Phenotypic and pathomechanistic overlap between tapasin and TAP deficiencies.

BACKGROUND: Human tapasin deficiency is reported to cause an autosomal-recessive inborn error of immunity characterized by substantially reduced cell surface expression of major histocompatibility complex class I (MHC-I). OBJECTIVE: We evaluated the immunologic and clinical consequences of tapasin deficiency. METHODS: A novel homozygous variant in TAPBP was identified by means of whole genome sequencing. The expression of tapasin and both subunits of the transporter associated with antigen presentation (TAP) were evaluated by Western blot analysis. Cell surface and intracellular expression of MHC-I were evaluated by flow cytometry. Small interfering RNAs were used for silencing TAPBP expression in HEK293T cells. RESULTS: We identified a deletion in TAPBP (c.312del, p.(K104Nfs∗6)) causing tapasin deficiency in a patient with bronchiectasis and recurrent respiratory tract infections as well as herpes zoster. Besides substantial reduction in TAP1 and TAP2 expression, peripheral blood mononuclear cells from this patient and TAPBP-knockdown HEK293T cells, displayed reduced cell surface expression of MHC-I, while reduction in intracellular expression of MHC-I was less prominent, suggesting a defect in MHC-I trafficking to the plasma membrane. IFN-α improved cell surface expression of MHC-I in tapasin deficient lymphocytes and TAPBP-knockdown HEK293T cells, representing a possible therapeutic approach for tapasin deficiency. CONCLUSION: Tapasin deficiency is a very rare inborn error of immunity, the pathomechanism and clinical spectrum of which overlaps with TAP deficiencies.

Novel hypermorphic variants in IRF2BP2 identified in patients with common variable immunodeficiency and autoimmunity.

The interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a transcriptional regulator, functioning a transcriptional corepressor by interacting with the interferon regulatory factor-2. The ubiquitous expression of IRF2BP2 by diverse cell types and tissues suggests its potential involvement in different cell signalling pathways. Variants inIRF2BP2have been recently identified to cause familial common variable immunodeficiency (CVID) characterized by immune dysregulation. This study investigated three rare novel variants inIRF2BP2, identified in patients with primary antibody deficiency and autoimmunity by whole exome-sequencing (WES). Following transient overexpression of EGFP-fused mutants in HEK293 cells and transfection in Jurkat cell lines, we used fluorescence microscopy, real-time PCR and Western blotting to analyze their effects on IRF2BP2 expression, subcellular localization, nuclear translocation of IRF2, and the transcriptional activation of NFκB1(p50). We found altered IRF2BP2 mRNA and protein expression levels in the mutants compared to the wild type after IRF2BP2 overexpression. In confocal fluorescence microscopy, variants in the C-terminal RING finger domain showed an irregular aggregate formation and distribution instead of the expected nuclear localization compared to the variants in the N-terminal zinc finger domain and their wildtype counterpart. Immunoblotting revealed an impaired IRF2 and NFκB1 (p50) nuclear localization in the mutants compared to the IRF2BP2 wildtype counterpart. LPS stimulation reduced IRF2BP2 mRNA expression in the variants compared to the wild type. Our findings significantly contribute to understanding the clinical significance of IRF2BP2 mutations in the pathogenesis of immunodeficiency and immune dysregulation. We observed impairment of the nuclear translocation of IRF2 and NFκB1 (p50) due to the upregulation of IRF2BP2, potentially affecting specific gene expressions involved in immune regulation.

The GAIN Registry - a New Prospective Study for Patients with Multi-organ Autoimmunity and Autoinflammation.

Patient registries are a very important and essential tool for investigating rare diseases, as most physicians only see a limited number of cases during their career. Diseases of multi-organ autoimmunity and autoinflammation are especially challenging, as they are characterized by diverse clinical phenotypes and highly variable expressivity. The GAIN consortium (German multi-organ Auto Immunity Network) developed a dataset addressing these challenges. ICD-11, HPO, and ATC codes were incorporated to document various clinical manifestations and medications with a defined terminology. The GAIN dataset comprises detailed information on genetics, phenotypes, medication, and laboratory values. Between November 2019 and July 2022, twelve centers from Europe have registered 419 patients with multi-organ autoimmunity or autoinflammation. The median age at onset of symptoms was 13 years (IQR 3-28) and the median delay from onset to diagnosis was 5 years (IQR 1-14). Of 354 (84.5%) patients who were genetically tested, 248 (59.2%) had a defined monogenetic cause. For 87 (20.8%) patients, no mutation was found and for 19 (4.5%), the result was pending. The most common gene affected was NFkB1 (48, 11.5%), and the second common was CTLA4 (40, 9.5%), both genetic patient groups being fostered by specific research projects within GAIN. The GAIN registry may serve as a valuable resource for research in the inborn error of immunity community by providing a platform for etiological and diagnostic research projects, as well as observational trials on treatment options.

CTLA-4 Insufficiency due to a Novel CTLA-4 Deletion, Identified through Copy Number Variation Analysis.

BACKGROUND: The diagnostic yield of next-generation sequencing (NGS) technologies in the diagnosis of monogenic inborn errors of immunity (IEI) remains limited, rarely exceeding 30%. Monoallelic pathogenic germline variants in cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) result in variable immunodeficiency and immune dysregulation. The genetic diagnosis of CTLA-4 insufficiency can affect follow-up procedures and may lead to consideration of treatment with CTLA-4-Ig. OBJECTIVES: The aim of the study was to identify the genetic cause of familial immunodeficiency and immune dysregulation in cases where single nucleotide variant analysis of short-read NGS data yielded no diagnostic result. METHODS: Analysis of copy number variants (CNVs) was applied on short-read NGS data. RESULTS: We identified a novel monoallelic deletion-insertion variant in CTLA-4 (c.445_568-544delinsTTTGCGATTG) resulting in familial autoimmunity. This is the second larger scale variant in CTLA-4, which despite consistently reduced expression of CTLA-4 displayed variable expressivity, ranging from typical juvenile idiopathic arthritis to common variable immunodeficiency-like immunodeficiency. CONCLUSIONS: Our report suggests the significance of integration of CNV analysis in routine evaluation of NGS, which may increase its diagnostic yield in IEI.

Novel aspects of regulatory T cell dysfunction as a therapeutic target in giant cell arteritis.

OBJECTIVES: Giant cell arteritis (GCA) is the most common primary vasculitis, preferentially affecting the aorta and its large-calibre branches. An imbalance between proinflammatory CD4+ T helper cell subsets and regulatory T cells (Tregs) is thought to be involved in the pathogenesis of GCA and Treg dysfunction has been associated with active disease. Our work aims to explore the aetiology of Treg dysfunction and the way it is affected by remission-inducing immunomodulatory regimens. METHODS: A total of 41 GCA patients were classified into active disease (n=14) and disease in remission (n=27). GCA patients' and healthy blood donors' (HD) Tregs were sorted and subjected to transcriptome and phenotypic analysis. RESULTS: Transcriptome analysis revealed 27 genes, which were differentially regulated between GCA-derived and HD-derived Tregs. Among those, we identified transcription factors, glycolytic enzymes and IL-2 signalling mediators. We confirmed the downregulation of forkhead box P3 (FOXP3) and interferon regulatory factor 4 (IRF4) at protein level and identified the ineffective induction of glycoprotein A repetitions predominant (GARP) and CD25 as well as the reduced T cell receptor (TCR)-induced calcium influx as correlates of Treg dysfunction in GCA. Inhibition of glycolysis in HD-derived Tregs recapitulated most identified dysfunctions of GCA Tregs, suggesting the central pathogenic role of the downregulation of the glycolytic enzymes. Separate analysis of the subgroup of tocilizumab-treated patients identified the recovery of the TCR-induced calcium influx and the Treg suppressive function to associate with disease remission. CONCLUSIONS: Our findings suggest that low glycolysis and calcium signalling account for Treg dysfunction and inflammation in GCA.

Diagnostic Yield and Therapeutic Consequences of Targeted Next-Generation Sequencing in Sporadic Primary Immunodeficiency.

INTRODUCTION: Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders characterized by increased susceptibility to infections, immune dysregulation, and/or malignancy. Genetic studies, especially during the last decade, led to a better understanding of the pathogenesis of primary immunodeficiencies and contributed to their classification into distinct monogenic disorders falling under one of the >430 currently known inborn errors of immunity (IEI). The growing availability of molecular genetic testing resulted in the increasing identification of patients with IEI. Here, we evaluated the diagnostic yield and the clinical consequences of targeted next-generation sequencing (tNGS) in a cohort of 294 primary immunodeficiency patients, primarily consisting of cases with sporadic primary antibody deficiency. METHOD: We have custom designed a tNGS panel to sequence a cohort of PID patients. Agilent's HaloPlex Target Enrichment System for Illumina was used for DNA target enrichment. RESULTS: tNGS identified a definite or predicted pathogenic variant in 15.3% of patients. The highest diagnostic rate was observed among patients with combined immunodeficiency or immune dysregulation, for whom genetic diagnosis may affect therapeutic decision-making. CONCLUSION: Next-generation sequencing has changed diagnostic assignment and paved the way for targeted therapeutic intervention with agents directed at reverting the disease-causing molecular abnormality or its pathophysiological consequences. Therefore, such targeted therapies and identifying the genetic basis of PID can be essential for patients with manifested immune dysregulation as conventional immunomodulatory regimens may exert an immunosuppressive effect, aggravating their immunodeficiency or may only inadequately control autoimmune or lymphoproliferative manifestations.

[Genetics of inborn errors of immunity]. / Zur Genetik angeborener Krankheiten des Immunsystems.

In the last 10 years there has been enormous progress in the field of inborn errors of immunity (IEI). The number of newly discovered diseases is growing exponentially, including not only rare but also frequent genetic defects. The spectrum of clinical phenotypes ascribed to IEI is also rapidly expanding. There is every reason to assume that this is only the tip of the iceberg and in the near future further IEI will be discovered with the help of genetic and immunological studies. Patients will benefit from the timely diagnostics as well as from the individualized treatment.

High frequency of variants in genes associated with primary immunodeficiencies in patients with rheumatic diseases with secondary hypogammaglobulinaemia.

OBJECTIVES: Treatment of rheumatic diseases requires immunomodulatory agents which can compromise antibody production. However, even in case of agents directly targeting B cells, a minority of patients develop hypogammaglobulinaemia, suggesting a genetic predisposition, which has not been investigated so far. The phenotypic overlap between primary immunodeficiency disorders (PIDs) and rheumatic diseases suggests a shared genetic basis, especially in case of patients with rheumatic diseases with hypogammaglobulinaemia. METHODS: 1008 patients with rheumatic diseases visiting the outpatient clinics of the Hannover University Hospital were screened for hypogammaglobulinaemia. Those with persistent hypogammaglobulinaemia and an equal number of patients without it underwent targeted next-generation sequencing, searching for variations in genes linked with hypogammaglobulinaemia in the context of PIDs. RESULTS: We identified 33 predicted pathogenic variants in 30/64 (46.9%) patients with persistent secondary hypogammaglobulinaemia. All 33 variants were monoallelic and 10 of them in 10/64 (15.6%) patients were found in genes associated with autosomal dominant PIDs. 2/64 (3.1%) patients harboured variants which were previously reported to cause PIDs. In the group without hypogammaglobulinaemia we identified seven monoallelic variants in 7/64 (10.9%), including a variant in a gene associated with an autosomal dominant PID. CONCLUSIONS: Approximately half of patients with persistent secondary hypogammaglobulinaemia harboured at least a variant in a PID gene. Despite the fact that previous immunomodulatory treatment is an exclusion criterion in the diagnosis of PIDs, we identified genetic variants that can account for PID in patients with clear rheumatic phenotypes who developed hypogammaglobulinaemia after the introduction of immunomodulatory treatment. Our data suggest the common genetic causes of primary and secondary hypogammaglobulinaemia.

Characterization of the clinical and immunologic phenotype and management of 157 individuals with 56 distinct heterozygous NFKB1 mutations.

BACKGROUND: An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes. OBJECTIVE: To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations. METHODS: In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional in vitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) signaling. RESULTS: We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-κB1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents. CONCLUSIONS: We present a comprehensive clinical overview of the NF-κB1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-κB1 pathway-targeted therapeutic strategies should be considered in the future.

A novel NFKBIA variant substituting serine 36 of IκBα causes immunodeficiency with warts, bronchiectasis and juvenile rheumatoid arthritis in the absence of ectodermal dysplasia.

Genetic studies have led to identification of an increasing number of monogenic primary immunodeficiency disorders. Monoallelic pathogenic gain-of-function (GOF) variants in NFKBIA, the gene encoding IκBα, result in an immunodeficiency disorder, typically accompanied by anhidrotic ectodermal dysplasia (EDA). So far, 14 patients with immunodeficiency due to NFKBIA GOF mutations have been reported. In this study we report three patients from the same family with immunodeficiency, presenting with recurrent respiratory tract infections, bronchiectasis and viral skin conditions due to a novel pathogenic NFKBIA variant (c.106 T > G, p.Ser36Ala), which results in reduced IκBα degradation. Immunological investigations revealed inadequate antibody responses against vaccine antigens, despite hypergammaglobulinemia. Interestingly, none of the studied patients displayed features of EDA. Therefore, missense NFKBIA variants substituting serine 36 of IκBα, differ from the rest of pathogenic GOF NFKBIA variants in that they cause combined immunodeficiency, even in the absence of EDA.

Evaluation of RAG1 mutations in an adult with combined immunodeficiency and progressive multifocal leukoencephalopathy.

Here we describe novel mutations in recombination activation gene 1 (RAG1) in a compound heterozygous male patient with combined T and B cell immunodeficiency (CID). Clinical manifestations besides antibody deficiency included airway infections, granulomatosis and autoimmune features. He died at the age of 37 due to PML caused by JC virus infection. By targeted next-generation sequencing we detected post mortem in this patient three mutations in RAG1. One allele harbored two novel mutations (c.1123C>G, p.H375D and c.1430delC, p.F478Sfs*14), namely a missense variant and a frameshift deletion, of which the latter leads to a truncated RAG1 protein. The other allele revealed a previously described missense mutation (c.1420C>T, p.R474C, rs199474678). Functional analysis of the p.R474C variant in an in vitro V(D)J recombination assay exhibited reduced recombination activity compared to a wild-type control. Our findings suggest that mutations in RAG1, specifically the p.R474C variant, can be associated with relatively mild clinical symptoms or delayed occurrence of T cell and B cell deficiencies but may predispose to PML.

Limited role of interferon-kappa (IFNK) truncating mutations in common variable immunodeficiency.

We used whole exome sequencing to determine the genetic background of CVID in two non-consanguineous German families. We identified IFNK (interferon-kappa) as the only candidate gene that harbored truncating mutations in affected members from both families. One family segregated c.30_31insTGTT, a known frameshift variant, while the other family segregated the novel IFNK mutation p.K199X that creates a premature stop codon. We sequenced the whole coding region of IFNK in a further series of 167 CVID patients and 192 healthy controls. Frameshift mutation c.30_31insTGTT was identified in 12 cases and 17 controls (OR 0.79, 95% CI 0.33-1.81, p=0.79), whereas the p.K199X mutation remained restricted to the original family. No additional truncating variants were found. We conclude that, given their frequent occurrence in non-diseased family members and controls, it is unlikely that truncating variants in IFNK constitute a major factor in the development of CVID.

Limitation of Simultaneous Analysis of T-Cell Receptor and κ-Deleting Recombination Excision Circles Based on Multiplex Real-Time Polymerase Chain Reaction in Common Variable Immunodeficiency Patients.

AIM OF STUDY: We used a triplex real-time polymerase chain reaction (PCR) to classify our common variable immunodeficiency (CVID) patients into distinct groups according to the amount of their T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs). MATERIALS AND METHODS: TREC and KREC analysis was performed using a multiplex real-time PCR assay. The T- and B-lymphocyte subsets were measured by flow cytometry. RESULTS: The copy number of TRECs and KRECs was significantly reduced in CVID patients compared to healthy controls. The TREC copy number was inversely correlated with age in both healthy subjects and patients; however, the KREC copy number was inversely correlated with age only in CVID patients. Moreover, no association was seen between TREC/KREC copy number and clinical manifestations such as bronchiectasis, splenomegaly, granulomata, autoimmune cytopenias, organ-specific autoimmunity, enteropathy and lymphoid hyperplasia. CONCLUSION: TREC and KREC quantification might be a useful tool to differentiate between CVID and combined immunodeficiency, but considering the results of this study a classification of CVID patients in certain groups is hardly possible.

JAK inhibitors to treat STAT3 gain-of-function: a single-center report and literature review.

Objective: The signal transducer and activator of transcription 3 (STAT3) gain-of-function (GOF) syndrome (STAT3-GOF) is an inborn error of immunity (IEI) characterized by diverse manifestations of immune dysregulation that necessitate systemic immunomodulatory treatment. The blockade of the interleukin-6 receptor and/or the inhibition of the Janus kinases has been commonly employed to treat diverse STAT3-GOF-associated manifestations. However, evidence on long-term treatment outcome, especially in the case of adult patients, is scarce. Methods: Clinical data, including laboratory findings and medical imaging, were collected from all seven patients, diagnosed with STAT3-GOF, who have been treated at the Hannover University School, focusing on those who received a Janus kinase (JAK) inhibitor (JAKi). Previously published cases of STAT3-GOF patients who received a JAKi were evaluated, focusing on reported treatment efficacy with respect to diverse STAT3-GOF-associated manifestations of immune dysregulation and safety. Results: Five out of seven patients diagnosed with STAT3-GOF were treated with a JAKi, each for a different indication. Including these patients, outcomes of JAKi treatment have been reported for a total of 41 patients. Treatment with a JAKi led to improvement of diverse autoimmune, inflammatory, or lymphoproliferative manifestations of STAT3-GOF and a therapeutic benefit could be documented for all except two patients. Considering all reported manifestations of immune dysregulation in each patient, complete remission was achieved in 10/41 (24.4%) treated patients. Conclusions: JAKi treatment improved diverse manifestations of immune dysregulation in the majority of STAT3-GOF patients, representing a promising therapeutic approach. Long-term follow-up data are needed to evaluate possible risks of prolonged treatment with a JAKi.

Reduced mRNA expression level of corticotropin-releasing hormone-binding protein is associated with aggressive human kidney cancer.

BACKGROUND: Significance of Urocortin (Ucn or UcnI), Ucn2, Ucn3 and their receptors, Corticotropin Releasing Factor Receptor 1 and 2 (CRFR1 and CRFR2), and the binding protein, Corticotropin-Releasing Hormone-Binding Protein (CRHBP) in oncology is growing rapidly. The objective of our study was to assess the expression of the CRHBP mRNA and protein in renal cancer. METHODS: Tumoral tissues of 78 patients with clear cell renal cell cancer and their corresponding normal tissues were analyzed using quantitative mRNA expression analysis for detection of mRNA expression level. Protein expression and tissue localization of CRHBP protein in renal specimens was evaluated using western blotting, immunohistochemistry and double immunofluorescence, respectively. RESULTS: We found an approx. 33 fold decrease of average CRHBP mRNA level in tumoral tissues compared to paired normal tissues (p<0.001). Diminished CRHBP mRNA expression was positively correlated with advanced, metastasized and higher stage of disease (p<0.001, p=0.026, p=0.028 respectively). CRHBP protein was detected in glomeruli and proximal tubules of normal kidney while none or weak immunopositivity was found in cc-RCC (p<0.001). CONCLUSIONS: The expression analysis of CRHBP shows that cc-RCC is characterized by a significant loss of CRHBP mRNA expression that furthermore is associated with a more aggressive state of tumors. Depletion of CRHBP proteins also indicate that the protein as part of the UCN system may be involved in renal carcinogenesis.

Corrigendum: Vulnerability to meningococcal disease in immunodeficiency due to a novel pathogenic missense variant in NFKB1.

[This corrects the article DOI: 10.3389/fimmu.2021.767188.].