Functional evaluation of novel heterozygous CARD11 mutations associated with diverse immunologic phenotypes in patients with Primary Immunodeficiencies (PID)

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

Congreso; Latinoamerican Society for Immunodeficiency; 2021

CARD11 encodes a lymphocyte-specific scaffold protein necessary inter alia forproper NF-κB activation in B- and T- cells. Germline CARD11 mutations havebeen 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 inpatients with PID is needed to characterize their functional impact.Our aim is to determine the functional relevance of heterozygous CARD11variants identified in pediatric PID patients treated in Hospital Garrahan. In thiswork we further analyse three novel variants in CARD11 p.T117P, p.E96K andp.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 differentmutant 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.E96Kresulted in LOF/DN activity, while p.R818Q behaved similar to CARD11 wildtype (WT) in a heterozygous transfection assay upon antigen receptor-inducedactivation of NF-κB. The results of immunofluorescence assays showed thatboth the GOF mutation p.G123S and the novel variant p.T117P causedmultimeric aggregation of CARD11 in cytoplasmic complexes in absence ofstimuli which were previously described as indicative of active signaling. Thisunique aggregation from GOF CARD11 mutants may collaborate with futureclinical management.The identification of novel CARD11 mutations in PID patients along with thebroad range of clinical manifestations point out the necessity for availablefunctional studies validating their potential pathogenic outcome and contributingto further understanding the mechanism that underlies the development of thedisease.