A Novel Gain-Of-Function Mutation In The CARD Domain Of CARD11 (C49Y) Results In Benta Disease

Background A growing family of disorders of immune system function is linked to genetic aberrations that impact nuclear factor (NF) kB activation. Caspase recruitment domain family member 11 (CARD11) is a scaffold protein that couples lymphocyte antigen receptor signaling to NF-kB activation. A recent report documented causative, activating germ-line CARD11 mutations associated with a novel congenital disorder known as B cell expansion with NF-kB and T cell anergy (BENTA) disease. Both mutations (E127G and G116S) resided within the coiled-coil domain of CARD11, a known hotspot for gain-of-function somatic mutations found in diffuse large B cell lymphomas. Here we describe a 16 year-old female with chronic sinusitis and splenomegaly who was noted to have polyclonal B cell lymphocytosis suggestive of BENTA disease. A novel gain-of-function mutation in CARD11 (C49Y) was documented involving the N-terminal CARD domain. Methods After IRB consent was obtained, blood was obtained and used to isolate RNA. RNA sequencing was completed to identify potential candidate mutations. The presence of a mutation was confirmed by traditional Sanger sequencing. CARD11 expression plasmids containing the C49Y plasmids were constructed for biochemical analysis in transfected lymphocyte cell lines. Results A 13 month-old Caucasian female was referred for evaluation of splenomegaly (10 cm) with splenic hilar adenopathy, leukocytosis (51,000/uL), neutropenia (3%) and lymphocytosis (91%). A bone marrow examination demonstrated a normocellular (100%) marrow with trilineage hematopoiesis. Cytogenetic evaluation demonstrated a normal female karyotype with no numerical or structural aberrations. Flow cytometric analysis of peripheral blood demonstrated a predominance of B cells 88% (CD19+, CD20+, CD21+, CD22+, CD23+ partial, CD24+, HLA-DR +). The kappa to lambda ratio was 1.1 and CD34 and TdT were negative. Infectious etiologies and other immune dysregulation states including autoimmune lymphoproliferative syndrome were excluded. Immunologic evaluations including quantitative immunoglobulin levels were normal with few exceptions (IgA level 27 mg/dL). Levels of antibodies specific to Tetanus Toxoid, Haemophilus influenzae Type b were normal; however, levels of antibodies to Pneumococcal Capsular Polysaccharide were low. Mitogen studies and antigen specific responses were normal with few exceptions (low ConA and Candida). She remains healthy at 16 years of age with persistent splenomegaly (22 cm) associated thrombocytopenia and persistent B cell lymphocytosis. Secondary to phenotypic similarity to BENTA disease, further analysis of CARD11 was completed. A gain-of-function mutation in CARD11 was documented: a single heterozygous missense mutation (C49Y) involving the N-terminal CARD. Similar to other BENTA-associated CARD11 mutants, ectopic expression of C49Y CARD11 in the BJAB B cell line resulted in increased expression of the NF-kB-dependent gene CD83. Comparable upregulation of NF-kB dependent green fluorescent protein (GFP) expression was noted when C49Y CARD11 was expressed in JPM50.6 cells, a CARD11-deficient reporter T cell line. Confocal microscopy analysis of these cells revealed spontaneous aggregation of CARD11 and co-localization with markers of an active CARD11 signalosome, including MALT1 and phosphorylated IkB kinase (IKK). Conclusion Germ-line activating CARD11 mutations are associated with BENTA disease. To date, only mutations within the coiled-coil domain have been described. Now we have documented the first germline gain-of-function mutation in CARD11 outside of the coiled-coil domain (C49Y) in a patient with a clinical phenotype consistent with BENTA disease. Indeed, cell transfection analysis confirmed C49Y can promote constitutive NF-kB activation in both B and T cells without requiring antigen receptor stimulation. Understanding the entire spectrum of CARD11 mutants and their associated clinical phenotypes will improve our understanding of BENTA and other NF-kB-related diseases, and hopefully illuminate potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.