Oncogene (2002) 21, 1461 ± 1468 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

Identi®cation of a region of homozygous deletion on 8p22 ± 23.1 in medulloblastoma

Xiao-lu Yin1, Jesse Chung-sean Pang1 and Ho-keung Ng*,1

1Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China

To identify critical tumor suppressor loci that are radiotherapy and chemotherapy, have greatly improved associated with the development of medulloblastoma, the outcomes of patients. In the past 30 years, the 5- we performed a comprehensive genome-wide allelotype year survival rate has increased from 10 to about 50%. analysis in a series of 12 medulloblastomas. Non-random However, long-term survival in children with advanced allelic imbalances were identi®ed on chromosomes 7q disease is only about 30% (Giangaspero et al., 2000; (58.3%), 8p (66.7%), 16q (58.3%), 17p (58.3%) and Heideman et al., 1997). Further enhancement of 17q (66.7%). Comparative genomic hybridization analy- survival will rely on a better understanding of the sis con®rmed that allelic imbalances on 8p, 16q and 17p biology of this malignant disease to improve current were due to loss of genetic materials. Finer deletion treatments or to develop novel therapy. mapping in an expanded series of 23 medulloblastomas Non-random loss of genetic material from chromo- localized the common deletion region on 8p to an interval somal loci is a common feature in the development of of 18.14 cM on 8p22 ± 23.2. We then searched within the tumors and is often indicative of the presence of critical region of loss on 8p for loci that might contain tumor suppressor genes in the given tumor. Inactivation homozygous deletion using comparative duplex PCR. of multiple tumor suppressor genes is typically required An overlapping homozygous deletion region was identi- for tumor formation. In medulloblastoma, deletion in ®ed in a 1.8 cM interval on 8p22 ± 23.1, between markers the short arm of chromosome 17 is the most common D8S520 and D8S1130, in two medulloblastomas. This genetic abnormality seen in up to 50% of tumors region of homozygous deletion also encompasses the (Bigner et al., 1988). This result suggests that loss of a 1.4 cM minimal deletion region detected on 8p in ductal tumor suppressor gene located on 17p plays an carcinoma in situ of breast. In conclusion, we reported important role in the pathogenesis of this malignant for the ®rst time a detailed deletion mapping on 8p in tumor. The TP53 gene was found to be infrequently medulloblastoma and have identi®ed a region of mutated in medulloblastoma, indicating that TP53 is homozygous deletion on 8p22 ± 23.1 that is likely to not the target on 17p (Saylors et al., 1991). Using contain a critical tumor suppressor gene involved in the comparative genomic hybridization technique, several development of medulloblastoma. groups have demonstrated the involvement of multiple Oncogene (2002) 21, 1461 ± 1468. DOI: 10.1038/sj/ chromosomes in medulloblastoma (Avet-Loiseau et al., onc/1205204 1999; Gilhuis et al., 2000; Nicholson et al., 1999; Reardon et al., 1997). Of those chromosomes that show Keywords: medulloblastoma; chromosome 8p; homo- deletions, losses of chromosomes 10q and 8p are zygous deletion; allelotyping recurrent genetic alterations, with about 30% of tumors showing such abnormalities. Other common genetic losses occurring at a lower frequency include deletion of Introduction chromosomes 3, 6, 9, 11, 16 and 22. We have previously performed a deletion mapping study on chromosomes Medulloblastoma is a malignant, invasive embryonal 10q, 11 and 16. Three regions of frequent loss were tumor that predominantly arises in the cerebellum. It is localized to the Deleted in Malignant Brain Tumor 1 the most common brain tumor in children and (DMBT1) locus on 10q25 ± 26, 11p13 ± 15.1 and 16q24 constitutes 10 ± 20% of pediatric central nervous (Yin et al., 2001). So far, no medulloblastoma- system neoplasms. Medulloblastoma has a propensity associated tumor suppressor genes have been identi®ed for cerebrospinal dissemination, which is present in in any of these aberrant chromosomes. one-third of patients at presentation. Signi®cant In this study, we aimed to search for critical tumor advances in a multimodal therapy, including surgery, suppressor loci that are important for the development of medullolastoma. We used high-resolution genome- wide allelotype analysis followed by ®ner deletion mapping to localize chromosomal regions with fre- *Correspondence: HK Ng; E-mail: [email protected] Received 21 August 2001; revised 9 November 2001; accepted 27 quent deletion. Non-random allelic deletions were November 2001 identi®ed on 8p, 16q and 17p. Further mapping has Chromosome 8p loss in medulloblastoma X-l Yin et al 1462 revealed a homozygous deletion region of 1.8 cM on (41.7%), 7p (33.3%), 8q (41.7%), 10q (41.7%), 13q 8p22 ± 23.1, which is likely to contain a tumor (33.3%), 14q (33.3%), and 20q (33.3%). suppressor involved in the tumorigenesis of medullo- The fractional allelic loss (FAL) for each tumor was blastoma. In addition, the Deleted in Liver Cancer 1 also determined. FAL is de®ned as the fraction of the (DLC1) gene, a recently cloned candidate tumor total number of informative chromosomal arms that suppressor gene located on 8p21.3 ± 22, was also displays allelic loss. In the series, the FAL values assessed for its involvement in medulloblastoma. ranged from 0.08 to 0.77. Interestingly, the tumor with the highest FAL (0.77) was a desmoplastic variant (case M15). The mean FAL was 0.26+0.19. There was no correlation between FAL and patient age, sex, or Results tumor type. Clinical data of patients Localization of common deletion region on 8p In the series, there were 24 classical, two desmoplastic and one anaplastic medulloblastomas. All tumors were To re®ne the common deletion region on the short arm cerebellar. No history of Gorlin or Turcot syndrome of chromosome 8, we evaluated the LOH pattern in an was associated with this series of tumors. There were expanded series of 21 primary and two recurrent three adult and 21 pediatric patients and their ages medulloblastomas using a separate panel of 19 ranged from 2 ± 69 years with a mean of 12.5 years. microsatellite markers mapping to 8p. Nineteen of 23 The male/female ratio was 2.8 : 1. One child (case M1) (83%) tumors showed allelic loss on at least two loci experienced two relapses over a period of 4 years. (Figure 3). The overall frequency of LOH on 8p was 51% (124 of 243 informative markers), comparable to that observed in allelotype analysis (58.8%, 30 of 51 Allelotype analysis informative markers). Two cases (M15 and M16) To localize critical tumor suppressor loci involved in revealed LOH on all informative markers examined, the tumorigenesis of medulloblastoma, we performed a suggesting the presence of monosomy 8 in these comprehensive genome-wide allelotype analysis on 12 tumors. The other 21 cases showed partial or medulloblastomas. All 22 autosomes were examined interstitial deletion on 8p. Combining all LOH data, for allelic imbalances. An average of 238 (62.3%) a common region of deletion was narrowed down from informative loci/case was detected in our series. Allelic 37 cM on 8p12 ± 23, as determined by allelotyping, to imbalances were seen in all 39 autosomal arms. The an interval of 18.14 cM on 8p22 ± 23.2, between highest frequencies of allelic imbalance were detected markers D8S262 and D8S1130. The frequency of on chromosomal arms 8p and 17q (66.7%), whereas LOH at this region was 70.7%. In addition, one tumor the arms with the lowest frequencies of allelic (M3) revealed microsatellite instability on the marker imbalances were seen on chromosomes 5p, 9p, 9q, D8S1721. Case M1 and its two recurrent tumors 11q, 16p, 18q, 19p, and 21p (8.3%). The mean showed the same allelic loss pro®le in all informative percentage of allelic imbalances was 26.7%+16.6%. markers examined. Figure 1 summarizes the frequency of allelic imbal- ances at each autosomal arm. In the present study, Detection of homozygous deletion on 8p22 ± 23.1 43.3% (mean percentage+s.d., 26.7+16.6%) was chosen to be a signi®cant percentage of allelic Identi®cation of homozygous deletion region is im- imbalances in our series. This represents the 99% portant for positional cloning of tumor suppressor con®dence upper limit for the overall rate for random gene. We therefore screened chromosomal loci, at chromosome loss or imbalance in tumors. Non-random which the heterozygous alleles were ¯anked by lost allelic imbalances above the baseline (43.3%) were alleles, on 8p for possible homozygous deletion using identi®ed on chromosomal arms 7q (58.3%), 8p comparative duplex PCR. Two tumors were found to (66.7%), 16q (58.3%), 17p (58.3%), and 17q (66.7%). have homozygous deletion: M2 at D8S520 and M3 at Representative results of allelic imbalances at selected D8S1130 (Figure 4). These two tumors shared an microsatellite loci are illustrated in Figure 2a. Using overlapping deletion region, with the two markers CGH analysis, we con®rmed that allelic imbalances being only 1.8 cM apart. The homozygous deletion detected by allelotyping on chromosomes 8p, 16q, and region was mapped to the junction of 8p22 and 8p23.1. 17p were due to chromosome loss, whereas those of 7q Thus we have further re®ned the common region of and 17q referred to allelic gain (Figure 2b). We have deletion on 8p to an interval of 1.8 cM. also delineated the common regions of deletion on chromosomes that are frequently lost in medulloblas- Molecular genetic investigations of DLC1 gene toma. These regions of loss on chromosomes 8p, 16q and 17p were mapped to 8p12-pter (37 cM), 16q22.3- A recently identi®ed candidate tumor suppressor gene pter (46.6 cM) and 17p13.1-pter (15 cM), respectively. termed DLC1, that is located on 8p21.3 ± 22, was In addition, chromosomal arms with frequencies of investigated for its involvement in medulloblastoma. allelic imbalances higher than the mean percentage of Using duplex PCR, we did not detect homozygous LOH were identi®ed on 3p (33.3%), 3q (33.3%), 4q deletion of the DLC1 gene in the tumor series (data

Oncogene Chromosome 8p loss in medulloblastoma X-l Yin et al 1463

Figure 1 Summarized results of allelic imbalances at each nonacrocentric chromosomal arm in 12 medulloblastomas studied. Black and white bars denote short (p) and long (q) arms, respectively. The dotted line represents the cut-o€ baseline and is de®ned as the mean percentage of allelic imbalance plus one standard deviation (26.7+16.6%=43.3%) not shown). We then employed a highly sensitive Although the number of medulloblastomas allelotyped method, CSGE, to detect somatic mutations in the in the present study is limited (12 cases), our results are DLC1 gene. Twenty-six medulloblastomas were consistent with the published data. We have previously screened. No heteroduplexes with aberrant gel shift mapped the regions of frequent loss on chromosomes were seen, indicating the absence of somatic mutations 11 and 16 to 11p13 ± 15.1 and 16q24.1 ± 24.3, respec- in the DLC1 gene in medulloblastoma (data not tively (Yin et al., 2001). The localization of these shown). critical loci is important for future ®ne mapping and cloning of candidate tumor suppressor genes. The DMBT1 locus on 10q25 ± 26 is also found to be closely Discussion associated with the development of medulloblastoma (Yin et al., 2001). In this study, we performed a high-resolution genome- The most striking ®nding in the present study is the wide allelotype analysis on medulloblastoma with an high incidence (67%) of allelic deletion on chromosome aim to localize putative tumor suppressor loci involved 8p in medulloblastoma. This result is in agreement with in this malignant neoplasm. Non-random allelic losses previous low-resolution allelotyping study (50%) but is were identi®ed on chromosomes 8p (66.7%), 16q about twofold higher than those determined by CGH (58.3%) and 17p (58.3%), strongly indicating that studies (32%) (Blaeker et al., 1996; Reardon et al., alterations of these chromosomes play critical roles in 1997; Avet-Loiseau et al., 1999; Nicholson et al., 1999; medulloblastoma. Further detailed mapping unveiled Gilhuis et al., 2000). The use of high-density markers on 8p22 ± 23.1 a novel region of homozygous deletion in the present microsatellite analysis probably explains in an interval of 1.8 cM, ¯anked by markers D8S520 the increased sensitivity of detecting allelic loss. and D8S1130. Frequent deletion of the chromosomal arm 8p has To compare our ®ndings with those of previous also been detected in a variety of tumors, such as investigations, we have reviewed genetic data from six prostate, breast, lung, colorectal, bladder, head and genome-wide studies covering a total of 107 medullo- neck, liver, ovarian and gastric cancers (Sun et al., blastomas (Blaeker et al., 1996; Reardon et al., 1997; 1999; Wang et al., 1999; Wistuba et al., 1999; Avet-Loiseau et al., 1999; Nicholson et al., 1999; Farrington et al., 1996; Takle and Knowles, 1996; Gilhuis et al., 2000; von Deimling et al., 2000). The Sunwoo et al., 1999; Pineau et al., 1999; Wright et al., summarized results revealed that deletions of 17p 1998; Ba€a et al., 2000). Deletion mapping has (39%), 10q (35%), 8p (32%), 11p (28%), 11q (28%), identi®ed several sub-chromosomal regions on 8p that and 16q (20%) are the most frequent genetic are important for cancer development. These regions abnormalities involving loss of genetic materials. are localized to 8p12, 8p21, 8p22, and 8p23-pter.

Oncogene Chromosome 8p loss in medulloblastoma X-l Yin et al 1464 a involvement of 8p loss in tumorigenesis (Nihei et al., 1996; Tanaka et al., 1996). In this study, we have for the ®rst time reported a detailed deletion mapping on chromosome 8p in medulloblastoma and have identi- ®ed a homozygous deletion region that encompasses an interval of 1.8 cM on 8p22 ± 23.1. Losses of 8p22 and 8p23.1 subregions are recurrent genetic aberrations in a number of tumors such as prostate, pancreas, breast, liver, gastric, ovarian and colorectal cancers (Arbieva et al., 2000; Pineau et al., 1999; Levy et al., 1999; Wang et al., 1999; Ba€a et al., 2000; Wright et al., 1998; Fujiwara et al., 1993; Lassus et al., 2001). Three critical deletion regions and several candidate tumor suppres- sor genes have been identi®ed within the locus of 8p22 ± 23.1 and their relative genetic locations are shown in Figure 4. One deletion region is located in an interval of 1.4 cM, de®ned by markers D8S520 and D8S261, in ductal carcinoma in situ of breast (Wang et al., 1999). This deletion region lies within the b homozygously deleted region identi®ed in medulloblas- toma, suggesting the presence of a novel tumor suppressor gene at this locus. It is likely that the same tumor suppressor gene from this region is operative in both breast cancer and medulloblastoma. A sequence- ready YAC/BAC clone contig has been constructed for *1.7 cM in this region and at least 10 known genes and expressed sequence tags are mapped (Wang et al., 1999). It remains to be determined whether any of these genes is the candidate tumor suppressor in the interval ¯anked by D8S520 and D8S1130. Another critical deletion region that maps to 8p22 is located proximal to the 1.8 cM interval identi®ed in this study. An overlapping region of homozygous deletion of 1 ± 1.5 cM was seen in a metastatic prostate tumor and a pancreatic tumor cell line (Arbieva et al., 2000; Bova et al., 1996; Levy et al., 1999). No candidate tumor Figure 2 Representative results of genome-wide allelotype and suppressor genes have yet been identi®ed from this CGH analyses. (a) Allelic imbalances (arrows) detected at selected microsatellite loci, D8S258 (8p21) and D8S514 (8q24), in case region. The third deletion region is located on 8p23.1 M7. N, normal blood control; T, tumor. (b) CGH pro®le of case between D8S262 and D8S520. This region is found M7 showing loss on chromosome 8p21-pter and gain on deleted in about 60% of ovarian tumors (Lassus et al., chromosome 8q23-q24.1, as indicated by red and green bars, 2001). respectively. CGH can be used to verify the allelic status of Recently, a novel tumor suppressor gene named polymorphic loci. In case M7, allelic imbalances at D8S258 and D8S514 are due to chromosomal loss and gain, respectively DLC1, which is associated with hepatocellular carcinoma, is isolated from 8p21.3 ± p22 (Yuan et al., 1998). Recent draft human genome sequence has mapped the DLC1 gene to 8p22. The DLC1 encoded Within the deletion loci, critical regions with homo- product is a member of the Rho family GTPases, zygous deletion have been identi®ed on 8p21, 8p22, which function as important regulators in the and 8p23 (Kagan et al., 1995; Prasad et al., 1998; organization of actin cytoskeleton. In tumors, Iswad et al., 1999; Levy et al., 1999; Sun et al., 1999; dysregulation of the RhoGTPases-associated signaling Arbieva et al., 2000). These results suggest that pathways may lead to motile and invasive phenotype multiple tumor suppressor genes are located on the (Schmitz et al., 2000). Wilson et al. (2000) detected short arm of chromosome 8. In addition, loss of 8p has one missense mutation of the DLC1 gene in a series been found to be associated with clinicopathological of 126 colorectal and 33 ovarian tumors and cell ®ndings. For instance, frequent deletion of 8p was lines. Ng et al. (2000) detected homozygous deletion correlated with invasive behavior in breast cancer and in two of six hepatocellular carcinomas and two with poor outcome in prostate cancer. (Yaremko et al., hepatoma cell lines. More importantly, they demon- 1996; Jenkins et al., 1998). Functional studies demon- strated that overexpression of DLC1 in DLC1- strated that restoration of normal chromosome 8p de®cient hepatoma cells resulted in suppressed growth suppressed tumorigenecity and metastasis in colon and (Ng et al., 2000). In the present study, we evaluated prostate tumor cells respectively, further supporting the whether DLC1 plays a role in the development of

Oncogene Chromosome 8p loss in medulloblastoma X-l Yin et al 1465 medulloblastoma. We examined the DLC1 gene by DLC1 gene sequence were detected in the series. Our comparative duplex PCR and mutation analyses. No results suggest that DLC-1 is not involved in homozygous deletion and somatic mutation of the medulloblastoma.

Figure 3 Summary of microsatellite analysis on chromosome 8p. A total of 25 polymorphic loci were evaluated for LOH in 23 medulloblastomas. A region of homozygous deletion (striped box) is localized to an interval of 1.8 cM on 8p22 ± 23.1, between loci D8S520 and D8S1130. Case numbers are indicated on top. Polymorphic loci examined in allelotype analysis are boxed. Filled circles, LOH; open circles, heterozygous; lines connecting loci, non-informative or not done; MI, microsatellite instability. Case M1 and its two recurrent tumors show identical allelic loss pattern and only results of primary tumor are shown

Figure 4 A genetic map displaying the relative locations of critical deletion regions and candidate tumor suppressor genes on 8p22 ± 23.1. The order of markers is arranged based on genetic map available at website http://research.marsh®eldclinic.org/genetics. The dotted box represents deletion region seen in ovarian cancer on 8p23.1. The striped box indicates the 1.8 cM homozygously deletion region detected in medulloblastoma on 8p22 ± 23.1. The gray box represents the region of homozygous deletion in the DLC1 gene detected in hepatocellular carcinoma. The dark box represents the region of homozygous deletion on 8p22 found in prostate and pancreatic cancer. The thumbnails show the allelic status of medulloblastoma cases M2 and M3 at loci D8S520 and D8S1130. M3 shows LOH (arrow) at locus D8S520 by microsatellite analysis and homozygous deletion (arrowhead) at D8S1130 by comparative duplex PCR. M2 shows homozygous deletion at D8S520 and LOH at D8S1130. N, normal blood control; T, tumor

Oncogene Chromosome 8p loss in medulloblastoma X-l Yin et al 1466 Another candidate tumor suppressor gene termed of three ¯uorescent dyes, FAM, HEX, or NED. PCR was LZTS1/FEZ1 (Leucine Zipper. Putative Tumor Sup- performed in a ®nal volume of 7.5 ml containing two primer pressor 1) is mapped to 8p22. Somatic mutation and pairs (2.5 pmoles of each primer), 60 ng of DNA, 10 mM altered transcript expression of LZTS1/FEZ1 have Tris-HCl (pH 8.3), 50 mM KCl, 0.2 mM deoxyribonucleoside been detected in several cancer including esophageal, triphosphates, 2.5 mM MgCl2 and 0.6 unit of AmpliTaq Gold DNA polymerase (Applied Biosystems). To facilitate prostate and gastric tumors (Ishii et al., 1999; high throughput of samples, all liquid handling and thermal Vecchione et al., 2001). A functional approach cycling were carried out in an ABI Prism 877 robotic demonstrated that overexpression of LZTS1/FEZ1 in workstation (Applied Biosystems). PCR was started, LZTS1/FEZ1-negative cancer cells resulted in suppres- according to manufacturer's recommendation, with 958C sion of tumorigenicity and reduced cell growth. for 15 min, followed by 10 cycles composed of 948C for Further investigation showed that LZTS1/FEZ1 is 15 s, 558C for 15 s and 728C for 30 s, and another 22 cycles involved in regulation of mitosis (Ishii et al., 2001). composed of 898C for 15 s, 558C for 15 s and 728C for These results strongly suggest that LZTS1/FEZ1 is a 30 s. Ampli®ed PCR products of multiple loci were pooled potential tumor suppressor involved in various human and electrophoresed in denaturing 5% polyacrylamide gels cancer. on an ABI Prism 377 automated DNA sequencer (Applied Biosystems). The data collected were analysed using In conclusion, our comprehensive and extensive GeneScan Analysis software version 3.1 (Applied Biosys- deletion mapping analysis has unveiled a region of tems). Allelic imbalance was de®ned by calculating the homozygous deletion on chromosome 8p22 ± 23.1 in allelic ratio (AR) of both normal (N) and tumor (T) DNA, medulloblastoma. This region is likely to contain a where AR was the ratio of peak height of the longer allele critical tumor suppressor important for the develop- (N2 or T2) to that of the shorter allele (N1 or T1), i.e., ment of this malignant neoplasm. The recent avail- AR=(N2/N1)/(T2/T1). Allelic imbalance was indicated ability of physical map and gene sequence within the when the ratio was greater than 1.5 or smaller than 0.5, homozygous deletion region will facilitate the identi®- representing loss of longer or shorter allele respectively. As cation of the putative tumor suppressor gene on 8p22 ± both allelic loss and gain exhibited LOH pattern in 23.1. microsatellite analysis, we therefore used comparative genomic hybridization (CGH) to verify the allelic status. The CGH analysis was performed according to protocol reported (Yin et al., 2000). To narrow down the deletion region on the short arm of Materials and methods chromosome 8, we performed ®ner deletion mapping using radioactively labeled microsatellite markers as described Patients and specimens previously (Yin et al., 2001). A separate set of 19 Twenty-seven medulloblastoma samples of 25 primary and polymorphic markers mapping to 8p was used. The order two recurrent tumors were collected from the Department of of markers was arranged according to the genetic map Anatomical and Cellular Pathology, Prince of Wales compiled by Broman et al. (1998) and is available at the Hospital, Hong Kong and four hospitals in China (An Hui website http://research.marsh®eldclinic.org/genetics. Medical University, Nan Jin Medical University, Hang Zhou Hospital, and Shan Xi Hospital). Informed consent was also Comparative duplex PCR for detection of homozygous deletion obtained from the patients. Each tumor was classi®ed according to current World Health Organization (WHO) Homozygous deletion was investigated by comparative criteria (Giangaspero et al., 2000). All investigations in this duplex PCR. The PCR reaction was optimized in a panel study were performed on frozen tissues, except for two cases of ®ve normal DNA samples, until equivalent levels of in which formalin-®xed paran-embedded materials were ampli®cation were detected in the target and reference loci. used. Samples were considered suitable for the study if tumor The PCR mix was prepared as described in the microsatellite cell content was histologically con®rmed to be greater than analysis. The ampli®cation conditions included an enzyme 80%. Tumor-matched blood samples were also collected as activation step at 958C for 10 min, followed by 26 ± 28 cycles constitutional controls. High molecular weight DNA was composed of 948C for 30 s, 56 ± 588C for 30 s, and 728C for isolated from both tumor and blood samples using the 1 min. Chromosomal loci were considered homozygously conventional proteinase K-phenol/chloroform extraction deleted when the peak height (in the ¯uorescence method) or method. the band intensity (in the radioactive method) of the target loci was less than 80% of that of the reference loci. Each locus of homozygous deletion was veri®ed with at least two Microsatellite analysis reference markers. Polymerase chain reaction (PCR) based-microsatellite ana- To assess homozygous deletion in the DLC1 gene, tumor lysis was used to evaluate allelic imbalances or loss of DNA was ampli®ed with primers speci®c for exon 3 or exon heterozygosity (LOH) in medulloblastoma. A high-resolu- 9 of DLC1 and D5S816, D5S1480 or the STS marker tion genome-wide allelotype analysis was performed accord- SGC33915 that are located on chromosome 5. The latter ing to reported protocols (Tong et al., 2001). Brie¯y, 382 markers were chosen as reference loci because genetic microsatellite loci derived from 22 autosomes were exam- aberrations on chromosome 5 are rarely detected in ined for allelic imbalances. The average interval of these medulloblastoma. loci is about 10 cM. The polymorphic microsatellite markers were obtained from the ABI Prism Linkage Mapping Set V. Conformation-sensitive gel electrophoresis 2 (Applied Biosystems, Foster City, CA, USA) and were originally selected from the Ge ne thon human linkage map. All 13 exons and splice junctions of the DLC1 gene were The set consists of primer pairs end-labeled with either one screened for somatic mutations by conformation-sensitive gel

Oncogene Chromosome 8p loss in medulloblastoma X-l Yin et al 1467 Table 1 DLC1 primers used in conformation-sensitive gel electrophoresis analysis Primers Sequences Exon Exon size (bp) Product size (bp) Annealing temperature (8C)

E1-F: ATA AGC ATG GCG AGG TAC 1 72 172 54 E1-R: AAG TTG GCA TAA TCA TAA TCC E2-F: ACA GTA GGT GCT TAG TAT 2 83 269 50 E2-R: AGC CTA TCT TTG TGA GAA E3-F: GTG TTT ATC CAC TCA TTA G 3 63 254 52 E3-R: AGA TGT AGA TGC TTA CGG E4-F1: TAC TGG CAT ATT ATG AGC AG 4 1423 422 56 E4-R1: GCC TGC CAA GTT GCC TG E4-F2: GAC TAA CTC CGT CAT CAG 4 1423 269 54 E4-R2: GTC TGC GTG GAG TTG GA E4-F3: CAG CTG CGT GGA GAT CTC 4 1423 387 56 E4-R3: GTC CTC CAG TTC ACA GAG E4-F4: AGC TCT CAC CAA TGG CAG 4 1423 282 56 E4-R4: TTC ACG TGG TAG AGG ATG T E4-F5: ATC TGG AGA ACG AGG ACA T 4 1423 368 56 E4-R5: AGC ACA GGC AAT GTC TTT C E5-F: GCT TCA CAC TCA TTT TCC 5 176 361 52 E5-R: GAG TGA TTG CCA TAG GAT E6-F: GGT TTG CCT GCA GAA TG 6 157 309 54 E6-R: GAA GCT GCA TGT AAT CCG E7-F: GCG TCC ACG TGA ATG TA 7 202 306 52 E7-R: AAG CTT CAG TTG GTC CC E8-F: GAT ATT TGC TCT GGG ACA T 8 213 351 54 E8-R AGT CCT AGG AAG AAT GTA G E9-F: GTT TCC TTG AAT AAT ACC TT 9 113 255 52 E9-R: AGC CTA AGA TTT TGT ACC T E10-F: GCA TTG AGA TTG AGT ACA G 10 219 328 54 E10-R: CCA AGC TTC GAC TCC TG E11-F: CTA GGT GTG TAT GGA TTG C 11 218 326 56 E11-R: GGC TTG GTT CAT ATA TGG AG E12-F: AGT GAA GAG TGA AGC ACG 12 175 301 54 E12-R: AAT TTC CTT CAT GAC CCT C E13-F: GCT CGG CCA TGA ATT CAA A 13 119 328 56 E13-R: CAT TCT TCA AGG ACT GGC A to stop

electrophoresis (CSGE) as described previously (Leung et al., SYBR Gold solution (Molecular Probes, Eugene, OR, USA), 2001). Seventeen pairs of primers were designed and their and visualized under UV illumination. sequences are listed in Table 1. DNA was ampli®ed by PCR with annealing temperature optimized at 50 ± 568C for 38 Acknowledgments cycles. PCR products were resolved on nondenaturing 15% This study was supported by a Direct Grant from the polyacrylamide gels at 40 W for 4 ± 6 h, stained with 1 mg/ml Research Grant Council of Hong Kong.

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