DMD transcripts in CRL-2061 rhabdomyosarcoma cells show high levels of intron retention by intron-specific PCR amplification

Abstract Background The DMD gene encoding dystrophin is mutated in Duchenne muscular dystrophy, a fatal progressive muscle wasting disease. DMD has also been shown to act as a tumor suppressor gene. Rhabdomyosarcoma (RMS) is a mesodermal sarcoma that shares characteristics of skeletal muscle precursors. Products of the DMD gene in RMS have not yet been fully clarified. Here, DMD products were analyzed in CRL-2061 cells established from alveolar RMS. Methods The 14-kb long DMD cDNA was PCR amplified as 20 separated fragments, as were nine short intron regions. Dystrophin was analyzed by Western blotting using an antibody against the C-terminal region of dystrophin. Results Sixteen of the 20 DMD cDNA fragments could be amplified from CRL-2061 cells as muscle cDNA. Three fragments included aberrant gene products, including one in which exon 71 was omitted and one each with retention of introns 40 and 58. In one fragment, extending from exon 70 to 79, no normally spliced product was obtained. Rather, six alternatively spliced products were identified, including a new product deleting exon 73, with the most abundant product showing deletion of exon 78. Although dystrophin expression was expected in CRL-2061 cells, western blotting of cell lysates showed no evidence of dystrophin, suggesting that translation of full-length DMD mRNA was inhibited by intron retention that generated a premature stop codon. Intron specific PCR amplification of nine short introns, showed retention of introns 40, 58, and 70, which constituted about 60, 25 and 9%, respectively, of the total PCR amplified products. The most abundant DMD transcript contained two abnormalities, intron 40 retention and exon 78 skipping. Conclusions Intron-specific PCR amplification showed that DMD transcripts contained high levels of introns 40, 58 and 70. Retention of these introns may have been responsible for the lack of dystrophin expression by CRL-2061 cells, thereby abolishing the tumor suppressor activity of dystrophin.