Nucleic Acids Research

CFromosome 7 long arm deletions (7q-) are recurring chromosome abnormalities in leukemic bone marrow cells. In four patients we have previously localized the breakpoints in band 7q22 between the erythropoietin (EPO) and plasminogen activator inhibitor type 1 (PLANH1) genes that map 3 cM apart. The proalpha2(I)collagen (COL1A2, in band 7q22) and T cell receptor beta chain genes (TCRB, in band 7q35) have been found undeleted in one patient with an interstitial deletion. Pulsed field gel electrophoresis was used to map the breakpoints more accurately in two patients with a 7qchromosome. The results suggested that lymphocytes and granulocytes give identical restriction patterns with several enzyme-probe combinations, and that a breakpoint possibly was within 195 kb of EPO in one patient but not in another. The gene order cen-COLlA2-EPO-breakpoint-tel was suggested but physical linkage between COL1A2 and EPO was not found. A new putative TCRB restriction fragment length polymorphism or inherited methylation site was detected. INTRODUCTION Monosomy for chromosome 7 and partial deletion of the chromosome 7 long arm (7q-) are among the common recurring clonal chromosome abnormalities observed in bone marrow cells in patients with acute nonlymphocytic leukemia (ANLL) or myelodysplastic syndrome (MDS) (1,2). The biological basis of this phenomenon is not understood. It might involve the loss of a gene as in retinoblastoma (3), the rearranged fusion of two genes as in translocation (9;22) resulting in the formation of a Philadelphia chromosome (4), or the dysregulation of a gene as in lymphoid diseases (5). The loss of a chromosome 7 long arm has been associated with susceptibility to infections, a granulocyte locomotor defect, rapid progression of the disease, and poor response to therapy (6-9). One step toward the understanding of these features is to determine the 7qchromosome breakpoint(s) in molecular terms. Molecular mapping studies have indicated that in at least some patients and deletion types a chromosomal breakpoint involves a narrow region in band 7q22. In 4 patients the breakpoint was localized between the erythropoietin ) IRL Press 1511 Nucleic Acids Research Volume 17 Number 4 1989 Nucleic Acids Research (EPO) and plasminogen activator inhibitor type 1 (PLANHK) genes mapping 3 cM apart, a distance comprising less than 2% of the total length of chromosome 7 (10-12). In one patient the proalpha2(I)collagen (COL1A2) gene in band 7q22 and a distal segment including the T cell receptor beta chain (TCRB) gene in band 7q35 were retained (13). EPO and COL1A2 are tightly linked with 0 cM genetic distance (11). Pulsed field gel electrophoresis (PFGE) techniques are novel methods for the separation of very large DNA fragments produced by restriction enzymes for rarely occurring sequences (14-16). PFGE analysis has been used to detect deletions in patients with Duchenne muscular dystrophy and to produce long range restriction maps in a few genomic regions, e.g. around the cystic fibrosis and Duchenne muscular dystrophy genes (17,18). Field inversion gel electrophoresis has also been used to enrich selected genomic regions in the cloning of new segments within a hundred-kilobase distance from a probe (19). This study had three objectives: first, to develop methods for the mapping of 7qbreakpoints by PFGE, second, to determine more accurately the breakpoints in two patients, and third, to study the physical distance of the tightly linked COL1A2 and EPO loci preserved in the 7qchromosomes. MATERIALS AND METHODS Patients Two patients with a 7qchromosome, 2 patients with other chromosomal abnormalities, and altogether 18 control subjects were studied. A diagram of the 7qchromosome in patients 1 and 2 based on previous results (10,13) is shown in Figure 1. Patient 1, a female aged 77, had had polycythemia vera and had received chemotherapy and radiophosphorus. At the age of 72 her disease turned to MDS. At the time of the study her bone marrow cytology was compatible with refractory anemia with excess of blasts (RAEB) as classified according to the proposal of the French-American-British group (20). The bone marrow karyotype was interpreted as 46,XX,del(7)(pter->q21::q32-35->qter)=16 (21). Patient 2, a male aged 82, had developed 2 years earlier a primary MDS compatible with FAB classification RAEB. His bone marrow karyotype was 46,XY,t(2;13),del(7)(pter->q22::q34->qter)=20. Patient 3, a male with no history of leukemogenic exposure, developed myelofibrosis at the age of 57. At diagnosis one clone with the karyotype 46,XY,del(13)(ql2ql4) was detected in phytohemagglutinin-stimulated blood