A NOVEL ARG(362)SER MUTATION IN THE STEROL 27-HYDROXYLASE GENE (CYP27) - ITS EFFECTS ON PRE-MESSENGER-RNA SPLICING AND ENZYME-ACTIVITY

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 ad renodoxin cofactor binding residue (362)Arg to (362)Ser (CGT (362)Arg to AGT (362)Ser), and was responsible for deficiency in the sterol 27- hydroxylase activity, as confirmed by expression of mutant cDNA into C OS-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 positio n of exon 6-intron 6 splice site) of the gene, we hypothesized that th e mutation may partially affect the normal splicing efficiency in exon 6 and cause alternative splicing elsewhere, which resulted in decreas ed transcript in the patient. Transfection of constructed minigenes, w ith or without the mutation, into COS-1 cells confirmed that the mutan t 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 alt ernative pre-mRNA splicing of the gene. To our knowledge, this is the first report regarding effects on pre-mRNA splicing of a mutation at t he -2 position of a 5' splice site.