Concomitant deletions of tumor suppressor genes MEN1 and AIP are essential for the pathogenesis of the brown fat tumor hibernoma Karolin H. Norda,1, Linda Magnussona, Margareth Isakssona, Jenny Nilssona, Henrik Lilljebjörna, Henryk A. Domanskib, Lars-Gunnar Kindblomc,d, Nils Mandahla, and Fredrik Mertensa aDepartment of Clinical Genetics, University and Regional Laboratories, Skåne University Hospital, Lund University, SE-221 85 Lund, Sweden; bDepartment of Pathology, University and Regional Laboratories, Skåne University Hospital, SE-221 85 Lund, Sweden; cDepartment of Musculoskeletal Pathology, Royal Orthopaedic Hospital National Health Service Foundation Trust, Birmingham B15 2TT, United Kingdom; and dDivision of Cancer Studies, Birmingham University, Birmingham B15 2TT, United Kingdom Edited* by Janet D. Rowley, University of Chicago, Chicago, IL, and approved October 26, 2010 (received for review September 10, 2010) Hibernomas are benign tumors with morphological features re- logical features are shown in Fig. S1). Chromosome banding had sembling brown fat. They consistently display cytogenetic rear- been performed in eight of the cases and rearrangement of 11q13, rangements, typically translocations, involving chromosome band or the neighboring 11q21 band, was found in all of them (Table S1). 11q13. Here we demonstrate that these aberrations are associated Genomic copy numbers were analyzed using SNP arrays and with concomitant deletions of AIP and MEN1, tumor suppressor deletions in 11q13 were detected in all but one of the 15 cases genes that are located 3 Mb apart and that underlie the hereditary (Table S2). Apart from these deletions, few or no other aberrations syndromes pituitary adenoma predisposition and multiple endo- were identified. One additional recurrent deletion was detected in crine neoplasia type I. MEN1 and AIP displayed a low expression in 14q11 in two cases, both of which presented translocations in- hibernomas whereas the expression of genes up-regulated in volving this chromosome band. Also included in the SNP analyses brown fat—PPARA, PPARG, PPARGC1A, and UCP1—was high. were normal blood DNA samples from four of the patients with Thus, loss of MEN1 and AIP is likely to be pathogenetically essen- hibernoma; none of them displayed the aberrations detected in the tial for hibernoma development. Simultaneous loss of two tumor corresponding tumor samples (Table S2). Deletions in 11q13 pri- MEDICAL SCIENCES suppressor genes has not previously been shown to result from marily clustered around the regions covering MEN1 and AIP (Fig. a neoplasia-associated translocation. Furthermore, in contrast to 1, Fig. S2,andTable S2). Losses of these genes were confirmed by the prevailing assumption that benign tumors harbor relatively multiplex ligation-dependent probe amplification (MLPA) analysis few genetic aberrations, the present analyses demonstrate that (Fig. S3). Using this technique, the tumor lacking alterations by a considerable number of chromosome breaks are involved in SNP array (case 2) also displayed loss of MEN1 and AIP. Fur- the pathogenesis of hibernoma. thermore, FISH analysis on metaphase spreads from short-term cultured tumor cells could be performed in five cases (cases 1, 3, 6, soft tissue tumor | lipoma | adipocytic tumor | adipose tissue | SNP array 7, and 15). This technique confirmed the SNP array findings for MEN1 in three cases and for AIP in two cases (Table 1 and Fig. 1). ibernoma is a benign neoplasm with morphological features In the remaining cases, the results from SNP array and FISH Hhighly similar to brown adipose tissue (BAT) (1). In contrast to analyses were discrepant. When SNP array data were interpreted as white adipose tissue (WAT), which stores energy, BAT enables showing hemizygous loss of the genes, FISH analysis displayed energy from oxidized lipids to dissipate as heat. This ability is de- homozygous deletion of AIP in three cases and of MEN1 in one pendent on the expression of uncoupling protein 1 (UCP1), a mi- case. This is likely explained by the fact that when tumor cells are tochondrial proton transporter that uncouples electron transport mixed with stromal cells FISH analysis is more accurate than from ATP production. Morphological similarities between BAT the corresponding SNP array. In case 7, homozygous deletion of MEN1 fi and hibernoma include a typical yellow to brown appearance par- was detected by SNP array but could not be con rmed by tially as a result of their rich vascularization (1). Microscopically, the FISH, as the homozygous deletion was too small to be detected BAT/hibernoma cells show a multivacuolated cytoplasm, numerous by the fosmid clone used for the FISH analyses. mitochondria, and a centrally located nucleus. Intermingled with To map further the deletions associated with translocations the brown fat cells of hibernoma are varying proportions of mature, affecting chromosome band 11q13 in hibernomas, FISH was A univacuolated white adipocytes with a peripherally located nucleus. performed in case 1 using 18 different fosmid clones (Fig. 2 ). The percentage of white adipocytes can be high and tumors with This analysis revealed complex translocation and deletion events only small clusters of brown fat are referred to as lipoma-like. To that could be explained only partly by the t(11;17;12)(q13;q12; avoid misdiagnosing hibernomas as ordinary lipomas or, more im- p13) detected at banding analysis. Both the der(12) and the der portantly, liposarcomas, clinical and morphological data can be (17) chromosomes displayed genetic material from band 11q13, complemented by cytogenetic analysis. The presence of trans- the der(11) chromosome had at least two interstitial deletions, locations affecting 11q13 with few or no other aberrations is a kar- yotypic signature of hibernoma. FISH analysis has indicated that these rearrangements are more complex than expected from the Author contributions: K.H.N., N.M., and F.M. designed research; K.H.N., L.M., M.I., J.N., H.L., and H.A.D. performed research; L.-G.K. contributed new reagents/analytic tools; karyotypes (2). Both hemi- and homozygous deletions have been K.H.N., L.M., M.I., J.N., H.L., H.A.D., and F.M. analyzed data; and K.H.N. and F.M. wrote described in the affected region, although so far without conclusive the paper. results regarding the target gene(s) (3). In the present study, we The authors declare no conflict of interest. wished to determine the genetic pathways associated with hiber- *This Direct Submission article had a prearranged editor. noma development. Data deposition: The data reported in this paper have been deposited in the Gene Expres- sion Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE19040). Results 1To whom correspondence should be addressed. E-mail: [email protected]. Fifteen hibernomas were available for genetic analyses (clinical This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. information is presented in Table S1 and representative morpho- 1073/pnas.1013512107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1013512107 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Genomic losses in hibernoma detected by SNP array and FISH analyses. (A) Global DNA copy numbers were evaluated by SNP array analysis and all cases with an aberrant SNP array profile displayed deletions in 11q13. In case 2, the genomic profile was normal. The vertical blue lines represent the MEN1 and AIP genes. Eight tumors showed homozygous deletion of MEN1 and three showed homozygous loss of AIP. The remaining cases demonstrated hemi- zygous deletion of MEN1 and all but one showed hemizygous loss of AIP. The genomic positions of all alterations are available in Table S2 and the aberrations affecting MEN1 and AIP are presented in detail in Fig. S2. Fluorescence in situ hybridization analysis with fosmid probes covering MEN1 and AIP was used to detect deletions in tumor cells, identified by rearrangements of chromosome 11. (B) As controls, signals from the probes were readily observed in normal cells from the same cases. (C) In case 1, homozygous loss of AIP and hemizygous deletion of MEN1 were detected, (D) case 3 showed homozygous loss of MEN1,(E) case 6 displayed homozygous deletions of both genes, (F) case 7 showed hemizygous deletion of MEN1 and homozygous loss of AIP, and (G) in case 15 both genes were homozygously deleted. The discrepancies between the SNP array and FISH analyses can be explained by differences in resolution of the tech- niques as well as normal cell contamination. and the karyotypically normal chromosome 11 was rearranged cases in which homozygous losses of MEN1 and/or AIP could not with loss of a segment greater than 3 Mb. Combined, the rear- be confirmed, the coding regions of the genes were sequenced rangements and deletions must have involved at least 11 different without detecting any mutations. chromosome breaks. Two of the breakpoints were cloned by using Transcriptional effects of the genomic losses were assessed in long-range PCR (Fig. 2B). One break was located in intron 1 of all but one of the hibernomas by using global gene expression MEN1 and the other was positioned 180 kb upstream of this gene. analysis. As references, 22 lipomas, three WAT, and two skeletal Thus, exon 1 of MEN1 was homozygously lost also in this case. In muscle (SM) samples were included. The expression of all genes summary, the whole or parts of MEN1 and AIP were homozy- located in the 3 Mb region in 11q13 to which the deletions clus- gously deleted in 10 and six of 15 cases, respectively. One case tered was extracted from the gene expression data set (Fig. 3A). showed normal copy number of AIP and in the remaining cases at This included 132 genes from NRXN2 in 11q13.1 to ALDH3B2 in least one of the alleles of both genes was deleted (Table 1). In all 11q13.2 (genomic position 64.13–67.20 Mb).