Germline CARD11 Mutation in a Patient with Severe Congenital B Cell Lymphocytosis

Abstract

Purpose: Activating germline mutations in CARD11 have recently been linked to a rare genetic disorder associated with congenital B cell lymphocytosis. We describe a patient with a similar clinical phenotype who had a de novo germline G123D CARD11 mutation.

Methods: Whole exome sequencing was performed on DNA from the patient and his biological parents. Laboratory studies examined characteristics of the patient's B and T lymphocytes. A CARD11 cDNA containing the mutation was transfected into a lymphocyte cell line to gain an understanding of its function. RNA sequencing was performed on samples from the patient and from patients with alternate germline CARD11 mutations and differential gene expression analysis was performed.

Results: The patient had a decade-long history of severe polyclonal B lymphocytosis in the 20,000-90,000 lymphocytes/mm(3) range, which was markedly exacerbated by EBV infection and splenectomy at different times. He had a heterozygous germline CARD11 mutation causing a G123D amino acid substitution, which was demonstrated to induce NF-κB activation in unstimulated lymphocytes. In contrast to previous patients with CARD11 mutations, this patient's B cells exhibited higher expression of several cell cycle progression genes, as well as enhanced proliferation and improved survival following B cell receptor stimulation.

Conclusions: This is the third reported germline and first de novo CARD11 mutation shown to cause congenital B cell lymphocytosis. The mutation was associated with a dramatically greater lymphocytosis than in previously described cases, disproportionate to the level of constitutive NF-κB activation. However, comparative review of the patient's clinical history, combined with additional genomic and functional analyses, underscore other important variables that may affect pathophysiology or regulate mutant CARD11 function in B cell proliferation and disease. We now refer to these patients as having BENTA disease (B cell Expansion with NF-κB and T cell Anergy).

Keywords: B-cell lymphocytosis; BENTA; CARD11; NF-κB; congenital.

Figure 1. Clinical history and pathology

(A) High power (500X) of peripheral blood smear showing circulating lymphocytes. (B) Total circulating lymphocytes (ALC) and B cell and T cell subsets from 3 months of age to present. Arrows mark key events (a = EBV infection; b=splenectomy; c= MTX started). (C) Micrographs of H&E stained sections from lymph node (top, 100X) and laparascopic splenectomy tissue (bottom, 40X) from a normal adult and the patient. (D) Low power (40X) CD4 (top) and CD8 (bottom)-stained lymph node biopsy taken during acute EBV infection. (E) CD4/CD8 ratio of peripheral T cells over time, with an arrow denoting the timing of an acute EBV infection. Shaded area represents normal range (1:1 – 4:1).

Figure 2. B and T cell activation in a suspected BENTA patient

(A) Proliferation of naïve, CD10⁻ B cells purified from the patient and a normal control donor was measured by ³H-thymidine incorporation in response to various B cell activating stimuli for 4 days. The polyclonal mitogen SAC + IL-2 served as a positive control. Patient responses were significantly higher than control for all stimuli (p < 0.02) except anti-IgM + anti-CD40. (B) Secreted IgM and IgG levels were measured by ELISA in cell supernatants following 6 days of activation with various B cell activating stimuli. (C) Plasmablast generation from control and patient naïve B cells was quantified by flow cytometry following 6 days in culture left unstimulated (top row) or activated with anti-IgM, anti-CD40, and IL-21 (bottom row). Gates denote the % of cells that differentiated into IgD⁻CD38^hi plasmablasts. (D) Proliferation of purified T cells was measured as in (A) following 4 day stimulation with soluble anti-CD3ε + anti-CD28 mAbs (gray) or bead-coupled anti-CD2/CD3/CD28 Abs (black) (left panel). IL-2 secretion was measured by ELISA in cell supernatants following 3 days of stimulation as described above (right panel). For (A–D), data show mean ± standard deviation of triplicate wells.

Figure 3. De novo, heterozygous missense CARD11 mutation (G123D) detected in new BENTA patient

(A) Summary of exome sequencing data at chr7:2984162. A C→T substitution in exon 5 of CARD11 was detected in reads obtained from DNA libraries derived from patient blood and saliva, with no mutation evident in parents’ samples. (B) Sanger dideoxy-DNA sequencing confirmation of the G123D missense mutation in genomic DNA derived from patient samples (blood, saliva, fibroblasts), but not mother and father. The reference sequence is shown in reverse complement orientation (G->A substitution), reflecting the direction of protein translation. (C) Schematic diagram of the CARD11 protein. The G123D mutation in the putative LATCH domain is denoted, relative to other BENTA-associated mutations.

Figure 4. Spontaneous aggregation and constitutive signaling of G123D CARD11 in B and T cell lines

(A) Ectopic expression of WT, G123D, and other BENTA-associated CARD11 mutants (E134G, G123S) as N-terminal Venus fusion proteins in transfected Jurkat T cells. Single, punctate spots of Venus-CARD11 mutants indicate aggregation, relative to cytoplasmic distribution of WT. Empty vector (Venus only) is shown for comparison; data are representative of at least 3 experiments. (B) Venus-CARD11 proteins were transfected and visualized as in (A) in BJAB B cells. Data are representative of at least 3 experiments. (C) Jurkat T cells transfected with WT or G123D CARD11-FLAG (blue) were analyzed by confocal microscopy using AlexaFluor647-anti-FLAG plus anti-MALT1 (green) and anti-phospho-IKKα/β Abs (red). MALT1 and phospho-IKK were detected using AlexaFluor488 and 594-conjugated secondary Abs, respectively. Similar results were obtained for transfected BJAB B cells. Data are representative of two separate experiments. (D) CARD11-deficient Jurkat cells (JPM50.6) harboring an NF-κB responsive GFP reporter cassette were transfected with empty vector (EV), WT or various mutant CARD11-FLAG constructs. At 48 hours post transfection, the percentage of GFP⁺ cells (indicating active NF-κB) was analyzed by flow cytometry. CARD11-FLAG expression was confirmed by immunoblotting of cell lysates 48 hours post-transfection; actin serves as a loading control. Data are representative of 3 separate experiments. (E) CD83 expression was measured on BJAB B cells 24 hours post-transfection with CARD11-FLAG constructs as in (D). Mean fluorescence intensity (MFI) of CD83 is listed for each construct, and CARD11-FLAG expression in cell lysates was confirmed by immunoblotting. Data are representative of 3 separate experiments. (F) CD83 expression on unstimulated, naïve B cells isolated from the patient and two independent controls. Data are representative of two independent blood draws.

Figure 5. Evidence of marginally elevated CARD11-induced NF-κB signaling in patient lymphocytes

(A) Confocal microscopy analysis of CARD11 expression (green) in purified B and T cells from the patient (Pt) and a normal donor (Ctrl). The percentage of cells with visible CARD11 aggregates (white arrowheads) was quantified by scoring ~200 cells from multiple fields. Scoring data represent mean ± standard deviation from two blinded scorers; p<0.03 (B cells), p<0.01 (T cells). Scalebars = 5 μm. (B) Confocal microscopy imaging of phospho-IKK, p65 (RelA) and RelB in control and patient lymphocytes. Scalebars = 5 μm. (C) Immunoblotting of total lysates from purified T cells and naïve B cells prepared from the patient (Pt) and a normal donor (Ctrl). Detected proteins are listed at right; β-actin serves as a loading control. Data are representative of two separate blood draws.

Figure 6. Comparative analysis of enhanced cell cycle progression and survival of patient B cells upon stimulation

(A) Heat map of 19 differentially expressed cell cycle-related genes in naïve B cells from the patient (P6) relative to other BENTA patients and B cell controls. Color intensity represents fragments per kilobase per million (FPKM) values derived from paired-end RNA-Seq analysis. (B) Quantitative PCR validation of the expression of selected genes in controls versus BENTA patients, derived from the cell cycle signature outlined in (A). (C) KREC analysis of in vivo replication history (displayed as # of cell divisions) for sorted immature and mature naïve B cells from P6, P3, and a normal control. (D) Cell cycle profiling of naïve B cells from control, P3, and P6. Markers represent the percentage of cells in G1 (M1), S (M2), and G2/M (M3) phases of the cell cycle. Data in (C) and (D) are representative of two experiments. (E) CFSE dilution analysis of naïve B cells left unstimulated (gray filled histogram) or activated with anti-IgM F(ab)₂ plus anti-CD40 (blue) or dextran-conjugated anti-IgM plus anti-CD40 (green) for 5 days. The percentage of divided cells and the proliferative index (total # of divisions/total # of dividing cells) are displayed at right for activated samples. Data are representative of three separate experiments. (F) Cell survival assays for naïve B cells purified from a normal control (black), P3 (gray), or P6 (blue) −/+ stimulation with anti-IgM F(ab)₂ plus anti-CD40. Cell viability was measured over 5 days in culture. Data are representative of two separate experiments. (G) Quantitation of apoptotic cells following BCR stimulation. The percentage of Annexin V⁺ apoptotic cells (inset numbers) within the actively dividing population (based on CFSE dilution) from (F) was determined on day 5 by flow cytometry.