on cohorts of BEST1 patients reporting phenotypes and genotypes. In addition, a summary of contributing variants is reported by region. RESULTSAcross regions BEST1 retinopathy patients are estimated to occur from 1 in 38K (in Germany) to 1 in 363K in Japan. In the western countries, bi-allelic patients are expected to contribute nearly 20% of the total BEST1 population, whereas one third of patients in East Asia are anticipated to be bi-allelic. CONCLUSIONSWhile patients are reported across the world, their prevalence and composition vary across geographies. In the literature bi-allelic patients are less often reported explicitly as ARB patients in the United States as compared to Europe. Variant diversity is reflected in regional reports and drives phenotypic distribution.

Antiphospholipid syndrome (APS) and systemic sclerosis (SSc) are immune-mediated multisystem autoimmune diseases with distinct clinical phenotypes but overlapping pathogenic themes, including immune dysregulation, chronic inflammation, and endothelial injury. Using peripheral blood transcriptome datasets from the Gene Expression Omnibus (GSE102215: 9 APS/9 controls; GSE231691: 49 SSc/18 controls), we performed differential expression analysis within each cohort (limma; |log2FC|>1, P<0.05) and identified 281 genes dysregulated in the same direction in both diseases (100 upregulated and 181 downregulated). Enrichment analyses highlighted interferon-related and cytokine/inflammatory signaling programs in APS and SSc. To derive a compact diagnostic signature, we combined random forest feature ranking with a single-hidden-layer artificial neural network, prioritizing five shared candidate biomarkers (S100A8, IER5L-AS1, LTK, PRR5-ARHGAP8, and PCDH1). Each gene showed consistent case-control differences in both cohorts (P<0.001) and achieved good discrimination (AUC>0.75), with S100A8 performing most consistently (AUC=0.98 in APS; AUC=0.88 in SSc). CIBERSORT deconvolution indicated a myeloid-skewed blood profile in both diseases, characterized by higher neutrophil and monocyte/macrophage signals; SSc additionally showed stronger inferred CD4+ T cell and NK cell signals. S100A8 expression correlated with inferred neutrophil abundance in both cohorts (APS r=0.62; SSc r=0.58; P<0.05). Finally, miRNA-target prediction and DSigDB drug-signature enrichment generated regulatory and pharmacologic hypotheses, including immune-regulatory miRNAs (e.g., miR-155 and miR-146a) and candidate compounds (celecoxib, tamibarotene, HMN-176, and XMD14-99). Overall, these results nominate shared blood transcriptional markers and immune correlates across APS and SSc for follow-up validation.

AimsGestational diabetes mellitus (GDM) is the most common pregnancy-related medical complication. GDM is linked to aberrant immune responses in both mothers and offsprings, specifically, the subsequent development of inflammatory diseases. Whereas prior research has focused on specific immune cell subsets, a comprehensive overview of the impacts of GDM on maternal and fetal immune landscape is lacking. Here, we aim to comprehensively decipher how GDM modulates various immune cell populations in mothers and offsprings. MethodsA prospective, longitudinal case-control study was carried out. Maternal blood from GDM-affected (GDM, n=18) and non-GDM-affected (Ctrl, n=21) mothers were collected at ante-(36-38 weeks of gestation) and post-partum (6-8 weeks post-partum) timepoints. Cord blood from GDM (n=7) and Ctrl (n=11) pregnancies were collected upon C-section. They were analyzed with the state-of-the-art cytometry by time of flight (CyTOF) with a 40-marker panel. Additionally, a publicly available RNA-seq dataset for cord blood mononuclear cells was re-analyzed to confirm results from CyTOF experiments. ResultsCompared to Ctrl, GDM was associated with more activated maternal T cell subsets ante-partum, including increased CD45RO+ and Ki67+ CD4+ T cell populations, which reverted post-partum. GDM-affected maternal innate lymphoid cell (ILC) also exhibited increased granzyme B production ante-partum. On the other hand, in GDM-impacted cord blood, fetal T and B cells were more activated, displaying less naive and more effector phenotypes, further supported by RNA-seq analyses. ConclusionsOur comprehensive analyses revealed that GDM is linked to profound changes in the immune landscapes of the mothers (ante-/post-partum) and foetuses (at birth), casting novel insights towards GDM pathophysiology. Longitudinal immune profiling might be warranted for early detection and stratification of risk, and development of targeted interventions to prevent inflammatory disorders in GDM mothers and their offspring. Research in contextO_LIWhat is already known about this subject? O_LIThe maternal and intrauterine environments are important contributors to long-term health outcomes of mothers and offsprings. C_LIO_LISome maternal and fetal immunity changes have been observed in gestational diabetes mellitus (GDM)-affected pregnancies. C_LIO_LIGDM is associated with increased risk of later-life metabolic and inflammatory diseases in mothers as well as offsprings. C_LI C_LIO_LIWhat is the key question? O_LIWhat are the longitudinal alterations in maternal and fetal immune landscapes in GDM-affected pregnancies? C_LI C_LIO_LIWhat are the new findings? O_LIHigh-dimensional immune profiling provided the most comprehensive overview of alterations in maternal and fetal immune landscapes associated with GDM. C_LIO_LIGDM is associated with skewing of maternal CD4+ T cell and ILC towards activated phenotypes ante-partum. C_LIO_LIGDM is linked to more activated fetal T and B cell profiles. C_LI C_LIO_LIHow might this impact on clinical practice in the foreseeable future? O_LIUnderstanding the complex alterations in the maternal and fetal immune landscape in GDM-affected pregnancy provides insights into the long-term impacts of GDM on the mother and offspring. C_LI C_LI

PurposeSepsis-associated immunothrombosis significantly contributes to high mortality, yet the role of N-glycosylation in this process remains poorly understood. This study aimed to comprehensively profile the plasma N-glycosylation landscape in sepsis and elucidate how its specific reprogramming in the complement and coagulation cascades influences immunothrombotic balance and patient outcomes. MethodsWe performed in-depth 4D-DIA proteomic and N-glycomic analyses on plasma from 43 sepsis patients and 9 healthy controls. Differential expression, weighted gene co-expression network analysis (WGCNA), and protein-glycosylation correlation analyses were used to characterize molecular features. Clinical relevance was assessed via correlation and survival analyses. ResultsExtensive N-glycosylation reprogramming was observed in sepsis plasma,with marked enrichment in complement and coagulation pathways(KEGG p=7.76x10- {superscript 2}{superscript 1}).Pro-coagulant proteins(eg,vWF,fibrinogen)showed increased abundance together with enhanced site-specific glycosylation,potentially amplifying their activity.In contrast,key anticoagulant proteins(eg,SERPINC1)displayed unchanged glycosylation at critical sites despite abundance changes,which may impair function.Survival analysis revealed distinct prognostic values of glycoproteins and specific glycosylation sites.For instance,high vWF protein levels predicted mortality(HR=2.83),whereas elevated glycosylation at vWF N211 was associated with improved survival(HR=0.135),suggesting a negative regulatory role.These glycosylation markers correlated closely with disease severity and prognosis,representing potential early-warning biomarkers independent of current clinical coagulation indicators. ConclusionOur study demonstrates widespread reprogramming of the plasma proteome and N-glycome in sepsis.We propose that decoupling of protein function from abundance through N-glycosylation in the complement-coagulation network contributes to immunothrombotic imbalance.Specific N-glycosylation sites may serve as novel prognostic biomarkers,offering new perspectives for early risk stratification and glycosylation-targeted therapies in sepsis. Key PointsO_LISepsis plasma exhibits specific N-glycosylation reprogramming overwhelmingly focused on the complement and coagulation cascade. C_LIO_LIA dominant "glycosylation-dominated co-upregulation" mode in procoagulant factors, coupled with a "silent" glycosylation state in key anticoagulants, drives prothrombotic imbalance. C_LIO_LISite-specific N-glycosylation levels provide prognostic information distinct from, and often superior to, their carrier protein abundance, offering novel early-risk biomarkers. C_LI

RIG-I is a cytosolic immune receptor that provides the first line of defense by detecting viral RNA and triggering antiviral responses. Its physiological role in humans remains unclear, as no patients with complete RIG-I deficiency have yet been reported. We identified a critically ill COVID-19 patient with severe RIG-I deficiency caused by heterozygous RIG-I G731R, a novel dominant loss-of-function variant. The G731R mutation in helicase motif VI disrupts the arginine finger, impairing the ATPase activity of RIG-I, but not its RNA-binding ability. However, viral RNA binding fails to expose the signaling domains, thereby impairing the IFN-{beta} response of G731R. Instead, G731R competes with wild-type RIG-I, exerting a dominant negative effect. The loss-of-function is caused by bulky-charged substitutions at G731, as alanine or leucine substitution results in an unexpected gain-of-function phenotype. These findings highlight the importance of uncompromised RIG-I function for human antiviral immunity and the pleiotropic effects of single mutations.

BackgroundSickle Cell Anaemia (SCA), a genetic blood disorder caused by a single mutation in the beta globin gene, displays a highly variable clinical course. Hydroxyurea (HU), an effective treatment, has an unclear mechanism of action. Plasma proteins can act as biomarkers for understanding disease states and response to HU treatment in SCA patients. MethodsPlasma proteome profiling of 31 healthy individuals and 76 SCA patients, including those with and without HU treatment, was performed using a high-performance liquid chromatography system and Orbitrap mass spectrometer. Statistical analysis was performed to identify differentially abundant proteins (DAPs) between SCA patients and healthy controls. Subgroup analyses were performed to look at the impact of HU treatment on plasma proteome. ResultsOur analysis yielded 43 DAPs in the plasma of SCA patients. Global correlation and protein-protein network analysis revealed that these proteins are part of a robust interaction network. Proteins showing higher abundance (LBP, ORM1 and TFRC) were primarily associated with immune response whereas those with reduced abundance (FBLN1 and F13B) were linked to blood coagulation and proteolysis. Differential abundance of several proteins such as CD14, FCN3, LFALS3BP, LAP and TGFBI was observed in either male or female patients indicating influence of gender. Importantly, HU treatment was associated with elevated levels of haptoglobin (HP) and hemopexin (HPX), key proteins involved in free hemoglobin scavenging. Notably, DAPs such as F10, LPA, and FCN3 overlapped with proteins previously reported to be differentially abundant in beta-thalassemia patients. Moreover, multiple proteins, including APOL1, AZGP1, FBLN1, GPLD1, HPX, LGALS3BP, and TFRC correlated with clinical parameters, such as blood transfusion frequency and, vaso-occlusive crisis, and WBC and platelet counts. ConclusionsThis study identifies novel differentially abundant plasma proteins in SCA, expanding the current repertoire of disease-associated biomarkers and proteins modulated by hydroxyurea therapy. The observed overlap with beta-thalassemia associated signatures reinforces shared pathophysiological mechanisms between these hemoglobinopathies. Several of these proteins show significant correlations with key clinical parameters and disease complications, offering insights into disease mechanisms and potential utility in disease management. Collectively, these findings provide a strong foundation for translational validation in larger, independent cohorts.

Immune checkpoint inhibitors (ICIs) have transformed cancer treatment but commonly cause immune-related adverse events (irAEs), whether administered as monotherapy or in combination with other oncological agents. We present the first reported case of ICI-induced granulomatous sialadenitis in a male patient in his mid-fifties with BRAF-V600E-mutated papillary thyroid carcinoma who received sequential treatment with BRAF/MEK inhibitors followed by pembrolizumab. The patient experienced acute-onset severe xerostomia and salivary hypofunction, prompting ICI cessation and salivary gland biopsy. Integrative analysis using histology, single-cell RNA sequencing, and spatial transcriptomics revealed macrophage- and T-cell-mediated epithelial damage driven by epithelial senescence and Th1-polarized inflammation. Corticosteroid therapy reduced granuloma burden and improved salivary flow rates and tissue architecture; however, extensive fibrosis persisted despite treatment. These findings underscore the critical importance of early irAE recognition and intervention to preserve glandular function and enable continuation of cancer therapy.

The Endoplasmic Reticulum AminoPeptidase 2 (ERAP2) gene encodes an aminopeptidase involved in antigenic peptide processing for the MHC-I pathway. Genetic variants in the ERAP2 gene are associated with autoimmune conditions and infectious diseases. The linkage between genetic variants in the ERAP2 gene has given rise to the prevailing assumption that a single ERAP2 allotype with invariant amino acid sequence accounts for all immunological functions of ERAP2. Here, we show by analyzing exon-sequencing data from 160,000 individuals that 15 missense amino acid variants across the ERAP2 gene result in an array of different protein allotypes that are maintained in the European population. These allotypes can be divided into three haplotype groups, defined by the genotypes of two common splice-altering variants. We found novel associations between newly identified protein allotypes and immune-mediated diseases, suggesting that ERAP2 functional variation modifies disease susceptibility at the population level. An MHC class I antigen presentation assay revealed that disease-associated ERAP2 allotypes differ in their capacity to generate antigenic peptides for MHC-I presentation, resulting in differential activation of an antigen-specific T-cell receptor compared to non-disease-associated allotypes. These findings provide strong evidence that ERAP2 function is allotype-dependent and demonstrate that ERAP2 diversity shapes MHC-I antigen presentation and T-cell immunity. Significance statementThe ERAP2 enzyme modulates adaptive immunity and plays a role in autoimmunity, infection, and cancer. The authors discovered that a variety of protein allotypes of ERAP2 are maintained in the human population. Allotypes that increase disease risk for autoimmune and cardiovascular conditions are functionally distinct in their capacity to activate T-cells. The results of this study demonstrate that ERAP2 is a functionally diverse immune modulator that contributes to immune variation and influences susceptibility to immune-mediated diseases.

BackgroundHFE p.C282Y (c.845G>A; rs1800562) is a common missense mutation in persons of European ancestry, but we found no comprehensive tabulation of p.C282Y allele frequencies in Iberia. MethodsWe performed computerized and manual searches to identify evaluable reports of p.C282Y alleles in population/control cohorts [&ge;]50 subjects in Iberia. We tabulated numbers of subjects, nominal geographic sites of cohort recruitment, cohort characteristics, corresponding latitudes and longitudes, and p.C282Y allele frequencies [95% confidence intervals]. We computed the aggregate p.C282Y allele frequencies of mainland Spain and mainland Portugal and compared the aggregate frequencies using the Chi-square test (two-tailed). Using combined mainland Spain and mainland Portugal data, we computed the aggregate p.C282Y allele frequency in Iberia. ResultsWe identified 25 cohorts in mainland Spain (12,297 subjects; 11 of the 15 autonomous communities) and nine cohorts in mainland Portugal (1,024 subjects; each of the five administrative regions). Cohorts were recruited in this region: latitude 43.4619 - 37.2299{degrees} N; longitude -9.1366 - 2.1899{degrees} W. The range of p.C282Y allele frequencies in the 34 cohorts was 0.0000 to 0.0517. The aggregate p.C282Y allele frequency in mainland Spain was 0.0291 (716/24,594) [0.0271, 0.0313] and that in mainland Portugal was 0.0303 (62/2048) [0.0237, 0.0386] (p = 0.8343). The aggregate p.C282Y allele frequency in Iberia was 0.0292 (778/26,642) [0.0272, 0.0313]. ConclusionsWe conclude that the aggregate HFE p.C282Y allele frequencies in mainland Spain and mainland Portugal do not differ significantly. The aggregate p.C282Y allele frequency of 34 population/control cohorts (13,321 subjects, 16 geographic regions) in Iberia is 0.0292.

Rhabdomyolysis is the acute breakdown of skeletal muscle resulting from failure of cellular homeostasis in response to metabolic stress. Recurrent forms are frequently linked to inherited defects affecting energy metabolism or calcium handling. Ryanodine receptor type 3 (RyR3) is an intracellular calcium release channel, expressed in skeletal muscle, that contributes to the fine-tuning of calcium signaling. Although variants in other calcium-handling proteins have been implicated in rhabdomyolysis, the role of RyR3 has not been established. In this study, we report rare compound heterozygous missense variants in RYR3 identified in two unrelated individuals with severe, fever-triggered recurrent rhabdomyolysis. Muscle biopsies revealed mild structural changes with triadic disorganization, mitochondrial alterations, lipid accumulation, and autophagic material, while overall muscle architecture was largely preserved. Structural modeling supports the pathogenicity of the variants, and calcium flux analysis demonstrated significantly reduced ryanodine receptor-mediated calcium release in patient-derived myoblasts. Functional analyses showed that RyR3 deficiency impaired starvation-induced autophagy, characterized by defective autophagosome formation and reduced autophagic flux, and increased susceptibility to metabolic stress. Mitochondrial bioenergetic profiling revealed reduced oxidative phosphorylation capacity and decreased membrane potential under stress conditions, consistent with compromised mitochondrial adaptation. In zebrafish, ryr3 knockdown resulted in structural and functional muscle abnormalities, including reduced myotome area and decreased locomotor activity, associated with impaired autophagic flux. This study establishes a novel association between recessive RYR3 variants and recurrent rhabdomyolysis and identifies RyR3 as a critical regulator of skeletal muscle stress adaptation through calcium-dependent control of autophagy and mitochondrial homeostasis. More broadly, our findings further highlight autophagy as a central determinant of muscle resilience in the context of rhabdomyolysis.

Hereditary ataxias are complicated neurological disorders with enormous genetic heterogeneity as well as the diverse genetic mechanism. Among different genetic mechanism, tandem nucleotide repeat expansion (TNRex) are the most common cause for genetic ataxias followed by single nucleotide variations in over 200 genes. The detection and the diagnosis of tandem nucleotide repeats in clinics and laboratories has been at large common in comparison with SNVs owing to the large number of the mutations in the respective genes they are found. The widely used platforms for detection of these mutations are capillary electrophoresis and Next generation sequencing based targeted gene panel or clinical or whole exome sequencing. Long read sequencers have been proven useful for detection of tandem nucleotide repeat expansions. We have evolved a method to detect in one experiment and on single platform the detection of TNRex and SNVs on Oxford Nanopre Technology using adaptive sequencing approach. We were able to optimize the target region sequencing of both TNR loci and SNV-loci and validate the capture of both by detection of FXN-GAA repeats and pathogenic SNVs in SETX

This exploratory analysis of PAX LC, a Phase 2, 1:1 randomized, double-blind, superiority, placebo-controlled trial examined whether treatment with nirmatrelvir/ritonavir (NMV/r) versus placebo/ritonavir (PBO/r) in individuals with Long COVID could reveal immune features associated with symptom improvement. Eighty-two participants (n=45 PBO/r; n=37 NMV/r) provided blood samples at baseline (Day 0) and post-treatment (Day 28). Baseline demographic and immunological phenotypes were similar in the two groups. No significant differences were observed in major immune cell populations or organ function markers between NMV/r vs. PBO/r groups, or before vs. after the treatment. Modest hematologic changes were noted in the NMV/r arm. SARS-CoV-2-specific IgG levels remained constant, with changes in total immunoglobulin subtypes and isotypes in both arms. Both arms showed similar shifts in cytokine levels. Notably, the levels of S1 and Spike proteins in circulation remained unchanged post-treatment. Regardless of the treatment arm, participants with self-reported symptom improvement showed reductions in the level of the inflammatory chemokine RANTES. Taken together, the findings of this study demonstrate limited virological and immunological changes in response to nirmatrelvir, contributing insights into the reason for the lack of benefit of the 15-day NMV/r treatment in Long COVID.

XRN1 encodes a highly conserved cytoplasmic 5-3 exoribonuclease involved in mRNA decay and quality control. It localizes to transient ribonucleoprotein aggregates, P-bodies and stress granules, where it interacts with other mRNA decay proteins and is involved in various cellular responses, including an emerging role in viral infection responses. Complete knockout of XRN1 in multicellular organisms is lethal, most likely due to its central role in mRNA homeostasis, with no prior human disease association reported. Here, we characterize seven individuals from six unrelated families with a lethal infantile form of mitochondrial cardiomyopathy caused by heterozygous de novo frameshift truncation variants clustering in the C-terminal region of XRN1, each predicted to evade nonsense-mediated mRNA decay. Each variant results in a near-identical XRN1 C-terminal sequence predicted to alter a characterized binding domain that interacts with the mRNA decapping enhancer EDC4. Biochemical investigations of striated muscle revealed combined oxidative phosphorylation deficiencies, demonstrated by decreased respiratory chain enzyme activities, decreased proteomics abundances, and abnormal histochemical reactivities. Despite having no established mitochondrial function in mammals, clinical and molecular findings across the cohort were consistent with mitochondrial disease. The precise mechanism by which the altered XRN1 proteins cause disease remains to be elucidated.

BackgroundImmune function alters with age, and is often accompanied by chronic, low-grade inflammation (inflammageing). In individuals with frailty, inflammageing is increased, reducing immune function and increasing susceptibility to serious outcomes from infection. In this study we investigated the changes that take place in human neutrophils during healthy ageing and ageing with frailty (FR) using RNAseq and functional assays. We also compared neutrophil phenotype in frailty with rheumatoid arthritis (RA) neutrophils. MethodsBlood neutrophil RNAseq data were analysed using IDEP2 and Ingenuity Pathway Analysis (IPA). Neutrophil phenotype was assessed for reactive oxygen species (ROS) production, neutrophil extracellular trap (NET) release, chemotaxis, and bacterial killing capacity. Experimental data were analysed by ANOVA in R (v4.5.1). ResultsRNAseq identified activation of G-protein coupled receptors, interferon and cytokine receptor signalling, and chemotaxis pathways in frail individuals (n=10) compared to healthy older (n=9) and healthy younger people (n=8, adj. p<0.05). FR neutrophils expressed more IL-8 receptors (CXCL1 and CXCL2) and CD177 on their surface (p<0.05). FR neutrophil phenotype was similar to RA, with significantly higher ROS production and impaired chemotaxis and bacterial killing capacity (p<0.05). FR neutrophils also released significantly more NETs in response to LPS (10ng/mL, p<0.05). ConclusionsThis work provides novel insight into the changes to neutrophil phenotype associated with ageing in good health and ageing with frailty, and highlights similarities between inflammageing in frailty and chronic inflammation in RA. This may be important in the development of future therapeutics and/or health management strategies to support healthy living as we age. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=148 HEIGHT=200 SRC="FIGDIR/small/25342352v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@15decc0org.highwire.dtl.DTLVardef@1627086org.highwire.dtl.DTLVardef@1c9982eorg.highwire.dtl.DTLVardef@262730_HPS_FORMAT_FIGEXP M_FIG In this study we have combined RNAseq with functional experiments to describe the phenotype of neutrophils from people with frailty. We found that frailty neutrophils had altered gene expression with activation of G-protein coupled receptors, interferon and cytokine receptor signalling, and chemotaxis pathways. This was associated with altered ROS and NET production, impaired chemotaxis and impaired bacterial killing capacity compared to healthy older neutrophils. Created in BioRender. Abdullah, G. (2026) https://BioRender.com/ze634lt and https://BioRender.com/7khpj2z. C_FIG

Sjogrens disease (SjD) is a chronic autoimmune disorder characterized by inflammation of the exocrine glands, leading to dry mouth and dry eyes. This study investigates the role of interleukin-9 (IL-9) and T helper 9 (Th9) cells in the pathogenesis of SjD. We found that serum IL-9 levels were significantly elevated in SjD patients and correlated with clinical laboratory parameters, including autoantibody production. In a mouse model of SjD, IL-9 and Th9-associated cytokines were also elevated, and Th9 cells were enriched in the salivary glands. Our results suggest that IL-9 is produced by multiple cell types, including macrophages, CD4+ T cells, and NK cells, and that Th9 cells contribute to the development of SjD by promoting inflammation and autoantibody production. We also found that Th9 and Th17 polarization conditions increased Th2 and Th17 cells in SjD mice, indicating a shared epigenetic program that renders T cells permissive to multiple differentiation pathways. Anti-IL-9 treatment had a sex-dependent effect, reducing autoantibody production in male mice but worsening focal glandular infiltration in female mice. Our findings suggest that IL-9 plays a complex role in SjD pathobiology, contributing to both local immunoregulation and systemic autoantibody response. Overall, this study offers new insights into the role of IL-9 and Th9 cells in SjD, highlighting the potential for therapeutic targeting of the IL-9/Th9 axis in the treatment of this disease.

Vaccination frequently elicits suboptimal immunogenicity in organ transplant recipients, particularly those on long-term immunosuppressive therapy, highlighting the need for improved understanding of immunosuppression mechanisms and optimized vaccination strategies. This study enrolled a cohort of 132 individuals and observed significantly lower antibody levels in kidney transplant recipients (KTRs) compared to non-transplant controls (non-KTRs). Antibody levels were inversely associated with both the dosage and duration of immunosuppressive therapy. Complementary small animal studies demonstrated that immunosuppressive treatment dosage-dependently and reversibly impaired antibody production, primarily by depleting immune cells, notably B cells. A single shot of adenoviral vector-based vaccines demonstrated enhanced immunogenicity relative to two shots of alum-adjuvanted protein vaccines, inducing potent neutralizing antibodies (NAbs) and a Th1-biased T-cell response even under continuous immunosuppression. The enhanced response was driven by reduced interference from pre-existing antibodies, sustained transgene expression, and the reprogramming of lipid metabolism to activate T and B cells. Our findings advocate for tailored vaccination strategies, positioning adenoviral vectors as a candidate modality for this vulnerable population.