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.

The wide genetic landscape of clinical frontotemporal dementia: systematic combined sequencing of 121 consecutive subjects

“…This established mutation has been demonstrated to lead to alternative pre-mRNA splicing and decreased sterol 27-hydroxylase activity, thereby causing cerebrotendinous xanthomatosis. 20 To confirm the pathogenicity of this variant, we confirmed the characteristic reduction of 27-OH-cholesterol (below the detection threshold), a sterol 27-hydroxylase product, and compensatory increases of 7-alpha-OH-cholesterol (1372 ng/ml) and cholestanol (3410 ng/dl) in our patient. This subject presented with impulsivity, disinhibition, apathy, and executive deficits at the age of 49 years, associated with pyramidal signs (for pedigree see Figure 3j ; for more subject and pedigree details, see Supplementary Material S7 ).…”

Novel splice-affecting variants in CYP27A1 gene in two Chilean patients with Cerebrotendinous Xanthomatosis

“…It is worthy of note that the mutation c.Arg395Ser is located in the same genomic position (penultimate nucleotide of exon 6) as the c.Arg395Cys (codon CGT to TGT; c.1183C > T) ( Chen et al (1998) . Given the special location of this mutation in the exon-intron boundary, Chen et al (1998) proposed that the c.Arg395Ser mutation may affect normal splicing and gene expression efficiency, implying that this specific genomic DNA position may also affect gene expression in other mutations located in the same genomic site. As far as we know, there are no reports assessing the possible impact of the mutation c.1183C>Ton gene expression or the splicing process.…”

Alternative Splicing in the Cytochrome P450 Superfamily Expands Protein Diversity to Augment Gene Function and Redirect Human Drug Metabolism

“…Some P450 SNPs are exceedingly rare, occurring in less than 1% of the population. These rare mutations are linked to congenital defects, including glaucoma (CYP1B1; Stoilov et al, 1998;Tanwar et al, 2009;Sheikh et al, 2014), 17-a hydroxylase deficiency (CYP17A1; Yamaguchi et al, 1998;Costa-Santos et al, 2004;Hwang et al, 2011;Qiao et al, 2011), congenital adrenal hyperplasia (CYP21A2; Robins et al, 2006;Lee, 2013;Szabó et al, 2013;Sharaf et al, 2015), spina bifida (CYP26A1; Rat et al, 2006), focal facial dermal dysplasia (CYP26C1; Slavotinek et al, 2013), and cerebrotendinous xanthomatosis (CYP27A1; Garuti et al, 1996;Verrips et al, 1997;Chen et al, 1998;Tian and Zhang, 2011). Other P450 polymorphisms that alter splicing are associated with neurologic and metabolic diseases, including Parkinson's disease (CYP2D6, Denson et al, 2005;CYP2J2, Searles Nielsen et al, 2013), hypertension (CYP4A11, Zhang et al, 2013;CYP17A1, Wang et al, 2011), breast cancer (CYP2D6, Huang et al, 1996;CYP19A1, Kristensen et al, 2000;) colon cancer (CYP2W1, Stenstedt et al, 2012), and lung cancer (CYP2D6, Huang et al, 1997;CYP2F1, Tournel et al, 2007).…”