This study has been approved by the institutional review board of Wuhan Children’s Hospital, Tongji Medical College, Huazhong University of Science & Technology. A patient presented with persistent upper respiratory illness and fever was recruited in this study. Upon obtaining informed consent, peripheral venous blood was withdrawn from the patient and his parents. Genomic DNA was extracted from leukocytes of whole blood samples using the QIAamp Blood DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was extracted using Trizol reagent (Invitrogen).

WES and subsequent data analysis were conducted with the help of the third party medical testing laboratory (Chigene Lab, Beijing China). Candidate variants were confirmed by Sanger sequencing using self-designed primers in the patient and his parents. A conservative analysis of mutation sites was conducted using MEGA software (https://www.megasoftware.net/).

The wildtype CARD11 plasmid (WT-CARD11) was a gift from Xin Lin (http://n2t.net/addgene:44431). To generate G126D-CARD11 and C49Y-CARD11 (included as a positive control), site-directed mutagenesis was performed with the oligonucleotides 5’- GCC CTA CCT GCG TCA GTA TAA GGT CAT TGA TG -3’ and 5’- GAA GGC CAC GAG GAC CTC ACG CAC TTC -3’ using overlap PCR. All positive clones were verified for the correct sequence by Sanger sequencing. Hela cells were grown in DMEM supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific), and cells cultured in 6-well plate were transfected with 2 μg plasmids (pcDNA3.1, WT, WT+G126D, G126D and C49Y) using Lipofectamine 3000 (Invitrogen), respectively, according to the manufacturer’s instructions.

Cells were lysed in 1% NP-40 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.5% sodium deoxycholate) for 30 min on ice, and then centrifuged at 12000rpm for 15 min at 4°C. Cell nucleoprotein was extracted using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Protein concentration was determined by BCA assay (Thermo Fisher Scientific), and 20 μg of total protein was separated by 12% SDS-PAGE and subsequently transferred to PVDF membrane. After blocking by 3% BSA, membranes were probed with the following antibodies: anti-CARD11(Proteintech, 21741-1-AP), anti-IKKγ (Cell Signaling Technology, #2685), anti-phosphorylation IKKγ (Cell Signaling Technology, #2689), anti-NF-κB p65(Cell Signaling Technology, #8242), anti-phosphorylation mTOR(Ser2448) (Cell Signaling Technology, #2971), anti-phosphorylation JNK(Tyr185) (Proteintech, 80024-1-RR). Bound antibodies were detected using appropriate HRP-conjugated secondary antibodies (Southern Biotech) and enhanced chemiluminescence (ECL, Thermo Fisher Scientific).

Cells adhered to poly-L-lysine–coated slides were fixed for 15 min with 3% paraformaldehyde, and then permeabilized for 15 min in 0.1% Triton X-100/PBS. After blocking for 60 min in 3% BSA/PBS, cells spots were incubated with the anti-CARD11 (Proteintech, 21741-1-AP) antibody for 60min. Cells were washed in PBS for 3 times and then incubated with goat anti–rabbit secondary Ab conjugated to Alexa Fluor 488 (Invitrogen) After washing in PBS, coverlips were attached with Fluoromount (SouthernBiotech). Fluorescent images were acquired on a fluorescence microscope using a 100× oil immersion objective.

DNA was extracted from peripheral blood using QIAamp Blood DNA mini kit (Qiagen, Hilden, Germany). PCR was performed using consensus fluorescent labeled primers to V_H framework region (FR I and II) and joining region(J_H) of the IgH gene. PCR products were separated by capillary electrophoresis on an ABI 3500DX Genetic Analyzer with electropherograms analyzed using GeneMapper software version 5.0 (Applied Biosystems).

Hela cells were plated in 12-well tissue culture plates (3 × 10⁵ cells/well) and were maintained for 24 h. The cells were then co-transfected with 500 ng of pcDNA3.1+, CARD11-expression vector (CARD11-WT, -C49Y- and -G126D) as needed, 300 ng of pNFκB-luc (Bayotime, Shanghai, China) containing NF-κB binding motifs (GGGAATTTCC), and 100 ng of pRL-TK control plasmid (containing Renilla reniformis luciferase gene, Promega, Madison, WI). After 36-48h, the cells were harvested in lysis buffer and were analyzed for luciferase activity using the Dual-Luciferase reporter Assay System (Promega, Madison, WI). Three independent experiments were performed to assess luciferase activity.

This assay was performed in the Beijing Friendship Hospital. Briefly, whole blood samples (5 mL) were collected from the patient and three age-matched healthy controls in EDTA-containing vials, and PBMCs were isolated. K562 cells stably expressing enhanced green fluorescent protein (EGFP) were constructed as target cells (EGFP-K562), and the effector cells were from healthy controls. The cells were incubated for 4 hours according to the effector/target cell ratio of 10:1, with the density of effector cells of 5×10⁶/ml. Meanwhile, EGFP-K562 cells only were included as the background control. After staining with Annexin V-PE and 7-ADD, NK cell activity was analyzed by evaluating the apoptosis ratio of targets cells after co-culture of effector with target cells. Detailed protocol was described previously (15).

CD107a degranulation assay was performed with the same target and effector cells. PBMCs were isolated and incubated overnight at 37°C in medium. Step by step procedures were according to the articles (16), and data were acquired on a FACSCalibur (BD Biosciences). The ratio of NK cells expressing CD107a were compared between co-cultured with K562 cells and medium alone. The ΔCD107a was defined as the difference in the percentage of NK cells expressing CD107a incubated in different conditions.

RNA was isolated from Hela cells transfected with CARD11-G126D and CARD11-WT, respectively, and 1 mg RNA was used for constructing cDNA libraries. The standard Illumina Pipeline for RNA-Seq was used by using paired-end 108-bp runs with each sample run in one sequencing lane, and yielded 20 million reads per sample, and the raw data was submitted to the database (https://submit.ncbi.nlm.nih.gov/subs/sra/SUB9299352/). Gene expression was calculated as transcripts per million (TPM) mapped reads by using the TopHat alignment program with redundant reads removed, and the expression values of reads were normalized using Lo g 2 TPM .

Total RNA was extracted from Hela cells and PBMCs isolated from the patient’s and healthy controls by using Trizol Reagent (Invitrogen, USA), respectively. The first complementary DNA was synthesized from RNA using reverse transcriptase (TAKARA, Dalian). Real-time PCR was performed in 7500 instrument (Applied Biosystems) using SYBR Green PCR Kit (TaKaRa, Dalian) and ACTIN was included as an internal control. The primer sequences of the genes are shown in Table 1 .

Statistical analyses were performed using Graphpad Prism 5 (Graphpad software, USA). Every experiment was repeated three times with duplicates, and data are reported as the mean ± SD. A Student’s t-test was used to compare two groups, and a one-way analysis of variance (ANOVA) was used when comparing three or more groups and statistical significance is indicated by *P<0.05; **P<0.01, ***P<0.001.

Our patient, an 8-month-old Chinese boy, is the only child from healthy nonconsanguineous parents without related personal or familial medical history ( Table 2 ). He was born at 39 weeks of gestation by cesarean delivery without complications. When 6-month old, the patient first presented with a persistent upper respiratory tract infection, fever with splenomegaly, lymphadenopathy, and anemia. The highest temperature was 39.1°C. As shown in Table 2 , clinical laboratory examination showed lymphocytosis with normal morphological lymphocytes and normal total white blood cells, increased glutamic-pyruvic transaminase and aspartate aminotransferase, positive perforin, granzyme, and soluble CD25, and normal CD3-CD56+NK cells of lymphocytes, increased ferritin, lower fibrinogen, and elevated triglycerides. The diagnosis of hemophagocytic lymphohistiocytosis (HLH) was considered according to the clinical characteristics and laboratory examinations.

In addition, he had a hypergammaglobulinemia, decreased serum IgA levels, and normal IgM levels. 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 ).

In order to identify the pathogenic gene and verify the diagnosis of HLH, trios WES was conducted. Bioinformatic analysis was performed to identify candidate pathogenic gene according to filtering strategy based on variant classification, population frequency, and variant functional damaging prediction, including Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), and Combined Annotation Dependent Depletion (CADD). 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. The novel germline mutation was submitted to the database(https://submit.ncbi.nlm.nih.gov/subs/variation_clinvar/SUB9923934/). The diagnosis of BENTA was made, based on the patient’s clinical presentation: polyclonal B cell lymphocytosis, splenomegaly, lymphadenopathy, and recurrent infection.

In order to establish the G126D as a pathogenic variant, in vitro functional study was performed. 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.

As reported previously, CARD11 plays an essential role in NF-κB activation (3). To investigate the effects of G126D mutation, we measured the levels of IKKγ and phosphorylated IKKγ in whole cell protein extracts, as well as NF-κB p65 in the nuclear fraction. We noticed that all of them were significantly increased in mutant CARD11 compared to WT CARD11 ( Figures 2D, E ). In addition, increased soluble CD25 was detected in our patient ( Table 2 ).

NF-κB activation was determined by measuring pNF-κB-luc-reporter gene expression in Hela cells transiently transfected with pcDNA3.1, CARD11-WT, -G126D, or -C49Y plasmids. As shown in Figure 2F , the luciferase activity in cells with mutated CARD11 was significantly higher than that in cells with CARD11-WT.

Taken together, these data indicated that G126D mutation is a GOF mutation and could spontaneously signal and promote NF-κB activation independently of antigen receptor stimulation.

As CARD11 serves as a bridge linking cell surface antigen receptor signaling with the activation of the NF-κB, JNK, and mTORC1 signaling, we also investigated the contribution of CARD11 mutation to the activation of JNK and mTOR pathways by assessing their extent of phosphorylation in Hela cells. As expected, phosphorylation of both JNK and mTOR substantially increased in cells with mutant CARD11compared to that in control cells ( Figure 2D ).

All these results suggest that G126D is a GOF mutation, and could enhance the expression of downstream effectors such as NF-κB, JNK, and mTORC1. To further examine the effect of G126D on downstream genes, RNA-seq was performed. KEGG pathway analysis showed that some genes involved in NF-κB pathway, were up-regulated in cells with G126D mutation, such as CCND3, RELB (encoding an NF-κB Subunit), NFKB2, and TNFAIP3 ( Figures 3A, B ), which is consistent with constitutive activation of NF-κB signaling.

Natural Killer (NK) cells are important effector lymphocytes that are best characterized for their antiviral and anticancer activities. NK cells can directly kill target cells through cytotoxic mechanism. Previous study revealed that constitutive activation of NF-κB driven by mutant CARD11 may be stimulatory in B cells, which contributes to polyclonal B cell lymphocytosis, but partially inhibitory in T cells, which render normal T cells hyporesponsive to antigen receptor stimulation (8). However, it is unclear whether G126D mutation of CARD11 exerts effects on NK cells, particularly their killing activity. To answer this question, we assayed NK cell activity by measuring the proportion of apoptosis in target cells (EGFP-K562) incubated with NK cells isolated from our patient or WT controls, respectively. 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 ). Furthermore, we evaluated NK activity by measuring the expressions of CD107a, a sensitive marker of NK cell activity, by performing degranulation assays using flow cytometry. Our results showed that the degranulation was dramatically decreased in the patient (P<0.001) ( Figures 4C, D ). Collectively, the G126D mutation of CARD11 is associated with decreased NK cell activity.

To identify potential mechanisms through which G126D mutation impaired NK cell activity, we analyzed the expression of genes involved in NK cell activity and degranulation by Gene Ontology (GO) enrichment analysis. We found significant differences in several genes, such as PRDX1 (peroxiredoxins), BAG6, IL18, and ITGB2 ( Figure 5A ). The expression of PRDX1, a gene positively regulating the activation and functions of NK cells, had the most significant decrease. 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.