The Ig heavy chain gene is frequently involved in chromosomal translocations in multiple myeloma and plasma cell leukemia as detected by in situ hybridization

Chromosome rearrangement of 14q32.33 has recurrently occurred with variable partner sites, including 11q13.3, 8q24.1, 18q21.3, and 6p21.1 in multiple myeloma (MM). To assess the actual incidence of 14q32.33 translocation and to elucidate its implication in the pathogenesis of MM, we studied 42 patients with MM, plasma cell leukemia, or plasmacytoma and 5 with monoclonal gammopathy with undetermined significance (MGUS) by G-banding and molecular cytogenetic methods. Using double-color fluorescence in situ hybridization (DCFISH) with 2 Ig heavy chain (IgH) gene probes, a yeast artificial chromosome (YAC) clone containing variable region, and a phage clone containing gamma constant region, 14q32.33 translocation was detected as split signals of the IgH gene in 31 patients with plasma cell malignancies and 3 with MGUS. In contrast, of 40 patients who were assessed by G-banding, 3 (7.5%) showed the 14q+ chromosome. DCFISH detected a split of the IgH gene on interphase nuclei in 34 (73.9%) of 46 patients analyzed, whereas on metaphase spreads, it was in 22 (51.2%) of 43 patients analyzed. Interphase DCFISH was particularly useful to detect 14q32.33 translocation in 17 (65.4%) of 26 patients with normal karyotypes. Donor sites were identified in 11 of 22 patients demonstrated as carrying 14q32.33 translocation by metaphase FISH. Chromosome t(11;14)(q13.3; q32.33) was detected in 5 patients, t(8;14)(q24.1;q32.33) in 2, t(14;18)(q32.33;q21.3) in 2, and t(7;14)(q32.1;q32.33) in 1. A complex 14q32.33 translocation involving 3q and 16q24 was detected in 1 patient. Myeloma cells with t(7;14) showed myelomonocytoid surface antigen. Because rearrangements of 14q32.33 were closely associated with translocation of proto-oncogenes into the IgH gene, our findings indicate that 14q32.33 translocation with various partner chromosomes is a critical event in the pathogenesis of MM and MGUS.     

Neuropeptide Y-mediated pressor responses following high-frequency stimulation of the rat sympathetic nervous system

Recombinations between c-myc and immunoglobulin (Ig) sequences that typically occur in pristane-induced mouse plasmacytomas were detected in secondary lymphoid tissues from normal mice, chiefly in the gut-associated lymphoid tissue. Based on the analysis of recombination sequences as clonotypic markers, migration of c-myc recombination-positive cells was observed between Peyer's patches and into the intestine. Treatment of plasmacytoma-susceptible BALB/cAn mice with pristane induced proliferation and migration of these cells into mesenteric lymph node, spleen, and oil granuloma within 7 days. Plasmacytoma-resistant strains of mice (DBA/2N, C3H/HeJ, C57BL/6) differed in that (1) they harbored fewer clones (Ig/c-myc recombinations were detected in 33% of resistant mice versus 91% of BALB/cAn mice after pristane treatment); (2) Ig/c-myc-positive cells were rarely detected in the oil granuloma, and (3) c-myc recombined predominantly with the Ig alpha locus in BALB/cAn mice (72%), but with the Ig mu locus in DBA/2N and in C57BL/6 (67%). The results demonstrate that normal mice generate a large number of lymphocytes with aberrant c-myc in intestinal tissues without developing tumors.     

Reciprocal homologous recombination in or near antibody VDJ genes

To determine if rearranged heavy chain variable (VDJ) genes can recombine with each other by crossing over of DNA strands, we constructed a transgene that contained a promoter, VDJ gene, reporter gene to detect crossover events, intron enhancer, matrix attachment region, and constant gene for IgM (C mu). Following immunization of transgenic mice, hybrid molecules were isolated from B cell DNA which contained the transgene recombined with the endogenous IgH locus. Reciprocal products of crossovers were detected by plasmid rescue and PCR amplification, and they were sequenced. Recombination occurred somewhere within 147 bp of homology that contained the JH4 gene segment and 3' flanking DNA. The recombined transgenes had a 20-fold increase in mutation in the VDJ region compared to nonrecombined transgenes, which indicates that DNA sequences 3' of the C mu gene in the endogenous IgH locus are necessary for full activity of the mutator mechanism. The recovery of recombinants between VDJ transgenes and endogenous VDJ genes raises the possibility that reciprocal recombination may somatically diversify rearranged genes between maternal and paternal alleles.     

Construction of a yeast artificial chromosome contig spanning the spinal muscular atrophy disease gene region

The childhood spinal muscular atrophies (SMAs) are the most common, serious neuromuscular disorders of childhood second to Duchenne muscular dystrophy. A single locus for these disorders has been mapped by recombination events to a region of 0.7 centimorgan (range, 0.1-2.1 centimorgans) between loci D5S435 and MAP1B on chromosome 5q11.2-13.3. By using PCR amplification to screen yeast artificial chromosome (YAC) DNA pools and the PCR-vectorette method to amplify YAC ends, a YAC contig was constructed across the disease gene region. Nine walk steps identified 32 YACs, including a minimum of seven overlapping YAC clones (average size, 460 kb) that span the SMA region. The contig is characterized by a collection of 30 YAC-end sequence tag sites together with seven genetic markers. The entire YAC contig spans a minimum of 3.2 Mb; the SMA locus is confined to roughly half of this region. Microsatellite markers generated along the YAC contig segregate with the SMA locus in all families where the flanking markers (D5S435 and MAP1B) recombine. Construction of a YAC contig across the disease gene region is an essential step in isolation of the SMA-encoding gene.     

Pravastatin sodium activates endothelial nitric oxide synthase independent of its cholesterol-lowering actions

The oncogenic potential of many nonacute retroviruses is dependent on the duplication of the enhancer sequences present in the unique 3' (U3) region of the long terminal repeat (LTR). In a molecular clone (MCF 247-W) of the murine leukemia virus MCF 247, a leukemogenic mink cell focus-inducing (MCF) virus, the U3 enhancer sequences are tandemly repeated in the LTR. We mutated the enhancer region of MCF 247-W to test the hypothesis that the duplicated enhancer sequences of this virus have a sequence-specific and/or a stereospecific role in enhancer function required for transformation. In one virus, we inserted 14 nucleotide bp into the novel sequence generated at the junction of the two enhancers to generate an MCF virus with an interrupted enhancer region. In the second virus, only one copy of the enhancer sequences was present. This second virus also lacked the junction sequence present between the two enhancers of MCF 247-W. Both viruses were less leukemogenic and had a longer mean latency period than MCF 247-W. These data indicate that the sequence generated at the junction of the two enhancers and/or the stereospecific arrangement of the two enhancer elements are required for the full oncogenic potential of MCF 247-W. We analyzed proviral LTRs within the c-myc locus in tumor DNAs from mice injected with the MCF virus with the interrupted enhancer region. Some of the proviral LTRs integrated upstream of c-myc contain enhancer regions that are larger than those of the injected virus. These results are consistent with the suggestion that the virus with an interrupted enhancer changes in vivo to perform its role in the transformation of T cells.     

Detection of c-myc translocation in human lung cancer cells by fluorescence in situ hybridization (FISH)

c-myc gene amplification has been found in lung cancer, however, it can not explain all cases of lung cancer with c-myc gene overexpression. Gene translocation is one of the ways by which oncogene is activated. But the old methods for detecting gene mutations are not so effective for the detection of gene translocation, especially in solid tumors. Fluorescence in situ hybridization (FISH) can be used to detect gene translocation more efficiently. Using FISH, we discovered c-myc gene translocation in a lung adenocarcinoma cell line GLC-82 and SV40T-transformed human bronchial epithelial cells. In GLC-82, c-myc gene translocated to the short arm of a C group marker chromosome. In the SV40T-transformed epithelial cells, c-myc gene translocated to 14q32, which was the same as that found in Burkitt's lymphomas. Translocation was related to oncogene activation. c-myc translocation may play an important role in the carcinogenesis of lung cancer.     

FISH detection of chromosome 14q32/IgH translocations: evaluation in follicular lymphoma

A FISH strategy capable of detecting chromosome 14q32 rearrangements involving the IgH locus, including in interphase nuclei, was developed using Ig variable and constant region cosmids from the extremities of the locus in a dual hybridization approach, using signal splitting as evidence of rearrangement. The large size of the locus (1.3 Mb) and the propensity for internal deletion due to physiological VDJ recombination and isotype switching complicate analysis of this locus. We used the Ig10 cosmid, which hybridizes to C epsilon and C alpha2 at the 3' end of the constant region, in order to minimize deletion and/or splitting of the constant region probe. Cos Ig10 and the IgV18 VH probes were compared with a specific IgH-BCL2 FISH dual hybridization approach in follicular lymphoma (FL). Both were capable of detecting the t(14;18) in interphase nuclei, including in cases with no apparent abnormality by classic karyotype analysis, although the sensitivity of the IgH approach was slightly lower. We have also successfully applied these probes to whole cell cytospin preparations, rendering analysis of cryopreserved material possible, although interpretation should be limited to frequent events, particularly following cell manipulation. Analysis of flow cytometric sorted bone marrow fractions from three FL patients by FISH and FICTION showed that the t(14;18) was present in a much lower proportion of CD34 positive than negative cells but that the higher level of background hybridization limits use of these techniques for the reliable quantification of rare events.     

YAC contigs of the Rab1 and wobbler (wr) spinal muscular atrophy gene region on proximal mouse chromosome 11 and of the homologous region on human chromosome 2p

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized the wobbler spinal atrophy gene wr to proximal mouse Chr 11, tightly linked to Rab1, a gene coding for a small GTP-binding protein, and Glnsps1, an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of the Rab1 region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescence in situ hybridization (FISH), and sequence-tagged site (STS) isolation and mapping. Rab1 and Glns-ps1 were found to be only 200 kb apart. A potential CpG island near a methylated NarI site and a trapped exon, ETG1.1, were found between these loci, and a new STS, AHY1.1, was found over 250 kb from Rab1. Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising the RAB1 locus, AHY1.1, and the human homologue of ETG1.1, indicating a high degree of conservation of this region in the two species. We mapped AHY1.1 and thus human RAB1 on Chr 2p13.4-p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the gene LMGMD2B for a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13-p16. The conservation between the mouse Rab1 and human RAB1 regions will be helpful in identifying candidate genes for the wobbler spinal muscular atrophy and in clarifying a possible relationship between wr and LMGMD2B.