A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro.
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 polymorphism. 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.
Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chuo, Kobe 650-0017, Japan.
Hoai Thu
HT
Thi Tran
Yasuhiro
Y
Takeshima
Agus
A
Surono
Mariko
M
Yagi
Hiroko
H
Wada
Masafumi
M
Matsuo
D001483:Base Sequence; D002648:Child; D004252:DNA Mutational Analysis; D016189:Dystrophin / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008297:Male; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*; D017354:Point Mutation; D022821:RNA Splice Sites; D012326:RNA Splicing; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016189:Dystrophin; D022821:RNA Splice Sites