Abstract
BackgroundThe 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.MethodsThe 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.ResultsSixteen 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.ConclusionsIntron-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.
Highlights
The DMD gene encoding dystrophin is mutated in Duchenne muscular dystrophy, a fatal progressive muscle wasting disease
Multiplex ligation-dependent probe amplification assays showed that all 79 DMD exons were present in genomic DNA of CRL-2061 cells, indicating that the genomic structure of the DMD gene is normal in this alveolar RMS (ARMS) cell line
To analyze DMD transcripts in CRL-2061 cells, fulllength DMD cDNA was RT-PCR amplified as 20 partially overlapping fragments. 16 of the 20 fragments were successfully amplified in CRL-2061 and muscle cDNA preparations (Fig. 1b)
Summary
The DMD gene encoding dystrophin is mutated in Duchenne muscular dystrophy, a fatal progressive muscle wasting disease. The DMD gene is the largest human gene, spanning more than 2.4 Mb on chromosome X and composed of 79 exons. This gene encodes a 14-kb transcript, which produce dystrophin, a 427 kDa protein [7]. Dystrophin deficiency caused by mutations in the DMD gene is a fundamental defect in Duchenne muscular dystrophy (DMD), one of the most common inherited muscular diseases. DMD is characterized by muscle weakness, leading to fatal progressive muscle wasting, as well as other complications [8, 9]. Two DMD patients have been reported to show complications of RMS [10, 11]
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