Journal
MOLECULAR GENETICS AND METABOLISM
Volume 85, Issue 3, Pages 213-219Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymgme.2005.03.006
Keywords
dystrophin; DMD/BMD; extra-exon; splicing; mutation
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The splicing pattern of pre-mRNA is unpredictable in genes harboring a single-nucleotide change within the consensus sequence of a splice-donor site. In the dystrophin gene, a transition from G to A at the fifth position of intron-32 (4518 + 5G > A) has been reported as a polymorphism within the consensus sequence or a mutation identified in Duchenne muscular dystrophy (DMD). Here, we report both in vivo and in vitro evidence that shows inactivation of the splice-donor site caused by this mutation. In one Japanese DMD case, two novel dystrophin mRNAs were identified in the patient's lymphocytes, one with a 98 bp deletion of the 3' end of exon-32 (dys32 - 98) and the other with a 28 bp intron retained between exons 32 and 33 (dys32 + 28). Genomic sequencing disclosed a single-nucleotide change from G to A at the fifth position of intron-32 (4518 + 5G > A). To demonstrate in vitro the inactivation of this splice-donor site by this nucleotide change, mini-dystrophin genes comprising three exons harboring either normal or mutant intron-32 sequences were expressed in HeLa cells, and the splicing products were analyzed by reverse-transcription PCR amplification. A normal transcript consisting of three exons was obtained from the normal construct. From the mutant, we obtained one product containing a 98 bp deletion at the 3' end of exon-32, indicating complete inactivation of the native splice-donor site. Thus, both in vivo and in vitro experiments demonstrate that 4518 + 5G > A causes a splicing error leading to transcript termination; it did not behave like a silent polymorphisrn. Our results indicate that the in vitro splicing system is a powerful tool for determining the underlying mechanism of a disease-causing mutation in a splicing consensus sequence. (c) 2005 Elsevier Inc. All rights reserved.
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