Biomedical subjects
Publications and source records attributed to M de Weers.
A 13-year-old boy with immunodeficiency with hyper-IgM was analyzed for mutations in the CD40L gene. An insertional mutation of an extra T in a run of four T's was found in the second exon of the gene, leading to a premature translation stop. Genetic counselling of the family was performed, based on mutation detection by PCR/oligohybridization.
X-linked agammaglobulinemia (XLA) is an inherited human immunodeficiency disease, characterized by an arrest in B-cell development, which results in a dramatic decrease in immunoglobulin production. The gene product defective in XLA has been identified as a cytoplasmic protein tyrosine kinase, named Bruton's tyrosine kinase (Btk). The dramatic XLA phenotype indicates a critical role for Btk in the regulation of B-cell development. However, neither external stimuli leading to Btk activation nor any of its in vivo substrates have thus far been identified, and the mechanism of disease induction remains unexplained. We report here that stimulation of the B-cell antigen receptor (membrane immunoglobulin) on mature B-cells induces tyrosine phosphorylation of Btk in vivo, accompanied by an increase in its kinase activity in vitro. These results place Btk in the B-cell receptor signal transduction pathway, which is known to be essential in driving B-cell differentiation.
X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease in man, reflecting an arrest in differentiation of pre-B cells to mature B cell stages. The gene defective in XLA has been identified as a cytoplasmic protein tyrosine kinase, named btk (Bruton's tyrosine kinase). Here we report the characterization of mutations in the btk gene of five unrelated XLA families. Amplified products were generated from cDNA, cloned and sequenced. Three single point mutations and two small insertions were identified. One of the point mutations and the two insertions created stop codons that would lead to truncated btk proteins. In one XLA patient we found a single basepair substitution that altered the highly conserved Arg288 within the SH2 domain and would therefore abrogate interactions with substrate phosphotyrosines. In another XLA patient a single basepair substitution was observed that altered the conserved Arg28 residue in the N-terminal unique region of unknown function. This residue is also mutated in the xid mouse, which has a different, less severe, B cell deficiency. We conclude that a similar mutation in the btk gene leads in man to an almost complete arrest at an early stage of B cell differentiation, but in the mouse to only limited B cell abnormalities.
X-linked agammaglobulinemia (XLA) is an immunodeficiency disease in man, resulting from an arrest in early B cell differentiation. The gene defective in XLA has recently been identified and encodes a cytoplasmic protein tyrosine kinase, named Bruton's tyrosine kinase (btk), essential for cell differentiation and proliferation at the transition from pre-B to later B cell stages. In this study we investigated btk expression by Northern blotting experiments in a series of human (precursor-) B cell lines, acute lymphoblastic leukemias and plasmacytomas. btk was found to be already expressed in very early stages of B cell differentiation, even prior to immunoglobulin (Ig) heavy (H) or light (L) chain gene rearrangements. Transcripts were also detected at the pre-B cell stage and in mature B cells, irrespective of the Ig H chain class expressed. Approximately at the transition from mature B cells to plasma cells, expression of the btk gene is down-regulated. In addition, the btk gene was found to be expressed in myeloid cell lines and acute myeloid leukemias. btk expression in myeloid cells is probably not a prerequisite for myeloid differentiation, since myeloid cells in XLA patients seem not to be affected. No btk expression was found in T-lineage cells. The btk expression profile, i.e. from early precursor-B cell stages preceding Ig rearrangement up to mature B cells, supports the hypothesis that the XLA defect resides in a critical step of B cell development which is independent of the Ig gene recombination machinery.
Mhc-DRB and -DQA1 second-exon and -DRB 3'-untranslated-region nucleotide sequences of three lowland gorillas with no known family relationship with each other and of two HLA homozygous typing cell lines were determined and compared with published primate Mhc-DRB and -DQA1 sequences. Eleven distinct MhcGogo-DRB second-exon sequences were found, which represent the gorilla counterparts of the HLA-DRB1*03, -DRB1*10, -DRB3, -DRB5, and -DRB6 allelic lineages. One Gogo-DRB second-exon sequence does not have an obvious human counterpart and is tentatively designated Gogo-DRBY*01. The gorilla equivalents of the HLA-DRB2 and -DRB8 loci were identified as judged on Mhc-DRB 3'-untranslated-region sequences. In addition, four different Gogo-DQA1 alleles belonging to three different allelic lineages were detected. The Mhc-DRB-DQA1 haplotypes of these gorillas were deduced based on the obtained Mhc-DRB and -DQA1 sequences and the two published Mhc-DRB haplotypes of the lowland gorilla Sylvia. All deduced Gogo-DRB-DQA1 haplotypes show gene constellations different from known HLA-DRB-DQA1 haplotypes, while some of the Gogo-DRB haplotypes presented here contain more DRB genes than the HLA-DRB haplotypes. Based on phylogenetic trees, bootstrap analyses, and the gorilla, chimpanzee, and human Mhc-DRB haplotypes described, we propose that at least two Mhc-DRB loci, here tentatively designated Mhc-DRBI and -DRBII, existed on an ancient primate Mhc-DRB haplotype. The Mhc-DRB1*01, -DRB1*02 (-DRB1*15 and -DRB1*16), -DRB1*03 (-DRB1*03, -DRB1*08, -DRB1*11, -DRB1*12, -DRB1*13, and DRB1*14), and -DRB1*10 allelic lineages and -DRB3 and -DRBY loci probably evolved from the hypothetical primate Mhc-DRBI locus, whereas the present primate Mhc-DRB2, -DRB4, and -DRB6 loci originate from the ancient Mhc-DRBII locus of this core primate Mhc-DRB haplotype.
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X-linked agammaglobulinemia (XLA) patients manifest a very low production of immunoglobulins (Ig) of all classes and plasma cells are virtually absent. The XLA gene plays a crucial role in the transition of pre-B cells to later B cell stages, as hardly any slg-positive B lymphocytes can be detected. In the bone marrow almost normal numbers of pre-B lymphocytes are present. These cytoplasmatic C mu+ pre-B lymphocytes appear to express truncated M heavy chain molecules lacking the variable region segment. The T lymphocyte compartment is intact: the numbers of mature T cell receptor (TcR) alpha beta expressing T lymphocyte populations and their proliferative responses to antigens are normal. That the B cells are primary and exclusively affected was proven by X-chromosome inactivation studies. There is no evidence that the XLA gene is directly involved in the Ig gene rearrangements since B lymphoblastoid cell lines (BLCLs) established from peripheral blood of XLA patients were found to produce IgM molecules composed of complete Ig heavy and light chains and were shown to contain normal VHDJH recombinations. The data do not exclude the involvement of the XLA gene in a B cell specific process that makes the Ig loci accessible for recombination. Investigations on the degree of diversity of immunoglobulins generated by XLA patients exposed no limitations in the VH family usage. Sequence analysis of expressed VH3 and VH4 rearrangements however revealed that some genetic elements of the Ig locus might be over-represented and that a high portion of rearrangements was generated by unconventional mechanisms. By restriction length polymorphism (RFLP) and pulsed field gel electrophoreses analyses the XLA gene was mapped to an 8- to 12-Mb DNA fragment located in the Xq22 region. The known location of the XLA gene on the X-chromosome with closely linked RFLP markers and the availability of X-chromosome inactivation assays provides methods for carrier detection and prenatal diagnosis.
The gene for the Wiskott-Aldrich syndrome, an X-linked immunodeficiency disease, has been mapped between the RFLP markers DXS7 and DXS14 on the short arm of the X-chromosome. Close linkage to these markers permits accurate carrier detection and prenatal diagnosis. In one family with WAS patients in two generations, RFLP analysis was applied to three women at risk. It could be determined with more than 98.5% accuracy that these women were not carriers.