The Putative Glioma Tumor Suppressor Gene on Chromosome 19Q Maps Between APOC2 and HRC1

[CANCER RESEARCH 54, 4760-4763, September 1, 1994] The Putative Glioma Tumor Suppressor Gene on Chromosome 19q Maps between APOC2 and HRC1

Mari-Paz Rubio, Katia M. Correa, Keisuke Ueki, Harvey W. Mohrenweiser, James F. Gusella, Andreas von Deimling, and David N. Louis2

Molecular Neuro-Oncology Laboratory, Neurosurgical Service and Department of Pathology (Neuropathology) [M-P. R., K. M. C., K. U., D. N. L.] and Molecular Neumgenetics Unii and Department of Genetics [J. F. G.], Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129; Human Genome Center, Lawrence Livermore National Laboratory, Livermore, California 94550 Â¡H.W. M.]: and Institute for Neuropathology, University Hospital, Bonn D-53105, Germany Â¡A.v. D.]

ABSTRACT astrocytomas (4). Ransom et al. (5) detected allelic losses of chromo some 19q in four of four informative oligodendrogliomas, and Bigner The frequent alleile loss of chromosome 19q in human gliomas suggests Ã©tal.(6) have shown that structural abnormalities of chromosome 19q that 19q harbors a tumor suppressor gene that is integral to glioma are common in malignant gliomas. Within fibrillary astrocytic tumors, tumorigenesis. Our initial deletion mapping of this gene localized the chromosome 19q is rarely lost in low-grade tumors but is lost in common region of deletion to the distal long arm, 19ql3.2-13.4. To bracket the putative tumor suppressor gene further, we have studied this approximately 50% of anaplastic astrocytomas, implying that this region in 55 gliomas, using loss of heterozygosity studies for 11 well locus may be important in anaplastic progression of astrocytomas (7). mapped, highly informative microsatellite polymorphisms that cover this To localize this putative tumor suppressor gene, we have recently area: D19S178; BCL3; APOC2; ERCC1; DM; D19S112; HRC; D19S246; used polymorphic markers spanning chromosome 19 to search for KLK; D19S180; and D19S254 (from centramene to telomeric). Twenty interstitial deletions using loss of heterozygosity studies. A common, astrocytic, oligodendroglial, and mixed gliomas had deletions affecting overlapping region of deletion was demonstrated on the distal portion this region. Of nine partial deletions, two cases maintained heterozygosity of the long arm, between the proximal marker D19SÃŒ78andthe distal at APOC2 while showing allelic loss at the more telomeric markers, marker DÃŒ9S180(8),suggesting that the gene maps to 19ql3.2-13.4. ERCC1 and DM, while five cases maintained heterozygosity at HRC but However, this region remains a large portion of the chromosome, lost the more centramene markers, D19S112 and DM. Nine cases lost the entire D19S178 to D19S254 region. Three astrocytic gliomas, including measuring between 21 and 29 cM (9, 10). To bracket this tumor one with an interstitial deletion, had terminal deletions of 19ql3.4. The suppressor gene further, we have performed fine deletion mapping of minimum area of overlap shared by the interstitial deletions is between the distal 19ql3.2-13.4 region using 11 well mapped, highly inform APOC2 and HRC, including ERCC1, DM, and D19S112. These findings ative microsatellite polymorphisms. suggest that the glioma tumor suppressor gene maps to an approximately 8-cM/5-megabase region on 19q 13.2-13.3 between the proximal marker APOC2 and the distal marker HRC. Among the DNA repair/DNA metab MATERIALS AND METHODS olism genes on chromosome 19q, ERCC1, LIGI, and perhaps ERCC2 are within the common area of deletion; XRCC1 is centramene and is there Tumor tissues and blood samples were obtained from patients operated on fore excluded as a candidate. at the Massachusetts General Hospital (Boston, MA), the University Hospital (Bonn, Germany), and the University Hospital (Zurich, Switzerland). All tumors were examined by a neuropathologist and graded according to WHO INTRODUCTION criteria (11). Tumors were excluded from the present study if they had Gliomas are the most common primary human brain tumors of previously shown loss of the entire long arm of chromosome 19. The 55 studied gliomas consisted of 13 WHO grade II astrocytomas, 15 grade III adults and can be divided primarily into astrocytomas (including GBM3), oligodendrogliomas, and mixed gliomas (oligoastrocytomas). anaplastic astrocytomas, 8 grade IV GBM, 1 grade IV gliosarcoma, 6 grade II oligodendrogliomas, 3 grade III anaplastic oligodendrogliomas, 4 grade II Loss of heterozygosity studies in human astrocytomas have suggested mixed gliomas, and 5 grade III anaplastic mixed gliomas. DNA was extracted the presence of tumor suppressor genes on chromosomes 9p, 10p, lOq, from frozen tumor tissues and blood samples according to standard phenol- lip, 13q, 17p, 19q, and 22q, while analyses of oligodendrogliomas chloroform procedures (12). and mixed gliomas have suggested tumor suppressor genes on chro For each blood-tumor DNA pair, PCR was performed on a Programmable mosomes Ip and 19q (1). However, only the p53 gene on chromo Thermal Controller (M. J. Research) according to a published protocol (13). some 17p and the retinoblastoma susceptibility gene on chromosome PCR was performed in 20-fj.l volumes with one of the oligonucleotide primers previously end-labeled using T4 polynucleotide kinase and [y-32P]ATP. The 13q have been implicated as the responsible genes at these chromo somal loci (2, 3). For the remaining chromosomes, ongoing studies are appropriate annealing temperatures, optimum magnesium concentrations, and references for oligonucleotide primer sequences and alÃelefrequencies are attempting to narrow the locations of these putative tumor suppressor listed for each locus in Table 1. PCR products were diluted in 10 jj.1 Â°f genes. formamide buffer, denatured, and separated on 6% polyacrylamide denaturing The chromosome 19q locus is of particular interest since it is gels at 80 W for 1-3 h. Dried gels were exposed to X-OMAT (Kodak) frequently involved in astrocytomas, mixed gliomas, and oligoden autoradiographic film for 2â€”24h. AlÃeleswere scored as suggested previously drogliomas. In our experience, allelic loss of chromosome 19q occurs (13). The polymorphic loci, their locations on chromosome 19q, and genetic in 57% of oligodendrogliomas, 67% of mixed gliomas, and 27% of distances between the loci are indicated in Fig. 1.

Received 4/21/94; accepted 7/6/94. The costs of publication of this article were defrayed in part by the payment of page RESULTS charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Allelic losses in the 19ql3.2-13.4 region, from D19S178 to ' Supported by N1H Grant CA 57683. Work was performed by the Lawrence Livermore National Laboratory under the auspices of the United States Department of D19S254, occurred in 20 of 55 gliomas. Losses were noted in 2 of 13 Energy under Contract W-7405-Eng-48. grade II astrocytomas, 6 of 15 anaplastic astrocytomas, 5 of 8 GBM, - To whom requests for reprints should be addressed, at Molecular Neuro-Oncology 3 of 6 oligodendrogliomas, 2 of 4 mixed gliomas, and 2 of 5 anaplastic Laboratory, CNY6, Massachusetts General Hospital, Charlestown, MA 02129. 3 The abbreviations used are: GBM, glioblastoma multiforme; PCR, polymerase chain mixed gliomas but were not detected in the 1 gliosarcoma or the 3 reaction. anaplastic oligodendrogliomas. As discussed below, however, these 4760

Table 1 Locus name, type of microsatellite repeat polymorphism, reference for in addition to an interstitial loss. These distal losses involved 19ql3.4, oligomicleotide primer sequences and alÃelefrequencies, and cycle conditions. either D19S180 and D19S254 or D19S254 alone (Fig. 2, right; Fig. 5).

LocusD19SI78BCL3APOC2ERCClDMDI9SII2HRCD19S246KLK1DI9SI80D19S254RepeatdinucdinucdinucdinuctrinucdinucdinuctetranucdinucdinuctetranucRef.92314249152592699r5558555558625550555555Mg(IHM)1.52.00.51.01.01.01.02.00.51.01.0Cycles3030303040303040303030 DISCUSSION By blanketing the 19ql3.2-13.4 region distal to D19S178 with numerous polymorphic markers, we have demonstrated that intersti tial losses in human gliomas target the 19ql3.2-13.3 area between the loci APOC2 and HRC. These data agree with and extend our earlier mapping of chromosome 19q deletions in gliomas, which suggested that deletions preferentially occurred in 19ql3.2-13.4, distal to the marker DÃŒ9SI78and proximal to the marker D19S180 (see Fig. 1) (4, " T, annealing temperature; Mg, optimum magnesium concentration; Cycles, number 8). The data also support previous cytogenetic studies of malignant of PCR cycles; dinuc, dinucleotide, trinuc, trinucleotide; tetranuc, tetranucleotide. gliomas, which have shown a clustering of breakpoints at 19ql3 (6). In a comprehensive allelotype of oligodendrogliomas, Ransom et al. (5) analyzed five oligodendrogliomas for chromosome 19q deletions 19pter cM using Southern blotting with probes to markers D19S7 (19ql2) and .D19S178â€¢ D19S8 (19q 13.2), both centromeric to DÃŒ9S178.These authors 13.3 BCL3 ' ! 1 detected a "possible homozygous deletion" involving D19S8. How 2.5 APOC2â€¢ ' 1 ever, since 4% of the population exhibits a null alÃeleat the D19S8 ERCC1 â€¢ DM- ;o.5 Sstl polymorphism recognized by probe pl7.1 (14), it is possible that D19S112- , 0.5 their finding represents loss of a single alÃelerather than a homozy gous deletion. Furthermore, the lack of any markers distal to D19S8 in the study by Ransom et al. (5) precludes comparison of their findings HRC. with the present data. 3 D19S246' Our current study greatly narrows the common region of deletion 3 and suggests that a glioma tumor suppressor gene resides between KLK1 â€¢ APOC2 and HRC. On the basis of recent maps of chromosome 19q, the genetic distance between these two markers is approximately 8 cM (9,10, 15,16) (see Fig. 1). Given that the genetic map of chromosome D19S180- 19 is approximately twice the size of the physical map,4 however, the entire region between APOC2 and HRC could be less than 5 mega- 16 bases. At least four DNA repair/DNA metabolism genes reside at D19S254. 19ql3.2-13.3: XRCC1; ERCC1; ERCC2; and LIGI. Since DNA re pair genes are emerging as an important class of tumor suppressors in 19qter human cancer (17, 18), these four genes could be candidates for the Fig. i. Map of chromosome 19 with expanded ql3.2-ql3.4 on right. The studied loci chromosome 19q glioma tumor suppressor gene. XRCd is the most and approximate genetic intervals are indicated on the expanded map. centromeric of these genes and maps between CEA and DÂ¡95178(19). Our present data therefore exclude XRCCl as a candidate, since its location is proximal to the common region of deletion. ERCC2 maps numbers do not reflect the true frequency of chromosome 19q losses approximately 250 kilobases centromeric to ERCC1, approximately in an unselected group of gliomas. halfway between APOC2 and ERCCl (see Fig. 1) (19). Because no Nine cases had interstitial deletions that affected part of the PCR-based polymorphism was available for ERCC2, we could not D19S178 to D19S254 region (Fig. 2, left). Of the tumors with partial determine whether ERCC2 itself falls within the common area of losses, two were oligodendrogliomas, one was a grade II astrocytoma, deletion in cases such as 2506 and 354 in which the deletion ends three were anaplastic astrocytomas, and three were GBMs. Two between APOC2 and ERCCl (see Fig. 2). Losses at ERCCl were gliomas (2506 and 354) maintained heterozygosity at APOC2 on common in our study and ERCCl is clearly within the common region 19ql3.2 but lost distal loci at ERCC1 or DM on 19ql3.3 (Fig. 3). Five of deletion (Fig. 2). LIGI maps within 19ql3.2-13.3, distal to ERCCl (20) and proximal to HRC,4 and is thus within the interval. Therefore, cases (354, 262, 420, 2012, and 438) maintained heterozygosity at HRC on 19ql3.3 but lost alÃelesat proximal loci at D19SÃŒ12or DM of the four DNA repair/DNA metabolism genes at 19ql3.2-13.3, on 19ql3.3 (Fig. 4). The remaining cases with partial deletions (534, ERCCl and LIGI remain candidates, ERCC2 is a possible candidate, 322, and 308) showed losses of larger areas within the D19SI78 to but XRCCl is excluded by its more centromeric location. It is also D19S254 region. The common region of overlap for these partial possible that other currently uncharacterized genes in this region may deletions involves 19ql3.2-13.3, telomeric to APOC2 and centro- be tumor suppressors. meric to HRC, and encompasses the loci ERCC1, DM, and D19S112 The 19ql3.2-13.3 tumor suppressor locus appears to be involved in (Fig- 2). tumorigenesis of astrocytic, mixed, and oligodendroglial gliomas, Nine cases showed loss of the entire area extending from D19S178 since all three glioma types suffered deletions in this region. Because to D19S254 (Fig. 2, middle). These tumors included one oligoden- the cases in this study were selected by excluding tumors which had droglioma, two mixed gliomas, two anaplastic mixed gliomas, one previously shown loss of the entire long arm of chromosome 19, the grade II astrocytoma, one anaplastic astrocytoma, and two GBMs. present analysis underestimates the frequency of 19q losses in glio- Two gliomas (both anaplastic astrocytomas: 62, 2754) had terminal deletions only, while one case (a GBM, 438) had a terminal deletion 4 H. W. Mohrenweiser, unpublished data. 4761

DNAÂ»

WHO AOA A O AOAGM AA GM OA OA AA AA

D19S178

BCL3

Fig. 2. Deletion map of allelic losses of chromo some 19ql3.2-13.4 in 20 gliomas. The two bold double line pairs on the left indicate the cases which define the common region of deletion between APOC2 and HRC. The loci are in order from centromeric (lop) to telomeric (bottom). D, loss of heterozygosity; Q, noninformative; â€¢maintenance of heterozygosity; A, astrocytoma; AA, anaplastic astrocytoma; GM, glioblastoma multiforme; O. oligoden- droglioma; OA, mixed glioma (oligoastrocytoma); AOA, anaplastic mixed glioma (anaplastic oligoastro cytoma).

KLK1

D19S1SO

D19S254

354 mas. Nonetheless, the finding of relatively frequent, interstitial 19ql3.2-13.4 losses in these selected tumors suggests that involve APOC2 ment of the 19q gene may be more common in human gliomas than previously suspected (4). NT Our previous mapping study of chromosome 19q losses in gliomas did not include any markers telomeric to D19SI80 (8). To assay the terminal region of 19q for deletions, we therefore included the marker D19S254, which lies substantially telomeric to D19SI80 (see Fig. 1). Three cases displayed telomeric deletions that involved DÃ•9S254 (Fig. 2); nine cases had losses of the entire region, including D19S254. The finding of some terminal deletions raises the possiblity that a second tumor suppressor locus exists at distal 19ql3.4. Candidate genes that map to distal 19ql3.4 include a number of genes encoding zinc finger proteins (16). While these appear to cluster near the q terminus, their exact locations relative to D19SI80 and DJ9S254 remain unclear. Protein kinase C-y, which maps to 19ql3.4 (21), is not a likely tumor suppressor candidate since this protein is expressed Fig. 3. Representative data that define the centromeric limit of the common region of in most GBM (22). If distal 19ql3.4 harbors a second glioma tumor deletion. Case 354 shows maintenance of heterozygosity at APOC2 with loss of heterozy gosity at ERCC1. N, constitutional DNA; T, tumor DNA; bars, alÃeles;arrow, loss of the suppressor gene, it is tempting to speculate that this gene is involved upper alÃeleat ERCCI. in the progression of astrocytic tumors, since distal telomeric losses

438 262 2012 HRC D19S112 HRC D19S112 D19S112 NT NT NT NT NT Fig. 4. Representative data that define the telo meric limit of the common region of deletion. Cases â€”â€¢r 438. 262, and 2012 all show loss of heterozygosity at D/95//2 with maintenance of heterozygosity at HRC. N, constitutional DNA; T, tumor DNA; bars, I alÃeles;arrows, lost alÃeles. *- l

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8. von Deimling, A., Nagel, J., Bender, B., Lenartz, D., Schramm, J., Louis, D. N., and 2754 Wiestler, O. D. Deletion mapping of a putative tumor suppressor gene on chromo some 19q associated with human gliomas. Int. J. Cancer, 57: 676-680, 1994. D19S180 D19S254 9. Weber, J. L., Wang, Z., Hansen, K., Stephenson, M., Kappel, C., Salzman, S., Wilkie, P. J., Keats, B., Dracopoli, N., Brandiff, B. F.. and Olsen, A. S. Evidence for human NT NT meiotic recombination interference obtained through construction of a short tandem repeat-polymorphism linkage map of chromosome 19. Am. J. Hum. Genet., 53: 1079-1095, 1993. 10. Buetow, K. H., Weber, J. L., Ludwigsen, S.. Scherpbier-Hedderma. T., Duyk, G. M., Sheffield, V. C., Wang, Z., and Murray. J. C. Integrated human genome-wide maps constucted using the CEPH reference panel. Nature Genet.. 6: 391-393, 1994. 11. Kleihues, P., Burger, P. C., and Scheithauer, B. W. Histological Typing of Tumours of the Central Nervous System. Berlin: Springer-Verlag, 1993. 12. Sambrook, J., Fritsch, E. F., and Maniatas, T. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. 13. Louis, D. N., von Deimling, A., and Seizinger. B. R. A (CA)n dinucleotide repeat assay for evaluating loss of ailelic heterozygosity in small and archival human brain tumor specimens. Am. J. Pathol., 141: 777-782, 1992. 14. Williamson, R., Bowcock, A., Kidd, K., Pearson, P., Schmidtke, J., Ceverha, P., Chipperfield, M., Cooper, D. N., Coutelle, C., Hewitt, J., Klinger, K., Langley, K., Beckmann, J., Tolley, M., and Maidak, B. Report of the committee DNA committee Fig. 5. Representative data for terminal deletions. Case 2754 shows loss of heterozy- and catalogues of cloned and mapped genes, markers formatted for PCR and DNA gosity at the distal locus D19S254 while maintaining heterozygosity on the more proximal polymorphisms. Cytogenet. Cell Genet., 58: 1190-1832, 1991. D19SI80; bars, alÃeles;arrow, lost alÃele. 15. Jansen, G., de Jong, P. J., Amemiya, C., Aslandis, C., Shaw, D. J., Harley, H. G., Brook, J. D., Fenwick, R., Korneluk, R. G., Tsilfidis, C., Shutler, G., Hermens. R., Wormskamp, N. G. M., Smeets, H. J. M., and Wieringa, B. Physical and genetic were restricted to high-grade astrocytic tumors (anaplastia astrocy- characterization of the distal segment of the myotonic dystrophy area on 19q. toma and GBM). On the other hand, while multiple tumor suppressor Genomics, 13: 509-517, 1992. 16. Mohrenweiser, H., Swartz, A. R., and Johnson. K. Report of the committee on the genes can occur on the same chromosome arm, these terminal dele genetic constitution of chromosome 19. Genome Priority Reports, in press, 1994. tions may be random and nonspecific, since similar telomeric losses 17. Kastan, M. B., Zhan, Q., El-Deiry. W. S.. Carrier, F., Jacks, T., Walsh, W. V., have been noted in deletion mapping studies that have definitively Plunkett, B. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1994 American Association for Cancer Research. The Putative Glioma Tumor Suppressor Gene on Chromosome 19q Maps between APOC2 and HRC

Mari-Paz Rubio, Katia M. Correa, Keisuke Ueki, et al.

Cancer Res 1994;54:4760-4763.

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