FUNCTIONAL EVALUATION OF NOVEL HETEROZYGOUS CARD11 MUTATIONS ASSOCIATED WITH DIVERSE IMMUNOLOGIC PHENOTYPES IN PATIENTS WITH PRIMARY IMMUNODEFICIENCIES (PID)

MARÍA FLORENCIA MERCOGLIANO; MARÍA MARTHA KATSICAS; SILVIA DANIELIAN; ERRA LORENZO; VERÓNICA GORIS; MATÍAS OLEASTRO; EMMA PRIETO; ALEJANDRO PALMA; MARÍA BELÉN ALMEJÚN

Congreso; LXVIII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI); 2020

SAI,SAIC,SAFIS

CARD11 encodes a lymphocyte-specific scaffold protein necessary inter alia for proper NF-κB activation in B- and T- cells. Germline CARD11 mutations have been described in PID patients with diverse clinical phenotypes including SCID (biallelic null mutations), BENTA (heterozygous, gain-of-function mutations, GOF) and severe atopic disease (heterozygous, loss-of-function, dominant-interfering mutations LOF/DN). Evaluation of novel and rare CARD11 variants in patients with PID is needed to characterize their functional impact.Our aim is to determine the functional relevance of heterozygous CARD11 variants identified in pediatric PID patients treated in Hospital Garrahan. In this work we further analyse three novel variants in CARD11 p.T117P, p.E96K and p.R818Q, and two previously reported pathogenic mutations p.R30W (LOF/DN) and p.G123S (GOF) carried by five unrelated Argentinean patients. To evaluate signalling and function of CARD11, transfection of the different mutant CARD11 constructs into HEK293T, Jurkat and JPM50.6 was performed. Our results showed that p.T117P drive to a GOF activity of the protein, p.E96K resulted in LOF/DN activity, while p.R818Q behaved similar to CARD11 wild type (WT) in a heterozygous transfection assay upon antigen receptor-induced activation of NF-κB. The results of immunofluorescence assays showed that both the GOF mutation p.G123S and the novel variant p.T117P caused multimeric aggregation of CARD11 in cytoplasmic complexes in absence of stimuli which were previously described as indicative of active signaling. This unique aggregation from GOF CARD11 mutants may collaborate with future clinical management.The identification of novel CARD11 mutations in PID patients along with the broad range of clinical manifestations point out the necessity for available functional studies validating their potential pathogenic outcome and contributing to further understanding the mechanism that underlies the development of the disease.