Loss of imprinting at the 14q32 locus in osteosarcoma
Jingmin Shu1, Lihua Li2, Branden Moriarity3, Venugopal Thayanithy2, Logan Spector4,
David Largaespada3,4, Clifford J Steer1,3 and Subbaya Subramanian2,*
1
Department of Medicine
Department of Surgery, Division of Basic and Translational Research
3
Department of Genetics, Cell Biology and Development
4
Department of Pediatrics
University of Minnesota, Minneapolis, MN 55455, USA
2
Abstract
Osteosarcoma is the most common primary bone malignancy affecting children and
adolescents. Although several genetic predisposing conditions have been associated
with osteosarcoma our understanding of its pathobiology is rather limited. We used
various cancer cell lines and osteosarcoma tumor tissue samples in this study. We
performed DNA Bisulfite pyrosequencing and TA cloning and sequencing for DNA
methylation analysis. Chromatin immunoprecipitation (ChIP) assays were carried out for
histone modification studies. The unpaired t-test was used to compare patient samples;
and the paired t-test for both the apoptosis and cell proliferation assays. Here, we show
that loss-of-imprinting at human 14q32-locus is highly prevalent (87%) and is
significantly associated with osteosarcoma in patients < 30 years of age. The average
demethylation at 14q32-locus was significantly different from the global demethylation.
Further, the 14q32-locus was enriched with both H3K4-me3 and H3K27-me3 histone
modifications.
Epigenetic alterations at this locus affect the expression of imprinted
genes DLK1, RTL1 and DIO3 with potential tumor suppressive role. Also, using buccal
DNA samples we found loss-of-imprinting at 14q32-locus in most osteosarcoma patients
and their biological parents. Furthermore, analysis of tumor tissues from spontaneous
OS mice model supported the notion that LOI at 12q32 locus (a homolog of 14q32 locus
in humans) is not a secondary genetic event. In conclusion, we have established the
presence of loss-of-imprinting at the 14q32-locus, which is predominantly associated
with early-onset and may contribute to the pathogenesis of osteosarcoma in humans.