Kinase mutant Btk results in atypical X-linked agammaglobulinaemia phenotype

Abstract

X-linked agammaglobulinaemia (XLA) is a B cell humoral abnormality arising from mutations in the gene encoding Bruton's tyrosine kinase (Btk). The phenotype of XLA can be variable, with some individuals having a less severe immunophenotype, although in most cases this cannot be correlated with the Btk mutation or expression of Btk protein. In this study we describe clinical and immunological heterogeneity within the same pedigree. Analysis of the genetic defect identified a missense mutation in the kinase domain of Btk which, unusually, preserved Btk protein expression but at reduced levels, and also considerably diminished autophosphorylation activity. Structural analysis of the effect of this mutation on the kinase domain suggests that this mutation is not an integral part of the ATP or substrate binding domains but may affect the interaction of the kinase domain with its own kinase domain and other substrates. Together, these data may provide an explanation for the variable XLA phenotype.

Fig. 1

Oligoclonal nature of immunoglobulin production in P2. Agar gel immunoelectrophoresis of immunoglobulin isotypes, IgG (G), IgA (A), IgM (M) and κ- and λ-chains in P2 (P) and controls (C). P2 shows production of IgG and IgM but a higher proportion of λ-chains than κ-chains (control κ > λ), indicating an oligoclonal IgG population.

Fig. 2

(a) P1 and P2 show expression of Btk protein. Western blot analysis of mononuclear cell lysates from P1 (lane 1) and P2 (lane 3) with H360B anti-Btk antibody detects an approx. 77-kD band similar to that seen in a healthy control (lane C). A cross-reactive smaller band in seen in both patient lysates (lanes 1 and 3). Immunoprecipitation of the mononuclear cell lysate from P1 with H360B and re-immunoblotting with the same antibody confirmed the expression of a normal sized Btk protein (lane 2). (b) P1 and P2 show increased levels of control protein β-actin. Immunoblotting of equivalent amounts of cell lysates from P1 and P2 (lanes 1 and 2) and a control sample (C) with a control protein β-actin shows increased amounts of protein in the patient samples.

Fig. 3

P1 and P2 show abnormal Btk autophosphorylation activity. Mononuclear cell lysates containing equivalent amounts of protein from a control sample, P1 and P2 (lanes C, 1 and 2, respectively) were incubated with anti-Btk serum and the immunoprecipitates washed and subject to in vitro kinase assay. Kinase activity is virtually absent in samples from P1 and P2.