A novel CARD11 germline mutation in a Chinese patient of B cell expansion with NF-κB and T cell anergy (BENTA) and literature review

Abstract

Germline gain-of-function (GOF) mutations in the CARD11 gene lead to a rare primary immunodeficiency disease known as B cell expansion with NF-κB and T cell anergy (BENTA). Affected patients present with a polyclonal expansion of B cells, lymphadenopathy, and splenomegaly. Herein, we report a novel germline in-frame three base-pair deletion (c.1030_1032del, p.K344del) in the CARD11 gene in a patient with atypical BENTA, presenting with a recurrent fever and B cell lymphocytosis. This mutation was inherited from his mother, who is clinically asymptomatic and had a recurrent respiratory tract infection in her childhood. In vitro functional analysis demonstrated that this variant decreased the expression level of the CARD11 protein and activated the NF-κB signal pathway, leading to a higher expression of several NF-κB target gene transcripts in HCT116 cells transfected with mutant CARD11 (K344del-CARD11) as revealed by RNA sequencing analysis. To our knowledge, only 23 BENTA patients have been identified and carried seven distinct GOF mutations in CARD11. The clinical manifestations of patients are highly heterogeneous and there was no significant correlation between genotype and phenotype. In summary, we identified a novel in-frame three base-pair deletion that may be responsible for the pathogenesis of atypical BENTA in a Chinese family. Our study expands the mutational spectrum of the CARD11 gene and may be helpful in the understanding of diseases caused by CARD11 mutations and the clinical management of BENTA.

Keywords: BENTA; CARD11; NF-κB; gain-of-function; lymphocytosis.

Figure 1

Characterization of our patient with CARD11 gene mutation and conserved features of the CARD11 protein. (A) Bone marrow smear from the patient showing microcytic hypochromic anemia; (B) Result of IgH rearrangement of our patient; (C) Pedigrees of this family and sanger sequencing of CARD11 mutation in this family; (D) Aligned amino acid sequence at this mutation among different species. The position at residue 344 is noted by a gray bar and highly conserved throughout all indicated species; (E) Scheme of the distribution of the CARD11 gain-of-function mutations, and the mutation noted in red was reported in this study. Gain of function mutations are highlighted in red, dominant negative mutations are in blue, and loss of function mutations are in black.

Figure 2

Expressions and Distribution of CARD11-WT and K344del in HCT116 cells. Expressions of RNA and protein of mutant CARD11 and its controls (A, B, respectively); (C) Distribution of CARD11-WT, -C49Y, and –K344del in HCT116 cells investigated by immunofluorescence.

Figure 3

Functional analysis of K344del mutation in HCT116 cells. (A) The expression levels of CARD11, NF-κB p65 in HCT116 cells transfected with wildtype or mutant CARD11 in whole cell lysates and nuclear lysates, GAPDH and Lamin B serve as a loading control; (B) The activity of NF-κB-dependent luciferase of cell extracts from each sample was measured and recorded as a fold increase compared to control cells with WT-CARD11 plasmid. (C) The expression levels of CARD11 in different ratios of plasmid transfection. (D) The luciferase activity of cells transfected with mutants was increased in the case of consistent expression levels. The results from three independent experiments are described as the mean + standard deviation (*P < 0.05; **P < 0.01, ***P < 0.001). (E) Heat map of differentially expressed genes involved in NF-κB signal pathway between WT and K344del by RNA-seq analysis in HCT116 cells, the color scales of heatmap refer to Log₂TPM.