PMC8453161_01
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.
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Bone marrow smear showed increased lymphocytes (40.5%) and decreased granulocytes (15.5%) (Figure 1A). Lymphocyte proliferation in response to phytohemagglutinin (PHA) was poor, and IgH rearrangement indicated that patient B cells are polyclonal (Figure 1B). Surprisingly, we did not find any pathogenic variant in HLH-associated genes after filtration, but instead one germline heterozygous missense mutation in exon 5 of CARD11 (NM_032415), c.377G>A (p.G126D) was identified. Sanger sequencing showed that this mutation was de novo in the patient and his parents did not carry this mutation (Figure 1C). The mutated site is conserved among different species (Figure 1D), located within the LATCH domain (Residues 112-130) of CARD11 protein (Figure 1E) in human, and has not previously been reported.
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, IKKgamma, p-IKKgamma anti-NF-kappaB 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-kappaB-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).
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WT-CARD11, G126D-CARD11, C49Y-CARD11 (positive control) pcDNA3.1 empty plasmids alone, were transfected into Hela cells, respectively (Figures 2A, B). Protein aggregates were observed in cells ectopically expressing C49Y- or G126D-CARD11 proteins, whereas the proteins dispersed throughout cytoplasm in the cells transfected with WT-CARD11 (Figure 2C), suggesting that both C49Y and G126D mutations result in aggregation of CARD11 proteins. We noticed that all of them were significantly increased in mutant CARD11 compared to WT CARD11 (Figures 2D, E). As shown in Figure 2F, the luciferase activity in cells with mutated CARD11 was significantly higher than that in cells with CARD11-WT. As expected, phosphorylation of both JNK and mTOR substantially increased in cells with mutant CARD11compared to that in control cells (Figure 2D).
G126D leads to upregulation of some downstream targets of NF-kappaB signal pathway. (A) Heat map of differentially expressed genes involved in NF-kappaB signal pathway between CARD11-WT and CARD11-G126D by RNA-seq analysis in Hela cells. The color scales of heatmap refer to . (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).
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KEGG pathway analysis showed that some genes involved in NF-kappaB pathway, were up-regulated in cells with G126D mutation, such as CCND3, RELB (encoding an NF-kappaB Subunit), NFKB2, and TNFAIP3 (Figures 3A, B), which is consistent with constitutive activation of NF-kappaB signaling.
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 DeltaCD107a was defined as the difference in the percentage of NK cells expressing CD107a incubated under different conditions. (D) DeltaCD107a 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.
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Our results showed that NK cell activity in the blood sample from the patient was obviously lower than those from three controls (P<0.001) (Figures 4A, B). Our results showed that the degranulation was dramatically decreased in the patient (P<0.001) (Figures 4C, D).
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 . (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).
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We found significant differences in several genes, such as PRDX1 (peroxiredoxins), BAG6, IL18, and ITGB2 (Figure 5A). Further, real-time PCR confirmed the decreased expression of PRDX1 gene in peripheral blood of this patient (Figure 5B) and in mutant Hela cells (Figure 5C) compared to their respective controls.