Gain and loss of function novel variants in CARD11 in patients with Inborn Errors of Immunity

FERNÁNDEZ, JULIETA BELÉN; URDINEZ LUCIANO; ERRA, LORENZO; PALMA ALEJANDRO; MERCOGLIANO FLORENCIA; PRIETO EMMA; GORIS VERÓNICA; VILLA MARIANA; BOUZO CAROLINA; CAPUTI LUCÍA; QUESADA BELÉN; SOLÍS DANIEL; AGUIRRE BRUZZO ANABEL; OLEASTRO MATÍAS; DANIELIAN SILVIA; ALMEJÚN, MARÍA BELÉN

Mar del Plata

Congreso; Reunión Conjunta SAIC. SAI. SAFIS FAIC 2022; 2022

Sociedad Argentina de Inmunología

CARD11 encodes a lymphocyte-specific scaffold protein necessary inter alia for proper NF-κB activation in B- and T- cells. Germline pathogenic variants in CARD11 have been linked to Inborn Errors of Immunity (IEI) with diverse clinical phenotypes: BENTA (heterozygous GOF), CADNIS (heterozygous LOF/DN) and SCID (biallelic null).Functional impact of novel CARD11 variants identified by NGS cannot be predicted with absolute certainty. Therefore, functional assessment must be used for a proper interpretation. Our aim is to evaluate two novel mutations in CARD11 identify in IEI patients NGS in the Hospital Garrahan: p.T43R and p.Q249P. Both CARD11 variants were absent from gnomAD and ExAC, presented a high degree of amino acid conservation by Clustal Omega and predicted to be damaging by PolyPhen2.We assessed the ability for the novel variants to alter TCR signaling by transfection with WT and mutant CARD11 expression constructs into JPM50.6 and Jurkat cell lines. We use the previously reported LOF (p.R30W) and GOF (p.G123S) mutations as controls. Compared to WT, the novel variant p.T43R barely induced NF-κB activation upon anti-CD3/CD28 stimulation and disrupted WT CARD11 ability to activate NF-κB when is co-transfected in JPM50.6, in similar levels as with the previously described p.R30W. On the other hand, p.Q249P induced a constitutive GFP expression in the absence stimulation like to the p.G123S GOF mutation. The immunofluorescence assays performed in transfected HEK293T revealed that p.Q249P variant and the GOF, p.G123S, exhibited cytoplasmic aggregates in absence of stimuli which were previously described as indicative of active signaling.Overall, novel variant p.Q249P was considered GOF while the new variant p.T43R behaved as LOF/DN. The identification of novel CARD11 mutations in IEI patients together with the functional studies contribute to better understanding the mechanisms underlying CARD11 pathways in the development of primary immunodeficiencies.