Novel heterozygous mutation in STX11 in a pediatric patient with Evans syndrome

ERRA LORENZO; BRUNELLO FRANCISCO; MARTÍNEZ MAYER JULIAN; OLEASTRO MATÍAS; BORGE MERCEDES; COLADO ANA; GORIS VERÓNICA; FELLIÚ AURORA; MARTÍ MARCELO; DANIELIAN SILVIA; FERNÁNDEZ JULIETA; VISHNOPOLSKA SEBASTIÁN; VILLA MARIANA; POZNER ROBERTO; ALMEJÚN MARÍA BELÉN

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Conjunta 2021; 2021

Sociedad Argentina de Inmunología

Natural killer (NK) and CD8 + T cells play an important role in the immune response. STX11 encodes a protein thatfunctions as a t-SNARE for the final fusion of lytic granules with the plasma membrane of these cells. Biallelicmutations in STX11 results to a ?particular? Familial Hemophagocytic Lymphohistiocytosis type 4. Heterozygousmutations have been identified in several patients, although the clinical/functional relevance of these mutations in theimmune system remains poorly understood.Our aim was to determine the functional relevance of an heterozygous STX11 variant (R129P) identified by massivesequencing and confirmed by Sanger in a pediatric patient diagnosed with Evans syndrome. Targeted sequencingshowed that the patient´s mother was heterozygous for the mutation.PBMC from healthy donors, the patient, the mother and a patient with Chediak Higashi syndrome (negative control)were used.We analysed the degranulation capacity of CD8+ T-cells and the degranulation and cytotoxicity ability of NK cells,using flow cytometry (FC) assays. We observed a reduction of all these functions in the R129P-STX11 patient andmother in comparison to healthy controls. Nevertheless, these reductions were less defective than observed for theChediack cells. The in vitro treatment with IL-2 restored these functions.Then, we measured the RNA (qRT-PCR) and protein (WB) expression. Interestingly, the RNA levels of the patient andmother were similar to healthy donors but the protein expression was reduced.The membrane-associated TLR4 recirculation was also evaluated, by stimulating monocytes with LPS and measuringTLR4 on membrane by FC. We found that TLR4 relocalization was impaired in the patient monocytes.Finally, we performed in-silico structural analysis of R129P substitution using available STX11:Munc18-2 complexstructure. Visual inspection shows that R129 is part of a helix in the NHabc domain of the STX11 protein displaying arich hydrogen bond network with Munc18-2. In this context, replacing it with a proline is expected not only tosignificantly impact helix stability, but also the protein-protein interaction.Altogether, we demonstrated that the novel R129P-STX11 mutation can play a pathogenic role by impairingdegranulatory activity of NK and CD8+ T cells and cytotoxic activity of NK cells. The aberrant functionality of NK cellshave been reported in several autoimmune disorders. This novel mutation may explain the clinical patient EvansSyndrome phenotype.