DMBT1 Polymorphisms: Relationship to Malignant Glioma Tumorigenesis1

[CANCER RESEARCH 62, 1790–1796, March 15, 2002] DMBT1 Polymorphisms: Relationship to Malignant Glioma Tumorigenesis1

Hikaru Sasaki, Rebecca A. Betensky, J. Gregory Cairncross, and David N. Louis2 Molecular Neuro-Oncology and Pathology Laboratories, Department of Pathology and Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129 [H. S., D. N. L.]; Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts 02115 [R. A. B.]; and Departments of Clinical Neurological Sciences and Oncology, University of Western Ontario and London Regional Cancer Centre, London, Ontario, N6A4L6 Canada [J.G.C.]

ABSTRACT 17). DMBT1, however, is composed of highly homologous repeating units (DMBT1 repeats; Ref. 18), and recent findings of intragenic The deleted in malignant brain tumors 1 (DMBT1) gene on 10q25-26 is deletion polymorphisms in a subset of normal individuals have given a candidate tumor suppressor gene in malignant gliomas, but its role is pause to accepting DMBT1 as a tumor suppressor (19). Indeed, some controversial, e.g., some DMBT1 homozygous deletions reflect unmasking of constitutional deletion polymorphisms by 10q loss. To clarify the role of of the homozygous deletions in tumors simply reflect allelic loss DMBT1 in gliomagenesis, we investigated three reported deletion hot unmasking a constitutional deletion polymorphism (20). spots. Homozygous deletions at DMBT1 repeat 2-4 to 2-7 were found in 10 DMBT1 encodes a secreted or membrane-linked protein belonging of 73 gliomas with 10q loss, but all 10 deletions reflected unmasking of to the SRCR superfamily. DMBT1 binds to surfactant proteins (col- constitutional hemizygous deletions. Alleles bearing deletion 2-4/2-7 were lectins), suggesting a role in mucosal immune defense (21, 22). not selected significantly for by 10q loss, with retention of only 10 of 16 Mollenhauer et al. (19, 22, 23) have proposed a possible role for ؅ deleted alleles. No homozygous deletion was detected at locus 74k in the 5 DMBT1 in cancer immune surveillance based on its stimulatory effect upstream region of DMBT1, and four tightly linked polymorphisms were on macrophages, as suggested for another SRCR protein, Mac-2-bp/ found around this region; chromosome 10q loss randomly affected alleles with or without the variant sequences around locus 74k. Moreover, no 90K. Specific binding and potent growth-inhibitory activity of man- significant selection pressure was detected for the haplotype with both nan-binding lectin, a collectin, to colorectal carcinoma cells also .(deletion 2-4/2-7 and 5؅ polymorphisms. There was no segregation of raises the possibility of an antitumor activity for DMBT1 (19, 22, 24 deletion 2-4/2-7 in glioma patients compared with unrelated individuals Three deletion hot spots have been suggested in the DMBT1 gene, from reference families but a suggestion of a difference in the distribution from centromeric to telomeric, at the 74k locus in the 5Ј upstream of the 5؅ polymorphisms between the reference individuals and glioma region of the gene; at DMBT1 repeat 2-4 to 2-7 (deletion 2-4/2-7); and patients. Constitutional polymorphisms at DMBT1 repeat 2-9/2-10 ap- at DMBT1 repeat 2-9 and 2-10 (deletion 2-9/2-10; Fig. 1). Because peared common in patients with both benign brain tumors and gliomas. A point mutations are only found rarely, such intragenic deletion appears homozygote for both the 2-4/2-7 deletion and the 5؅ polymorphisms had a the predominant mechanism of DMBT1 alteration. In the present glioma arise at a typical age and without an apparent family cancer predisposition. These data suggest that DMBT1 polymorphisms are not study, we investigated these deletion hot spots using a large panel of likely primary targets of 10q loss in malignant gliomas and do not support malignant gliomas to clarify the role of DMBT1 in glioma tumorigen- a major role for DMBT1 in gliomagenesis. esis. Specifically, we evaluated whether tumor-specific deletions occur during tumorigenesis, if selective pressure favors loss of the INTRODUCTION wild-type allele in the presence of constitutional deletion polymorphisms, and if the deletion polymorphisms are related to glioma Allelic loss of the long arm of chromosome 10 is one of the most predisposition. frequent genetic alterations in malignant gliomas, commonly affecting all or most of the chromosomal arm. Although the PTEN gene, the sole established tumor suppressor on chromosome 10q at the present MATERIALS AND METHODS time, has been identified on 10q23 based on allelic deletion analysis in advanced prostate carcinoma (1) and by representational difference Tissues and DNA. A total of 126 malignant gliomas, all from different analysis (2), 10q25-26 has been suggested as the primary target region patients and all with paired constitutional-tumor DNA samples, were initially of 10q losses in gliomas (3–7). Moreover, PTEN gene alterations have included. Of these cases, 99 had sufficient constitutional DNA quality for only been found rarely in some tumors associated with 10q loss, such successful amplification of long-range PCR products and were therefore stud- as lung cancer and pancreatic cancer (8), indicating another possible ied further: 78 glioblastomas, 9 anaplastic astrocytomas, 5 anaplastic oligodendrogliomas, 2 anaplastic oligoastrocytomas, and 5 high-grade gliomas that target for 10q loss. 3 were not easily classified into the above common groups. To evaluate DMBT1 The DMBT1 gene is located between D10S209 and D10S587, an alterations in a control population, constitutional DNA from 64 apparently interval included in some of the putative suppressor regions for unrelated and unaffected individuals4 (25, 26) in the CEPH reference families gliomas and other cancers (4, 6, 7, 9–11). Although only a few was obtained from Coriell Cell Repositories.5 For polymorphisms at DMBT1 potential point mutations have been identified to date (12–14), fre- 2-9/2-10, constitutional DNA was studied by Southern blotting from 17 quent intragenic homozygous deletions and lack of expression have patients with benign intracranial tumors (9 meningiomas, 7 schwannomas, and suggested DMBT1 as a candidate tumor suppressor in glioblastoma, 1 hemangioblastoma) and 16 patients with gliomas (3 glioblastomas, 2 ana- medulloblastoma, lung cancer, and gastrointestinal cancer (12, 15– plastic astrocytomas, 8 anaplastic oligodendrogliomas, and 3 low-grade oligodendrogliomas; no overlap with 126 cases mentioned above); these were selected randomly from the anonymous DNA bank in our laboratory. Tumor Received 9/28/01; accepted 1/14/02. The costs of publication of this article were defrayed in part by the payment of page DNA was extracted from either frozen tumor tissue or formalin-fixed, paraffin- charges. This article must therefore be hereby marked advertisement in accordance with embedded sections, and constitutional DNA was extracted from blood leuko- 18 U.S.C. Section 1734 solely to indicate this fact. cytes (27). The present investigations have been approved by the Massachu- 1 Supported by NIH Grants CA57683 and MRC-MOP-37849. 2 To whom requests for reprints should be addressed, at Molecular Neuro-Oncology setts General Hospital Subcommittee on Human Studies and the Review Board Laboratory, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. for Health Science Research Involving Human Subjects at the University of Phone: (617) 726-5690; Fax: (617) 726-5684; E-mail: [email protected]. Western Ontario. 3 The abbreviations used are: DMBT1, deleted in malignant brain tumors 1; SRCR, scavenger receptor cysteine rich; CEPH, Center d’Etude du Polymorphisme Human; LOH, loss of heterozygosity; SSCP, single-strand conformational polymorphism; CI, 4 Internet address: http://locus.umdnj.edu/nigms/ceph/ceph.html. confidence interval; RT-PCR, reverse transcriptase-PCR. 5 Internet address: http://locus.umdnj.edu/nigms/. 1790

Fig. 1. The DMBT1 gene. Top: exon-intron structure of DMBT1. Except for the first and the last exons, only exons encoding SRCR domains are numbered (referenced sequence: GenBank AJ243211). RsaI fragments that contain any of the first 13 SRCR domains and hybridize with Southern blot probe DMBT1/sr1sid2 are also described. Middle: distribution of the highly homologous DMBT1 repeats. Bottom: the reported three deletion hot spots. Long-range PCR primers for deletion 2-4/2-7, the expected size of long-range PCR products, and the position of the 74k locus primers are depicted. STS positions, which had reportedly undergone frequent homozygous deletions, are also shown.

LOH Studies. Allelic loss of chromosome 10q was assessed by LOH Southern Blotting. Southern blotting for the 2-4/2-7 and 2-9/2-10 dele- assays using microsatellite markers at 10q25-26 (D10S187, D10S221, tions was performed as described previously with minor modification (18). D10S587, and D10S1723) as described previously (20, 27). Briefly, 12–15 ␮g of genomic DNA from either peripheral blood leukocytes or Multiplex PCR. Three sets of multiplex PCR with different target/refer- frozen tumor tissues were digested with 200 units of RsaI. After ethanol ence sequence pairs were used to detect deletion 2-4/2-7 (see Fig. 1): (a) g14 precipitation, the digested DNA solubilized in 25 ␮l of Tris-EDTA was (150 bp, intron 18)/c12 (190 bp; Ref. 15); (b) intron 20 unique sequence (150 separated for 36 h on a 1.1% Tris acetate-EDTA-agarose gel at 45 V and bp)/desmin sequence (171 bp, chromosome 2q); and (c) g14/APEX nuclease transferred with 0.4 M NaOH to Hybond Nϩ (Amersham Pharmacia Biotech, sequence (187 bp, chromosome 14q). All primer sequences have been pub- Little Chalfont, United Kingdom). The hybridization was carried out for 20 h lished previously except primers for intron 20 sequence (L1865: 5Ј-GCCCCT- at 65°C in 25 ml of modified Church and Gilbert buffer [0.5 M phosphate GTCTTTTTCACAT-3Ј and L1866: 5Ј-GAATGAGGATCTTTACTGAT-3Ј; buffer (pH 7.2) containing 7% SDS; Ref. 28], with a cDNA probe DMBT1/ Refs. 15 and 20). PCR was performed for 29 cycles with an annealing sr1sid2 covering exons 2–12 (generous gift from Dr. Jan Mollenhauer; refer- temperature of 58°C to amplify g14/c12, for 30 cycles at 50°C to amplify enced sequence: GenBank AJ243211) labeled with the Megaprime DNA intron 20 sequence/desmin, and for 32 cycles with gradually decreasing an- labeling system (Amersham Pharmacia Biotech) using [␣-32P]dATP and nealing temperature from 63°Cto56°C to amplify g14/APEX. The products [␣-32P]dCTP. Because of the high homology of the first 13 SRCR domains, were separated on 3% Tris-borate EDTA agarose gel and visualized by RsaI fragments that contain any of these SRCR domains hybridize with ethidium bromide staining. DMBT1/sr1sid2, which contains SRCR domains 1 and 2 (exons 7 and 10; Fig. The 74k locus was investigated using two sets of multiplex PCR assays 1). The total copy numbers of each fragment representing DMBT1 repeat 2-4 covering the entire original 74k locus (293 bp; Fig. 1; Ref. 15). Novel target to 2-7 (1.8-, 2.1-, and 3.9-kb fragments) or 2-9/2-10 (4.1-kb fragment) were sequences were designed to allow amplification of DNA extracted from estimated by visual inspection. For the 4.1-kb fragment, only cases with archival materials, 74k-1 (165 bp, L1930: 5Ј-TCAGGAAACACTTGG- estimated copy numbers clearly less than or more than the reported wild-type GAGTC-3Ј; 74kr1; Ref. 15) for 3Ј end of the locus and 74k-5 (156 bp, L1935: configuration were judged abnormal (19). 5Ј-AAAACCATCCCAAAGAGATG-3Ј and L1936: 5Ј-AAGTGTTTCCT- For the 74k locus, a 414-bp PCR probe (nt12698–13111 in GenBank GAATACAGG-3Ј) for 5Ј end of the locus (15). PCR was performed for 30 AJ243211) was generated with primer L1975 (5Ј-CATGCCTTCT- cycles with annealing temperatures of 60°C and 50°C for 74k-1/APEX and GCTCTCTGA-3Ј) and the published antisense primer for the original 74k 74k-5/desmin, respectively. locus (74kr1), using genomic DNA from glioma cell line IN157, which is Long-range PCR. The possibility of a constitutional 2-4/2-7 deletion, homozygous for the wild-type sequence, as a template. Genomic DNA (12–15 spanning 12.7 kb from intron 14 to exon 25 (reference sequence: GenBank AJ ␮g) from glioma cell lines IN157 and NHG was digested with 200 units of 243211), was assessed by long-range PCR (primers: L1595 in intron 10 and KpnIorEcoRI and separated on a 1.2% Tris acetate-EDTA-agarose gel at 45 L1629 in intron 26) flanking the deletion as described previously (20). With V. Hybridization was carried out as described above. this long-range PCR, the wild-type product would be Ͼ18 kb, and, therefore, RT-PCR. Gli 13, a glioma cell line that is homozygous for the four variant only alleles with intragenic deletions are amplified (5.5 kb; Fig. 1). Blood sequences in the 5Ј upstream region of the DMBT1 gene, was investigated by DNA quality was verified by successful amplification of an 8-kb, long-range RT-PCR for expression of DMBT1. Total RNA extraction and reverse tran- PCR product with primers L1595 and L1864 (in intron 17, 5Ј-GAGGGCCAG- scription was performed as described (20). The published DMBT1-specific GAACGAGGTCGAATA-3Ј); this control long-range PCR was performed at primers were used to amplify a 947-bp DMBT1 cDNA sequence spanning the same time as PCR L1595/L1629 using common PCR reagents, such as exons 45–51 (GenBank AJ243211; Ref. 15). deoxynucleotide triphosphates and Taq DNA polymerase. The presence of a SSCP Analysis. The presence or absence of the variant sequences in the 5Ј constitutional deletion polymorphism was next confirmed by another long- upstream region of the DMBT1 gene was assessed using two overlapping range PCR with a different antisense primer (L1591 in intron 25, 4.2 kb; Ref. SSCP analyses (primers: L1952: 5Ј-CCTCGTTCTTCCAAGGTGAG-3Ј; 20). Because reliability of the selection pressure analysis depends largely on L2045: 5Ј-GGGGAGTTCAAATTCACCTG-3Ј, 74kf2; Ref. 15; and L1933: the accuracy of this assay, the sensitivity and specificity of the long-range PCR 5Ј-GAGGACTCCCAAGTGTTTCC-3Ј) as described (29). assay were verified by comparison with Southern blot data in 48 cases (see DNA Sequencing. The sequence of the proximal 5Ј upstream region of the below) using the probe DMBT1/sr1sid2. DMBT1 gene was investigated by direct sequencing of PCR products (primers: 1791

which had homozygous deletion (Fig. 3). On the other hand, alleles bearing the deletion polymorphism were lost in 6 cases. The identity of the lost allele was indeterminate in a single case. Southern blotting with DMBT1/sr1sid2 as a probe confirmed constitutional deletions in 7 of the 17 cases and lost alleles by 10q loss in 4 of the 7 cases. In all 17 tumors, allelic loss at DMBT1 locus was confirmed by LOH at flanking markers. Alleles with constitutional deletions at DMBT1 2-4/2-7 were not significantly selected for by 10q loss in malignant gliomas (the rate of wild-type allele loss is 62.5%; CI: 35 and 85%, P ϭ 0.45). In addition, there was no significant difference in the frequency of constitutional 2-4/2-7 deletions between malignant glioma patients with (17 of 73) and without LOH (5 of 26, including 1 homozygote; P ϭ 0.79; Table 1). Because Mollenhauer et al. (19) found the constitutional hemizygous deletion at 2-4/2-7 in only 1 of 36 normal individuals (3%), we Fig. 2. Long-range PCR assays for constitutional deletions at DMBT1 2-4/2-7. Top and next investigated the frequency of constitutional DMBT1 deletions in middle: long-range PCRs L1595/L1629 (top) and L1595/L1591 (middle) flanking the 64 unrelated individuals from CEPH reference families to compare an deletion. Amplification of truncated 5.5- and 4.2-kb products indicates constitutional unaffected population with a group of glioma patients. Constitutional deletions in cases 6346, 6364, and 6490 (see Fig. 1). Arrowhead, probable wild-type PCR product (18.2 kb) is occasionally shown. Bottom: long-range PCR L1595/L1864 confirm- hemizygous deletions at 2-4/2-7 were found in 17 of the 64 CEPH ing DNA quality. M, 1-kb ladder marker; U343, a cell line with homozygous deletion at individuals, with a frequency (26.5%; CI: 16 and 39%) not signifi- 2-4/2-7; C, a control constitutional DNA for long-range PCR L1595/L1864; N, normal cantly different from the 22.2% (22 of 99; CI: 14 and 32%) in leukocyte DNA. malignant glioma patients (P ϭ 0.57; Table 1). Moreover, the age at onset of primary glioblastoma patients with constitutional deletions L1952 and 74kr1) with the fmol DNA cycle sequencing system (Promega, and those homozygous for the wild-type copy at 2-4/2-7 was not Madison, WI) or at the Massachusetts General Hospital Genomics Core significantly different (P ϭ 0.96). Therefore, the data do not support Facility. the possibility that carriers of the constitutional DMBT1 2-4/2-7 Statistical Analysis. Exact binomial tests and 95% CIs were used to deletions are predisposed to glioma. analyze the deviation of rates of allelic selection by 10q loss from the null ؅ value of 50%. Fisher’s exact test was used to compare proportions between two Common Polymorphisms in the 5 Upstream Region of the groups. The Wilcoxon rank-sum test was used to compare the distributions of DMBT1 Gene. The 74k locus, which had been noted to undergo age at onset among subgroups of glioma patients. homozygous deletion in 4/21 glioblastomas by PCR (16), was investigated by two multiplex PCR assays covering the entire original 74k RESULTS locus (Fig. 1; Ref. 15). The 74k-1/APEX multiplex PCR assay did not show any tumor with 10q LOH that had an under representation of the Absence of Selection Pressure for Constitutional Deletion 2-4/ target sequence, which would have been compatible with homozygous 2-7. In 99 malignant gliomas with good quality constitutional DNA, deletion. On the other hand, the 74k-5/desmin multiplex PCR dem- 73 had allelic loss at 10q25-26 and were included in an analysis to onstrated under representation of the target sequence in 21 of the 73 determine whether there is selection pressure for loss of the intact versus the deleted DMBT1 allele. All 73 tumors were analyzed suc- cessfully by both multiplex PCR g14/c12 and intron 20 sequence/ desmin assays for homozygous deletion at DMBT1 2-4/2-7. Homozy- gous deletion was found in 10 of the 73 malignant gliomas with LOH at 10q25-26. The possibility of constitutional hemizygous deletion was investigated by two long-range PCRs, using primer pairs L1595/ L1629 and L1595/L1591 flanking the deletion, in which the presence of truncated 5.5- and 4.2-kb products would indicate a constitutional Fig. 3. Selection pressure by 10q loss for DMBT1 deletion 2-4/2-7, assessed by multiplex PCR intron 20 sequence/desmin. All tumors had allelic loss at 10q25-26. deletion (Figs. 1 and 2). Blood DNA quality was verified by success- Homozygous deletions were noted in tumors 1670, 1672, and 1688, suggesting losses of lost alleles were confirmed by Southern blot ,ء .ful amplification of an 8-kb product in a control long-range PCR, alleles bearing the wild-type copy L1595/L1864, which was performed at the same time as PCR L1595/ analysis. N, normal leukocyte DNA; T, tumor DNA. L1629 (Figs. 1 and 2). These strategies eliminated the possibility that constitutional deletions would be missed because of poor DNA qual- Table 1 DMBT1 constitutional polymorphisms in patients with malignant gliomas and ity or rare polymorphisms in primer target sequences. Long-range reference individuals PCRs L1595/L1629 and L1595/L1591 demonstrated constitutional Cases with Cases with hemizygous deletion at 2-4/2-7 in 17 of the 73 cases whose tumors Cases deletion 2-4/2-7a,b 5Ј polymorphismsc had 10q LOH. These 17 cases included all 10 cases whose tumors Patients with malignant glioma (99 cases) d showed homozygous deletion, indicating that all homozygous dele- 73 cases with LOH 17 (0) 38 (7) 26e cases without LOH 5 (1) 9 (3) tions at DMBT1 repeat 2-4/2-7 in tumors occurred as a result of 10q Reference individualsf (64 cases) 17 (0) 36 (2) loss, unmasking the constitutional deletion polymorphism. a No significant difference between malignant glioma patients with and without LOH The possibility of selection pressure for the deleted versus intact (P ϭ 0.79). b allele was then assessed by comparing the DMBT1:control ratio of No significant difference between patients with malignant gliomas and reference individuals (P ϭ 0.57). tumor DNA to those of paired constitutional DNA using multiplex c Suggestion of a difference in the distribution between patients with malignant PCR g14/c12, intron 20 sequence/desmin, and g14/APEX. Of the 17 gliomas and reference individuals (P ϭ 0.08). d Number of homozygotes are indicated in parentheses. cases with constitutional hemizygous deletions, chromosome 10q loss e Twenty-five cases were examined for 5Ј polymorphisms. affected alleles bearing the wild-type DMBT1 gene in 10 cases, all of f Apparently unrelated and unaffected individuals from the CEPH reference families. 1792

pressed in a glioma cell line (Gli 13) homozygous for the four polymorphisms, in contrast to glioma cell lines that lacked DMBT1 expression, such as IN157, NHG, and U343 (data not shown). In 64 unrelated CEPH individuals, two were homozygous, and 34 were heterozygous for the polymorphisms by SSCP, with a suggested difference in the distribution of genotypes between glioma patients and CEPH individuals (P ϭ 0.08; Table 1). The age at onset of primary glioblastoma patients homozygous for the polymorphisms was not significantly different from heterozygotes and those homozygous for the wild-type copy at the 74k locus (P ϭ 0.10). The haplotype with both deletion 2-4/2-7 and the two 5Ј polymorphisms within the region analyzed by SSCP (2-bp deletion and G to T missense) were not significantly selected for by 10q loss, with 10 of 14 alleles bearing the haplotype selected (the rate of wild-type allele loss is 71%; CI: 42 and 92%, P ϭ 0.18). Insertion/Deletion Polymorphisms at DMBT1 Repeat 2-9/2-10. Deletion polymorphisms at DMBT1 repeat 2-9/2-10 have been found in 9 of 36 (25%) normal individuals by Southern blotting (19). Fig. 4. A, multiplex PCR 74k-5/desmin. N4, leukocyte DNA homozygous for the Because it was not possible to design a unique PCR assay within the wild-type sequence at 5Ј upstream of DMBT1. Cases 1576, 8086, 6368, 6320, 6140, 1670, and 1672 are heterozygotes for the linked polymorphisms whose tumors had allelic loss putative deleted region at 2-9/2-10 to assess the possibility of selec- of chromosome 10q. The primer target sequences are unique to the wild-type sequence, tion pressure, to address the possibility of predisposition of deletion and loss of alleles with the wild-type sequence demonstrates apparent homozygous carriers to gliomas, we compared the frequency of these polymor- deletions in cases 1670 and 1672. w, allele with the wild-type sequence retained; v, allele with the variant sequences retained. N, normal leukocyte DNA; T, tumor DNA. B, PCR phisms in patients with benign intracranial tumors and patients with products flanking two of the four polymorphic sites (2-bp deletion and G to T missense) malignant gliomas. Southern blot analysis with DMBT1/ sr1sid2 as a separated on a sequencing gel. Cases 7048 and 8046 lack the deletion and are homozygous for the wild-type copy. Cases 6912 and 6180 are homozygous for the 2-bp deletion and probe demonstrated aberrations in the DMBT1 gene in 10 of 17 were homozygous for the G to T polymorphism as well (data not shown). Cases 6368, patients with benign intracranial tumors, relative to the reportedly 6320, and 1670 are heterozygous for the polymorphisms, as illustrated by the presence of wild-type G1 configuration (19). These included 8 patients with a gain both alleles with and without the 2-bp deletion. In tumors 6368 and 6320, the wild-type ϭ ϭ alleles were selectively retained after 10q loss. In tumor 1670, the allele with the variant (n 2) or loss (n 6) at DMBT1 repeat 2-9/2-10 and 2 with a sequences was selected for by 10q loss, as illustrated by loss of the wild-type allele. N, deletion at 2-4/2-7 (Fig. 5). In 16 patients with gliomas, 6 had a gain normal leukocyte DNA; T, tumor DNA. Bars, bands specific for wild-type and variant (n ϭ 1) or loss (n ϭ 5) at DMBT1 repeat 2-9/2-10, 1 had a deletion sequences. at DMBT1 2-4/2-7, and 2 had both deletions at 2-9/2-10 and 2-4/2-7 tumors with 10q LOH, and the 74k-5 sequence was not amplified even in corresponding blood DNA in 7 of the 21 cases. However, on Southern blotting with a probe covering the 74k locus, fragments containing exon 1 sequence were detected in both cell lines: (a) IN157, which had amplification of 74k-5; and (b) NHG, which lacked 74k-5 amplification (data not shown). This suggested that the 74k locus 5Ј upstream of DMBT1 exon 1 was present in both cell lines. Sequencing of the proximal 5Ј region in 9 samples (3 cell line DNA, 2 blood DNA, and 4 tumor DNA) demonstrated at least four polymorphic sites: (a) 12816–12817delAA; (b) 12878G Ͼ T; (c) 12942T Ͼ C; and (d) 12969C Ͼ T (GenBank AJ243211). These four polymorphisms appeared linked, based on sequencing and two overlapping SSCP analyses for three of the polymorphisms. Two of the polymorphisms (2bp deletion and T to C missense) were on primer target sequences for 74k-5, making the 74k-5 PCR unique to the wild-type sequence. SSCP analysis, including two of these four polymorphic sites (2-bp deletion and G to T missense), demonstrated that, in 98 malignant glioma patients, 10 were homozygous, and 37 were heterozygous for the apparently linked polymorphisms (data not shown). All 21 patients whose tumors showed apparent homozygous deletions at 74k-5 locus had at least one allele with the polymorphisms in their constitutional DNA, suggesting that under represen- Fig. 5. Southern blot analysis with DMBT1 cDNA probe. RsaI-digested constitutional tation of the 74k-5 sequence in tumors was attributable to unmasking DNA was hybridized with DMBT1/sr1sid2, and fragments containing SRCR domains are of alleles with the variant sequences by 10q loss. The possibility of shown. a, a glioma patient with probable wild-type DMBT1 configuration. The total copy numbers of each fragment are depicted on the right (see Fig. 1). About 2-fold increased selection pressure to retain the polymorphic alleles was assessed using intensities are shown for the 4.1- and 3.9-kb fragment, because of duplication. DMBT1 SSCP analysis and the 74k-5/desmin multiplex PCR. Only 14 alleles repeats 2-4 to 2-7 are represented by 1.8-, 2.1-, and 3.9-kb fragments, and DMBT1 repeats with the 5Ј polymorphisms were selected for by 10q loss in 31 2-9 and 2-10 are represented by 4.1-kb fragments. For 4.1-kb fragments, the distinction between three and four copies was difficult and not attempted. b, a meningioma patient heterozygotes whose tumors had 10q LOH (Fig. 4), suggesting that who lacks the entire DMBT1 repeat 2-9/2-10 in both alleles; c, a glioma patient who lacks there is no significant selection pressure to retain the polymorphic three of the four copies at 2-9/2-10 and one of the two copies at 2-4/2-7; d, a glioma patient who lacks two of the four copies at 2-9/2-10. An extra band ϳ1.6 kb with a single allele (the rate of wild-type allele loss is 45%; CI: 27 and 64%, copy intensity was noted occasionally. Arrows, aberrations, with the copy numbers of the P ϭ 0.72). In addition, DMBT1 was shown by RT-PCR to be ex- remaining fragments noted. 1793

tumors6 (12–14). Furthermore, Mollenhauer et al. reported constitutional hemizygous deletion 2-4/2-7 involving the g14 locus in 1 of 36 normal individuals (3%), indicating that some homozygous deletions in tumors may reflect allelic loss unmasking preexistent, constitutional deletions. In malignant gliomas, we have shown that at least some homozygous DMBT1 deletions indeed reflect constitutional deletions and allelic loss (20). In the present study, we identified 10 tumors with homozygous deletion at DMBT1 2-4/2-7 in a series of 73 malignant gliomas with 10q loss; significantly, all of these deletions represented constitutional hemizygous deletions coupled with allelic loss. Taken together, the above data argue against DMBT1 as a classical tumor suppressor gene after the somatic “two-hit” model. Nonetheless, there is increasing evidence of associations between common polymorphisms and diseases, including cancer (31, 32). It remains possible that constitutional DMBT1 deletion polymorphisms are tumorigenic once loss of the wild-type allele takes place. To address this possibility, we determined whether allelic loss preferen- tially affected the wild-type versus the deleted DMBT1 copy. We hypothesized that, if these polymorphisms contributed to tumorigenesis, there would be selection to delete the wild-type allele. However, no significant selection pressure was apparent for alleles with either the 2-4/2-7 deletion, polymorphisms in the 5Ј upstream region of the gene, or the haplotype with both. If these polymorphisms play a major Fig. 6. A, Southern blot analysis of the family members of a glioma patient homozy- role in tumorigenesis, alleles with the polymorphisms should be gous for constitutional deletion 2-4/2-7. a, a glioma patient with probable wild-type 7 DMBT1 configuration for direct comparison; b, a patient with an anaplastic oligodendro- selected exclusively for when 10q is lost. Therefore, these data glioma who is constitutionally homozygous for deletion 2-4/2-7. This patient also has only suggest that DMBT1 polymorphisms are not likely primary targets of a single copy of a 4.1-kb fragment, indicating deletion polymorphism at DMBT1 2-9/2-10. 10q loss in gliomas. Nonetheless, we cannot exclude the possibility c, father of the patient; d, mother of the patient. Arrows, hemizygous losses of 1.8-, 2.1-, and 3.9-kb fragments, representing DMBT1 repeats 2-4/2-7, in both parents. B, SSCP that a specific haplotype, such as a combination of three polymor- analysis flanking two of the four polymorphism sites (G to T missense and T to C phisms (5Ј upstream, 2-4/2-7, and 2-9/2-10) or polymorphisms with a missense) in the 5Ј upstream region of DMBT1. a, leukocyte DNA homozygous for the wild-type sequence; b, father of the patient; c, mother of the patient; d, patient. Both particular parental inheritance, might be selected for by 10q loss. parents are heterozygous, and the patient is homozygous for the polymorphisms. Bars, Because polymorphic sequences in putative cancer susceptibility bands specific for wild-type and variant sequences. Bottom band, nondenatured, double- genes may be associated with an increased risk of cancer (33, 34), it strand DNA. remained possible that constitutional DMBT1 polymorphisms predis- pose to glioma formation. Although there was a suggestion of a difference in the distribution of the 5Ј upstream polymorphisms be- (Fig. 5). Three patients with benign tumors (2 meningiomas and 1 tween patients with malignant gliomas and CEPH individuals, a schwannoma) and a patient with glioblastoma lacked the whole constitutional 2-4/2-7 deletion was found at a similar frequency in DMBT1 repeat 2-9/2-10 in both alleles (Fig. 5). The age of tumor CEPH individuals (26.5%) and in patients with malignant gliomas presentation in these 4 patients was typical (47–52 years of age for (22.2%). In patients with benign brain tumors, aberrations at 2-9/2-10 benign tumors and 46 years of age for glioblastoma). appeared as common as in patients with gliomas. Furthermore, the age Constitutional Homozygotes for DMBT1 Deletion 2-4/2-7. In of glioblastoma presentation for patients who had the constitutional the course of our previous (20) and present studies, which have 2-4/2-7 deletion and for patients who were homozygous for the 5Ј included 190 patients with gliomas, 17 with benign intracranial tu- polymorphisms was not significantly different from those of patients mors, and 64 CEPH individuals, we have encountered 2 persons with with two wild-type copies at each of the polymorphic sites. All of constitutional homozygous deletions at DMBT1 2-4/2-7. These 2 these results, except for the possible difference in the distribution of Ј persons were also constitutionally homozygous for the three 5 poly- the 5Ј polymorphisms between glioma patients and CEPH individuals, morphisms within SSCP region. Both were patients with malignant argue against DMBT1 as a potential glioma susceptibility gene. glioma: (a) a 32-year-old woman with a glioblastoma; and (b)a Strikingly, two patients were homozygous for the constitutional 35-year-old man with an anaplastic oligodendroglioma. The family 2-4/2-7 deletion, as well as for the 5Ј polymorphisms. These patients history was available from the latter patient; although both parents are apparently had no history of developmental abnormalities, arguing healthy, his brother has Crohn’s disease, and his sister had multiple against an essential role for DMBT1 in development. In addition, the sclerosis. Southern blotting with DMBT1/ sr1sid2 as a probe and lack of other known tumors in these individuals, and their relatively SSCP analysis demonstrated that both parents are heterozygous for typical age of clinical presentation, as well as the lack of family cancer Ј both the 2-4/2-7 deletion and the 5 polymorphisms, suggesting Men- predisposition in the patient for whom family history was available, delian inheritance of these polymorphisms (Fig. 6). Constitutional all also argue against a role for these polymorphisms in tumorigenesis. DNA from the two siblings was not available. Interestingly, however, this family history raises the intriguing possibility that DMBT1 alterations could relate to other diseases, because DISCUSSION the patient’s siblings suffered from inflammatory bowel disease and multiple sclerosis, both considered autoimmune diseases. Because Homozygous deletion at the g14 locus is present in a significant fraction of various cancers and has suggested DMBT1 as a candidate 6 A. von Deimling, personal communication. tumor suppressor (12, 13, 15–17, 30). However, only rare somatic 7 The 95% upper confidence limits for the rates of wild-type allele loss are 85, 64, and point mutations of uncertain significance have been found in human 92% for deletion 2-4/2-7, the 5Ј polymorphisms, and the haplotype, respectively. 1794

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