Identification and Characterization of a Germline Mutation in CARD11 From a Chinese Case of B Cell Expansion With NF-κB and T Cell Anergy

Abstract

B cell expansion with NF-κB and T cell anergy (BENTA) is a rare primary immunodeficiency disorder caused by gain-of-function (GOF) mutations in the CARD11 gene. Affected patients present with persistent B cell lymphocytosis in early childhood paired with lymphadenopathy and splenomegaly. Until now only six activating mutations from 14 patients have been reported in CARD11. Here we report a patient from China with polyclonal B cell lymphocytosis and frequent infections in early life. A heterozygous mutation (c.377G>A, G126D) in exon 5 of CARD11 gene (NM_032415) was identified by whole exome sequencing. In vitro functional studies showed that the G126D mutation is associated with increased expression of CARD11 and NF-κB activation in Hela cells. Flow cytometry analysis indicated NK cell activity and CD107a degranulation of the patient were decreased. RNA sequencing analysis showed that a number of genes in NF-κB pathway increased while those involved in NK cell activity and degranulation were down-regulated. In summary, our work identified a de novo germline GOF mutation in CARD11 with functional evidence of BENTA.

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

Figure 1

Characterization of our patient with mutated CARD11 gene and conserved features of CARD11 protein. (A) Bone marrow smear from the patient, showed increased lymphocytes and decreased granulocytes; (B) Result of IgH rearrangement of our patient, it shows polyclonal rearrangement in FR1-JH region (above) and FR2-JH region (below); (C) Sanger sequencing of CARD11 mutation in the family; (D) Conservation analysis of CARD11 protein among different species. The position of the mutations at residue 126 is indicated by a gray bar and highly conserved throughout all indicated species; (E) Scheme of the distribution of GOF CARD11 mutations, and the mutation in red was reported in our patient.

Figure 2

Functional study of G126D mutation in Hela cells. The expression levels of wild-type and mutant CARD11 gene in Hela cells were detected by Real-time PCR (A) and western blot (B). (C) Distribution of CARD11-WT, -C49Y, and -G126D, respectively, investigated by immunofluorescence. (D) The expression levels of CARD11, IKKγ, p-IKKγ anti-NF-κB p65, p-MTOR, p-JNK in Hela cells transfected with wildtype or mutant CARD11, respectively, in whole cell lysates. GAPDH serves as a loading control; (E) Phosphorylated p65 increased in nuclear lysate of cells with mutant CARD11, and lamin B serves as a loading control. (F) 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 empty pcDNA3.1 plasmid. The results from three independent experiments are expressed as the mean + standard deviation (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3

G126D leads to upregulation of some downstream targets of NF-κB signal pathway. (A) Heat map of differentially expressed genes involved in NF-κB signal pathway between CARD11-WT and CARD11-G126D by RNA-seq analysis in Hela cells. The color scales of heatmap refer to $\text{Lo}{\text{g}}_2^{\text{TPM}}$. (B) Realtime PCR was performed to measure the expression level of RELB, BCL2, NFKB2, TNFAIP3 and CCND3 gene in Hela cells. WT, wild type; M126, G126D mutation; TPM, Transcripts per million in RNA seq. The results of three independent experiments are expressed as the mean + standard deviation (*P < 0.05; **P < 0.01).

Figure 4

Decreased NK cell activity and degranulation in our patient. (A) NK cell activity was measured as the proportion of apoptosis in target cells (EGFP-K562) incubated with NK cells isolated from our patient or normal control, and bivariate distribution was set, with Annexin V-PE and 7-AAD on the horizontal and vertical axes. (B) The proportion of apoptosis in EGFP-K562 cells was decreased when incubated with NK cells of this patient compared with that of control. The results showed that the activity of NK cells decreased significantly. (C) The NK cells expressing CD107a were compared between these co-cultured with K562 cells and those incubated with medium alone. The ΔCD107a was defined as the difference in the percentage of NK cells expressing CD107a incubated under different conditions. (D) ΔCD107a was decreased after stimulation and abnormal degranulation was observed in this patient compared with control, The results suggested degranulation function of NK cells decreased significantly. (***P < 0.001). HD, Healthy donor.

Figure 5

Impact of CARD11 G126D on Natural Killer cell activity. (A) Heat map of differentially expressed genes involved in NK cell activation between CARD11-WT and CARD11-G126D, as measured by RNA-seq analysis in Hela cells. The color scales of heatmap refer to $\text{Lo}{\text{g}}_2^{\text{TPM}}$. (B) Expression level of PRDX1 gene in peripheral blood by Realtime PCR. (C) The mRNA level of PRDX1 in Hela cells by Realtime PCR and protein level by western blot. P, patient; N, normal controls; WT, wild type; M126, G126D mutation; TPM, Transcripts per million in RNA seq; H, Healthy donor. The results of three independent experiments are expressed as the mean + standard deviation (*P < 0.05; **P < 0.01).