MALT B cell lymphomas with t(1;14)(p22;q32) showed a recurrent breakpoint upstream of the promoter of a novel gene, Bcl10. Bcl10 is a cellular homolog of the equine herpesvirus-2 E10 gene: both contain an amino-terminal caspase recruitment domain (CARD) homologous to that found in several apoptotic molecules. Bcl10 and E10 activated NF-kappaB but caused apoptosis of 293 cells. Bcl10 expressed in a MALT lymphoma exhibited a frameshift mutation resulting in truncation distal to the CARD. Truncated Bcl10 activated NF-kappaB but did not induce apoptosis. Wild-type Bcl10 suppressed transformation, whereas mutant forms had lost this activity and displayed gain-of-function transforming activity. Similar mutations were detected in other tumor types, indicating that Bcl10 may be commonly involved in the pathogenesis of human malignancy.

Gene duplication followed by adaptive evolution is one of the primary forces for the emergence of new gene function. Here we describe the recent proliferation, transposition and selection of a 20-kilobase (kb) duplicated segment throughout 15 Mb of the short arm of human chromosome 16. The dispersal of this segment was accompanied by considerable variation in chromosomal-map location and copy number among hominoid species. In humans, we identified a gene family (morpheus) within the duplicated segment. Comparison of putative protein-encoding exons revealed the most extreme case of positive selection among hominoids. The major episode of enhanced amino-acid replacement occurred after the separation of human and great-ape lineages from the orangutan. Positive selection continued to alter amino-acid composition after the divergence of human and chimpanzee lineages. The rapidity and bias for amino-acid-altering nucleotide changes suggest adaptive evolution of the morpheus gene family during the emergence of humans and African apes. Moreover, some genes emerge and evolve very rapidly, generating copies that bear little similarity to their ancestral precursors. Consequently, a small fraction of human genes may not possess discernible orthologues within the genomes of model organisms.

A recently described atypical myeloproliferative disorder is invariably associated with reciprocal translocations involving 8p11-12. The most common rearrangement is a t(8;13)(p11;q11-12). Here we determine that this translocation results in the fusion of the fibroblast growth factor receptor 1 gene (FGFR1), a member of the receptor tyrosine kinase family at 8p11, to a novel gene at 13q11-12 designated RAMP . The predicted RAMP protein exhibits strong homology to the product of a recently cloned candidate gene for X-linked mental retardation, DXS6673E . We also provide the first report of a novel, putative metal-binding motif, present as five tandem repeats in both RAMP and DXS6673E. RT-PCR detected only one of the two possible fusion transcripts, encoding a product in which the N-terminal 641 amino acids of RAMP become joined to the tyrosine kinase domain of FGFR1. Receptor tyrosine kinases are not commonly involved in the formation of tumour-specific fusion proteins. However, the previous reports of involvement of receptor tyrosine kinases in fusion proteins in non-Hodgkin's lymphoma, chronic myelomonocytic leukaemia and papillary thyroid carcinoma described similar rearrangements. By analogy with these, we propose that the RAMP-FGFR1 fusion product will contribute to progression of this myeloproliferative disorder by constitutive activation of tyrosine kinase function.

The ZNF198- FGFR1 fusion gene arises as a result of the t(8;13)(p11;q12) in the 8p11 myeloproliferative syndrome. To determine the transforming properties of this chimeric protein we transfected ZNF198-FGFR1 into the interleukin (IL)-3 dependent cell line Ba/F3. Growth factor independent subclones were obtained in which ZNF198-FGFR1, STAT1, and STAT5 were constitutively tyrosine phosphorylated, as determined by immunoprecipitation and Western blot analysis. To test the hypothesis that constitutive activation of ZNF198-FGFR1 tyrosine kinase activity is a result of self-association of the fusion protein, we in vitro transcribed and translated ZNF198-FGFR1 and a derivative construct, ZNF198- FGFR1deltaC-myc, in which the C-terminal FGFR1 epitope was replaced by a c-myc tag. As expected, an anti-FGFR1 antibody immunoprecipitated ZNF198-FGFR1 but not ZNF198-FGFRdeltaC-myc. However when both products were translated together, both were coimmunoprecipitated by anti-FGFR1 antisera. Similar results were obtained by using an anti-myc antibody and demonstrated a physical interaction between the two proteins. Analysis of COS-7 cells transfected with ZNF198-FGFR1 demonstrated that the fusion gene, in contrast to normal FGFR1, is located in the cytoplasm. We conclude that ZNF198-FGFR1 is a cytoplasmic protein that self-associates and has constitutive transformation activity. These data suggest that ZNF198-FGFR1 plays a primary role in the pathogenesis of the t(8;13) myeloproliferative syndrome and is the first report to implicate STAT proteins in FGFR1-mediated signaling.

Clonal rearrangements of the Ig heavy chain (IGH ) locus consisting of either intrachromosomal (VDJ ) rearrangements or interchromosomal translocations are a consistent feature of all B-cell malignancies and may be used both diagnostically and to monitor response to therapy. Many of these rearrangements are targeted to the IGHJ segments, but only some can be amplified with regular polymerase chain reaction (PCR) techniques. To permit PCR amplification of potentially all IGHJ rearrangements, we have devised a method incorporating self-ligation of restriction endonuclease-digested DNA fragments with long-distance PCR (long-distance, inverse PCR [LDI-PCR]). We show here, using only 4 nested oligonucleotide primers, the successful amplification and DNA sequencing of all IGHJ rearrangements up to 5.4 kb in length from a panel of 13 cases and cell lines of various types of B-cell malignancy. In all cases, both VDJ and DJ IGH rearrangements and translocation breakpoints were amplified. Six cases exhibited t(14; 18)(q32; q21). All translocation breakpoints were cloned and sequenced. Three cases exhibited a rearrangement to the BCL2 major breakpoint region (MBR). However, 2 other cases exhibited rearrangements between the MBR and the minor cluster region (mcr). These 2 cases broke within 44 bp of each other, confirming the presence of an additional 3′ BCL2 breakpoint cluster region. The final case fell immediately 3′ of the 3′ UTR of the BCL2 gene adjacent to an Alu repeat. No other BCL2 breakpoints within this region have been reported. Four cases exhibited t(11; 14)(q13; q32). All 3 cases with translocations targeted to the IGHJ segments were successfully amplified and sequenced, including 1 case in which the BCL1 translocation could not be detected by DNA blot using the currently available probes. All three translocation breakpoints fell outside the BCL1 major translocation cluster between 20 and 40 kb telomeric and showed no clustering. Two of the three fell within or adjacent to Alu repeat regions. LDI-PCR is a simple and robust technique that allows PCR amplification of nearly all IGHJ rearrangements.