Molecular delineation of the smallest commonly deleted region of chromosome 5 in malignant myeloid diseases to 1–1.5 Mb and preparation of a PAC-based physical map (myeloid leukemiaytumor suppressor genesychromosomal deletionsytherapy-related leukemia)

Loss of a whole chromosome 5 or a deletion of the long arm, del(5q), is a recurring abnormality in malignant myeloid diseases. In previous studies, we delineated a commonly deleted segment of '4 Mb within band 5q31 that was f lanked by IL9 on the proximal side and D5S166 on the distal side. We have generated a physical map of P1 (PAC), bacterial (BAC), and yeast artificial chromosome (YAC) clones of this interval. The contig consists of 108 clones (78 PACs, 2 BACs, and 28 YACs) to which 125 markers (5 genes, 11 expressed sequence tags, 12 polymorphisms, and 97 sequence-tagged sites) have been mapped. Using PAC clones for f luorescence in situ hybridization analysis of leukemia cells with a del(5q), we have narrowed the commonly deleted segment to 1–1.5 Mb between D5S479 and D5S500. To search for allele loss, we used 7 microsatellite markers within and flanking the commonly deleted segment to examine leukemia cells from 28 patients with loss of 5q, and 14 patients without cytogenetically detectable loss of 5q. In the first group of patients, we detected hemizygous deletions, consistent with the cytogenetically visible loss; no homozygous deletions were detected. No allele loss was detected in patients without abnormalities of chromosome 5, suggesting that allele loss on 5q is the result of visible chromosomal abnormalities. The development of a stable PAC contig and the identification of the smallest commonly deleted segment will facilitate the molecular cloning of a myeloid leukemia suppressor gene on 5q. Recurring chromosomal abnormalities are characteristic of human malignant diseases, particularly the leukemias and lymphomas (1–3). To date, the major emphasis of the molecular analysis of chromosomal abnormalities in tumors has involved the recurring translocations, which result in the activation of a cancer-related gene in a dominant fashion (4). In addition to the recurring translocations, the loss of genetic material has been identified in many tumor types. Loss of genetic material is the hallmark of tumor suppressor genes and may result from loss of a whole chromosome or part of a chromosome (deletion) as well as by other genetic mechanisms. The consequence of these abnormalities is the unmasking of a recessive allele on the structurally ‘‘normal’’ homolog (5–6). The identification of recurring chromosomal deletions in the hematological malignant diseases suggests that as for a number of solid tumors, tumor suppressor genes may be involved in the pathogenesis of some leukemias (3, 7, 8). With respect to the myeloid disorders, recurring deletions include the del(5q), del(7q), del(9q), del(11q), del(12p), del(13q), and del(20q) (3, 7). With the exception of the del(9q), a commonly deleted segment has been identified by molecular mapping for each of these rearrangements (ref. 9; see ref. 10 for references). The occurrence of a myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) is a late complication of cytotoxic therapy used in the treatment of both malignant and nonmalignant diseases (11, 12). Therapy-related MDS or AML (t-MDSyt-AML) typically presents '5 years after treatment. Frequently all three hematopoietic cell lines (erythroid, myeloid, and megakaryocytic) are involved in the myelodysplastic process. Survival times of patients with t-AML are usually short (median, 8 months) (13). At the cytogenetic level, t-MDSyt-AML is characterized by loss of an entire chromosome 5 or 7, or a deletion of the long arm of these chromosomes [del(5q)ydel(7q)]. In our recently updated series of 257 consecutive patients with t-MDSytAML, 242 (94%) had a clonal chromosomal abnormality (ref. 13; M.M.L.B., R.A.L., E. M. Davis, J. D. Rowley, and J. W. Vardiman, unpublished work), and 183 patients (71%) had a clonal abnormality leading to loss or deletion of chromosome 5 andyor 7. Among these 183 patients, 34 had loss of chromosome 5, 52 had a del(5q), and 23 had loss of 5q following an unbalanced translocation. Overall, 109 patients (42%) had abnormalities of chromosome 5, and 132 (51%) had abnormalities of chromosome 7. Fifty-eight patients had abnormalities of both chromosomes 5 and 7. A del(5q) was the most common structural abnormality. In addition to t-MDSyt-AML, a 25ydel(5q) has also been observed in 10% of patients with AML or MDS de novo (14–16). Many of these patients have had significant occupational exposure to potential carcinogens, suggesting that abnormalities of chromosomes 5 or 7 may be a marker of mutagen-induced leukemia. A distinct clinical disorder, termed the ‘‘5q2 syndrome,’’ is observed in a subset of patients with MDS de novo (16–18). This disorder is characterized by refractory anemia and a relatively mild course that usually does not progress to acute leukemia. These patients typically have the del(5q) as the sole abnormality. We and others have proposed that 5q contains a tumor suppressor gene. In previous studies, we delineated a segment of 5q, within band q31, that was deleted in all patients examined; this commonly deleted segment was '4 Mb in size, and was flanked by IL9 (proximal) and D5S166 (distal) (9). We have prepared a P1 (PAC), bacterial (BAC), and yeast artificial chromosome (YAC) contig of this interval. By using fluoresThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y946948-6$2.00y0 Abbreviations: AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; t-MDSyt-AML, therapy-related MDS or AML; PAC, P1 artificial chromosome; BAC, bacterial artificial chromosome; YAC, yeast artificial chromosome; FISH, fluorescence in situ hybridization; STS, sequence-tagged site; EST, expressed sequence tag; STRP, short tandem repeat polymorphism. *To whom reprint requests should be addressed at: Section of HematologyyOncology, The University of Chicago, 5841 S. Maryland Ave., MC2115, Chicago IL 60637. e-mail: [email protected].