Clinical and Immunological Phenotype of Patients With Primary Immunodeficiency Due to Damaging Mutations in NFKB2

Abstract

Non-canonical NF-κB-pathway signaling is integral in immunoregulation. Heterozygous mutations in NFKB2 have recently been established as a molecular cause of common variable immunodeficiency (CVID) and DAVID-syndrome, a rare condition combining deficiency of anterior pituitary hormone with CVID. Here, we investigate 15 previously unreported patients with primary immunodeficiency (PID) from eleven unrelated families with heterozygous NFKB2-mutations including eight patients with the common p.Arg853^* nonsense mutation and five patients harboring unique novel C-terminal truncating mutations. In addition, we describe the clinical phenotype of two patients with proximal truncating mutations. Cohort analysis extended to all 35 previously published NFKB2-cases revealed occurrence of early-onset PID in 46/50 patients (mean age of onset 5.9 years, median 4.0 years). ACTH-deficiency occurred in 44%. Three mutation carriers have deceased, four developed malignancies. Only two mutation carriers were clinically asymptomatic. In contrast to typical CVID, most patients suffered from early-onset and severe disease manifestations, including clinical signs of T cell dysfunction e.g., chronic-viral or opportunistic infections. In addition, 80% of patients suffered from (predominately T cell mediated) autoimmune (AI) phenomena (alopecia > various lymphocytic organ-infiltration > diarrhea > arthritis > AI-cytopenia). Unlike in other forms of CVID, auto-antibodies or lymphoproliferation were not common hallmarks of disease. Immunophenotyping showed largely normal or even increased quantities of naïve and memory CD4⁺ or CD8⁺ T-cells and normal T-cell proliferation. NK-cell number and function were also normal. In contrast, impaired B-cell differentiation and hypogammaglobinemia were consistent features of NFKB2-associated disease. In addition, an array of lymphocyte subpopulations, such as regulatory T cell, Th17-, cTFH-, NKT-, and MAIT-cell numbers were decreased. We conclude that heterozygous damaging mutations in NFKB2 represent a distinct PID entity exceeding the usual clinical spectrum of CVID. Impairment of the non-canonical NF-κB pathways affects function and differentiation of numerous lymphocyte-subpopulations and thus causes a heterogeneous, more severe form of PID phenotype with early-onset. Further characteristic features are multifaceted, primarily T cell-mediated autoimmunity, such as alopecia, lymphocytic organ infiltration, and in addition frequently ACTH-deficiency.

Keywords: ACTH-deficiency; CID; CVID; DAVID-syndrome; NF-kappaB signaling; NF-kappaB2 clinical cases; autoimmunity; deficiency of anterior pituitary function and variable immunodeficiency.

Figure 1

PID-associated C-terminal NFKB2 mutations. (A) Pedigrees of the family members who participated in this study. Circles, female; squares, male; filled symbols, affected individual with PID; open symbol, healthy family member; slash, deceased individual. Family members who underwent genetic testing are indicated by IDs and sequence variants. (B) Sanger sequencing confirms that all NFKB2 mutations cluster within exons 22–23 and affect the C-terminus of the 900 amino acids precursor protein p100. The mutation in Fam689 is shown as a representative for all families harboring the p.Arg853^* hot-spot mutation.

Figure 2

Decreased T follicular helper (cTFH), but normal NK and T cell function in NFKB2-mutated patients. (A) Reduced frequency of circulating T follicular helper cells (CXCR5posCD45RAneg) in CD4 T cells of patients with NFKB2 mutation. FACS plots shows example staining of a healthy donor (HD) and P41. (B) Percentage of CXCR5pos in CD45RA+ (left) and memory (CD45RAneg, right) CD4 T cells. (A,B) Dotted lines in graphs represent 95% percentile of 47 healthy donors. Values of healthy donors are depicted as means ± SD. Statistical significance was assessed by Mann-Whitney test (**P < 0.01, ****P < 0.0001). (C) Normal NK and T cell function. (C,D) PBMCs were stimulated either with medium alone or K562 cells for 2.5 h in presence of anti-CD107a-PE. Lytic exocytosis of NK cells (CD56⁺) are measured by CD107a staining. (D) NK cytotoxicity was analyzed by standard 51Cr release assay. Target cell lysis by NK cells: PBMC were incubated with K562 target cells in a standard 51Cr release assay. The percentage of NK cells among PBMC was measured by flow cytometry to determine the NK:target cell ratio. The gray shaded area represents the range of normal values (5th−95th percentile). (E) T cell proliferation was measured by CFSE dilution after 1 week of stimulation with PHA (1.25 ug/ml) or CD3 (100 ng/ml) with CD28 (200 ng/ml).

Figure 3

The clinical phenotype in NF-κB2 haploinsufficiency is characterized by early-onset antibody deficiency, clinical signs of T cell dysfunction, and autoimmunity. (A) Fraction of affected patients relative to the whole cohort of patients of whom information was available with different clinical manifestations depicted on the Y-axis. Manifestations below the solid line are relative to the 46 NFKB2-mutated patients with PID. (B) Absolute numbers of patients who are not affected by different clinical manifestations are depicted on the left (open white bars), while the absolute number of reported patients affected are depicted to the right side (solid gray bars).

Figure 4

Preserved T/NK cell numbers and function, but disturbed B cell differentiation, hypogammaglobulinemia and decreased lymphocyte subpopulations are consistent features of NF-κB2 haploinsufficiency. (A) Fraction of affected patients relative to the whole cohort of patients of whom information was available regarding decreased values in the immunological phenotype depicted on the Y-axis. (B) Absolute numbers of patients with decreased values in different immunological assays are depicted on the left (solid gray bars), while the absolute number of patients with either normal or increased values are depicted to the right side (open white bars).