(C) Expression of p105/50 in the PBMCs of healthy control (HC) and mutation carriers (S1-S3). with wild type NFB1 and mutant (p.R230C) showed a reduction in the p50 by immunoblotting. Both p105 and p50 were drastically reduced in the mutant compared to the wild type. (C) Expression of p105/50 in the PBMCs of healthy control (HC) and mutation carriers (S1-S3). Densitometry analysis revealed lowered expression in all subjects compared to the control. (D) PBMCs from HC and S1, S2 and S3 were stimulated with PMA; 50 ng/ml and ionomycin;1 g/ml and the expression of p105/50 examined. There was no significant changes in the p105/50 expression after stimulation. DataSheet_1.docx (732K) GUID:?3D09588E-8BE2-4545-BED8-9ED9EDE72E23 Data Availability StatementThe original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA788443. Abstract NF-B1 deficiency is suggested to be the most common cause of common variable immunodeficiency (CVID). encodes for the p105 precursor protein of NF-B1, which is usually converted into the active transcriptional subunit p50 through proteasomal processing of its C-terminal half upon stimulation and is implicated in the canonical NF-kB pathway. Rare monoallelic variants have been shown to cause (haplo) insufficiency. Our report describes a novel missense variant (c.691C T, p.R230C; Xanthatin allele frequency 0.00004953) in a family vulnerable to meningitis, sepsis, and late-onset hypogammaglobulinemia. We investigated the pathogenic relevance of this variant by lymphocyte stimulation, immunophenotyping, overexpression study and immunoblotting. The ectopic expression of p50 for c.691 C T restricted transcriptionally active p50 in the cytoplasm, and immunoblotting revealed reduced p105/50 expression. This study shows that the deleterious missense variant in NFKB1 adversely affects the transcriptional and translational activity of NFB1, impairing its function. Patients immunological parameters show a progressive course of Xanthatin hypogammaglobulinemia, which may partially account for the incomplete Xanthatin disease penetrance and suggest the need for closer immunological monitoring of those mutation carriers. and have been identified in CVID (1, 5, 6). Recently KLF11 antibody NF-B1 haplo (insufficiency) has been described as a novel monogenic cause of CVID (6). Several independent studies have reported that loss-of-function variants in are probably the common cause of antibody deficiency with a highly variable clinical and immunological presentation (7). The nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-B) is usually a family of closely related ubiquitous transcription factors that regulate an extensive array of genes involved in different immune and inflammatory responses. This family is composed of five structurally related proteins including NF-B1 (p50/p105), NF-B2 (p52/p100), RelA (p65), c-Rel, and RelB that mediates transcription of target genes by binding to a specific DNA element, B enhancer, as various hetero- or homo-dimers (8). The NF-B proteins are usually sequestered in the cytoplasm by a family of inhibitory proteins, including IB family members and related proteins defined by the presence of ankyrin repeats. Two different signal pathways have been proposed for NF-B activities, the classical/canonical or non-canonical pathway. Upon stimulation of classical pathway by Toll-like-receptors, or B and T cell receptors, the IKB subunit phosphorylates and polyubiquitinates, leading to its degradation by the 26S proteasome (8) and translocation of p50 into the nucleus to exert its function as a transcription factor (9). We have recently identified the pathogenic impact of identified deleterious variants in our PAD Xanthatin cohort; we have shown that missense mutation in the causes late-onset PAD by impairing the function of the transcriptionally active p50 (10). We identified further a previously uncharacterized missense variant in a family with a history of meningococcal meningitis and late-onset hypogammaglobulinemia by next-generation sequencing (NGS) as the only predicted deleterious variant within genes of inborn errors of immunity. Hence in the current work, we mainly focused on the relevance of this NFKB1 missense variant (c.691 C T, p.R230C) by immunophenotyping, immunoblotting, and ectopic expression assays. Materials and Method Ethical Aapproval The institutional medical ethical committee at Hannover Medical School approved the study (ethics approval number: Nr.8875_BO_K_2020). The written consent of all study participants was obtained. Isolation of Genomic DNA and Sequencing Methods Genomic DNA (gDNA) was isolated from peripheral blood of patients and healthy donors with QIAamp Kit (QIAamp DNA Blood Midi Kit; Lot# 16902455variant and its co-segregation with disease phenotype in this family using the primers: forward; 5′-GTCTATTCTTGGTGTGCCCC-3′ and reverse; 3′-TGCAGCAGACCAAGGAGATG-5′. PBMCs Isolation Whole blood was collected from both patients and healthy control. PBMCs were isolated using the standard centrifugation method. Briefly, whole blood was mixed with PBS in a 1:2 ratio, and the diluted cell suspension was gently layered around the Ficol-plaque separation gradient. Centrifugation was carried out at 1000xg for 20 mins with no break at 21C. The mononuclear cell layer was carefully removed, transferred into new 50 ml falcon tubes, and washed with PBS. Cells were either stored in 10% DMSO or immediately used. Lymphocyte Stimulation Assays For stimulation experiments, PBMCs were treated with phorbol Xanthatin 12-myristate 13-acetate (PMA; 50 ng/ml) and ionomycin (1 g/ml) and incubated for 30 min at.
