[CANCER RESEARCH 62, 4100 – 4108, July 15, 2002] Transcript Map of the 3.7-Mb D19S112–D19S246 Candidate Tumor Suppressor Region on the Long Arm of Chromosome 19 1 Christian Hartmann, Loki Johnk, Gaspar Kitange, Yanhong Wu, Linda K. Ashworth, Robert B. Jenkins, and David N. Louis 2 Molecular Neuro-Oncology Laboratory, Department of Pathology and Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129 [C. H., L. J., D. N. L.]; Division of Laboratory Genetics, Mayo Clinic and Foundation, Rochester, Minnesota [G. K., Y. W., R. B. J.]; and Lawrence Livermore National Laboratory, Livermore, California and Department of Energy Joint Genome Institute, Walnut Creek, California [L. K. A.] ABSTRACT Allelic losses of the q13.3 region of chromosome 19 have been docu- mented in malignant gliomas, neuroblastomas, and ovarian carcinomas, strongly suggesting the presence of a 19q13.3 tumor suppressor gene. Deletion mapping in tumors over the past decade has narrowed the candidate region considerably but has produced partially conflicting re- sults, with some small candidate regions defined only by isolated tumors with deletions. Mutation and expression screening of genes from the most likely candidate regions has failed to identify the gene of interest, perhaps because of the conflicting deletion mapping data. The recently increased public availability of human genomic sequence, combined with improved bioinformatics capabilities, has now made it possible to map much larger candidate regions in considerable detail. We have manually generated a transcript map that spans most of the 19q13.3 tumor suppressor candidate region, from D19S219 to D19S246, with a resolution and quality superior to that of computer-generated maps. These results are presented in the hope that an improved map of the candidate region will facilitate further widespread screening and eventual identification of the gene or genes deleted in human gliomas, neuroblastomas, and ovarian cancers. INTRODUCTION Allelic losses of the long arm of chromosome 19 in a variety of human malignancies strongly suggest that 19q harbors a tumor sup- pressor gene. Loss of 19q is found commonly in all three major types of diffuse human malignant gliomas (1–5) as well as in epithelial ovarian cancers and neuroblastomas (6 – 8). Indeed, this region is already of clinical importance, because 19q loss in combination with 1p loss is associated with a greater likelihood of chemosensitivity, more durable chemotherapeutic responses, and longer overall survival in patients with anaplastic oligodendrogliomas (9 –11). There has, therefore, been considerable interest in identifying the putative tumor suppressor gene from this candidate region. Deletion mapping studies of malignant gliomas have gradually narrowed the candidate region over the past decade (Fig. 1). A common region of loss in astrocytomas was initially found between 19q13.11 and 19q13.4 (12, 13) and was then reduced to a 9.5-Mb area between D19S178 on 19q.13.2 and D19S180 on 19q13.4 (14) and to a 4-Mb region on 19q13.3 between APOC1 and HRC (15). Subse- quently identified 1.4-Mb deletion areas between D19S412 and STD (16) and between D19S241E and D19S596 (17) defined a minimal common deletion region of 150 kb (17). Sequencing of this 150-kb candidate region revealed GLTSCR1, EDH2, GLTSCR2, and SW, but no mutations or expression abnormalities were found in these genes (18, 19). Recent deletion mapping in neuroblastomas has demon- strated a commonly deleted region between D19S412 and D19S606, overlapping the larger glioma candidate regions but excluding the 150-kb glioma candidate region (7). On the other hand, one glioma has been reported with an interstitial loss of a 425-kb region between D19S219 and D19S112 at 19q13.3 (20), centromeric to the probable glioma/neuroblastoma locus. Conversely, epithelial ovarian cancer deletions apparently overlap more telomerically on 19q13.3, between D19S246 and the KLK gene family (8). There is thus controversy in assigning the most likely location for the 19q tumor suppressor, with some candidate regions based only on a small number of deletions in genetically unstable tumors, and it remains possible that the region contains more than one tumor suppressor locus. These difficulties have thwarted identification of the gene, or genes, of interest. Although traditional gene-hunting strategies required mapping a region of interest to a few hundred kilobases, the public availability of extensive draft sequences from the human genome and of computer bioinformatics programs has made it possible to map much larger candidate regions. This event has in turn raised the possibility that a larger transcript map could be probed more effectively for the 19q gene than smaller maps following more traditional approaches. Here we present a manually generated transcript map that spans most of the 19q13.3 tumor suppressor candidate regions, from D19S219 to D19S246, with a resolution superior to that of currently available computer-based maps. The map covers a candidate region defined by many tumor breakpoints, reducing the likelihood that genomic insta- bility in individual tumors could direct gene identification strategies to an incorrect locus. This large transcript map thus lays the groundwork for the prioritization of 19q tumor suppressor gene candidates and the determination of their involvement in these important human malig- nancies by further high-throughput screening studies. MATERIALS AND METHODS Physical Map. An approximately 3.7-Mb physical map spanning the region from D19S112 to D19S246 was generated using 28 BACs 3 and 19 cosmids/fosmids sequenced by LLNL (Table 1). Publicly available scaf- folds GA_x2KMHMR58TM, GA_x2KMHMR58UK, GA_x2KMHMR58JG, GA_x2KMHMR58W5, GA_x2KMHMR58UK, GA_x2KMHMR592L, GA_x2KMHMR58HM, GA_x8WDQ41, GA_x8WDQ6N, GA_x8WDQ6T, and GA_x8WDQ7U from Celera (Celera Genomics, Rockville, MD) were then applied to control for sequence ordering and quality and also to fill gaps. Results from an earlier shotgun sequencing project of the minimal deletion region based on BAC 284K17 were also included (18). BAC and cosmid/ fosmid order was also accomplished using the chromosome 19q arm metric physical map from LLNL. 4 Discrepancies were resolved within the human genome BAC clone map, based on shared restriction fragments from the Genome Sequence Center of Washington University School of Medicine, St. Louis. 5 Sequence assembly was accomplished with SeqMan II 5.0 (DNAstar, Madison, WI) and Staden-Package 2000.0. 6 Identification of Genes, CpG Islands, and Promoter Regions. Genes were identified by two different strategies. Nucleotide similarity searches with Received 3/1/02; accepted 5/9/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants CA69285 (to D. N. L.) and CA85799 (to R. B. J.). 2 To whom requests for reprints should be addressed, at Molecular Neuro-Oncology Laboratory, CNY6, Massachusetts General Hospital, 149 Thirteenth Street, Charlestown, MA 02129. Phone: (617) 726-5510; Fax: (617) 726-5079; E-mail: louis@helix.mgh.harvard.edu. 3 The abbreviations used are: BAC, bacteria artificial chromosome; LLNL, Lawrence Livermore National Laboratory; hEST, human expressed sequence tag. 4 Internet address: http://greengenes.llnl.gov/genome-bin/loadmap?region = mq. 5 Internet address: http://genome.wustl.edu/gsc/human/Mapping/index.shtml. 6 Internet address: http://www.mrc-lmb.cam.ac.uk/pubseq/staden_home.html. 4100 Research. on October 30, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from