CANCER RESEARCH 55. 6038-6039. December 5, 1995] Advances in Brief Description of a Novel Fusion Transcript between HMGI-C, a Gene Encoding for a Member of the High Mobility Group Proteins, and the Mitochondrial Aldehyde Dehydrogenase Gene Bernd Kazmierczak, Yvonne Hennig, Sylke Wanschura, Piere Rogalla, Sabine Bartnitzke, Wim Van de Ven, and Jörn Bullerdiek' Centerfor Human Genetics and Genetic Counseling, University of Bremen, Leobenerstrafie, ZHG, D-28359 Bremen, Germany fB. K., Y. H.. S. W., P. R., S. B., J. B.], and Center for Human Genetics, Universir,' of Leuven, Herestraat 49, 8-3000 Leuven, Belgium fW. V. d. V.] Abstract Aberrations involving the chromosomal region 12q24 are a nonrandom cytogenetic abnormality in frequent benign tumors mainly of mesenchy mal origin, e.g., uterine leiomyomas, pleomorphic adenomas of the sail vary gland, lipomas, or hamartomas of the lung. Mostly, these 12q24 abnormalites occur as a result of inversions also affecting chromosomal region 12q14—15. In addition to the frequent tumors mentioned above, these abnormalities have also been found in rare mesenchymal tumors, e.g., hemangiopericytomas. Although recentiy the molecular basis of the aberrations of chromosomal region 12q14—15, i.e., a rearrangement of the HMGI-C gene has been identified, the molecular roots of the 12q24 changes still remain to be elucidated. Herein we report on 3' rapid amplification ofcDNA ends PCR results on cDNA from a primary uterine leiomyoma. As an ectopic sequence fused to exon 3 of the HMGI-C gene, we have identified a cDNA sequence that revealed 100% homology to exon 13 of the human mitochondrial aldehyde dehydrogenase gene (ALDH 2). Because ALDH 2 maps to 12q24.1, this fusion transcript is a good candi date underlying the chromosomal rearrangements involving 12q24. Introduction A huge variety of human benign tumors mainly of mesenchymal origin, e.g., pleomorphic adenomas of the salivary gland (1, 2), uterine leiomyomas (3—5), lipomas (6—8),or pulmonary chondroid hamarto mas (9—1), show specific chromosomal abnormalities involving the region 12ql4—l5 leading to rearrangements of HMGJ-C,2 a gene encoding for the high mobility group family of DNA binding proteins (12—13). HMGI-CproteinscontainthreeDNAbindingdomainsbind ing to the minor grooves of AT-rich DNA (14). Another chromosomal band non-randomly involved in these chromosomal rearrangements is l2q24, often as inversions also involving l2ql4—l5 (15, 16). Thus, in addition to the known HMGI-C rearrangements, band l2q24 represents another interesting target for investigations at the molecular level. To find a candidate gene in 12q24 rearranged by the chromosomal translocations, we used a primary uterine leiomyoma to characterize a fusion transcript between HMGI-C and an ectopic sequence belonging to a the mitochondrial aldehyde dehydrogenase 2 (ALDH 2) gene mapping to 12q24.l (17). Materials and Methods Tumor Material. A primary uterine leiomyoma from a 46-year-old woman was surgically removed. Immediately after surgery, one-third of the Received 10/10/95; accepted 11/2/95. 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 18 U.S.C. Section 1734 solely to indicate this fact. I To whom requests for reprints should be addressed. Phone: 49-421-218-2907; Fax: 49-421-218-4039. 2 The abbreviations used are: HMGI-C, high mobility group 1-C; ALDH, aldehyde dehydrogenase; FISH, fluorescence in situ hybridization; RACE, rapid amplification of cDNA ends. tumor was frozen in liquid nitrogen for RNA isolation, another one-third was used for cytogenetic analysis, and the remaining one-third was histologically examined. As a control for the RACE-PCR, the human hepatocellular cell line HEP 3B was used. CytogeneticAnalysis. Chromosomepreparation followed routinemethods described earlier (2). Chromosomes were GTG banded. FISH. To identify the chromosomes unambiguously, FISH analysis was performed after GTG banding of the same metaphase spreads. Treatment of metaphase spreads, subsequent FISH experiments, and cosmid labeling (with biotin-14-dATP) was performed according to the protocol of Kievits et a!. (18). As DNA probes, a pool ofcosmids, 27E12 and 142H1, flanking the third exon of the HMGI-C gene (12), was used. The slides were analyzed on a Zeiss (Oberkochem, Germany) Axioplan fluorescence microscope. Results of GTG banding and FISH were processed and recorded with a Power Gene karyotyp ing system (PSI, Halladale, Great Britian). 3'RACE-PCR. One hundred mg of tumor tissue were homogenized, and RNA was isolated using the trizol reagent (BRL, Gaithersburg, MD) contain ing phenol and isothiocyanate. For cDNA synthesis, we used the adapter primer (AP2) AAG GAT CCG TCG ACA TC (T)17. For first and secondary rounds of PCR, the universal amplification primer (UAP2) CUA CUA CUA CUA CUA AAG GAT CCG TGG ACA IC was used as a “reversed primer.― In the first round of PCR, we used the specific forward primer 5'-CTT CAG CCC AGG GAC AAC-3' (exon I), and in the second round of PCR, the following nested primer was used: 5'-CAU CAU CAU CAU CGC CTC AGA AGA GAG GAC-3' (exon 1). PCR [thermal cycling: 94°C, 3 mm; (94°C, 1 rain; 55°C, 45 s; 72°C, 3 mm) X 30, 72°C, 10 minI was performed with the Amplilaq polymerase (Perkin Elmer, Weiterstadt, Germany). The PCR prod ucts were separated in 0.8% agarose. Cloning of the gel-extracted fragments was performed using the CloneAmp cloning system (BRL). Therefore, the nested specific primers and the universal amplification primer were CUAJCAU tailed. Sequencing was performed on a 373 DNA sequencer (Applied Biosys tern, Weiterstadt, Germany). Results A total of 11 GTG-banded metaphases of the primary tumor was cytogenetically studied. All showed an apparently normal karyotype 46, XX, but the band resolution was rather poor. In addition, FISH studies using a pool of cosmids belonging to the third exon of HMGI-C were performed. The results revealed a rearrangement of this region in all metaphases analyzed. Using the cosmid pool, we observed a signal on a normal chromosome 12, a split signal on l2q 15 and l2q24 on an obviously inverted chromosome 12, and a weak signal on a cytogenetically normal chromosome 14. To characterize possible aberrant fusion transcripts between HMGI-C and ectopic sequences, 3'RACE PCR on the cDNA of the uterine leiomyoma was performed, resulting in the occurrence of bands of normal sizes (identical to the control) and one additional band of about 650 bp. The amplified DNA of this band was extracted from the gel, cloned, and sequenced. The analyzed sequence contained ectopic sequences fused to exon 3 of HMGI-C. A homology search revealed a 100% identity of the ectopic sequence to the thirteenth exon of the mitochondrial 6038 Research. on December 9, 2021. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from