title: A novel Arg362Ser mutation in the sterol 27-hydroxylase gene (CYP27): its effects on pre-mRNA splicing and enzyme activity.
abstract: A novel C to A mutation in the sterol 27-hydroxylase gene (CYP27) was identified by sequencing amplified CYP27 gene products from a patient with cerebrotendinous xanthomatosis (CTX). The mutation changed the adrenodoxin cofactor binding residue 362Arg to 362Ser (CGT 362Arg to AGT 362Ser), and was responsible for deficiency in the sterol 27-hydroxylase activity, as confirmed by expression of mutant cDNA into COS-1 cells. Quantitative analysis showed that the expression of CYP27 gene mRNA in the patient represented 52.5% of the normal level. As the mutation occurred at the penultimate nucleotide of exon 6 (-2 position of exon 6-intron 6 splice site) of the gene, we hypothesized that the mutation may partially affect the normal splicing efficiency in exon 6 and cause alternative splicing elsewhere, which resulted in decreased transcript in the patient. Transfection of constructed minigenes, with or without the mutation, into COS-1 cells confirmed that the mutant minigene was responsible for a mRNA species alternatively spliced at an activated cryptic 5' splice site 88 bp upstream from the 3' end of exon 6. Our data suggest that the C to A mutation at the penultimate nucleotide of exon 6 of the CYP27 gene not only causes the deficiency in the sterol 27-hydroxylase activity, but also partially leads to alternative pre-mRNA splicing of the gene. To our knowledge, this is the first report regarding effects on pre-mRNA splicing of a mutation at the -2 position of a 5' splice site.
authors.affiliation: Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Japan.
authors.forename: W
authors.initials: W
authors.lastname: Chen
authors.forename: S
authors.initials: S
authors.lastname: Kubota
authors.forename: H
authors.initials: H
authors.lastname: Ujike
authors.forename: T
authors.initials: T
authors.lastname: Ishihara
authors.forename: Y
authors.initials: Y
authors.lastname: Seyama
mesh_terms: D017398:Alternative Splicing; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics*; D019556:COS Cells; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D017354:Point Mutation; D012322:RNA Precursors / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000235:genetics*; D013250:Steroid Hydroxylases / Q000032:analysis / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D019294:Xanthomatosis, Cerebrotendinous / Q000201:enzymology / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D012322:RNA Precursors; D012694:Serine; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase