A platform for complementation and characterization of familial haemophagocytic lymphohistiocytosis 3 mutations

Abstract

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.