title: Naturally occurring Bruton's tyrosine kinase mutations have no dominant negative effect in an X-linked agammaglobulinaemia cellular model.
abstract: X-linked agammaglobulinaemia (XLA) is characterized by absence of mature B cells because of mutations in the Bruton's tyrosine kinase (Btk) gene. Btk-deficient early B cell precursors experience a block in their differentiation potentially reversible by the addition of an intact Btk gene. Btk expression was measured in 69 XLA patients with 47 different mutations and normal expression was detected in seven. We characterized these Btk mutant forms functionally by transfection into a lymphoma cell line that lacks endogenous Btk expression (Btk-/- DT40 cells) and analysed the calcium flux in response to B cell receptor stimulation. To test whether co-expression of a mutated form could compromise the function of the intact Btk transfection, studies in wild-type (WT) DT40 cells were also performed. Study reveals that none of the seven Btk mutants analysed was able to revert the absence of calcium mobilization upon IgM engagement in Btk-/- DT40 cells, as does intact Btk. In addition, calcium mobilization by anti-IgM stimulation in DT40 Btk+/+ cells was unaffected by co-expression with Btk mutants. These results suggest that gene addition would be feasible not only for patients with XLA and mutations that prevent Btk expression, but for those with expression of a mutant Btk.
authors.affiliation: Signal Transduction Group, Spanish National Cancer Research Centre (CNIO), Madrid, and Immunology Unit, Unviersity Hospital La Paz, Madrid, Spain. rperez@cnio.es
authors.forename: R
authors.initials: R
authors.lastname: Pérez de Diego
authors.forename: E
authors.initials: E
authors.lastname: López-Granados
authors.forename: J
authors.initials: J
authors.lastname: Rivera
authors.forename: A
authors.initials: A
authors.lastname: Ferreira
authors.forename: G
authors.initials: G
authors.lastname: Fontán
authors.forename: J
authors.initials: J
authors.lastname: Bravo
authors.forename: M C
authors.initials: MC
authors.lastname: García Rodríguez
authors.forename: S
authors.initials: S
authors.lastname: Bolland
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000361:Agammaglobulinemia / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D002648:Child; D002675:Child, Preschool; D040181:Genetic Diseases, X-Linked / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology; D007223:Infant; D008297:Male; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020125:Mutation, Missense; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D007075:Immunoglobulin M; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D002118:Calcium