Deficiencies of Bruton's tyrosine kinase (Btk) have been implicated in the pathogenesis of human X-linked agammaglobulinemia (XLA). The distinctive phenotype observed in B-cell deficiency indicates the crucial role of Btk in B-cell development. This report describes a nationwide study of Btk deficiency in Japan, covering 51 XLA patients (35 independent families). Along with the identification of mutations, the resulting protein products were characterized by an in vitro kinase assay and a Western blot analysis. Thirty-one of the families were found to have mutations in the coding region of Btk. Although mutations were not found in the cDNA of 4 families, the Btk transcripts of these patients were greatly reduced. The identification of several novel missense mutations, in combination with the result of other studies, clarified the presence of two (missense) mutation hot spots, one in the SH1 and the other in the PH domain. The absence of kinase activity seen in 32 of the families underscored the importance of Btk protein analysis as a diagnostic indicator of XLA. The protein analysis also clarified the different effects of missense mutations on kinase activity and protein stability.

The t(11;22)(q24;q12) and t(21;22)(q22;q12) are specific chromosomal translocations found in the Ewing family of tumors including ES, PNET and Askin tumors. In these translocations, the amino-terminal portion of the EWS gene located in 22q12 fuses to the carboxyl-terminal portion of the FLI-1 gene located in 11q24 or the ERG gene located in 21q22, which belong to the ets oncogene superfamily of transcription activators. We investigated the chimeric mRNAs of 15 ESs (7 cell lines and 8 tumor samples) and 7 PNETs (3 cell lines and 4 tumor samples) using the RT-PCR method and sequencing. We detected 2 types of EWS-ERG chimeric mRNA in 2 ES cell lines and 1 PNET tumor sample in addition to 4 types of EWS-FLI-1 chimeric mRNA in 11 ESs (4 cell lines and 7 tumor samples) and 4 PNETs (2 cell lines and 2 tumor samples). There seemed to be no association between the type of chimeric mRNA and clinical features such as sex, age, primary site and histopathology of the patients. All of the chimeric mRNAs are generated from in-frame junctions and are thought to encode fusion proteins that may be the molecular mechanism involved in the Ewing family of tumors.

Although macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine that inhibits the migration of macrophages, its ubiquitous expression suggests it may have a role beyond the immune system. Here we report a detailed characterization of MIF expression during mouse embryogenesis. The MIF expression pattern was found to parallel tissues specification and organogenesis.

We have investigated the alterations of p53 and ras genes including H-, K-, and N-ras genes in 22 acute lymphoblastic leukemia (ALL) cases and five cell lines carrying t(1;19) by use of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis and direct sequencing. The mutations of the p53 gene were found in 2 of 20 t(1;19)-ALL cases at diagnosis (10%), all of 4 cases at relapse (100%), and 4 of the 5 cell lines (80%). Four of the five patients who died had missense mutations at codons 49, 177, 179, and 248. In cases examined sequentially, one had the same point mutation at codon 179 at both diagnosis and relapse, and another had the same p53 gene mutation at codon 240 both in leukemic cells at relapse and in a cell line derived at that time. The other case had no mutation at diagnosis but had the mutation at codon 177 at relapse and cell lines derived from blast cells at diagnosis, suggesting that a small number of leukemic cells with the p53 gene mutation at diagnosis might have escaped PCR-SSCP analysis. In cell lines, SCMC-L9 had three point mutations in the p53 gene at codons 175, 248, and 358, whereas SCMC-L10 had frame shift at codons 209–211. One case had a rare polymorphism at codon 11. We found only one mutation of the N-ras gene that was a 2-bp substitution of GGT(Gly) to GTC(Val) at codon 13 among 22 t(1;19)-ALL cases and five cell lines. This case showed no mutation of the p53 gene and has had a good course. These results suggest that in t(1;19)-ALL, mutations of the p53 and ras genes are infrequent at diagnosis and that p53 gene alterations may be associated with relapse phase or progression of t(1;19)-ALL.

Alterations of the Bruton's tyrosine kinase (Btk) gene are responsible for X-linked agammaglobulinemia (XLA). Although mutations in various regions were reported mainly in the Caucasian population, correlation between the locations of mutation and the clinical phenotypes remains unclear. We report 12 abnormalities of the Btk gene found in 12 unrelated families out of 14 XLA families in Japan and their clinical features. We utilized Southern blotting and single-strand conformation polymorphism (SSCP) analysis. Gene rearrangement in the kinase domain was identified in two patients by Southern blotting. Seven point mutations, two small deletions, and one small insertion were detected by SSCP and sequencing. The SSCP analysis also provided information about the carriers in these families. We found some clinical heterogeneity in the affected family members with the same gene mutation. Moreover, there is considerable inconsistency between the locations of gene aberrations and the immunological phenotypes. Some patients with a nonsense mutation, which may result in the lack of kinase domain, have detectable B cells and immunoglobulins. These identified alterations will provide valuable clues to the Btk protein function and the pathogenesis of XLA.