Cell base assays to investigate the functional relevance of HLH genetic variants.

Student thesis: Doctoral thesis


Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disorder caused by an uncontrolled and dysfunctional immune response. Biallelic mutations in involved genes leads to the familial form of HLH (FHL) which is potentially lethal in the first weeks of life. HLH can also develop in the context of infections, malignancies, autoinflammatory or metabolic diseases but without mutations. This is the so called secondary HLH (sHLH). Nevertheless, there is an increasing evidence that typical FHL cases with biallelic mutations and sHLH without genetic and immunological defects represent only the extremes of the wide spectrum of HLH. To deepen the understanding in the genetics of HLH we analyzed the molecular and functional impact of previously unidentified biallelic and monoallelic mutations in patients diagnosed with HLH. In this study we diagnosed 12 patients with HLH, of which 75% (n=9) presented biallelic mutations and 25% (n=3) monoallelic mutations. The diagnosis of these patients allowed the novel description of 4 mutations not previously identified. Among those, we describe one mutation in perforin (p. Pro477X in homozygosity), two mutations in STXBP2 (L243R as compound heterozygous with other mutation and R190C as monoallelic) and three in UNC13D (P271S, R928C and R1075Q as monoallelic). Moreover, we also included into the characterization eleven mutations in STXBP2 reported in the literature as monoallelic but not yet characterized. Here, we describe that the novel biallelic mutation L243R is found in an evolutionarily highly preserved site acting as the center of a rich network of interactions important for stabilizing domains 2 and 3 of the STXBP2 protein. Moreover, on the study of monoallelic mutation we assessed protein structural destabilization and expression levels by transfecting different constructs containing monoallelic mutations on STXBP2 and UNC13D. STXBP2 mutations showed an hypomorphic effect while UNC13D protein expression was not affected by any of the studied mutations. As so, we evaluated the effect of these mutations into protein-to-protein interactions and found that interaction capacity was maintained within the studied partners. For mutations with no effects on the expression levels and protein-to-protein interaction, we addressed their effect as dominant negative by a degranulation assay. We show that mutant STXBP2R190C acts as a dominant negative, producing a decrease in degranulation compared to the controls. Finally, we studied whether the described mutations that involve genes in the cytotoxic pathway had functional impact on macrophages and B lymphocytes. For that, we analyzed the role of STXBP2 in the cellular traffic process. Our data demonstrate that in the absence of STXBP2 there is an accumulation of CD103 on the surface of LPS + IFNg activated cells compared to controls, demonstrating an active disruption of endosomal trafficking. Moreover, we also show reduction of CD107a dependent degranulation when using KD cells. Thus, mutations in STXBP2, which is involved in the cytotoxic pathway, appear to have a functional impact on macrophages and B lymphocytes at endosomal trafficking level. Overall, this work describes several previously unidentified mutations related to FHL and sHLH and unveils their effects at a protein level. Moreover, these results represent a primary approach in the understanding of STXBP2 role in macrophages and B lymphocytes.
Date of Award29 Nov 2019
Original languageEnglish
SupervisorMonica Martinez Gallo (Director), Joan Sayós Ortega (Director) & Ricardo Pujol Borrell (Tutor)

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