Leukemia (2001) 15, 371–376  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Deletion of chromosomal region 13q14.3 in childhood acute lymphoblastic leukemia H Cave ´ 1,2 , H Avet-Loiseau 3 , I Devaux 2 , G Rondeau 4 , P Boutard 5 , E Lebrun 6 , F Me ´chinaud 7 , E Vilmer 8 and B Grandchamp 2 1 Laboratoire de Biochimie Ge ´ne ´tique, Ho ˆ pital Robert Debre ´, Paris; 2 INSERM U409, Faculte ´ de Me ´decine Xavier Bichat, Paris; 3 Laboratoire de Cytoge ´ne ´tique, Ho ˆ pital de Nantes, Nantes; 4 INSERM U463, Centre Hospitalier Universitaire, Nantes; 5 Unite ´ d’Onco-He ´matologie Pe ´diatrique, CHU, Caen; 6 Etablissement de Transfusion Sanguine, Caen; 7 Service d’He ´matologie clinique, Ho ˆ tel Dieu, Nantes; and 8 Service d’He ´matologie Pe ´diatrique, Ho ˆ pital Robert Debre ´, Paris, France Deletion of the 13q14 chromosomal region is frequent in B cell chronic lymphocytic leukemia (B-CLL) and is believed to inacti- vate a tumor supressor gene (TSG) next to RB1. We studied microsatellite markers spanning the 13q14 chromosomal region in 138 children with acute lymphoblastic leukemia (ALL). Allelic loss was demonstrated in six cases (4.3%). Deletion did not include RB1 in two cases. In five patients, the deleted region overlapped that described in B-CLL. A sixth patient harbored a smaller deletion, slightly more telomeric than mini- mal deleted regions reported in B-CLL. Apparent differences in the delineation of the minimal deleted region could be due to the fact that the putative TSG is a very large gene, with some deletions affecting only a part of it. Our present findings sug- gest that at least some of its exons lie within a region of less than 100 kb more telomeric that previously thought. Leukemia (2001) 15, 371–376. Keywords: chromosome 13q14.3; tumor suppressor gene; loss of heterozygosity; childhood acute lymphoblastic leukemia Introduction Most of the alterations that have been characterized so far in childhood acute lymphoblastic leukemia (ALL) are non-ran- dom translocations that give rise to oncogenic fusion proteins. In contrast, very few instances of inactivation of tumor sup- pressor genes (TSG) have been reported. However, cytog- enetic and loss of heterozygosity (LOH) data point to the exist- ence of several recurrently deleted chromosomal regions in leukemia blasts. 1–3 These chromosomal regions are likely to harbor TSG whose inactivation is involved in leukemogenesis. The 13q14 chromosomal region is known to contain the reti- noblastoma susceptibility gene, RB1, which is frequently inac- tivated in solid tumors. 4 RB1 gene deletions are rarely seen in ALL but exceptional cases have been reported. 5,6 More recently, a locus slightly telomeric to RB1 was shown to be the target of heterozygous or homozygous deletion in 25 to 60% cases of B cell chronic lymphocytic leukemia (B-CLL). 7,8 This locus might be involved, although to a lesser extent, in other hematologic malignancies such as mantle cell lym- phoma, 9,10 myeloid leukemia 11 and multiple myeloma. 12 These findings have led to the hypothesis that a second TSG exists in the 13q14 chromosomal region and is located close to RB1. However, despite strong evidence for another TSG distal to RB1, the precise location of this putative TSG is still controversial and studies aimed at identification of this gene have so far proved inconclusive. In the present study, we assessed LOH of microsatellite mar- kers spanning the chromosomal region 13q14 to determine the frequency of subkaryotypic deletions and to delineate the region of interest in childhood ALL. Correspondence: H Cave ´, Laboratoire de Biochimie ge ´ne ´tique, Ho ˆ pi- tal Robert Debre ´, 48, Boulevard Se ´rurier, 75019 Paris, France; Fax: 33-(0)1 40 03 22 77 Received 22 August 2000; accepted 15 November 2000 Patients and methods Patients We studied 138 children (aged 3 months to 15 years) with ALL (116 B-lineage ALL, 22 T-ALL). Six of these children were studied both at diagnosis and at marrow relapse. Children were consecutive unselected patients except that inclusion required the presence of at least 70% of blasts in marrow at the time of diagnosis. Cases of ALL with near haploid kary- otype were excluded from the study. T-ALL was defined as presence of at least two of the T cell antigens CD2, CD5, and CD7. The diagnosis of B-precursor ALL was based on the expression of B cell-associated antigens (CD19 + , CD22 ± , CD10 ± ) and the lack of surface membrane immunoglobulins (Ig). B-precursor ALL displaying cytoplasmic -heavy chains (10% of cells) but lacking surface membrane Ig were referred to as pre B-ALL. All others were designated early pre B-ALL. Karyotype studies and search for the ETV6-AML1 fusion transcript associated with t(12;21) were systematically performed at diagnosis. Bone marrow (BM) was collected on EDTA before induction therapy and during clinical remission according to the EORTC 58 881 follow-up protocol. Mononuclear cells were separated and DNA was prepared as previously described. 13 Informed consent was obtained from the patients, their par- ents, or both, as appropriate. An extensive allelotype was previously reported for 55 of these patients, the only marker in common with the present study was D13S262. 3 Markers Twelve simple tandem repeat (STR) markers were used that span the chromosome band 13q14. The order of these mark- ers from centromere to telomere is according to cartography data 14 (Figure 1): D13S153, D13S273, D13S272, D13S319, AFM206xf12, AFMa301wb5, D13S1269, D13S25, D13S262, D13S294, D13S288, and D13S284. Sequences were obtained from the Genome Database (http://www.gdb.org). Identification of new microsatellite polymorphisms Cosmids P0445, A1124, O0064 and K1028 14 were digested with SauIIIa, Southern blotted and hybridized with a CA repeat probe (Pharmacia, Uppsala, Sweden) to reveal the pres- ence of new potentially polymorphic markers within the region. Digested bands positive for hybridization were excised from the gel, ligated in PGEM11Z, and subjected to a second hybridization screening after bacterial transformation and fil- ter transfer. Inserts from positive clones were sequenced. Two new CA repeats named O13 and K16 were characterized. Pri-