A novel cryptic exon identified in the 3′ region of intron 2 of the human dystrophin gene

Abstract

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), is the largest known human gene and is characterized by the huge size of its introns. Intron 2, the second largest intron, is 170-kb long and has been shown to include a 140-bp cryptic exon (exon 2a) in its 5¢ region. The rest of this intron has no known function. In this study, we find that another cryptic exon, located in the 3¢ region of intron 2, is activated in a promoter-or tissue-specific manner. An unknown 98-bp insertion precisely between exons 2 and 3 was identified in one of the dystrophin mRNAs from lymphocytes of a DMD patient with a duplication of exon 2. This 98-bp sequence, located in the 3¢ region of intron 2, was found to possess a branch point, acceptor and donor splice-site consensus sequences, and an exonic splicing enhancer sequence, and thus is a novel exon, which we named ''exon 2b.'' In lymphocytes, exon 2b incorporation was detected in the muscle-specific, promoter-driven transcript. Five of 20 normal human tissue mRNAs, including cardiac and skeletal muscle mRNAs, were confirmed to contain a fragment extending from exon 1 to exon 2b by reverse transcription PCR amplification, indicating that exon 2b is activated in a tissue-specific manner. This provides a clue to a novel cause of dystrophinopathy.

Gene Therapy of Some Genetic Diseases by Transferring Normal Human Genomic DNA into Somatic Cells and Stem Cells from Patients

“…Ahn & Kunkel, 1993;Black, 2003;Dwi Pramono et al, 2000;Muntoni et al, 2003;Nishio et al, 1994;L. Song et al, 2003;V. Tran et al, 2005;Zhang et al, 2007).…”

“…The human nebulin gene has 147 introns. Some introns like the human dystrophin gene intron 44 can be more than 250,000 bp in length (Hawkins, 1988;Lodish et al, 2008;Sakharkar et al, 2004;V. Tran et al, 2005).…”

“…99% of the dystrophin gene sequences are present in introns. The human dystrophin gene locates at locus Xp21.2, and is mutated in patients with Duchenne and Becker muscular dystrophies (Dwi Pramono et al, 2000;Golubovsky & Manton, 2005;Koenig et al, 1987Koenig et al, , 1988Nishio et al, 1994;Roberts, 2001;V. Tran et al, 2005;Zhang et al, 2007).…”

Insertion of the IL1RAPL1 gene into the duplication junction of the dystrophin gene

“…20 Five fragments encompassing each exon from 2 to 6 of the IL1RAPL1 gene and one fragment encompassing exon 22 of the dystrophin gene were co-amplified in one PCR reaction using six sets of primers (Table 1). PCR products were separated by capillary electrophoresis (Agilent 2001 Bioanalyzer with DNA 1000 Lab Chips, Agilent Technologies, Palo Alto, CA, USA).…”

“…The genomic dosage of the amplified intron regions was assessed by semiquantitative multiplex PCR as described earlier. 20 The insertion breakpoints were assumed roughly in the region between fragments with single and double amounts of the PCR products. Again, distributed fragments within the breakpoint region assumed from the preceding PCR amplification study were PCR amplified to localize the limit between single and double amounts of the PCR products.…”