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Identification of a Novel Gene NCRMS on Chromosome 12Q21 with Di Oncogene (2002) 21, 3029 ± 3037 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Identi®cation of a novel gene NCRMS on chromosome 12q21 with dierential expression between Rhabdomyosarcoma subtypes Agnes S Chan1,2, Paul S Thorner1,2, Jeremy A Squire1,3 and Maria Zielenska*,1,2 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1L5; 2Department of Paediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; 3Department of Oncologic Pathology, Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2M9 Rhabdomyosarcoma (RMS) is a malignant soft tissue detection of signs of skeletal muscle dierentiation. tumor showing varying degrees of skeletal muscle This can include evidence at the light microscopic level, dierentiation. Two major histologic subtypes exist, i.e. cross-striations, or immunohistochemical detection alveolar and embryonal, each with associated molecular of structural proteins such as a-actin, desmin, and genetic changes. We have used Representational Dier- regulatory proteins such as myogenin and MyoD1. ence Analysis (RDA) to compare gene expression Other than this apparent common myogenic lineage, between the two RMS subtypes and have identi®ed the RMS is a heterogeneous tumor with varying histolo- novel gene NCRMS (non-coding RNA in RMS) that has gical and clinical presentations. Two major histological increased expression in the alveolar subtype relative to subtypes are de®ned, namely embryonal (RMS-E) and the embryonal subtype. Multiple alternatively spliced alveolar (RMS-A) with RMS-A being clinically more forms of NCRMS were identi®ed through library aggressive. RMS with mixed embryonal and alveolar screening, RACE, and comparison to human expressed histology occasionally occurs and behaves more like sequence tags (ESTs). Northern blot analysis indicated RMS-A. Despite great improvement over past decades the transcript size to be 1.25 kb in alveolar RMS. There with multi-modal therapy, the 5-year survival rate of was no sequence homology to any of the known genes in RMS remains at about 65 ± 70% with most metastatic GenBank, but extensive homology to ESTs from various cases still fatal. Conventional therapeutic modalities species. Comparison to human genomic sequences maybe approaching their limit in bringing about identi®ed at least 11 exons mapping to chromosomal further improvements in RMS outcome. However region 12q21. Dierential expression of NCRMS was oncogenomic methodologies now oer opportunities noted between various tumor types. Since NCRMS RNA for identifying new classes of tumor-speci®c genes that possesses limited potential for protein coding, yet with may lead to more targeted therapeutic approaches conserved sequences between dierent species, it is likely (reviewed in Merlino and Helman (1999); Dagher and that NCRMS is a functional non-coding RNA. Known Helman (1999) and Anderson et al. (1999b)). genes in its proximity include myogenic regulators Myf5 RMS heterogeneity is also re¯ected on the molecular and Myf6, growth factor Igf1, and another potential level. RMS-E is commonly associated with a loss of dierentially expressed gene (ATP2B1) in RMS isolated heterozygosity (LOH) on chromosomal region 11p15.5 by RDA. suggesting that one or more tumor suppressor genes Oncogene (2002) 21, 3029 ± 3037. DOI: 10.1038/sj/ may map to this region. Putative candidate tumor onc/1205460 suppressor gene(s) in this region include H19, p57KIP2, KVLQT1, GOK, TSSC1, BWR1A and BWR1C Keywords: Rhabdomyosarcoma; non-coding tran- (reviewed in Anderson et al. (1999b)). Comparative scripts; chromosome 12q21; alternative splicing; novel genomic hybridization (CGH) and spectral karyotyping gene (SKY) analysis of RMS genome showed that numeric changes were more common in RMS-E, while structural changes were more prominent in RMS-A Introduction (Pandita et al., 1999). In RMS-A, the most obvious structural changes are the t(2;13) and t(1;13) transloca- Rhabdomyosarcoma (RMS) is the most common tions. The majority of RMS-A possess one of these malignant soft tissue tumor in children. Found in translocations which involve the in-frame fusion of diverse anatomic sites, diagnosis of RMS is based on PAX3 or PAX7 gene to the FKHR transcriptional activator. Resultant chimeric proteins were found to be more potent transcriptional regulators. The potential gain-of-function was thought to contribute to the more *Correspondence: M Zielenska, Department of Paediatric aggressive behavior of RMS-A (reviewed in Barr Laboratory Medicine, Hospital for Sick Children, Toronto, (1997)). Ontario, Canada M5G 1X8; E-mail: [email protected] Received 11 July 2001; revised 15 November 2001; accepted 11 Recent data are beginning to suggest that LOH at March 2002 11p15.5 is not exclusive to RMS-E (Anderson et al., Non-coding transcript NCRMS on 12q21 in Rhabdomyosarcoma AS Chan et al 3030 1999a; Visser et al., 1997), and that molecular changes MAGE (Lucas et al., 1998), as well as the isolation of in RMS between, as well as within, subtypes are more herpesvirus-like DNA sequences in AIDS-associated complex and heterogeneous than previously believed. Kaposi's sarcoma (Chang et al., 1994). As RDA The use of genomic screening technologies is increas- requires only minute amount of starting RNA, it is ingly proving to generate new candidate markers suited ideal for screening a rare tumor such as RMS in which for diagnostic and prognostic purposes. In this study, tumor tissue is often limited. we have used Representational Dierence Analysis (RDA) to compare gene expression between RMS-E and RMS-A. RDA allows the isolation of novel genes Results at a manageable scale and oers greater sensitivity than the traditional subtractive hybridization. It is a Difference products isolated by RDA PCR-based subtractive hybridization technique that had been employed successfully in identifying dier- To compare gene expression by RDA, RMS-A and ences between two very similar DNA or cDNA RMS-E cDNA was used as tester in the forward and populations (Hubank and Schatz, 1994; Lisitsyn et reverse subtraction, respectively. After three rounds of al., 1993). Its application in cancer research includes RDA subtraction and ampli®cation, 44 clones were the identi®cation of the tumor suppressor gene PTEN selected for initial dot-blot screening. Of these, 13 (Li et al., 1997), the family of tumor speci®c genes clones showed dierential signal intensity (Figure 1). Sequencing of these 13 clones revealed ®ve unique sequences, designated C1 to C5. C1 was obtained from the forward subtraction suggesting higher expression in RMS-E. C2 to C5 were products of the reverse subtraction and thus were likely to have higher expression in RMS-A. Sequence identities of C1 to C5 were shown in Table 1. Semi-quantitative analysis was performed to con®rm dierential expression in the four samples originally used in RDA (Figure 2). Follow-up analysis was focused on C2 and C3 which showed the greatest dierence in signal strength between RMS-A and RMS-E. As indicated in Table 1, C2 and C3 showed no nucleotide sequence homology to any known gene in GenBank. However, matches to human ESTs and high throughput genomic sequences (HTGS) were found. Homologies to mouse and rat ESTs were also present. Figure 1 Reversed dot-blot array screening of RDA products. After the cloning of RDA products, 44 clones were individually 32 dot-blotted onto duplicate membranes. P-labeled RDA 3rd Confirmation of differential expression of selected RDA dierence products were used as complex probes on each membrane. Membrane on left was probed with cDNA enriched products by semi-quantitative RT ± PCR with RMS-A sequences, while the one on right was probed with To assess relative expression level of C2 and C3 in RMS cDNA enriched with RMS-E sequences. Thirteen clones (under- lined) with dierential signal intensity were detected, of which 12 tumors, a series of 53 RMS tumors were screened with gave stronger signals with RMS-A probe semi-quantitative RT ± PCR with sequence speci®c Table 1 Sequence information from GenBank (June 11, 2001) for RDA products C1-5 Name Length Chromosomal location Sequence identity Sequences homologies C1 216 bp 12q21 ± q23 Human plasma membrane calcium (1 ± 216 bp, 99%) ATPase isoform 1 (ATP2B1) gene C2 341 bp 12q21 Unknown Mouse ESTs (1 ± 136 bp, 95%) (279 ± 341 bp, 87%) Rat ESTs (1 ± 136 bp, 94%) HTGS (1 ± 341 bp, 99+%) C3 371 bp 12q21 Unknown Human ESTs (23 ± 152 bp, 90%) (146 ± 371 bp, 98%) Mouse ESTs (1 ± 63 bp, 87%) (120 ± 240 bp, 80%) (218 ± 285 bp, 92%) Rat ESTs (1 ± 61 bp, 88%) HTGS (1 ± 371 bp, 99+%) C4 422 bp 18q12.3 Unknown KIAA0427 ± gene with protein product, function unknown (1 ± 422 bp, 95%) C5 264 bp mitochondrion Human mitochondrion (1 ± 264 bp, 96%) Oncogene Non-coding transcript NCRMS on 12q21 in Rhabdomyosarcoma AS Chan et al 3031 primers. Expression was noted in all samples, including a review, this case was reclassi®ed as a solid form RMS-A. sample of normal muscle. Comparing with RMS-E, For statistical purpose, band intensity of C2, C3 and RMS-A tumors and those with mixed alveolar and PBGD PCR products were measured and relative embryonal histology demonstrated an overall higher expression levels calculated as ratios of C2/PBGD and expression of both C2 and C3 (Figure 3). One case C3/PBGD (Table 2). Dierences in expression level of (denoted by *) in the RMS-E panel was noted to have both C2 and C3 between RMS-A group and RMS-E strong expression of C2 and C3. Upon blinded pathology group of tumors were found to be statistically signi®cant (P50.02, Mann-Whitney Rank Sum statistics). Isolation of full-length cDNA with various splice forms To determine transcript size, Northern blot was performed with one RMS-A and one RMS-E total RNA.
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