Abstract
A recently identified G to A mutation at the last nucleotide of exon 6 of the sterol 27-hydroxylase gene (CYP 27) in a patient with cerebrotendinous xanthomatosis (CTX) was shown here to cause alternative pre-mRNA splicing of the gene. Northern blot analysis of the patient's RNA revealed a broadened band in the human CYP 27 mRNA region compared to that of the normal sample, indicating that there may exist differently spliced mRNA species in the patient. RT-PCR produced three fragments in the patient, one was full-length size and the other two were of smaller sizes. Sequence analysis confirmed that the nucleotide of the full-length size was identical to that of the normal full-length cDNA, except for the G to A mutation at codon 362, which corresponds to the last nucleotide of exon 6. One of the smaller size species lacked exon 6 and the other was absent from the 3′ terminal 88 bp of exon 6 due to the use of an activated cryptic 5′ splice site in exon 6. The correctly spliced mRNA harbouring the G to A mutation was responsible for the deficiency of the sterol 27-hydroxylase activity, as confirmed by transfection experiment. Transfection of constructed minigenes, with or without the mutation, showed that correctly spliced mRNA was observed in the normal minigene while the mutant minigene was differently spliced. This is the first report of a G to A substitution at the last nucleotide of an exon resulting in both normal and abnormal pre-mRNA splicings, including exon skipping and activating of a coding region cryptic 5′ splice site. The results reveal a new molecular basis for the CTX and provide information on aberrant splicing of pre-mRNA in multi-exon genes. —Chen, W., S. Kubota, and Y. Seyama. Alternative pre-mRNA splicing of the sterol 27-hydroxylase gene (CYP 27) caused by a G to A mutation at the last nucleotide of exon 6 in a patient with cerebrotendinous xanthomatosis (CTX).
Highlights
A recently identified G to A mutation at the last nucleotide of exon 6 of the sterol 27-hydroxylase gene (CYP 27) in a patient with cerebrotendinous xanthomatosis (CTX) was shown here to cause alternative pre-mRNA splicing of the gene
We report here the first example of a G to A substitution at the last nucleotide of exon 6 in the CYP 27 gene that leads to alternative splicings of pre-mRNA, including exon 6 skipping, activating of a coding region cryptic 5Ј splice site, and correct splicing of pre-mRNA carrying the G to A mutation
As the mutation occurred at the last nucleotide of exon 6, its effect on pre-mRNA transcription was first analyzed in vivo
Summary
A recently identified G to A mutation at the last nucleotide of exon 6 of the sterol 27-hydroxylase gene (CYP 27) in a patient with cerebrotendinous xanthomatosis (CTX) was shown here to cause alternative pre-mRNA splicing of the gene. Transfection of constructed minigenes, with or without the mutation, showed that correctly spliced mRNA was observed in the normal minigene while the mutant minigene was differently spliced This is the first report of a G to A substitution at the last nucleotide of an exon resulting in both normal and abnormal pre-mRNA splicings, including exon skipping and activating of a coding region cryptic 5Ј splice site. Alternative premRNA splicing of the sterol 27-hydroxylase gene (CYP 27) caused by a G to A mutation at the last nucleotide of exon 6 in a patient with cerebrotendinous xanthomatosis (CTX). We analyzed the transcription products of this mutant gene in vivo and confirmed that the aberrant pre-mRNA splicings were caused by the mutation in vitro
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