A platform for complementation and characterization of familial haemophagocytic lymphohistiocytosis 3 mutations.
SourceJournal of Immunological Methods, 365, 1-2, (2011), pp. 58-66
Article / Letter to editor
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Journal of Immunological Methods
SubjectN4i 1: Pathogenesis and modulation of inflammation NCMLS 1: Infection and autoimmunity
Mutations in UNC13D cause the severe immune disorder familial haemophagocytic lymphohistiocytosis type 3 (FHL3). The gene product munc13-4 is expressed in hematopoietic cells and is essential for degranulation. Little information is available on genotype-phenotype relationships of UNC13D mutations. Some mutants may have residual functionality which qualifies them as promising targets for attempts to enhance function pharmacologically. A problem for such analysis is the scarcity of patient material. We established assays in the RBL-2H3 cell line to assess functionality of lentivirally transduced munc13-4 mutants. The basic principle of which is to silence endogenous rat munc13-4 and replace it with siRNA resistant YFP-tagged human variants. Localization, degranulation, and membrane binding kinetics can now easily be analyzed quantitatively. Such a system might also be useful to screen small molecular weight compounds for their ability to rescue degranulation in cells with reduced functional munc13-4.
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