Finbarr E. Cotter

Rapid isolation of human chromosome-specific DNA probes from a somatic cell hybrid.

A strategy for the rapid isolation from rodent hybrids of human chromosome-specific probes by enzymatic amplification is described. Synthetic oligonucleotide primers based on the consensus Alu sequence were used to amplify inter-Alu sequence from total human genomic DNA and from a somatic cell hybrid, PNTS-1, containing one homolog of chromosome 5 as its only human complement. Direct sequence analysis of the products from human genomic DNA confirmed their inter-Alu structure and provided a novel means for the examination of the 5 end of the Alu consensus. The amplified sequences from the somatic cell hybrid DNA were cloned into a plasmid vector by blunt-end ligation, yielding clones with inserts in the range 300 to 1000 bp. More than 80% of these clones carried inserts that behaved essentially as single-copy human sequences. Hybridization of a selection of these clones to human DNA, hamster DNA, and the original hybrid DNA confirmed that they were derived from chromosome 5. Direct sequence analysis of the vector/insert boundaries in two clones confirmed that inter-Alu sequences had been cloned. This approach has significant advantages over other methods of isolating chromosome-specific probes from hybrid cells, enabling direct separation and cloning of human DNA probes that can be readily used for mapping studies.

Fluorescent in situ identification of human marker chromosomes using flow sorting and Alu element-mediated PCR

A novel approach to the identification of human chromosomes has been developed. Chromosomal in situ hybridization (or chromosome painting) has been performed using Alu element-mediated PCR products from small quantities (250-500) of flow-sorted normal and abnormal chromosomes. Chromosome paints for various normal chromosomes, including 5, 6, 7, 14, 18, 19, 21, and 22, were generated and shown to be effective in the identification of the appropriate chromosomes. In addition, certain abnormal chromosomes, including a mental retardation-associated deletion chromosome 11 (q22-q23), the products of the constitutional translocation t(11;22), and the CML-associated t(9;22), were used to generate region-specific paints. In each case, the appropriate regions of the chromosomes were highlighted and this strategy is, therefore, well suited to the identification of previously unidentified marker chromosomes. A further direct consequence of this work is that chromosome paints specific for the common aberrant chromosomes, such as the Philadelphia chromosome, can be generated and made widely available. These may find particular use in the analysis of complex or masked chromosomal translocations.

Gene mapping by enzymatic amplification from flow-sorted chromosomes

A new approach to gene mapping which combines enzymatic amplification with high-resolution flow sorting of human chromosomes has been devised. Reliable amplification from as few as 200 chromosomes has been demonstrated. This method, with particular application to mapping the position of chromosomal translocations, has been used to show that the breakpoint for the constitutional translocation t(11;22)(q23;q11) lies proximal to the genes c-ets-1, Thy-1, and T3 delta and distal to the int-2 gene. The mapping was confirmed by Southern analysis to much larger numbers of chromosomes sorted from the same cell line. Control reactions for the bcl-2 gene on chromosome 18 and the C alpha gene of the IGH locus on chromosome 14 demonstrated the discrimination which can be achieved.

THE ROLE OF THE bcl-2 GENE IN LYMPHOMA

Non-random chromosome abnormalities have been associated with B-cell non-Hodgkin’s lymphoma (NHL), often related to a particular histological group (Levine et al, 1985; Koduruetal, 1987; Yunisetal, 1987; Cabanillasetal, 1988). Translocations involving the q32 region of chromosome 14, the site of the immunoglobulin (Ig) heavy chain gene, are most often observed (Kirsch et al, 1982). Rearrangement of one of the two alleles of this gene, under the influence of a DNA recombinase enzyme, is essential for development of the pre-B-cell into a functional B-cell (Tonegawa, 1983). During lymphoma development, c-myc, bcl-1 and bcl-2 oncogenes respectively on chromosomes 8, 11 and 18 may translocate to the Ig heavy chain region on chromosome 14 (Tsujimoto et al, 1984, 1985; Erikson et al, 1984; Hayday et al, 1984), possibly under the influence of recombinase (Tycko & Sklar, 1990). Alterations in the expression of c-myc and bcl-2 occur following these translocations and may play a role in oncogenesis (Croce & Nowell, 19 8 5; Nunez et al, 19 89). The t(8:14)(q24;q32) translocation is found in the majority of Burkitt’s and some other high grade lymphomas (DallaFavera et nl, 1982). Small cell lymphocytic lymphomas and chronic lymphocytic leukaemia are described with the t(11;14)(q13;q32) translocation (Tsujimoto et al, 1984; Erikson et al, 1984). The most commonly associated chromosomal abnormality in NHL is the t( 14;18)(q32;q21) translocation found in 8 5% of follicular centroblastic/centrocytic lymphomas (FCC) and 28% of high grade NHL (Weiss et al, 198 7). High levels of bcl-2 gene expression at chromosome segment 18q21 occur during pre-B cell development but are normally down regulated with B cell maturation (Graninger et al, 1987). Involvement of the bcl-2 gene during the t( 14: 18) translocation associated with FCC is of interest due to its increased expression following the translocation (Hua et al, 1988). dueto a failure of down regulation in the lymphoma cell (Nunez et al, 1989).

The generation of DNA probes to chromosome 11q23 by Alu PCR on small numbers of flow-sorted 22q- derivative chromosomes

A strategy for the isolation of DNA probes from small numbers of flow-sorted human chromosomes has been developed. A lymphoblastoid cell line carrying the 22q- derivative chromosome product of the constitutional t(11;22) translocation was used as the source of chromosomes. Synthetic oligonucleotide primers, based on the consensus Alu sequence, were used to amplify inter-Alu sequence from 500 flow-sorted 22q- derivative chromosomes. The amplified sequences were cloned into a plasmid vector by blunt-end ligation, yielding clones with inserts in the range of 400 to 1000 bp. Approximately 70% of these clones hybridized to human DNA as single-copy probes. To identify clones derived from chromosome 11, the library was screened with a heterogeneous probe prepared by Alu-PCR amplification from the DNA of a somatic cell hybrid containing one homology of chromosome 11. All the positive clones found were mapped to within the q23-q25 region of chromosome 11 known to be translocated onto the 22q- derivative chromosome. Further mapping studies showed that most of these probes (7/8) lay between the breakpoints for the t(4;11) translocation of acute lymphocytic leukemia and the t(11;22) of Ewing sarcoma. Thus, the use of Alu-PCR on the small derivative chromosome 22q- has provided a greatly enriched source of probes to region 11q23, a part of the genome that is currently of great interest. This approach will be particularly appropriate to small numbers of chromosomes when high specificity rather than total representation is required.

Mycosis fungoides in childhood: An unusual presentation

Mycosis fungoides, a cutaneous T cell lymphoma, is rare in childhood and adolescence. We report a case of mycosis fungoides in a 9-year-old boy initially at a site of benign reactive lymphoid hyperplasia.

Co-incident N and K ras gene mutations in a case of AML, restricted to differing cell lineages.

Peripheral blood from a patient with acute myeloid leukaemia (AML) of M5 FAB classification, was shown to have mutations to both the N and K ras genes. Leucophoresed blood was separated on a discontinuous Percoll density gradient to provide fractions enriched for different cell lineages. DNA extracted from these fractions was amplified using the polymerase chain reaction (PCR) technique, and hybridized with oligonucleotide probes specific for the single base mutations previously demonstrated. The N‐ras mutation was shown to be restricted to the blast and monocytic cell fractions, concordant with the FAB subtype of M5. The K‐ras mutation, however, was present in all fractions, suggesting it had occurred in a multi‐potential stem cell representing an earlier stage in the generation of the leukaemia, or possibly an incidental background phenomenon.

STERNAL‐CLAVICULAR PLASMACYTOMAS WITH ATYPICAL MORPHOLOGY

Islam et al(l990) described a patient with multiple myeloma in whom bizarre cellular morphology was associated with a poor prognosis, death occurring within 6 months of diagnosis. Zuckerberg et aJ (1990) had previously described six similar patients, all presenting with advanced disease and dying shortly after diagnosis. We recently observed similar morphological features in a patient with a localized plasmacytoma. A previously fit 61year-old Caucasian man presented with a 5-month history of pain in his sternum and shoulder. On examination he was tender over the sternum but otherwise well. Blood tests showed Hb 14.8 g/dl, WBC 5.8 x 109/1, platelets 185 x 109/l. ESR 4 mm/h; urea and electrolytes and liver function tests normal; no paraprotein demonstrable in serum either by isoelectric focusing or zone electrophoresis: serum immunoglobulins normal; serum beta-2 microglobulin normal. No Bence Jones protein was demonstrable in his urine. An isotope bone scan showed patchily increased uptake over the sternum. A chest X-ray showed osteolysis of the sternum and clavicles. A skeletal survey and computerized tomographic scans of thorax and abdomen failed to reveal any other bony or extra-osseous tumours. A bone marrow aspirate and trephine biopsy from the right iliac crest was normal (plasma cells 3%). Bone marrow aspirated from the sternum showed a dense infiltrate of pleomorphic cells, ranging from identifiable plasma cells with deeply basophilic cytoplasm, to large cells with abundant cytoplasm and cleaved, convoluted or multilobed nuclei. Some of these cells contained PAS negative nuclear and/or cytoplasmic vacuoles. The morphology (Fig 1 ) is similar to that reported by Islam et a1 (1 990) and Zuckerberg et al(l990). Immunophenotypic analysis of cell suspensions and cytospin preparations suggested a B-cell lineage, with strong cytoplasmic kappa light chain expression. This was confirmed by clonal immunoglobulin heavy chain gene rearrangement on Southern blots prepared with several different restriction enzymes using a JH probe. The diagnosis of a plasma cell tumour (Jackson et al, 1988) was supported by the following immunophenotype reactions: BU-11: 96% cells positive: CD-38: 54% cells weakly positive. The cells were non-reactive with antibodies directed against the following markers: (Surface) HLA-DR, CD34, CD13, CD33, CD15, CD14. CDllc. CD10, CD19. CD37, CD22, CD61, CD7. CD2. (Cytoplasmic) CD3, CD22. IgM. IgG. IgA. (Nuclear) TdT. CD45 was expressed on 15% ofcells. The cytogenetic analysis was normal. The immunophenotype and gene rearrangement data, together with the morphological descriptions