title: 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.
authors.affiliation: Department of Cell Biology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.
authors.forename: Edo
authors.initials: E
authors.lastname: Elstak
authors.forename: Arjan
authors.initials: A
authors.lastname: de Jong
authors.forename: Peter
authors.initials: P
authors.lastname: van der Sluijs
mesh_terms: D000818:Animals; D001483:Base Sequence; D015550:Cell Degranulation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D036681:Fluorescence Recovery After Photobleaching; D005816:Genetic Complementation Test / Q000379:methods*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051359:Lymphohistiocytosis, Hemophagocytic / Q000235:genetics* / Q000503:physiopathology; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D009154:Mutation; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D014161:Transduction, Genetic
chemical_list: D018076:DNA, Complementary; D008565:Membrane Proteins; D034741:RNA, Small Interfering; C507969:UNC13D protein, human