Fig 2: 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.

Fig 3: G126D leads to upregulation of some downstream targets of NF-?B signal pathway. (A) Heat map of differentially expressed genes involved in NF-?B signal pathway between CARD11-WT and CARD11-G126D by RNA-seq analysis in Hela cells. The color scales of heatmap refer to Log2TPM. (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).

Fig 4: 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 Log2TPM. (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).

Fig 5: 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, IKK?, p-IKK? anti-NF-?B 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-?B-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).