Subclass restriction pattern of antigen-specific antibodies in donors with defective expression of IgG or IgA subclass heavy chain constant region genes

We have developed a method for the measurement of the IgG and IgA subclass distribution of antigen-specific human antibodies. The controls for the specificity of the assay include the use of a number of monoclonal human antibodies and sera from individuals with deletions of particular immunoglobulin heavy chain constant region genes. The system was used to determine the shift in immunoglobulin subclass patterns of specific antibodies against a variety of protein and polysaccharide antigens in individuals with a regulatory deficiency of a given IgG or IgA subclass. Normally, the pattern is quite distinct and antibodies against protein antigens are mainly of the IgG1 subclass, whereas antibodies against polysaccharide antigens are mainly of the IgG2 subclass. The results on serum from an IgG1 deficient donor suggested that IgG3 and IgG4 appear to compensate for a lack of IgG1, whereas isolated deficiencies of IgG3, IgG4, or IgA2 do not markedly influence the expected distribution of specific antibodies. In IgG2-deficient individuals a more complex pattern was observed where antibodies against protein antigens were retained, whereas levels of antibodies against polysaccharide antigens could vary markedly between donors, which appeared to be dependent on whether the IgG2 deficiency was an isolated defect or combined with IgG4/IgA deficiency. However, all the IgG2-deficient donors had a skewed pattern of anti-polysaccharide antibodies with a shift to IgG1 to IgG3

Identification of Btk mutations in 20 unrelated patients with X-Iinked agammaglobulinaemia (XLA)

X-linked agammaglobulinaemia (XLA) is an inherited immunodeficiency resulting from mutations in the gene for a cytoplasmic protein tyrosine kinase (Btk). We have utilised reverse-transcription-based PCR in combination with the chemical cleavage and mis match technique (CCM) to screen for Btk mutations in 42 unrelated patients having classical XLA or ‘leaky’ XLA-like phenotypes. A variety of mutations, including point mutations, large deletions and splicing defects were detected using this strategy. In total, 20 mutations were found in these patients. All the mutations were different with the exception of three unrelated patients who all showed the same Arg→His amino acid substitution (R641 H) at a highly-conserved residue in the kinase domain. We have also used structural modelling of the Btk kinase domain to predict how two different amino acid substitution mutations at highly-conserved residues are likely to affect the Btk kinase activity

Clinical and molecular analysis of patients with defects in μ heavy chain gene

Autosomal recessive disorders of B cell development are rare and heterogeneous. To determine the proportion of affected patients who have defects in the μ heavy chain (IGHM) gene, we used single-stranded conformational polymorphism analysis to screen genomic DNA from 40 unrelated patients with early onset infections, profound hypogammaglobulinemia, and absent B cells. All of the patients were genotypically normal in BTK, the gene that underlies X-linked agammaglobulinemia. Eight different mutations in the μ heavy chain were identified in 19 members of 12 unrelated families. Four of the mutations were large deletions that removed more than 40 kb of DNA in the IGHM locus. In six of the 12 families, the affected patients had an identical single base pair substitution, a G→A, at the –1 position of the alternative splice site. Immunoglobulin haplotype analysis showed that this mutation occurred on at least three different haplotypes, indicating that this is a hot spot for mutations. Compared with patients with mutations in Btk, patients with defects in the μ heavy chain had an earlier onset of disease and more complications. Our study indicates that at least 20–30% of patients with autosomal recessive defects in B cell development have mutations in the μ heavy chain

BTKbase: a database of XLA-causing mutations

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled, and its use and the resultant information are discussed in this article

Genomic organization of mouse and human Bruton's agammaglobulinemia tyrosine kinase (Btk) loci

Btk is a cytoplasmic protein tyrosine kinase (PTK) that has been directly implicated in the pathogenesis of X-linked agammaglobulinaemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. We have isolated phage and cosmid clones that allowed us to deduce the genomic structure of mouse and human Btk loci. The mouse and human genes are contained within genomic regions that span approximately 43.5 kb and 37.5 kb, respectively. Both loci contain 18 coding exons ranging between 55 and 560 bp in size with introns ranging in size from 164 bp to approximately 9 kb. The 5'-untranslated regions are encoded by single exons located approximately 9 kb upstream of the first coding exon. Exon 18 encodes for the last 23 carboxyl-terminal amino acids and the entire 3'-untranslated region. The location of intron/exon boundaries in the catalytic domains of the mouse and human Btk loci differs from that found in other described sub-families of intracellular PTKs, namely that of Src, Fes/Fer, Csk, and Abl/Arg. This observation is consistent with the classification of Btk together with the recently characterized kinases, Tec and Itk, into a separate sub-family of cytoplasmic PTKs. Putative transcription initiation sites in the mouse and human Btk loci have been determined by using the rapid amplification of cDNA ends assay. Similar to many other PTK specific genes, the putative Btk promoters lack obvious TATAA and CAAAT motifs. Putative initiator elements and potential binding sites for Ets (PEA-3), zeste, and PuF transcription factors are located within the 300 bp which are located upstream of the major transcription start site in both species. These sequences can mediate promoter activity when placed upstream of a promotorless chloramphenicol acetyl transferase reporter gene in an orientation-dependent manner. The present analysis will significantly facilitate the mutational analyses of patients with XLA and the further characterization of the function and regulation of the Btk molecule