Identification of NFIB As Recurrent Translocation Partner Gene of HMGIC in Pleomorphic Adenomas
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Oncogene (1998) 16, 865 ± 872 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 Identi®cation of NFIB as recurrent translocation partner gene of HMGIC in pleomorphic adenomas Jan MW Geurts1, Eric FPM Schoenmakers1, Eva RoÈ ijer2, Anna-Karin AstroÈ m2,GoÈran Stenman2 and Wim JM van de Ven1 1Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven & Flanders Interuniversity Institute for Biotechnology, B-3000 Leuven, Belgium; 2Laboratory of Cancer Genetics, Department of Pathology, GoÈteborg University, Sahlgrenska University Hospital, S-41345 GoÈteborg, Sweden Approximately 12% of all pleomorphic adenomas of the guished, i.e. tumors with rearrangements of 8q12 or salivary glands are characterized by chromosome 12q13 ± 15. Whereas the t(3;8)(p21;q12) is the most aberrations involving the chromosome segment 12q13 ± frequently observed translocation, many dierent 15. Several chromosomes have been found as transloca- chromosome segments can act as translocation tion partners of chromosome 12, and some of these partners of both 8q12 and 12q13 ± 15 (Sandros et al., recurrently. Recently, the HMGIC gene was identi®ed as 1990; Bullerdiek et al., 1993). The short arm of the target gene aected by the 12q13 ± 15 aberrations. chromosome 9, with breakpoints in the segment Here, we report the identi®cation and characterization of 9p12 ± 24, is the preferential translocation partner of a new translocation partner gene of HMGIC in chromosome 12q13 ± 15 (Stenman et al., 1994). The pleomorphic adenomas. 3'-RACE analysis of a primary reason for the variability of the breakpoints in 9p is adenoma with an apparently normal karyotype revealed likely to be due to the limited resolution of the banding an HMGIC fusion transcript containing ectopic se- pattern in 9p12 ± 24 and 12q13 ± 15, making it almost quences derived from the human NFIB gene, previously impossible to determine the exact locations of the mapped to chromosome band 9p24.1. The HMGIC breakpoints with certainty. NFIB fusion transcript was also con®rmed by RT ± Recently, HMGIC was identi®ed as the gene PCR. Since the chromosome segment 9p12 ± 24 is consistently aected in pleomorphic adenomas of the repeatedly involved as translocation partner of chromo- salivary glands and in a variety of other benign some 12q13 ± 15 in pleomorphic adenomas, we tested mesenchymal tumor types characterized by 12q13 ± 15 whether NFIB might be a recurrent partner of HMGIC. aberrations (Schoenmakers et al., 1995b; Ashar et al., RT ± PCR analysis of a second adenoma with an 1995). The location of the breakpoints with respect to ins(9;12)(p23;q12q15) as the sole anomaly, revealed that HMGIC is variable and occur 5' and 3' to HMGIC as also in this tumor an HMGIC/NFIB hybrid transcript well as recurrently in the very large, third intron of the was present. The reciprocal NFIB/HMGIC fusion gene, resulting in the separation of the three predicted transcript, however, could not be detected in any of DNA binding domains in the amino-terminal region these tumors. Nucleotide sequence analysis of the fusion from the acidic, carboxy-terminal domain of HMGIC. transcripts indicated that the genetic aberration in both HMGIC is a member of the high mobility group tumors resulted in the replacement of a carboxy-terminal (HMG) protein gene family which comprises proteins segment of HMGIC by the last ®ve amino acids of that are heterogeneous, nonhistone components of NFIB. In conclusion, our results reveal the recurrent chromatin (Grosschedl et al., 1994). HMGIC expres- involvement of the NFIB gene as translocation partner sion is tightly regulated, with the highest expression in gene of HMGIC in pleomorphic adenomas. fetal tissues and undetectable expression by Northern blot analysis of the normal full-length transcript in Keywords: NFIB; pleomorphic adenoma of the salivary normal adult tissues. HMGIC binds in the minor glands; HMGIC groove of DNA and has been suggested to act as an architectural factor (Wole, 1994) in the nuclear scaold (Saitoh and Laemmli, 1994; Tjian and Maniatis, 1994). The causal relationship between Introduction genetic aberrations aecting HMGIC and aberrant growth control in benign tumors remains to be Approximately 3 ± 6% of the tumors of the head and established. Of interest to note in this context is the neck region occur in the major and minor salivary observation that inactivation of both HMGIC alleles in glands (Shah and Ihde, 1990) and of these, the benign mice results in the so-called Pygmy phenotype with as pleomorphic adenoma is the most common tumor most important feature, disrupted growth leading to (Waldron, 1991). Previous studies have shown that dwar®sm (Zhou et al., 1995). These data demonstrate about 50 ± 80% of the pleomorphic adenomas display the important role of the HMGI proteins in clonal chromosomal abnormalities (Sandros et al., mammalian growth and development. 1990; Bullerdiek et al., 1993). Two major cytogenetic So far, four dierent fusion partner genes of subgroups with abnormal karyotypes can be distin- HMGIC have been identi®ed. The ®rst one is the LPP gene (Schoenmakers et al., 1995b; Ashar et al., Correspondence: WJM van de Ven 1995; Petit et al., 1996) which is located on Received 16 June 1997; revised 30 September 1997; accepted 30 chromosome 3q27 ± q28 and encodes a protein contain- September 1997 ing LIM domains. The second one is a postulated gene NFIB, recurrent translocation partner gene of HMGIC JMW Geurts et al 866 on chromosome 15q24 which has not yet been centromeric and telomeric of HMGIC, respectively. characterized in detail but which is predicted to Both YACs gave only signals on the two chromosomes encode a protein with a serine ± threonine-rich domain 12, demonstrating that the second chromosome 12 (Ashar et al., 1995). The third one is the mitochondrial breakpoint is distal to SAS ± CDK4. As expected from aldehyde dehydrogenase ALDH2 gene on chromosome cytogenetics, the SAS ± CDK4 signal in CG732 was 12q24.1, which was found as translocation partner gene transposed to 9p while the MDM2 signal remained on of HMGIC in a uterine leiomyoma (Kazmierczak et the der(12) (data not shown). al., 1995). In pleomorphic adenoma, the ®rst known fusion partner gene of HMGIC is the FHIT gene, Detection of an HMGIC fusion transcript which has been mapped to chromosome 3p14.2 (Geurts et al., 1997a). The pathogenetic relevance of the fusion In an attempt to determine whether the target gene on partners remains to be elucidated. The diversity in 12q is indeed the HMGIC gene, 3'-RACE analysis was chromosomal segments that have been found to performed. Analysis of adenoma CG575 resulted in the participate in translocations could indicate that the isolation of a 814 bp PCR product, 773 bp of which critical sequences are abundantly present in the human were deposited at GenBank (accession number genome. However, it has not yet been possible to AF022215), representing the 3'-RACE product devoid reliably identify a common structural or functional of non-speci®c cloning and PCR tails. Nucleotide denominator for the limited number of known sequence analysis of this product revealed that it translocation partner genes. contains 21 nucleotides of exon 4 of HMGIC Here, we present the results of 3'-RACE and RT ± (nucleotides 1073 ± 1093 in wild-type HMGIC acces- PCR experiments leading to the identi®cation and sion number U28749) and 752 nucleotides from the last subsequent characterization of a novel fusion partner exon of NFIB and part of its 3'-UTR (nucleotides gene of HMGIC in pleomorphic adenomas of the 1455 ± 2206 in wild-type NFIB as described in this salivary glands. manuscript and also available under accession number U85193). The sequence diversion point was thus found immediately downstream of HMGIC exon 4, suggest- ing a rearrangement within intron 4 of the HMGIC Results gene. As a result, the acidic tail of HMGIC was separated from the three DNA binding domains and Cytogenetic and FISH analysis the spacer domain. In phase with the HMGIC reading Conventional cytogenetic analysis of adenoma CG575 frame, the ectopic sequences appeared to contain a revealed an apparently normal karyotype (Figure 1a). stop codon after ®ve codons, adding only ®ve, ectopic However, detailed analysis of two metaphases with amino acid residues (SWYLG) to the truncated elongated chromosomes suggested that one of the two HMGIC protein. chromosome 12 homologs was somewhat shorter than the other (data not shown). The banding pattern of the Identi®cation of NFIB as fusion partner gene of HMGIC 12q-marker suggested an interstitial deletion of band 12q15. Hybridization with a chromosome 12 painting To establish the nature and origin of the ectopic probe did not reveal translocation of chromosome 12 sequences, genomic clones corresponding to these were material to any other chromosome. Adenoma CG732 isolated from a phage library using PCH203 as had a pseudodiploid karyotype with an ins(9;12) molecular probe. This probe was synthesized with the (p23;q12q15) as the sole anomaly (Figure 1a). PCR primer set PCH203-upper and -lower, which were Hybridization with painting probes for chromosomes developed on the basis of the ectopic sequences. Three 9 and 12 con®rmed the insertion of 12q material into 9p. clones were obtained which were characterized by To test whether both cases with 12q13 ± 15 restriction enzyme and partial nucleotide sequence abnormalities belonged to the group of pleomorphic analysis. A routine BLAST search by e-mail at adenomas with rearrangements of HMGIC we used [email protected] revealed that the ectopic se- two YAC clones, 341C1 and 452E1 (data not shown), quences, fused to HMGIC sequences, were 95% which contain the complete HMGIC gene. FISH identical to hamster clone pNFI ± Red1 which encodes analysis using these YACs revealed signals on the nuclear factor I (NFI) protein (Gil et al., 1988), normal chromosome 12 as well as on the der(9) and suggesting that the ectopic sequences were derived der(12) chromosomes, demonstrating that the HMGIC from the human NFIB gene.