Expanding spectrum, intrafamilial diversity, and therapeutic challenges from 15 patients with heterozygous CARD11-associated diseases: A single center experience

Abstract

CARD11-associated diseases are monogenic inborn errors of immunity involving immunodeficiency, predisposition to malignancy and immune dysregulation such as lymphoproliferation, inflammation, atopic and autoimmune manifestations. Defects in CARD11 can present as mutations that confer a complete or a partial loss of function (LOF) or contrarily, a gain of function (GOF) of the affected gene product. We report clinical characteristics, immunophenotypes and genotypes of 15 patients from our center presenting with CARD11-associated diseases. Index cases are pediatric patients followed in our immunology division who had access to next generation sequencing studies. Variant significance was defined by functional analysis in cultured cells transfected with a wild type and/or with mutated hCARD11 constructs. Cytoplasmic aggregation of CARD11 products was evaluated by immunofluorescence. Nine index patients with 9 unique heterozygous CARD11 variants were identified. At the time of the identification, 7 variants previously unreported required functional validation. Altogether, four variants showed a GOF effect as well a spontaneous aggregation in the cytoplasm, leading to B cell expansion with NF-κB and T cell anergy (BENTA) diagnosis. Additional four variants showing a LOF activity were considered as causative of CARD11-associated atopy with dominant interference of NF-kB signaling (CADINS). The remaining variant exhibited a neutral functional assay excluding its carrier from further analysis. Family segregation studies expanded to 15 individuals the number of patients presenting CARD11-associated disease. A thorough clinical, immunophenotypical, and therapeutic management evaluation was performed on these patients (5 BENTA and 10 CADINS). A remarkable variability of disease expression was clearly noted among BENTA as well as in CADINS patients, even within multiplex families. Identification of novel CARD11 variants required functional studies to validate their pathogenic activity. In our cohort BENTA phenotype exhibited a more severe and expanded clinical spectrum than previously reported, e.g., severe hematological and extra hematological autoimmunity and 3 fatal outcomes. The growing number of patients with dysmorphic facial features strengthen the inclusion of extra-immune characteristics as part of the CADINS spectrum. CARD11-associated diseases represent a challenging group of disorders from the diagnostic and therapeutic standpoint, especially BENTA cases that can undergo a more severe progression than previously described.

Keywords: CARD11; atopic; benta; cadins; dominant negative; gain of function; inborn error of immunity; lymphoproliferation.

Figure 1

(A) Family Pedigrees, with CARD11 variants found and affected family members. (B) Schematic representation of CARD11 protein, with variants found in our cohort.

Figure 2

Reporter gene assays of CARD11 variants in JPM50.6,Jurkat T cells and HEK293T cell lines. (A) Quantification of Flow cytometric NF-κB-driven GFP reporter expression in JPM50.6 cells transfected with empty vector (EV), WT or mutant CARD11 constructs, unstimulated or stimulated with anti-CD3 and anti-CD28 for 24 h. (mean fluorescence intensity (MFI) of GFP+ cells). Data are mean ± s.e.m. (EV and WT n=16; p.R30W and p.G123S n=12, p.T117P and p.R818Q n=8; p.T43R, p.E96K and p.Q249P n=4) Two-way ANOVA with Sidak’s multiple t-test. *p<0.05 for each mutant versus WT with or without stimulation (EV<0.0001, <0.0001; p.R30W p=0.0110, <0.0001; p.T43R p=0.0485, <0.0001; p.E96K p=0.4627, <0.0001; p.T117P p<0.0001, <0.0001; p.G123S p<0.0001, <0.0001; p.Q249P <0.0001, <0.0001; p.R818Q p<0.0001, p=0.3119). (B) Quantification of Flow cytometric analysis (GFP MFI) of JPM50.6 cells transfected with WT CARD11 plus WT or mutant constructs and stimulated as in (A). Data are mean ± s.e.m. (EV and WT n=12; p.R30W and p.G123S n=9, p.T117P and p.R818Q n= 6; p.T43R, p.E96K and p.Q249P n=3). Two-way ANOVA with Sidak’s multiple t test. *p<0.05 for each mutant versus WT with or without stimulation (EV p=0.0255, 0.0864; p.R30W p=0.0097, <0.0001; p.T43R p=0.1358, <0.0001; p.E96K p=0.1760, <0.0001; p.T117P p<0.0001, <0.0001; p.G123S p<0.0001, <0.0001; p.Q249P p<0.0001, <0.0001; p.R818Q p=0.7581, p>0.9999). (C) Left: Quantification of relative specific activity of Luciferase assay in transfected Jurkat T cells with empty vector (EV), WT or mutant CARD11 and reporter luciferase constructs, and stimulated as in (A). Data represent fold change in luciferase activity normalized to renilla activity. Data are mean ± s.e.m. (EV, WT, p.R30W andp.G123S n=12; p.T117P andp.R818Q n=6; p.T43R, p.E96K and p.Q249P n=3). Two-way ANOVA with Sidak’s multiple t test. *p<0.05 for each mutant versus WT with or without stimulation (EV p<0.0001, <0.0001; p.R30W <0.0001, <0.0001; p.T43R p=0.0333, <0.0001; p.E96K p=0.0384, <0.0001; p.T117P p<0.0001, <0.0001; p.G123S <0.0001, <0.0001; p.Q249P <0.0001, <0.0001; p.R818Qp=0.0712, p=0.1365). Rigth: Quantification of relative specific activity of Luciferase assay in transfected HEK293T cells with empty vector (EV), WT or mutant CARD11 and reporter luciferase constructs. Data represent fold change in luciferase activity normalized to renilla activity. Data are mean ± s.e.m. Wilcoxon signed rank test *p<0.05, **p<0.01, ***p<0.005. (A–C) Immunoblot for CARD11-Flag expression in transfected cell lysates are at the bottom of each graphic β-Actin serves as a loading control. (D) HEK293T transfected with WT and CARD11 mutants constructs were analysed by confocal microscopy using anti-FLAG and Alexa-Fluor488-conjugated secondary Abs, nuclei were stained with DAPI. ns, not statistically significant.

Figure 3

Lymph node histology from BENTA patient 2B: (A) several secondary follicles with scant interfollicular tissue. HE 10X. (B) Secondary follicles with expanded interfollicular areas were seen (there is some starry sky, arrow). 20X HE. (C) CD20: positive lymphocytes in most of the components of the follicle (germinal center and mantle). (D) CD3: occasional positive lymphocytes in the germinal center of a large secondary follicle (arrow).