Bruton's tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling

Abstract

Bruton's tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk is a member of the Tec family of protein tyrosine kinases (PTKs) and plays a vital, but diverse, modulatory role in many cellular processes. Mutations affecting Btk block B-lymphocyte development. Btk is conserved among species, and in this review, we present the sequence of the full-length rat Btk and find it to be analogous to the mouse Btk sequence. We have also analyzed the wealth of information compiled in the mutation database for XLA (BTKbase), representing 554 unique molecular events in 823 families and demonstrate that only selected amino acids are sensitive to replacement (P < 0.001). Although genotype-phenotype correlations have not been established in XLA, based on these findings, we hypothesize that this relationship indeed exists. Using short interfering-RNA technology, we have previously generated active constructs downregulating Btk expression. However, application of recently established guidelines to enhance or decrease the activity was not successful, demonstrating the importance of the primary sequence. We also review the outcome of expression profiling, comparing B lymphocytes from XLA-, Xid-, and Btk-knockout (KO) donors to healthy controls. Finally, in spite of a few genes differing in expression between Xid- and Btk-KO mice, in vivo competition between cells expressing either mutation shows that there is no selective survival advantage of cells carrying one genetic defect over the other. We conclusively demonstrate that for the R28C-missense mutant (Xid), there is no biologically relevant residual activity or any dominant negative effect versus other proteins.

BTKbase: the mutation database for X-linked agammaglobulinemia

“…According to many recent studies, no simple pattern of genotype-phenotype correlations has been established in patients with XLA [Broides et al, 2006;Lindvall et al, 2005;López-Granados et al, 2005;Vihinen and Durandy, 2006]. Highly significant differences appear in the frequency of missense mutations as compared to frameshifts, and sites that seem to be essentially resistant to substitutions were previously reported by us [Lindvall et al, 2005].…”

“…This is a highly significant difference, po0.0001, suggesting that there is a genotype-phenotype correlation. We previously reported that missense mutations as compared to frameshift mutations (including nonsense) only affect a limited number of amino acid positions in the BTK gene, in analogy with the F9 gene, which encodes a coagulation factor [Lindvall et al, 2005]. Moreover, we found that for TGA codons, which when mutated either form stop codons, or result in amino acid substitutions, residues R13 as well as R255 were exclusively mutated to stop codons (po0.001).…”

“…A case in point are XLA-causing missense mutations affecting proline residues; this will be discussed further below. Proline substitutions are highly overrepresented, amounting to close to 40% of all mutations at positions that are not evolutionarily conserved, such as in loops connecting functional domains in the Btk protein [Lindvall et al, 2005]. Most of the insertions are duplications of a short DNA segment or of a single nucleotide.…”

“…The ratios of nucleotide substitutions have remained very constant, although the database has grown, e.g., when compared to version 4 with 318 unrelated families [Vihinen et al, 1997a]. It has also been shown previously that mutations in CpG dinucleotides are the most common mutation events in XLA [Lindvall et al, 2005;Ollila et al, 1996;Vihinen et al, 1997a]. Amino acid substitution frequencies were analyzed for all the possible substitution combinations (Table 3) similar to KinMutBase [Ortutay et al, 2005] and IDbases [Piirilä et al, 2006].…”

“…Btk has been shown to interact with several partners [Lindvall et al, 2005;Smith et al, 2001]. The PH domain binds to phosphatidyl inositols and can function as a membrane localizing module.…”