Proc. Nati. Acad. Sci. USA Vol. 84, pp. 9069-9073, December 1987 Genetics The breakpoint of an inversion of chromosome 14 in a T-cell leukemia: Sequences downstream of the immunoglobulin heavy chain locus are implicated in tumorigenesis (T-cell receptor/ataxia-telangiectasia) R. BAER*t, A. HEPPELLt, A. M. R. TAYLORt, P. H. RABBITTS§, B. BOULLIER§, AND T. H. RABBITTS* *Medical Research Council Laboratory of Molecular Biology and §Ludwig Institute for Cancer Research, Hills Road, Cambridge, CB2 2QH, England; and tUniversity of Birmingham, Cancer Research Laboratories, Department of Cancer Studies, the Medical School, Birmingham, B15 2TJ, England Communicated by C. Milstein, August 11, 1987 (received for review July 13, 1987) ABSTRACT T-cell tumors are characterized by inversions this alternative view. Cytogenetic studies of inv(14) chromo- or translocations of chromosome 14. The breakpoints of these some, by high resolution banding, have identified two differ- karyotypic abnormalities occur in chromosome bands 14qll ent break-reassociation points involved in inv(14) chromo- and 14q32-the same bands in which the T-cell receptor (TCR) somes (15). Notably, the 14q32 breakpoints of nonmalignant a-chain and immunoglobulin heavy chain genes have been clone inversions associated in ataxia-telangiectasia (A-T) are mapped, respectively. Patients with ataxia-telangiectasia are distinct from the 14q32 breakpoints of sporadic inversions particularly prone to development of T-cell chronic lympho- from normal subjects. A similar dichotomy of 14q32 cytic leukemia with such chromosomal abnormalities. We now breakpoints was found in clonal and sporadic t(14;14)(qll;- describe DNA rearrangements of the TCR a-chain gene in an q32) translocations (16). Furthermore, we have analyzed by ataxia-telangiectasia-associated leukemia containing both a in situ hybridization techniques, a clonal expansion ofT cells normal and an inverted chromosome 14. The normal chromo- some 14 has undergone a productive join of TCR a-chain containing translocation t(14;14)(qll;q32) in an A-T patient variable (Va) and joining (Ja) gene segments. The other allele and found the break at 14q32 on the centromeric side of the ofthe TCR a-chain gene features a DNA rearrangement, about IGH locus at 14q32.1 (17) (i.e., downstream of IGH locus). 50 kilobases from the TCR a-chain constant (Ca) gene, that A-T is a complex disorder in which patients are apparently represents the breakpoint of the chromosome 14 inversion; this at higher risk of cancer and in particular T-cell leukemia (18) breakpoint is comprised of a TCR Ja segment (from 14qll) associated with marker chromosomes 14 [e.g., inv(14) and fused to sequences derived from 14q32 but on the centromeric t(14;14)] (19, 20). A T-cell chronic lymphocytic leukemia side of C.. These results imply that 14q32 sequences located at (CLL) has been previously described in a patient with A-T in an undetermined distance downstream of the immunoglobulin which an inv(14) chromosome was present (3). The evolution C,, locus can contribute to the development of T-cell tumors. of this malignant clone was studied over a number of years from the apparently preleukemic state until overt cancer (20). The association of chromosomal abnormalities with human Similar studies have been carried out on an A-T associated tumors indicates that such abnormalities may play a caus- t(14;14) (21) where a preleukemic clone apparently developed ative role in cancer. In human T-cell leukemias, a number of into CLL. The tumor cells of the former patient, designated recurring abnormalities involving chromosome 14 band qll AT5B1, provided an opportunity to study the inv(14) chro- have been reported. The most frequent are inversion (inv) of mosomal abnormality in A-T. We now report an analysis of chromosome 14 (qil ;q32), the analogous translocation the inv(14) chromosome from this tumor$ and show that the t(14;14)(qll;q32) (1-5), and t(8;14)(q24;qll) (6-9). Recent 14q32 breakpoint occurs downstream of the immunoglobulin work on the molecular cloning ofbreakpoints associated with ,u-chain constant (C) region gene, whereas the breakpoint at these abnormalities has shown that the 14q11 region involved 14q11 occurs in TCR Ja. These data strongly support the view contains the T-cell receptor (TCR) a-chain gene and that that sequences located downstream of immunoglobulin C, breakpoints specifically occur within the joining (J) region of region at 14q32 can be important for T-cell tumorigenesis this locus (6, 10-12). The inv(14) chromosome is especially (12). interesting since the cytogenetic breakpoints coincide with the chromosome bands in which the TCR a-chain gene maps (14q11) and the immunoglobulin heavy chain genes (IGH; MATERIALS AND METHODS 14q32) are localized. Indeed, it has been shown that the Molecular Cloning and Hybridizations. A library ofgenom- inv(14) chromosome in a T-cell lymphoma cell line (SUP-Ti) ic DNA prepared from leukemic cells (unfractionated periph- was generated by recombination between TCR Ja and im- eral blood) from the AT5B1 patient was constructed in X2001 munoglobulin heavy (H) chain variable (V) region genes (10, (22). The percentage of leukemic cells bearing inv(14) in the 11, 13). Such an association of the a-chain TCR and immu- sampled blood was about 90%. X clones containing rear- noglobulin VH loci was also reported in a rare B-cell tumor were with carrying an inv(14) chromosome (14). ranged segments isolated by screening (23) JaSP The possibility that such gene fusions constitute tumori- Abbreviations: TCR, T-cell receptor; J, joining; V, variable; C, genic events must be considered. However, a different constant; A-T, ataxia-telangiectasia; CLL, T-cell chronic lympho- possibility is that two forms of inv(14) chromosome occur, cytic leukemia; H, heavy. one or both of which might be involved in T-cell tumor tPresent address: University of Texas Health Science Center, 5323 development. There are several lines of evidence to suggest Harry Hines Boulevard, Dallas, TX 75235. 9The sequences reported in this paper are being deposited in the EMBL/GenBank data base (Bolt, Beranek, and Newman Labora- The publication costs of this article were defrayed in part by page charge tories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg) payment. This article must therefore be hereby marked "advertisement" (accession no. J03597, rearranged normal chromosome 14; acces- in accordance with 18 U.S.C. §1734 solely to indicate this fact. sion no. J03598, rearranged inverted chromosome 14). 9069 Downloaded by guest on September 29, 2021 9070 Genetics: Baer et al. Proc. Natl. Acad. Sci. USA 84 (1987) (24) and JaBB (see Fig. 2) probes, and subclones were kilobases (kb) from the Ca gene. Recombinant clones were prepared from the X phages in pUC or M13 vectors (25). obtained from a genomic library of AT5B1 DNA, which Genomic hybridization was conducted by fractionation of corresponded to these rearrangements. One set of clones 10 ,ug of digested DNA on 0.8% agarose followed by transfer covered the rearrangement at about 5 kb from Ca and was to cellulose nitrate filters (26). Hybridizations were carried isolated using the previously described JaSP probe (24). A out and monitored as described (27) using probes labeled by representative clone (XA87) is shown in Fig. 1A. Comparison random oligonucleotide primer procedures (28). Somatic cell ofthe restriction map from XA87 with that ofunrearranged Ja hybrids of t(X;14) have been previously described, and the clones located the point of rearrangement near a Ja segment hybridization characteristics of JH and Ja probes have designated JsP. Nucleotide sequence analysis at this position been assessed (12). Somatic cell hybrids of Raji cells revealed that a TCR Va segment had joined to JsP. This Va [carrying t(8;14)(q24;q32)] and Chinese hamster ovary cells (V'-3) segment is identical to a previously described unrear- were prepared using hypoxanthine/aminopterin/thymidine ranged segment (35) belonging to the Va family expressed in growth medium supplemented with ouabain (29). Segregant JM cells. Furthermore, the rearrangement is productive at somatic cell clones were isolated by ring cloning, DNA was the genomic level, indicating the potential ofthe AT5B1 cells prepared, and the presence of the relevant chromosomes in to produce TCR a-chain protein. This is consistent with the hybrids was determined by the hybridization ofimmunoglob- observed surface phenotype of the tumor cells, which in- ulin VH and JH and TCR Ja. probes (data not shown). A clone cludes the TCR-associated protein CD3. (SH-9-10) containing the 8q- chromosome [which has 14q32 Molecular Cloning of the inv(14) Breakpoint. Two further -- qter from the JH locus (30)] but not normal 14 or 14q+ TCR Ja rearrangements occur in AT5B1 DNA. A recombi- chromosomes was selected for use, and another clone (SH4- nant X phage clone (XA23) was isolated using the JaBB probe, 9-1) with only 8q- and 14q+ chromosomes (but not normal and this phage DNA encompasses both rearrangements. One chromosome 14) was utilized as a control. rearrangement represents an 8.1-kb deletion in the Jcr locus of Nucleotide Sequencing. Dideoxynucleotide sequencing was this T cell (R.B. and T.H.R., unpublished results), and the carried out using M13 as described (31, 32). Sequence other rearrangment (approximately 50 kb from Ca) was alignment was facilitated using computer comparisons (33). located near the end of XA23. Preliminary genomic hybrid- In Situ Hybridization. In situ hybridization to metaphase ization placed the latter rearrangement within the region of chromosomes was carried out as described (17). 3H-labeled probes were prepared by nick-translation (34). For grain the A23HR fragment since we found a rearranged DNA analysis, chromosome 14 was divided into four arbitrary fragment in HindIII digests but not in EcoRI digests of sections, and grains falling within each section were summed AT5B1 DNA using the probe JaSH (shown in Fig.