Fusion Transcripts Involving HMGA2 Are Not a Common Molecular Mechanism in Uterine Leiomyomata with Rearrangements in 12Q151

[CANCER RESEARCH 63, 1351–1358, March 15, 2003] Fusion Transcripts Involving HMGA2 Are not a Common Molecular Mechanism in Uterine Leiomyomata with Rearrangements in 12q151

Bradley J. Quade, Stanislawa Weremowicz, David M. Neskey, Roberta Vanni, Carll Ladd, Paola Dal Cin, and Cynthia C. Morton2 Departments of Pathology [B. J. Q., S. W., P. D. C., C. C. M.] and Obstetrics, Gynecology, and Reproductive Biology [D. M. N., C. C. M.], Brigham and Women’s Hospital and Harvard Medical School [B. J. Q., S. W., P. D. C., C. C. M.], Boston, Massachusetts 02115; Sezione di Biologia e Genetica, Dipartimento Scienze Applicate ai Biosistemi, University of Cagliari, 09042 Monerrato (Cagliari), Italy [R. V.]; and Connecticut State Police Forensic Science Laboratory, Meriden, Connecticut 06451 [C. L.]

ABSTRACT The chromosome 12 breakpoint region has been mapped by FISH4 in many of the involved tumor types. In each case, it is located in or Uterine leiomyomata are one of several benign tumors characterized by near the DNA architectural factor HMGA2 (formerly HMGI-C), sug- frequent chromosomal rearrangement involving 12q15. The 12q15 rear- gesting that HMGA2 participates in a wide spectrum of benign mes- rangement in leiomyomata typically is manifested as t(12;14)(q15;q23-24), enchymal tumors. In lipomas, balanced translocations create fusion which has been hypothesized to create pathobiologically significant fusion genes between HMGA2 and translocation partners such as lipoma- transcripts derived from HMGA2 and RAD51L1. To explore further this preferred partner and lipoma HMGIC fusion partner (1, 2). Three hypothesis, we mapped chromosomal breakpoints in 38 uterine leiomyomata DNA binding domains encoded in the first three exons of HMGA2 are with rearrangements involving 12q15 using fluorescence in situ hybridization. .harbored der(14)t(12;14)(q15;q23-24), whereas chro- present in these fusion transcripts (26 ؍ Most tumors (n mosomes 1, 5, 8, and 10 were involved in rearrangements with 12q15 in six Involvement of 14q23-24 in rearrangements with 12q15 is rela- myomas. An additional six cases had more complex rearrangements, includ- tively specific for those mesenchymal tumors with full or partial ing breakpoints other than 12q15 or 14q23-24, inversions of chromosome 12, smooth muscle differentiation. Rearrangements of 14q23-24 are found insertions of 12q15 into chromosome 14, or additional translocation partners. in endometrial polyps and pulmonary chondroid hamartomata. The Breakpoints were mapped either 5؅ (centromeric) or 3؅ (telomeric) in the chromosome 14 breakpoint region in uterine leiomyomata has been HMGA2 locus in 24 and nine cases, respectively; one tumor was a mosaic of mapped within the DNA repair gene RAD51L1, which spans a large cells with either 5؅ or 3؅ breakpoints. Breakpoints flanking the gene in both genomic region of ϳ680 kb. It has been hypothesized that a HMGA2- ,5؅ and 3؅ regions were found in six cases. Analysis of one tumor by 3؅ rapid RAD51L1 fusion transcript, the reciprocal fusion transcript, or both amplification of cDNA ends showed altered transcripts in which either exons play an important role in leiomyomata (3–5). We mapped the chro- 1–3 of HMGA2 were aberrantly spliced to cryptic sites in chromosome 12 or mosome 12 breakpoints relative to the HMGA2 locus in a series of 38 transcripts encompassing the full coding sequence of HMGA2 through a uterine leiomyomata with 12q15 rearrangements and found that 12q15 portion of the 3؅ untranslated region were fused to sequence from chromo- rearrangements mapped predominantly in the 5Ј region of HMGA2.In some 14. A panel of 10 uterine leiomyomata with t(12;14) was specifically a subset, we tested for the presence of reciprocal fusion transcripts tested for fusion transcripts. RAD51L1-HMGA2 transcripts were not de- involving HMGA2 and RAD51L1. tected. HMGA2-RAD51L1 transcripts, however, were detected in four tumors; two of these tumors had uncommon rearrangements in the 3؅ region of MATERIALS AND METHODS HMGA2 and two had 5؅ rearrangements. Although the mechanism of fusion ؅ transcripts derived from tumors with 5 breakpoints is unclear, these findings Cytogenetics and FISH. Tissues from leiomyomata were obtained in indicate that formation of a fusion transcript is not the principle pathobio- accordance with institutional guidelines concerning use of human tissues. logical mechanism in uterine leiomyomata. The pattern of rearrangements Conventional cytogenetic analysis was performed as described previously (6). suggests dysregulated expression of HMGA2, most often by translocation of Karyotypes for the analyzed tumors are provided online.5 Cosmid probes chromosome 14 sequence 5؅ to this gene. located 5Ј (centromeric) in the HMGA2 locus, specifically 245E8, 145E1, and 142H1 (7), were labeled for FISH with biotin using the BioNick Labeling System (Invitrogen) and detected using the Oncor Kit (Oncor). Cosmids INTRODUCTION located 3Ј in the HMGA2 locus, namely 46G3, 59A1, 27E12, and 50D7, were labeled with digoxigenin-11-dUTP and detected with antidigoxigenin-rhoda- Benign uterine smooth muscle tumors (commonly known as mine. BAC 475C2 was labeled with biotin and detected using a fluorescein- leiomyomata, myomas or fibroids) occur in ϳ80% of females of avidin conjugate. FISH with ␭ clones H121, H206, H2, H4, H311, H738, reproductive age, arguably making them the most frequent human H501, H111, H528, H403, and H409 has been reported previously (8). neoplasm. Approximately 40% of leiomyomata have cytogenetic ab- Reverse Transcription and PCR Analysis. Total RNA from each sample was first transcribed with Superscript II reverse transcriptase (Invitrogen) and normalities. One of the most frequent rearrangements is t(12;14)(q15; an oligo-dT primer. After confirmation of RNA integrity by RT-PCR for q23-24). This translocation is present in 59 of 383 (or 15.4%) uterine GAPDH, cDNA samples were subjected to a variety of PCR assays. Primers leiomyomata at the Mitelman Database of Chromosome Abnormali- for HMGA2 and RAD51L1 were named according to their starting nucleotide ties web site (August 14, 2002 release).3 Rearrangements of 12q15 are in either of GenBank U28749 for HMGA2 or GenBank U92074 for RAD51L1, also found in a number of other benign mesenchymal tumors. the letter U or L to denote whether the primer was derived from the upper or lower strand, and the primer length in bases, respectively. Primer sequences also may be found online.5 Assays for HMGA2 transcripts were performed Received 9/23/02; accepted 1/31/03. with PCR conditions using 1 min of denaturation at 94°C, followed by 30 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with cycles of amplification (45 s at 94°C and 90 s at 68°C). Conditions for 18 U.S.C. Section 1734 solely to indicate this fact. amplifying RT-PCR products from RAD51L1 were modified to include a 30 s 1 Supported by the National Cancer Institute, NIH Grants R01 CA078895 (to C. C. M.) step at 60°C for annealing. and K08 CA72594 (to B. J. Q.) and a grant from Assessorato Igiene e Sanita´Regione Autonoma Sardegna (to R. V.). 2 To whom requests for reprints should be addressed, at Department of Pathology, 4 The abbreviations used are: FISH, fluorescence in situ hybridization; RT-PCR, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. Phone: (617) 732- reverse transcription-PCR; RACE, rapid amplification of cDNA ends; BAC, bacterial 7980; Fax: (617) 738-6996; E-mail: [email protected]. artificial chromosome. 3 Internet address: cgap.nci.nih.gov/Chromosomes/Mitelman. 5 Internet address: fibroids.net/research/hmga2_supplement.htm. 1351

Two sets of assays to detect a fusion transcript involving HMGA2 and region of HMGA2 and the chromosome 12 breakpoint was variable RAD51L1 were performed. The first reaction involved using 5Ј RAD51L1 relative to the 5Ј end of HMGA2. In many myomas (e.g., 112427 and upper primers 20U25 or 536U23 with 3Ј HMGA2 lower primers 1115L22 or 153759), the chromosome 12 breakpoint was centromeric to the most 1047L25. After this, products were subjected to nested amplification using 5Ј 5Ј clone tested by FISH, indicating that the 5Ј breakpoint was Ͼ100 Ј HMGA2 upper primers 712U24 or 959U25 with 3 RAD51L1 lower primers kb upstream. In five cases, the breakpoint was localized to within a 976L24 or 1040L24. All reactions were performed using the PCR conditions genomic clone or between clones (Fig. 2). Our mapping of ST90-194 described above for amplification of RAD51L1 RT-PCR products. ϳ Ј -3؅ RACE. Total RNA isolated from tumor 105274 was used for 3Ј RACE 10 kb 5 of HMGA2 has been confirmed independently (9), repre Ј Ј (Invitrogen). The 5Ј HMGA2 primer in the first round of amplification was senting the tumor with a 5 breakpoint closest to the 5 end of HMGA2 849U19. The hot start PCR profile was as follows: 3 min at 94°C for in this series. denaturation; 7 min at 80°C to allow for addition of eLONGgase polymerase Mapping data from tumor 166931 revealed the presence of re- mix (Invitrogen); 5 cycles of 30 s at 94°C and 4 min at 70°C; 5 cycles of 30 s arrangements both 5Ј and 3Ј in the HMGA2 locus (Figs. 1B and 2). at 94°C,30sat65°C and 4 min at 70°C; and 30 cycles of 30 s at 94°C, 30 s Specifically, the chromosome 12 breakpoint was mapped between at 60°C and 4 min at 70°C. The nested 5Ј HMGA2 primer in the second round clones 145E1 and 142H1 in 90% of cells (top and middle rows in Fig. was 909U19. The second round product was cloned into pCR2.1-TOPO vector 1B), whereas the rearrangement was mapped within clone 27E12 in (Invitrogen). the remaining 10% of cells (bottom row in Fig. 1B). These results Radiation Hybrid Mapping. The novel sequence in the 3Ј RACE product indicated that this myoma contained two lineages, each with re- B11 was selected for radiation hybrid mapping. The sequences for the upper (ect1U) and lower (ect1L) primers were 5Ј-ATTACTGCTTAAAATCTCA- arrangements at opposite ends of the HMGA2 locus. To exclude the CACTG-3Ј and 5Ј-CAAACCATATGTAGAAAATGCTC-3Ј, respectively. unlikely possibility of admixture with cells from another tumor, STR DNAs from monochromosomal hybrid cell lines for chromosomes 12 polymorphisms were compared from myomas 166931 and 105274, (GM10868) and 14 (GM10479 and GM11535) were obtained from the Na- which both have a 3Ј rearrangement. Results of DNA profiling tional Institute of General Medical Sciences Human Genetic Cell Repository showed at most two alleles for each polymorphism tested. Therefore, (Coriell Cell Repositories). Radiation hybrid mapping of the amplicon was the mapping results in myoma 166931 are due to two independent performed using the GeneBridge 4 radiation hybrid mapping panel (Invitro- chromosomal translocations flanking HMGA2. Whether this case rep- gen), and results were submitted to the Whitehead Institute Center for Genome resents two independent tumors that grew together or the result of 6 Research Mapping Server for chromosomal assignment. secondary rearrangements after neoplastic transformation is unknown. Isolation and Characterization of BAC 475C2. A BAC clone containing Some Chromosome 12 Breakpoints Are 3؅ (Telomeric) in the genomic DNA corresponding to the novel chromosome 14-specific sequence in plasmid B11 was isolated by screening the CITB Human BAC DNA Pools HMGA2 Locus. Nine myomas, including four with simple t(12;14) Ј (B&C Libraries), Release III (Invitrogen). The PCR primers and profile used rearrangements, were mapped to the 3 region of HMGA2 (Figs. 1C to screen the pools were the same as that for radiation hybrid mapping and 2). Five myomas had breakpoints clearly 3Ј of HMGA2 because described above. Partial sequencing of this clone was performed by high the breakpoint mapped telomeric to cosmid 27E12. Three of these throughput sequencing of subclones generated by digestion with Sau3A in the breakpoints were located even further 3Ј because they were also Collis Genome Laboratory at Brigham and Women’s Hospital. telomeric to cosmid 50D7. Of note, the chromosome 12 breakpoint STR Analysis. DNA samples isolated from tumors 166931 and 105274 was located within clone 27E12 in four myomas. Cosmid 27E12 were typed for 13 unique polymorphic STR loci using the AmpFlSTR Profiler includes a small portion of intron 3 through the end of the 3Ј untrans- Plus and COfiler kits (Applied Biosystems) and the amelogenin locus. STR lated region of HMGA2, and breakpoints mapping within this clone polymorphisms were resolved on an ABI 377 DNA Sequencer and analyzed potentially may disrupt HMGA2. using GeneScan and Genotyper software packages. A Minority of Uterine Leiomyomata Harbor more Com- plex Rearrangements of 12q15. Mapping of eight cases with more RESULTS complex chromosomal rearrangements suggests that two breakpoints involved the HMGA2 locus. One of these breakpoints mapped cen- Cytogenetic Analysis of 12q15 Breakpoint in Uterine Leiomy- tromeric to the 5Ј region of HMGA2 and the other breakpoint mapped omata. Thirty-eight uterine leiomyomata were analyzed by FISH to within or telomeric to the 3Ј region. determine the location of chromosome 12 breakpoints relative to Myoma ST93-220 is an example of such a tumor with a complex HMGA2. Most tumors (n ϭ 26) studied harbored the der(14) chro- chromosomal rearrangement. The karyotype of ST93-220, mosome from a reciprocal balanced t(12;14)(q15;q23-24). In six 46,XX,t(12;14)(q13;q32), was notable because the breakpoints were cases, chromosomes 1 (ST92-224 and 119620), 5 (188319), 8 located in distinctly different bands on both derivatives in comparison (99923), and 10 (ST99-658 and ST99-175) were observed in variant to the typical t(12;14)(q15;q23-24). Hybridization with whole chro- rearrangements with 12q15. More complex rearrangements, including mosome painting probes wcp12 and wcp14 revealed a complex re- inversion of 12q15, insertion of 12q15 into chromosome 14, and arrangement and indicated four breakpoints [two on der(12) and two three-way translocations, were observed in five additional myomas on der(14)]. As shown in Fig. 1D, a segment of chromosome 12 was (ST01-901, ST00-348, ST00-370, 178605, and 190464). One myoma inserted into chromosome 14 at q23-24, and the remaining part of (ST93-220) had an unusual translocation by GTG-banding involving chromosome 12 (q15-qter) was reciprocally translocated to 14q32. 12q13 and 14q32. FISH with probes from the 5Ј and 3Ј regions of HMGA2 (Fig. 1D, Most Chromosome 12 Breakpoints Are 5؅ (Centromeric) in the inset) indicated that the gene was uninterrupted during this complex HMGA2 Locus. Mapping of myomas with chromosome 12 re- rearrangement. In another FISH experiment (data not shown), BAC arrangements identified a breakpoint located 5Ј (centromeric) in the 475C2 colocalized with cosmid 27E12 on the der(14). HMGA2 locus in 24 of 38 (63%) cases. In all of these tumors, both 5Ј Another example of a complex rearrangement involving chromo- and 3Ј hybridization signals were colocalized on the derivative chro- somes 10, 12, and 14 was revealed by FISH of myoma 190464. Whole mosome (e.g., the der(14)t(12;14)(q15;q23-24) in 22 of 27 myomas), chromosome painting with wcp12, wcp14, and wcp10 confirmed indicating that HMGA2 had remained intact during rearrangement insertion of a segment of chromosome 12 into 14q23-24 (Fig. 1E) and (Figs. 1, A and B, and 2). The distance between the 5Ј untranslated revealed a reciprocal translocation between the der(14) and the short arm of chromosome 10 (Fig. 1F). Interestingly, two sets of signals 6 Internet address: www-genome.wi.mit.edu/cgi-bin/contig/rhmapper. from cosmid 27E12 were detected on the der(14) at the insertion site 1352

Fig. 1. Chromosome breakpoint mapping by FISH. Partial metaphases from short-term cultures of uterine leiomyomata with rearrangements involving 12q15 are shown in A–H. FISH probes are indicated in green and red lettering for fluorescein and rhodamine-detected signals, respectively. 4Ј,6-Diamidino-2-phenylindole was used as counterstain. The location of cosmids relative to HMGA2 (7) is shown at the top; hatch marks drawn in the large HMGA2 intron between exons 3 and 4 indicate that this intron is not drawn to scale. A–C show chromosomes 12, der(12), 14 or der(14) from rep- resentative tumors with reciprocal t(12;14)(q15;q23-24), as determined by GTG-banding. A, in tumor 153759, the chromosome 12 breakpoint was mapped 5Ј to cosmids 142H1 (green in top row) and 145E1 (green in middle row). B, tumor 166931 was mosaic. The majority of cells (90%) harbored breakpoints telomeric on 12q to 145E1 (top row) and centromeric to 142H1 (middle row), and the minority showed a 3Ј breakpoint located within 27E12 (bottom row). C, in tumor 105274, the chromosome 12 breakpoint mapped in the 3Ј region of HMGA2 within cosmid 27E12 (top row), and the chromosome 14 breakpoint was located within BAC 475C2 (bottom row), corresponding to the RAD51L1 locus. FISH analysis of cases with rearrangements other than reciprocal t(12;14) are illustrated in D–H. D, hybridization of tumor ST93-220 with wcp12 and wcp14 was performed. The der(14) contained two segments of chromosome 12 separated by chromatin from chromosome 14. The inset of D shows that only a single intact copy of HMGA2 was present in the der(14). E and F, E and F are from the same metaphase of tumor 190464, but sections of the image in F have been moved to accommodate the inset. The GTG-banded karyotype was 46,XX,der(3)t(3;?10)(p14;q32),der(10)t(10;?14), del(12)(q15q21),der(14)ins(14;12)(q24;q15q21)t(3;14) (p14;q24) [15]. Rearrangements involving chromosomes 10, 12, and 14 are illustrated by FISH with wcp12 and wcp14 (E) and by wcp10 (F). The inset in F shows two signals for cosmid 27E12 on the der(14). Another complex chromosomal rearrangement was observed in tumor 119620. The GTG-banded karyotype was 46,XX,t(1; 12)(p32;q14-15) [20]. FISH with wcp12 (G) suggested an apparently simple t(1;12). However, FISH with probes from the 5Ј (cosmids 142H1 and 145E1 in green in the top and lower rows, respectively) and 3Ј (cosmid 27E12 and 50D7 in red in the top and lower rows, respectively) regions in the HMGA2 locus (H) indicated a more complex rearrangement. These results are compatible with the model in I in which an inversion of chromosome 12 reversed the order of cosmids on the der(12), followed by a translocation involving chromosomes 1 and 12 (within cosmid 27E12).

of the chromosome 12 material (Fig. 1F, inset) and indicated dupli- of the inv(12) in 27E12 (Fig. 1I). GTG-banding and FISH with BAC cation of HMGA2 at either end of the inserted segment. Another 475C2 (data not shown) indicated that RAD51L1 was not rearranged. experiment with clone 142H1 (data not shown) confirmed that In the tumors with complex rearrangements, we never observed a HMGA2 remained intact during this complex rearrangement. hybridization pattern in which 142H1 and 27E12 (5Ј and 3Ј probes in Myoma 119620 also had a complex rearrangement involving chro- the HMGA2 locus, respectively) mapped to wholly different deriva- mosome 12 but not chromosome 14. FISH with wcp12 was consistent tive chromosomes. Such an observation would have been clear evi- with a simple translocation (Fig. 1G). FISH with single copy probes, dence that the breakpoint had fallen within HMGA2. however, suggested a more complex rearrangement (Fig. 1H). Cosmid Analysis of a Uterine Leiomyoma with a Breakpoint 3؅ in 27E12, which corresponds to the 3Ј region of HMGA2, hybridized to HMGA2 by 3؅ RACE. FISH mapping of myoma 105274 showed that both der(1) and der(12) chromosomes, indicating that a breakpoint the chromosome 12 breakpoint was within cosmid 27E12, which occurred in the 3Ј region of HMGA2. Paradoxically, the most telo- contains a portion of intron 3 through the 3Ј untranslated region of meric probe (cosmid 50D7) hybridized to the der(12) chromosome, HMGA2. This finding raised the possibility that the exons encoding whereas the most centromeric probes (cosmids 142H1 and 145E1) the DNA binding domains were fused to another gene such as hybridized to the der(1) chromosome. This hybridization pattern could RAD51L1.3Ј RACE was performed to search for such transcripts. Of be explained by paracentric inversion of chromosome 12 with break- five cloned RACE products characterized (Fig. 3A), none were full- points flanking HMGA2 centromeric to 145E1 and telomeric to 50D7, length transcripts of HMGA2, and all contained unusual (ectopic) followed by a reciprocal translocation to chromosome 1 with a break sequence replacing either the fifth (last) or fourth and fifth exons. 1353

Fig. 2. Summary of chromosome 12 breakpoints mapped by FISH in uterine leiomyomata with rearrangements involving 12q15. The order and location of cosmid (top row) and ␭ (bottom row) clones are shown (in black) relative to the five exons of HMGA2 (in blue) given at the top (7, 8). Hatch marks in the large intron between exons 3 and 4 indicate that this intron is not drawn to scale. The identifiers of leiomyomata studied are shown in the vertical column on the left border. The location of breakpoints in 12q15 are shown in the central area. Horizontal lines indicate the interval in which the breakpoint has been mapped for any given tumor. Triple arrowheads denote instances where breakpoints mapped centromeric (left-facing) or telomeric (right-facing) to the indicated clones. Red lettering, lines, and arrowheads refer to breakpoints 5Ј in the HMGA2 locus, whereas green lettering, lines, and arrowheads refer to breakpoints 3Ј in the HMGA2 locus. Myoma 166931 is listed twice, reflecting cytogenetic mosaicism. Absence of hybridization for cosmid 142H1 with was associated with poor quality metaphases and limited material. Myomas indicated by black lettering are those in which breakpoints were (ءtumor ST96-692 (denoted as No Signal found both 5Ј and 3Ј in the HMGA2 locus.

Thus, these transcripts included the three DNA binding domains. sequently leaves HMGA2 coding sequence uninterrupted but trun- Novel sequences fused to HMGA2 were analyzed by BLAST and four cated the 3Ј untranslated region. Amplification using primers ect1U (B1, B3, B8, and B12) were identical to intronic sequence in HMGA2. and ect1L derived from the 241-bp novel sequence from transcript One 174-bp sequence (B1) was identical to a transcript (GenBank B11 and genomic DNA from monochromosomal hybrid cell lines U29115) previously detected in a different leiomyoma cell line indicated that the novel sequence was present on chromosome 14 (LM-30.1/SV40; Ref. 3). A second 763-bp sequence (B3, GenBank (data not shown). This amplicon was mapped with a LOD score of AF533652) contained a 271-bp segment identical to another previ- Ͼ3.0 to chromosome 14 between WI-5202 and D14S277 with the ously reported novel transcript (GenBank U29113) from leiomyoma GeneBridge 4 panel. BAC 475C2 was identified by screening pooled LM-30.1/SV40 (3). In addition, the first 357-bp of the 467-bp ectopic BAC clones and hybridized to the normal chromosome 14 and both sequence in transcript B8 (GenBank AF533652) was identical to derivative chromosomes in cell line 105274 (Fig. 1C). Subsequently, another previously reported sequence (GenBank U29112) found in it was determined that BAC 475C2 is located within the RAD51L1 leiomyoma LM-538/SV40 (3). The sequences in transcripts B3 and locus. The breakpoint on chromosome 14 maps between exons 7 and B8, however, extend an additional 492 and 110 bp, respectively. The 8inanϳ405-kb intron based on partial sequencing of the BAC clone longer ectopic sequences in B3 and B8 and their correspondence with and comparison to genomic sequence. genomic sequence suggested that both transcripts were the result of alternative splicing and use of different polyadenylation sites com- Assessment of HMGA2-RAD51L1 and RAD51L1-HMGA2 Fu- pared with those described previously. The ectopic sequences B1, B3, sion Transcripts. Although a fusion transcript between HMGA2 and Ј B8, and B12 would result in translation of an additional 24, 13, 23, RAD51L1 was not detected in 105274 by a generic approach (i.e., 3 and 1 novel amino acid residues each, respectively, beyond HMGA2 RACE), aberrant splicing using apparently cryptic sites within exon 4 for B1 and exon 3 for B3, B8, and B12 (see web site for HMGA2 raised the possibility that other patterns of aberrant splicing translations).5 might occur. We developed a panel of RT-PCR reactions with paired One transcript (B11, GenBank AF533653), however, contained gene-specific primers to evaluate directly mRNA from 10 leiomyo- sequence from all five exons, but the sequence from exon 5 was mata with t(12;14). Fig. 4 shows the primer pairs used to test for interrupted at nucleotide 1912 (based on GenBank U28749) with HMGA2, RAD51L1, RAD51L1-HMGA2, and HMGA2-RAD51L1 novel sequence before the polyA tail (Fig. 3A). This transcript con- transcripts. In all myomas, transcripts for HMGA2 and RAD51L1 were 1354

Fig. 3. Characterization of HMGA2 fusion transcripts found in four leiomyomata with t(12;14). A depicts five altered transcripts of HMGA2 (green) from one leiomyoma (105274) with a 3Ј rearrangement in the HMGA2 locus on chromosome 12. 3Ј RACE clones B1 (GenBank U29115), B3 (GenBank AF533651), B8 (GenBank AF533652), and B12 (GenBank AF533654) revealed previously undescribed sequence from chromosome 12 (blue) fused with exons 3 or 4 of HMGA2; clone B11 (GenBank AF533653) was joined to undescribed chromosome 14 sequence (red). B illustrates two fusion transcripts between HMGA2 (green) and RAD51L1 (purple) from one leiomyoma (166931) harboring both 5Ј and 3Ј rearrangements of chromosome 12 relative to HMGA2. In transcript D15 (GenBank AY138860) the 3Ј UTR of HMGA2 (nucleotide 1222 of GenBank U28749) is joined to exon 8 of RAD51L1 (nucleotide 827 of GenBank 92074.1), leaving the entire HMGA2 coding sequence intact. Alternatively, fusion transcript D30 (GenBank AY138858) joins exon 3 of HMGA2 (nucleotide 1060) to exon 8 of RAD51L1 (nucleotide 827), leaving the DNA binding motifs of HMGA2 intact but not the acidic 3Ј region of the protein. C shows fusion transcripts A5 and C93 (GenBank AY138859 and AY138857, respectively) in which exon 3 of HMGA2 (green) was joined to exon 8 of RAD51L1 (purple) in two leiomyomata (ST94-114 and ST98-773) harboring 5Ј rearrangements of HMGA2.

detected regardless of the location of the chromosome 12 breakpoint. expected (ϳ500 bp) was observed in addition to the expected product Fusion transcripts were found in only four samples. HMGA2- (data not shown). This larger product was only detected by Southern RAD51L1 fusion transcripts were detected in both myomas with blotting (data not shown). Both products were cloned from a nested rearrangements in the 3Ј region of HMGA2. As noted above, both PCR and subsequently sequenced. The expected product (D30 in Fig. tumors 105274 and 166931 also expressed transcripts that included 3B, GenBank AY138858) showed a fusion product joining exon 3 of the complete coding sequence of HMGA2. The reciprocal fusion HMGA2 to exon 8 of RAD51L1 in the correct reading frame. The transcript (i.e., RAD51L1-HMGA2) was not detected in three different novel 381-bp product (D15, GenBank AY138860; Fig. 3B) joined a reactions for either tumor. From the RT-PCR product obtained from truncated 3Ј UTR of HMGA2 to exon 8 of RAD51L1. Similar to myoma 166931, an additional product (ϳ650 bp) of larger size than myoma 105274, this latter transcript would leave the coding sequence 1355

Fig. 4. RT-PCR of 10 leiomyomata with breakpoints 5Ј and/or 3Ј in the HMGA2 locus. Eight tumors had breakpoints 5Ј to HMGA2, one (105274) had a breakpoint 3Ј to HMGA2, and one tumor (166931) was a mosaic in which cells contained either breakpoints 5Ј or 3Ј in the HMGA2 locus. The green schematic represents the HMGA2 gene (GenBank U28749) with exons 1–5(dark green) labeled along with the 5Ј and 3Ј UTRs (light green) in exons 1 and 5, respectively. The purple schematic represents the RAD51L1 gene (GenBank U92074) with exons 1–11 (dark purple) labeled along with the 5Ј and 3Ј UTRs (light purple) in exons 1 and 11, respectively. The numbers to the left of each gene schematic correspond to the bp location, strand orientation, and length of each primer. The location of each primer relative to the gene’s exons and the primer’s direction can be determined from the arrows above or below the schematic of the gene. A (ϩ) indicates the presence of a RT-PCR product when visualized on a 1% agarose gel or on an autoradiogram, whereas a (Ϫ) indicates the lack of product. NT denotes an untested sample. HMGA2-RAD51L1 fusions were detected in four tumors (red ϩ). RAD51L1-HMGA2 fusions were not detected in any tumor in this series. of HMGA2 intact and suggests that one of the chromosome 12 HMGA2 coding sequence. We found no evidence by FISH for local- breakpoints in tumor 166931 resides within the 3Ј UTR. ization of the 5Ј and 3Ј probes in the HMGA2 locus to different Most surprisingly, fusion transcripts were detected by RT-PCR in derivative chromosomes. These mapping results provide evidence that two of eight tumors (ST94-114 and ST98-773) studied with 12q15 fusion transcripts of HMGA2 and RAD51L1 are neither required nor breakpoints 5Ј in the HMGA2 locus. Evidence of the fusion product frequent in uterine leiomyomata. The observation that chromosomes was found in two independent reactions, each with different primers other than 14 may occasionally participate in rearrangements with (Fig. 4). Products of nested reactions were cloned and sequenced (Fig. 12q15 additionally supports this hypothesis. 3C), showing that they were identical to D30, which was isolated from Interestingly, fusion transcripts in which the 5Ј region of HMGA2 tumor 166931. was joined to the 3Ј region of RAD51L1 were detected in both tumors tested in which an uncommon 12q15 breakpoint was 3Ј to HMGA2. DISCUSSION Sequence derived from novel transcripts suggests that the breakpoints reside within the 3Ј untranslated region of HMGA2, and fusion tran- In this large series of uterine leiomyomata studied with 12q15 rearrangements, cytogenetic analysis of GTG-banded chromosomes scripts containing the full coding sequence of HMGA2 were detected. and FISH mapping substantiated that the t(12;14) is a reciprocal Fusion transcripts involving HMGA2 and RAD51L1 were not de- Ј apparently balanced rearrangement. The majority of 12q15 break- tected in six of eight (75%) myomas with 5 breakpoints, but HMGA2 points in these tumors are located 5Ј in the HMGA2 locus. A minority and RAD51L1 transcripts were present in all tumors tested with of 12q15 breakpoints are found either in the 3Ј region or with 5Ј and t(12;14). With the breakpoint 5Ј (centromeric) relative to HMGA2, the 3Ј breakpoints flanking HMGA2. Of myomas with chromosome 12 entire gene should be translocated to the der(14), and all of the breakpoints mapping in the 3Ј region, analysis by FISH shows that sequence telomeric to HMGA2 should be derived from chromosome over half (five of nine) had breakpoints mapping well beyond the end 12. Exons 8–11 of RAD51L1 included in a potential HMGA2- of HMGA2. Mapping of the remaining myomas with 3Ј breakpoints RAD51L1 fusion product would be located either upstream (i.e., also may be consistent with rearrangement sites outside of the centromeric) to HMGA2 on the der(14) or on the der(12) remote from 1356

HMGA2, depending on the position of the chromosome 14 breakpoint. In addition to fusion transcripts containing the first three exons of HMGA2 and RAD51L1 would be present in an orientation that pre- HMGA2 joined to RAD51L1, we observed several truncated HMGA2 cludes transcription of fusion products in either case. Yet we detected transcripts lacking the fourth and fifth exons or only the fifth exon. All such fusion transcripts in two different leiomyomata (ST94-114 and of these transcripts were found in the same tumor (105274), which ST98-773). Aside from technical factors, we considered three possible also made transcripts capable of encoding a complete HMGA2 explanations. First, cytogenetic mosaicism can produce populations of polypeptide. Two of the fusion transcripts were similar to those cells with breakpoints both 5Ј and 3Ј in the HMGA2 locus within a previously reported by Schoenmakers et al. (3). All of these truncated single tumor. An example of this mechanism is illustrated by tumor transcripts included exons 1–3, encoding the AT-hook DNA binding 166931. However, no evidence of such mosaicism was noted in FISH domains translated in the HMGA2 polypeptide (3, 16, 17). The 3Ј experiments in either myoma. Nevertheless, a very low level of sequence of these transcripts was found in chromosome 12, suggest- mosaicism might be detectable by RT-PCR. Second, an additional ing the possibility that they are cryptic exons hidden in the large third rearrangement might position the two genes in the requisite orienta- or smaller fourth introns or illegitimately spliced products (18). These tion. We found evidence of multiple breakpoints in 7 of 38 (18.4%) sequences contribute as little as one and as many as 23 additional myomas with rearrangements involving 12q15. There was, however, amino acid residues to the truncated polypeptide. The biological no evidence of secondary rearrangements in either tumor. Lastly, activity of HMGA2 polypeptides in which the acidic tail has been transcripts from HMGA2 and RAD51L1 may combine by trans- replaced by a variable number of seemingly random amino acid splicing (10). On the basis of the available data, it is not possible to residues, either in the presence or the absence of the normal polypep- make a compelling argument for one mechanism over another. De- tide, is not understood presently in human uterine leiomyomata (15). spite these exceptions, formation of fusion transcripts in leiomyomata Overexpression of similar transcripts in transgenic mice, however, is with the most frequent rearrangement of 12q15 [i.e., breakpoints 5Ј in not associated with leiomyomata but rather is associated with atypical HMGA2 in t(12;14)] is not the most common finding. FISH mapping lipomatous proliferations (19). of karyotypically complex cases reveals two breakpoints on chromo- Two molecular mechanisms might account for aberrant expression some 12 flanking HMGA2 and raises for consideration the biological of HMGA2 after t(12;14) in uterine leiomyomata. First, a regulatory consequence of inserting HMGA2 into a wholly new chromosomal element that ordinarily negatively regulates HMGA2 expression in context. adult myometrial tissue might be separated from the gene by chro- Previously, fusion transcripts between HMGA2 and RAD51L1 have mosomal rearrangement. This hypothetical mechanism would be plau- been reported by several groups. Some have hypothesized that the key sible if the regulatory element was located 5Ј to HMGA2. Analysis of pathobiological mechanism operating in leiomyomata with t(12;14) the genomic sequence 5Ј to HMGA2 does not indicate such an involve fusion products between HMGA2 and RAD51L1. Schoenmak- element, but gaps persist in the genomic sequence and the features by ers et al. (3) first recognized that the chromosome 14 breakpoint fell which such an element might be recognized are not well defined. A within the large RAD51L1 locus and detected reciprocal fusion prod- more serious criticism of this model is that it is not comprehensive. ucts between the two genes in five SV40 transformed leiomyoma cell Specifically, simple or complex rearrangements with breakpoints 5Ј lines. In contrast, Ingraham et al. (4) did not find fusion transcripts in with respect to HMGA2 would remove the gene from the putative leiomyoma GM10964, which is the same tumor as ST90-194. Of note, regulator, but the infrequent rearrangements located 3Ј relative to HMGA2 is uncommonly fused to several other sequences, including HMGA2 would not alter the relationship between the gene and its COX6C on chromosome 8, a RTVL-H 3Ј LTR and mitochondrial regulator. It is unlikely that rearrangements on either side of the gene dehydrogenase gene (ALDH2) on chromosome 12, and enhancer of could abrogate a single regulatory element. invasion 10 (HEI10) on chromosome 14 at band q11 (11–14). Several A second mechanism would be the placement of a foreign regula- other leiomyomata in our series involved other chromosomal partners, tory element near HMGA2 that overcomes silencing of this gene suggesting that various genes potentially may infrequently be fused observed in normal adult tissues. Such regulatory elements might with HMGA2. include an enhancer of gene expression. The paradigm for this mech- It is possible that such rearrangements could still form a fusion anism is MYC and the reciprocal translocations between chromosome transcript in which 5Ј exons from RAD51L1 could be spliced to exons 8 and chromosomes that harbor immunoglobulin loci expressed in 2–5ofHMGA2, skipping over exon 1 of HMGA2. Indeed, Takahashi hematopoietic cells. In the case of MYC, modest increases in mRNA et al. (5) have detected such transcripts in 2 of 81 myomas. Tumors in can have profound biological consequences (20). Another interesting that series were not karyotyped but only ϳ10% would have been paradigm is the t(3;8) found in pleomorphic adenoma of the salivary expected to have had 12q15 rearrangements. The resulting polypep- gland. This rearrangement occurs in the 5Ј noncoding regions of tides would lack either the first or first and second AT hooks of the PLAG1 and ␤-catenin (CTNNB1) and results in exchanging promoters HMGA2 protein. HMGA2 transcripts, however, were detected in both and upstream elements between a developmentally regulated gene leiomyomata, raising a question about the pathobiological signifi- (PLAG1) and a constitutively expressed gene (CTNNB1; Ref. 21). cance of truncated HMGA2 or RAD51L1-HMGA2 transcripts. Inter- Consequently, PLAG1 is inappropriately expressed. Such promoter estingly, HMGA2-RAD51L1 fusion transcripts were not reported in swapping is unlikely in uterine leiomyomata because 3Ј rearrange- that series. In myomas specifically selected for presence of t(12;14), ments, even if uncommon, would not involve the promoter for we did not detect such anomalous splicing in RT-PCR reactions HMGA2. FISH mapping of 14q23-24 breakpoints in leiomyomata designed to detect RAD51L1-HMGA2 fusion transcripts. The most with 12q15 breakpoints 5Ј to HMGA2 also suggests that the interval frequent finding in leiomyomata with chromosomal rearrangements between the RAD51L1 promoter and the HMGA2 transcriptional start involving 12q15 is aberrant expression of transcripts containing the site is impressively large for promoter swapping. A mechanism in- full coding sequence for HMGA2. The resultant polypeptides would voking an enhancer, however, would not be constrained by variation be indistinguishable from normal by size and presence of specific in the orientation or distance between the chromosome 12 breakpoint epitopes, as has been shown by Klotzbucher et al. (Ref. 15 and data and HMGA2. This suspected enhancer may potentially be a specific not shown). Furthermore, dysregulated expression of the HMGA2 discrete feature contained within RAD51L1 before rearrangement. polypeptide also is the expected molecular mechanism underlying Examination of the genomic sequence of RAD51L1 does not reveal myomas with trisomy 12. presence of another gene within RAD51L1 that might supply an 1357

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2003 American Association for Cancer Research. HMGA2 AND UTERINE LEIOMYOMATA WITH 12q15 REARRANGEMENTS enhancer. Alternatively, enhancement of HMGA2 after translocation 6. Sornberger, K. S., Weremowicz, S., Williams, A. J., Quade, B. J., Ligon, A. H., may be a result of altered chromatin structure transmitted from the Pedeutour, F., Vanni, R., and Morton, C. C. Expression of HMGIY in three uterine leiomyomata with complex rearrangements of chromosome 6. Cancer Genet. Cyto- translocation partner. genet., 114: 9–16, 1999. RAD51L1 per se does not play an exclusive pathobiological role in 7. Schoenmakers, E. F. P. M., Wanschura, S., Mols, R., Bullerdiek, J., Van Den Berghe, uterine leiomyomata. In most cases, the 12q15 breakpoint occurs 5Ј H., and Van de Ven, W. J. M. Recurrent rearrangements in the high mobility group protein gene, HMGI-C, in benign mesenchymal tumours. Nat. 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Bradley J. Quade, Stanislawa Weremowicz, David M. Neskey, et al.

Cancer Res 2003;63:1351-1358.

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