Leukemia (1997) 11, 2097–2104 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Evidence for two molecular steps in the pathogenesis of myeloid disorders associated with deletion of chromosome 7 long arm S Kiuru-Kuhlefelt1, P Kristo1, T Ruutu2, S Knuutila1 and J Kere1 1Department of Medical Genetics, Haartman Institute, University of Helsinki; and 2Division of Haematology, Department of Medicine, Helsinki University Central Hospital, Finland Partial deletion of the long arm of chromosome 7 (7q−)isa many cases, the deletions have been characterized.16,17,19 In frequent chromosomal aberration in many neoplasias, includ- AML or MDS, the granulocyte–monocyte lineage contains ing acute myeloid leukemia (AML) and myelodysplastic syn- dromes (MDS). Recurrent deletions, leading to loss of hetero- affected cells, and LOH can thus be studied by comparing the zygosity (LOH), may be indicative of a tumor suppressor gene heterozygosity between DNAs extracted from granulocytes nearby. We studied eight AML or MDS patients with 7q−,in and lymphocytes.20–23 order to define the commonly deleted area in more detail. Sep- The biological significance of the acquired partial loss arated blood lymphocytes and granulocytes were typed with of the long arm of chromosome 7 remains unknown. 48 polymorphic microsatellite markers, and the heterozygosity One or more tumor suppressor genes have been suspected between the two cell lineages was compared. The minimum residing there, but definitive evidence for such genes is commonly deleted region spanned from D7S658 to D7S655. 3,6–8,17,19,21 Unexpectedly, four of the patients showed remarkable homo- lacking. zygosity in both lymphocytes and granulocytes around the The high resolution linkage maps that have recently commonly deleted area, and thus no deletions could be demon- become available and provide densely spaced, highly poly- strated by comparing the two cell lineages. Comparison to leu- morphic microsatellite markers,24 permit a much more accur- − kemic patients without 7q and to normal individuals revealed ate analysis of these molecular changes. This study was that the homozygosity was restricted to patients with 7q−.We suggest that a specific mechanism, such as mitotic recombi- designed to test the use of PCR amplification of the highly nation in bone marrow stem cells, leading to homozygosity in polymorphic microsatellite markers in identifying deletions of both granulocytes and lymphocytes, represents a leukemo- 7q in AML and MDS patients, and to characterize the genetic step in these patients. deletions in more detail. Surprisingly, we observed that in Keywords: leukemia; myelodysplastic syndromes; 7q, loss of some of these patients, both lymphocytes and granulocytes heterozygosity are highly significantly more often homozygous for chromo- some 7 long arm markers than was expected. To determine whether this phenomenon was restricted to bone marrow- Introduction derived cells we typed two patients’ mesenchymal organ DNA with markers that were homozygous in their lymphocytes Neoplastic hematologic cells often contain numerical or struc- and granulocytes. tural cytogenetic abnormalities that occur either alone or In addition to 7q− patients, 28 AML patients with no visible together.1,2 In acute myeloid leukemia (AML) and myelodys- aberrations of chromosome 7 were studied to ascertain pos- plastic syndromes (MDS), the most common solely occurring sible LOH or exceptional homozygosity. No LOH was found, aberrations include monosomy 7 (−7) and the deletion of the and the heterozygosity of this population was also close to long arm of chromosome 7 (7q−).3 Both terminal and inter- that of the normal population. We interpret these findings to stitial deletions as well as inversions and translocations of 7q suggest two consecutive or alternative molecular steps in the have been described, and the breakpoints vary among the pathogenesis of myeloid disorders involving chromosome 7 patients.4–8 The most commonly deleted segment is 7q22– abnormalities. q36, where two distinct critical regions have been postulated; 7q22 and 7q32–q34.3,6–10 Aberrations with 7q− or −7 are present in up to 5% of all AML or MDS cases, and their fre- quency is even higher in therapy-related AML or MDS.4,5,9 Materials and methods Clinically, monosomy 7 and 7q− are associated with a rela- tively poor response to chemotherapy and short survival,11 Patients and specimens with 7q− and cytogenetic studies have suggested the presence of a gene(s) that is responsible for the poor prognosis in 7q31 or Eight patients with the clinical diagnosis of AML or MDS, 7q32.12,13 Deletions of 7q are also common in many solid whose chromosome analysis had revealed deletion of the long tumors, including breast tumors, prostate carcinomas and arm of chromosome 7 exclusively or among other aberrations, colon carcinomas, where loss of heterozygosity (LOH) at the were selected for this study. Cytogenetic studies of unstimu- band 7q31 is a frequent finding.14–17 In these tumors, LOH is lated bone marrow and phytohemagglutinin-stimulated blood sometimes associated with poor survival.14 In solid tumors, it cells were performed as described elsewhere.25 The clinical is possible to study LOH by comparing the heterozygosity of and original cytogenetic findings of the patients are summar- DNAs extracted from the tumor and normal tissue,18 and in ized in Table 1. Red blood cells were removed by precipitation with 0.6% dextran from heparinized blood samples. Mononuclear cells Correspondence: S Kiuru-Kuhlefelt, Department of Medical Genetics, and granulocytes were separated from the remaining white Haartman Institute, University of Helsinki, PO Box 21 (Haartmaninkatu 3), FIN-00014 Helsinki, Finland; Fax: blood cell rich plasma by Ficoll gradient centrifugation. Purity 358 9 1912 6677 of the fractions has been consistently better than 80%. DNA Received 24 April 1997; accepted 9 September 1997 was extracted from lymphocyte and granulocyte cell fractions Chromosome 7 in myeloid disorders S Kiuru-Kuhlefelt et al 2098 Table 1 Diagnosis and cytogenetic characteristics of the patientsa Patient No. Diagnosis/Age at Sample type Bone marrow karyotype at time of diagnosisb diagnosis/Sex 1 RAEB/72/F LY,GR 46,XX,del(7)(q21q32-35),del(20)(q11) 2 RAEB/80/M LY,GR 46,XY,t(2;13)(p21;q12),del(7)(q22q33-34) 3 RAEB/40/F LY,GR 44,XX,del(5)(q13q22),del(7)(q22),del(13)(q12q21),−17,del(20)(q11),−21/46,XX 4 RAEB/73/F LY,GR 45,XX,−5,del(7)(q32),del(12)(p12)/55,XX,+3,+5,+7,+9,+11,+15,+15,+19,+21 5 AML M2/63/M BM 52,XY,−2,+t(2;12)(q10;q10),+del(5)(q13q22),+6,del(7)(q22),add(11)(q23), −12,+t(12;16)(q10;10),+13,−16,+17,+18,+2mar 6 AML M4/40/F LY,GR 46,XX,del(7)(q31.2) or del(7)(q22q34),inv(16)(p13q22)/46,XX 7 MDS/59/F LY,GR 46,XX,del(7)(q?)/46,idem,t(1;6)(q23;p22) 8 RAEB/57/M LY,GR 47,XY,del(7)(q22),+21 aThe diagnoses are according to the FAB classification, where applicable.26,27 bThe designations are based on original cytogenetic interpretations. Most chromosome 7 deletions are, however, interstitial (Table 2). RAEB, refractory anemia with excess of blasts; MDS, unspecified myelodysplastic syndrome; F, female; M, male; LY, lymphocytes; GR, granulocytes; BM, bone marrow. separately using a standard proteinase K digestion, phenol reactions that were slightly varied to optimize for each marker. extraction and ethanol precipitation method. Typical conditions consisted of 30–35 cycles of 94°C, 55°C The granulocytes of patients 1 and 4 were studied by the and 72°C each for 30–45 s. Template DNA was varied comparative genomic hybridization (CGH) method, described between 5–100 ng to check for consistency of interpretation. elsewhere,28 in order to exclude any changes between deleted After PCR amplification, the samples were electrophoresed in chromosome 7 material and other chromosomes. 6% polyacrylamide-7M urea sequencing gels. Films were Tissue specimens taken during operation or autopsy were exposed from 20 min to 4 days. Alleles present in the two cell obtained from patients 4 (gall bladder) and 6 (liver). The fractions were compared for each patient. Gall bladder and samples had been fixed in buffered formaldehyde and embed- liver DNA from patients 4 and 6, respectively, were typed ded in paraffin. DNA was extracted from tissue slices as with markers D7S486 and D7S514. described.29 Fluorescent markers D7S531, D7S507, D7S493, D7S519, D7S515, D7S486, and D7S636, part of the ABI PRISM Link- age Mapping Set, panel 12 (Applied Biosystems Division of Perkin Elmer (PE/ABI), Foster City, CA, USA), were used for AML patients with no abnormalities of chromosome 7 microsatellite analyses of AML patients without chromosome 7 aberrations. The PCR assays were performed according to In addition to patients with 7q−, 28 patients with AML and the manufacturer’s instructions in the ABI Catalyst 800 Labst- no cytogenetic abnormalities of chromosome 7 were studied. ation, and analyzed together with Tamra-500 as an internal Four of them had other than chromosome 7 abnormalities and size standard on a 373 Stretch DNA Sequencer using Gene- 24 had normal karyotypes in the cytogenetic analysis. DNA Scan software (PE/ABI). Allele sizing was performed with the samples from both blood cells and bone marrow cells were Genotyper 1.1 program. The degree of heterozygosity was obtained from 17 patients, blood cells alone from 10 patients, determined for each marker. In cases where both bone mar- and bone marrow cells alone from one patient. row and blood cell DNA was available, the heterozygosity of the two samples was compared. Non-leukemic controls Results The population frequency of heterozygosity for chromosome 7 markers was estimated from samples used to map recessive Comparative genomic hybridization disease genes in the laboratories of Department of Medical Genetics, University of Helsinki.