Abstract. Parental imprinting of differentially methylated regions (DMRs) contributes to appropriate expression of several developmentally important genes from paternally or maternally derived chromosomes. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and is asso- ciated

Parental imprinting of differentially methylated regions (DMRs) contributes to appropriate expression of several developmentally important genes from paternally or maternally derived chromosomes. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and is associated with altered expression of certain parentally imprinted genes. As previously reported, RMS cells display loss of imprinting (LOI) of the DMR at the IGF2-H19 locus, resulting in insulin-like growth factor 2 (IGF2) transcription from both paternally and maternally inherited chromosomes, and overall IGF2 overexpression. As the DLK1-GTL2 locus is structurally similar to the IGF2-H19 locus, the status of parental imprinting of the DLK1-GTL2 locus was studied in RMS. We observed that while both embryonal and alveolar rhabdomyosarcomas (ERMS and ARMS, respectively) show LOI of the DMR at the IGF2-H19 locus, imprinting of the DMR at the DLK1-GTL2 locus varies in association with the histological subtype of RMS. We found that, while ERMS tumors consistently show LOI of the DMR at the DLK1-GTL2 locus, ARMS tumors have erasure of imprinting (EOI) at this locus. These changes in imprinting status of the DLK1-GTL2 locus result in a higher GTL2/DLK1 mRNA ratio in ARMS as compared to ERMS. This difference in imprinting elucidates a novel genetic difference between these two RMS subtypes and may provide a potential diagnostic tool to distinguish between these subtypes. Introduction The delta-like homolog 1 (DLK1) and gene trap locus 2 (GTL2, also known as MEG3) gene cluster is located on human chromosome 14. The importance of the DLK1-GTL2 locus in skeletal muscle development is supported by the fact that a single-nucleotide polymorphism at this locus deregulates its expression and leads to DLK1-mediated skeletal muscle hypertrophy, as seen in callipyge sheep (1). Rhabdomyosarcoma (RMS), a family of cancers related to the skeletal muscle lineage, is the most common softtissue sarcoma in children. From a histological and clinical perspective, there are two major histological subtypes of RMS: embryonal rhabdomyosarcoma (ERMS) and alveolar rhabdomyosarcoma (ARMS) (2). Clinical evidence indicates that ARMS is more aggressive, shows more enhanced metastatic potential, and has a significantly worse outcome than ERMS. It has been postulated that there are different cellular origins for these tumors (3) but the molecular mechanisms responsible for the development of RMS are not well understood. Evidence has accumulated that imprinted genes play a role in RMS pathogenesis (4,5). In the human genome, there are ~80 genes that are imprinted and expressed only from the maternally or paternally derived chromosomes. This mechanism regulates the appropriate dosage and expression level of developmentally important genes in mammalian cells (6). The expression of imprinted genes is regulated by the imposition of epigenetic marks (DNA methylation) within differentially methylated regions (DMRs), which are regulatory CpG-rich regions in the gene locus (7-9). Most imprinted genes are methylated on maternally derived chromosomes and only a few (e.g., IGF2 and DLK1) are methylated within DMRs on paternally derived chromosomes. According to the parent-offspring conflict theory, while paternally expressed imprinted genes enhance embryo growth and size of the offspring, maternally expressed genes inhibit cell proliferation and negatively affect cell size (7-9). Based on this theory, during pregnancy, the paternal allele promotes increased body size, including muscle mass of the developing fetus, through expression of paternally imprinted genes. By contrast, the maternal allele conserves resources by epigenetic modulation of genes bearing maternal imprinting marks. The paternally imprinted DLK1-GTL2 locus is differentially methylated in embryonal and alveolar rhabdomyosarcomas GABRIELA SCHNEIDER1, MARK J. BOWSER2,4, DONG-MYUNG SHIN1, FREDERIC G. BARR3 and MARIUSZ Z. RATAJCZAK1 1Stem Cell Institute at the James Graham Brown Cancer Center, University of Louisville, Louisville, KY; 2Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA; 3Laboratory of Pathology, National Cancer Institute, Bethesda, MD, USA Received August 16, 2013; Accepted October 1, 2013 DOI: 10.3892/ijo.2013.2153 Correspondence to: Dr Mariusz Z. Ratajczak or Dr Gabriela Schneider, Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, 500 S. Floyd Street, Rm. 107, Louisville,