Analysis of a T-cell tumor-specific breakpoint cluster at human chromosome 14 q 32 ( inversion 14 / translocation 14 / ataxia-telangiectasia / T-cell leukemia / 14 q 32 . 1 proximal locus )

Cytogenetic abnormalities involving chromosome 14 band q32 are consistently observed in human T-cell tumors. Patients with ataxia-telangiectasia (AT) are especially prone to development of these tumors, which frequently carry either inversion inv(14)(qll;q32) or translocation t(14;14) (qll;q32) chromosomes. We have previously shown that the cytogenetic breakpoints of one t(14;14)(qll;q32) chromosome and two inv(14)(qll;q32) chromosomes in T-cell tumors from AT and non-AT patients join the T-cell receptor a chain locus, at chromosome band 14qll, with a region(s) at 14q32 centromeric of the immunoglobulin heavy chain variable region (VH) gene IGHV. We now show that these two inv(14) breakpoints are linked by 2.1 kb of germ-line 14q32 DNA and that the three breakpoints define, by in situ hybridization analysis, a single locus at chromosome band 14q32.1 located about 15-20 million base pairs on the centromeric side of the IGH locus. Sequence analysis of the 14q32.1 breakpoint regions indicates that abnormal recombination does not universally result from mistaken V-D-J joining (D = diversity region; J = joining region). Therefore, we invoke a tumor selection model to describe the role of the 14q32.1 locus in tumor development. Nonrandom karyotypic abnormalities (translocations, inversions, and deletions) are consistently found in many human lymphoid tumors. The frequency and phenotypic specificity of these cytogenetic aberrations suggest a causative role in lymphoid cell oncogenesis, presumably by interfering with the normal function of a gene near the site of chromosomal recombination. Many T-cell tumors carry cytogenetic changes involving chromosome 14 and, in particular, band q32. A large proportion of patients with T-cell chronic lymphocytic leukemia (T-CLL), T-cell prolymphocytic leukemia (T-PLL), T-cell lymphoma, and HTLV-I-associated adult T-cell leukemia carry 14q32 abnormalities, most often the inversion inv(14)(qll;q32) or the translocation t(14;14)(qll;q32) (1-4). In addition, patients with ataxia-telangiectasia (AT) show an increased incidence ofT-cell malignancies with inv(14) or t(14;14) chromosomes (5). Molecular analysis of the inv(14)(qll;q32) and t(14;14) (qll;q32) cytogenetic junctions from several AT and nonAT-derived T-cell tumors has shown that the cytogenetic breakpoint at 14q11 occurs within the 85-kilobase (kb)joining (J) region of the T-cell receptor (TCR) a chain locus designated TCRA (6-9). However, the breakpoints at 14q32 are more heterogeneous. In one T-cell tumor (SUP-Ti), an inv(14)(qll;q32) chromosome has a break at 14q32 within the immunoglobulin heavy chain locus (IGH) (6,7) resulting from site-specific recombination of IGH variable (V) region (IGHV) and TCRA Ja gene segments, to form a TCR-IGHV fusion gene. This implicates recombinase-mediated joining of specific signal sequences in the mechanism of recombination. However, we and others have described two inv(14)(q11;q32) and two t(14;14)(qll;q32) chromosomes from AT cells and from non-AT T-CLL and T-PLL cells with 14q32 breakpoints centromeric to the IGHV region (8-10). In addition, a t(7;14)(q35;q32) translocation in an AT patient joins the TCR ,8 chain locus, TCRB, at 7q35 to a region centromeric of the IGH locus (11). It is unclear whether these centromeric breakpoints constitute a single locus or multiple loci at 14q32. Additionally, the relationship of 14q32 breakpoints in T-cell tumors from AT and non-AT patients has not yet been studied. Here, we report the linkage of inv(14) and t(14;14) breakpoints at 14q32.1 in AT and non-AT-derived T-cell tumors. $ MATERIALS AND METHODS Molecular Cloning, Sequencing, and Hybridization. A human B-cell genomic library in phage A2001 (12) was screened with inv(14) and t(14;14) breakpoint-region probes to isolate A clones containing the germ-line regions surrounding the breakpoints at 14q32. Southern filter hybridization was carried out as described (8) with random hexamer-labeled probes (13). DNA sequence analysis was carried out by using the dideoxy chain-termination method (14) in M13 phage vectors. The various probes used in this work are detailed in Table 1 and also indicated in figures when relevant. In Situ Hybridization. In situ hybridization to metaphase spreads from normal human lymphocytes was performed with biotin-labeled probes detected by immunofluorescence (17, 18). This method for in situ hybridization using biotin labeling is fully described elsewhere (19). In brief, probes were nick-translated in the presence of 5-allylaminobiotinlabeled deoxyuridine triphosphate. The labeled products were hybridized (17, 18) to metaphase spreads, and the specific signal was detected with rabbit anti-biotin and Texas red-labeled goat anti-rabbit 1g. Images were analyzed by an Intellect 100 image-processing system as described (19). A heterologous ribosomal DNA marker is included for accurate alignment of the fluorescent images of the chromosomes stained with Hoechst 33258 and for the Texas red immunofluorescence detection of the site of hybridization. RESULTS AND DISCUSSION Molecular Cloning of a Breakpoint Cluster Region from 14q32. We previously reported the molecular cloning of (qll;q32) chromosomal rearrangement junctions from two inv(14) chromosomes (designated Linv and AT5B1) (8, 9) and Abbreviations: AT, ataxia-telangiectasia; TCR, T-cell receptor; V, variable; J, joining; D, diversity; C, constant; T-CLL, T-cell chronic lymphocytic leukemia; T-PLL, T-cell prolymphocytic leukemia. tPresent address: Monsanto Co., 700 Chesterfield Village Parkway, Saint Louis, MO 63198. $The sequence reported in this paper is being deposited in the EMBL/GenBank data base (accession no. J04136). 9171 The 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. 9172 Genetics: Mengle-Gaw et al. Table 1. Summary of DNA probes used in the in situ and filter hybridization experiments Probe Fig. Tumor origin Ref.