Loss-of-function nuclear factor κB subunit 1 (NFKB1) variants are the most common monogenic cause of common variable immunodeficiency in Europeans

Abstract

Background: The genetic cause of primary immunodeficiency disease (PID) carries prognostic information.

Objective: We conducted a whole-genome sequencing study assessing a large proportion of the NIHR BioResource-Rare Diseases cohort.

Methods: In the predominantly European study population of principally sporadic unrelated PID cases (n = 846), a novel Bayesian method identified nuclear factor κB subunit 1 (NFKB1) as one of the genes most strongly associated with PID, and the association was explained by 16 novel heterozygous truncating, missense, and gene deletion variants. This accounted for 4% of common variable immunodeficiency (CVID) cases (n = 390) in the cohort. Amino acid substitutions predicted to be pathogenic were assessed by means of analysis of structural protein data. Immunophenotyping, immunoblotting, and ex vivo stimulation of lymphocytes determined the functional effects of these variants. Detailed clinical and pedigree information was collected for genotype-phenotype cosegregation analyses.

Results: Both sporadic and familial cases demonstrated evidence of the noninfective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%), and autoimmune disease (48%), features prior studies correlated with worse clinical prognosis. Although partial penetrance of clinical symptoms was noted in certain pedigrees, all carriers have a deficiency in B-lymphocyte differentiation. Detailed assessment of B-lymphocyte numbers, phenotype, and function identifies the presence of an increased CD21^low B-cell population. Combined with identification of the disease-causing variant, this distinguishes between healthy subjects, asymptomatic carriers, and clinically affected cases.

Conclusion: We show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID, which results in a temporally progressive defect in the formation of immunoglobulin-producing B cells.

Keywords: B cells; common variable immunodeficiency; nuclear factor κB1.

Graphical abstract

Fig 1

Overall BeviMed results showing that NFKB1 has the highest posterior probability of association with disease in the NIHRBR-RD PID cohort. Genes with variants previously reported to cause PIDs are highlighted in red. Genes with posterior probabilities of greater than .05 are shown.

Fig 2

Plot of rare missense, truncating, and gene deletion NFKB1 variants identified in the NIHRBR-RD genomes of unrelated subjects and their location relative to NFKB1 domains. Tracks from left to right show the following: number of unrelated case (red) and control (black) subjects in whom each variant was observed; the 4 major NFKB1 domains; each exon in transcript ENST00000226574 (gray bars); variant annotation relative to transcript ENST00000226574 and genomic location of large deletions, with VEP high-effect variants and large deletions highlighted in blue; Combined Annotation Dependent Depletion (CADD) scores of all nonsense, frameshift, splice and missense variants; Exome Aggregation Consortium (ExAC) allele frequencies; and conditional probability of variant pathogenicity inferred by using BeviMed. Only variants labeled as being of moderate or high effect relative to the canonical transcript ENST00000226574 are shown. The initial inference that formed part of the genome-wide analysis included variant chr4:103423325G>A, which was observed in 1 control sample. This variant is intronic (low effect) relative to ENST00000226574 but is a splice variant (high effect) relative to the minor transcript ENST00000505458. Because variants were filtered based on the highest-effect variant annotation against any Ensembl transcript, this variant was originally included in the inference. For this plot, the inference was rerun, including only missense, truncating, and gene deletion variants relative to the canonical transcript.

Fig 3

NFKB1 LOF variants lead to haploinsufficiency of the p50 protein. A, Localization of RHD substitutions with a high Combined Annotation Dependent Depletion (CADD) score (>20) within the structure of the NF-κB p50 monomer. Shown is a solid (top panel) and a transparent (bottom panel) sphere representation of the NF-κB p50 monomer. Perturbed residues indicated in green were observed in a control data set and are located on the outside of the structure, whereas residues shown in red were perturbed exclusively in the PID cohort and are buried inside the structure. B, Western blot analysis targeting p50, IκBα, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of NFKB1 variant carriers. Left, Representative blot of a healthy control subject and patient B-II:1; right, summary of 16 NFKB1 variant carriers showing haploinsufficiency expressed as a percentage of healthy control subjects on the same blot corrected for GAPDH (mean ± SEM).

Fig 4

Pedigrees of familial NFKB1 cases. Six affected families for which pedigree information and additional family members were available. P, Proband/index cases.

Fig 5

Decreased class-switched memory B-cell and increased CD21^low B-cell counts in NFKB1 LOF variant carriers. A, Absolute numbers of CD19⁺ B cells; each dot represents a single subject and his or her age. Age-dependent reference values are shown in gray. B-E, Percentages within CD19⁺CD20⁺ B lymphocytes of CD27⁺IgD⁺ (nonswitched memory or marginal-zone B cells) and CD27⁺IgD⁻ (switched memory B cells; Fig 5, B) or CD27⁺IgG⁺ (Fig 5, C), CD27⁺IgA⁺ (Fig 5, D), or CD21^lowCD38^low/dim (Fig 5, E). CA, Clinically affected subjects with an LOF variant in NFKB1; CU, clinically unaffected subjects with an LOF variant in NFKB1; HD, healthy donor. The gating strategy is shown in Fig E6, A. Only subjects with sufficient B cells could be analyzed. P values were determined by using 1-way (Fig 5, E) or 2-way (Fig 5, B) ANOVA with the Bonferroni post hoc test or unpaired Student t test (Fig 5, C and D). ns, Not significant. **P ≤ .01 and ***P ≤ .001.

Fig 6

Ex vivo class-switch recombination defect of subjects carrying NFKB1 LOF variants is linked to the more extreme phenotype. Six-day culture of CFSE-labeled lymphocytes normalized for B-cell numbers: unstimulated, CpG/IL-2 stimulated (T cell–independent activation), or anti-IgM/anti-CD40/IL-21 stimulated (T cell–dependent activation). A, Percentage of divided B cells, as measured based on CFSE dilution. B, Percentage of CD27⁺⁺ plasmablasts. The gating strategy is shown in Fig E7, A. C and D, IgM and IgG production in supernatants of 6-day cultures. CA, Clinically affected subjects with an LOF variant in NFKB1; CU, clinically unaffected subject; HD, healthy donor. Only subjects with sufficient B cells could be analyzed. P values were determined by using 2-way ANOVA with the Bonferroni post hoc test. ns, Not significant. **P ≤ .01 and ***P ≤ .001.

Fig E1

Fig E2

Fig E3

Fig E4

Fig E5

Fig E6

Fig E7

Fig E8

Fig E9

Fig E10