Chromosomal Rearrangements Leading to Abnormal Splicing Within Intron 4 of HMGIC? Sven Hauke, Volkhard Rippe, and Jo ¨ rn Bullerdiek * Center for Human Genetics, University of Bremen, Germany Fusion of the high-mobility group protein gene HMGIC to other genes due to chromosomal rearrangements occurs in a variety of human benign tumors. In contrast to genes clearly derived from other chromosomes, some of the ectopic sequences fused to HMGIC have been assigned to chromosome 12 by CASH (chromosome assignment using somatic cell hybrids) analyses and thus can be assumed either to result from alternative splicing or to represent true ectopic sequences derived from other genes on chromosome 12. In an attempt to identify the ectopic sequences fused to this exon, we have sequenced the entire intron 4. Four of seven ectopic sequences previously described to be fused to exon 4 of HMGIC in different tumors were found to be located within intron 4 of the gene and thus are due to abnormal splicing. As for a mechanism explaining this observation, it can be suggested that breakpoints of chromosomal aberrations not directly disrupting HMGIC may induce small genomic alterations in their vicinity and thus facilitate abnormal splicing. The latter mechanism may underlie the development of part of the neoplasms characterized by 12q14 –15 rearrangements. © 2001 Wiley-Liss, Inc. Fusion of the high-mobility-group protein gene HMGIC to other genes due to chromosomal rear- rangements occurs in a variety of human benign tumors, e.g., lipomas, uterine leiomyomas, aggres- sive angiomyxomas, pleomorphic adenomas of the salivary glands, endometrial polyps, and pulmonary chondroid hamartomas (Ashar et al., 1995; Kaz- mierczak et al., 1995, 1996; Schoenmakers et al., 1995; Bol et al., 1996). The high incidence of some of these tumors makes HMGIC the most frequently involved fusion partner of chimeric genes in human tumors. HMGIC rearrangements often result in chimeric genes with transcripts consisting of the first three exons of HMGIC followed by ectopic sequences from other genes, such as ALDH2 (Kazmierczak et al., 1995), LPP (Petit et al., 1996), and FHIT (Geurts et al., 1997). In contrast, only seven tran- scripts consisting of exons 1– 4 of HMGIC followed by sequences of either known ectopic origin, e.g., NFIB (Geurts et al., 1998), or unknown origin (Schoenmakers et al., 1995; Kazmierczak et al. 1996) have been described. In the latter cases, either no cytogenetic aberrations that might lead to fusion transcripts were visible (Kazmierczak et al., 1996) or the ectopic sequences fused to HMGIC have been assigned to chromosome 12 by CASH (chromosome assignment using somatic cell hy- brids) analysis, although the results of the cytoge- netic studies showed translocations to other chro- mosomes (Schoenmakers et al., 1995). Thus, the ectopic sequences can be assumed either to result from alternative splicing with the sequences de- rived from intron 3 or 4 (Schoenmakers et al., 1995) or to represent true ectopic sequences derived from other genes on chromosome 12, as suggested by Kazmierczak et al. (1998), for a sequence fused to exon 3 of HMGIC. In the latter case, fusion genes might be a direct result of chromosomal aberra- tions, e.g., small deletions or inversions. In contrast, the origin of sequences from intron 3 or 4 would indicate abnormal splicing. To address that question, we have sequenced the entire intron 4 of HMGIC, allowing the identi- fication of the origin of the reported ectopic se- quences fused to it. Screening of a human PAC library (Genome Sys- tems, St. Louis, MO) for a PAC containing the entire intron 4 of HMGIC was performed using the primer set 5'-ACGGCTTTTGTCAGTATG- GCTTTTA-3' and 5'-CCAGAGATGGAAAT- CACTCGGTGCA-3'. A clone of about 96 kbp was obtained. The DNA was isolated following the instruction of the manufacturer (Genome Systems). Briefly, cell pellets from 50-ml cultures grown after induc- tion with IPTG were resuspended in 5-ml GTE (50-mM glucose, 10-mM EDTA, 25-mM Tris- HCl, pH 8.0). Cells were carefully lysed by adding Supported by: To ¨ njes-Vagt Stiftung, Bremen, Germany. *Correspondence to: Dr. Jo ¨ rn Bullerdiek, Center for Human Ge- netics, University of Bremen, Leobenerstrasse ZHG, D-28359 Bre- men, Germany. E-mail: bullerdiek@uni-bremen.de Received 17 April 2000; Accepted 17 August 2000 Published online 10 January 2001 GENES, CHROMOSOMES & CANCER 30:302–304 (2001) BRIEF COMMUNICATION © 2001 Wiley-Liss, Inc.