Functional studies in novel mutation emerged by NGS in patients with Inborn Errors of Immunity in Argentina

ALMEJÚN MARÍA BELÉN

Mar del Plata

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

Inborn Errors of Immunity (IEI) are a heterogeneous immune disease with alteration in different components of the immune system. Next Generation Sequencing (NGS) has revolutionized the diagnosis of genetic diseases making it possible to improve diagnostic possibilities in patients with IEI, especially in those cases where the clinical presentation is complex, and the candidate gene strategy has not been successful. We have evaluated different novel mutations emerged by NGS in IEI patients in Argentina through functional studies. We have evaluated a novel heterozygous variant R129P in STX11 in a pediatric patient diagnosed with Evans syndrome. We observed a reduction of NK-cells and T-cells functionality in the R129P-cells and a lesser protein expression. TLR4 re-localization was impaired in the patient’s monocytes. Structural analysis showed an impact in helix stability and in the protein-protein interaction.We demonstrated that the novel R129P-STX11-mutatio can play a pathogenic role. This novel mutation may explain the clinical patient Evans Syndrome phenotype. We assessed novel variants in CARD11 by functional analysis using JPM50.6, Jurkat and HEK293T cells transfected with a wild type CARD11 construct and/or with mutated constructs. Genetic defects in CARD11 can present as a loss of function (LOF) or contrarily, a gain of function (GOF) of the affected gene product. A total of 6 novel heterozygous CARD11 variants were identified. Two variants resulted in LOF/Dominant Negative by ex vivo evaluation, defining them as causative of CARD11-associated atopy with dominant interference of NF-kB signaling (CADINS). Two variants showed a GOF effect as well a spontaneous aggregation in the cytoplasm, leading to B cell expansion with NF-κB and T cell anergy (BENTA) diagnosis. The remaining variants showed neutral functional assays. Our results, underlines the importance of studying novel mutations, not only for the early and accurate diagnosis of IEI patients, but also to understand a protein function.