Silent Nucleotide Substitution in the Sterol 27-Hydroxylase Gene (CYP 27) Leads to Alternative Pre-mRNA Splicing by Activating a Cryptic 5‘ Splice Site at the Mutant Codon in Cerebrotendinous Xanthomatosis Patients

Abstract

A functionally silent nucleotide substitution of the sterol 27-hydroxylase gene (CYP 27), identified in two families with cerebrotendinous xanthomatosis (CTX), was confirmed to cause alternative pre-mRNA splicing of the gene. Full-length RT-PCR analysis of the CYP 27 gene in a patient from one of the CTX families revealed one major and an additional faint band. Sequence analysis of the cloned RT-PCR product showed three species of cDNA: 3' terminal 13 bp of exon 2 deleted cDNA, exon 2 skipped cDNA, and full-length cDNA with a functionally silent G to T mutation at codon 112 (GGG 112Gly to GGT 112Gly). Only a single base change was identified by genomic DNA sequence analysis of the CYP 27 gene in the patient: T replaced G at the third position of codon 112, 13 bp upstream from the 3' terminus of exon 2. Transfection of constructed minigenes, with or without the mutation, confirmed that this silent mutation resulted in alternative pre-mRNA splicing by activating a cryptic 5' splice site around the mutant codon. The mutation was also identified in two patients from another CTX family, with a compound heterozygous pattern of A for G substitution at codon 372, a mutation reported previously by our group. The results elucidate a novel molecular basis for the CTX and suggest the significance of a silent nucleotide substitution with regard to pre-RNA splicing.

Analysis of exon 1 mutations in the androgen receptor gene

“…This different tRNA may, however, be present in the cytoplasm in much lower concentrations than the original tRNA; in turn, this could result in reduced synthesis of protein. Well documented consequences of silent mutations have included large reductions in mRNA and protein levels [Deana et al, 1998], alterations of pre-mRNA splicing [Chen et al, 1998], induction of exon skipping [Liu et al, 1997], and a translated protein with an internal deletion [Du et al, 1998]. Cases 51391, 39989, 9323, 40183, 812309 It is interesting to note that in the one 211 G→A case we examined (51391), levels of mRNA and hAR were both negligible.…”

Homozygous single nucleotide polymorphism of the complement C1QA gene is associated with decreased levels of C1q in patients with subacute cutaneous lupus erythematosus

“…Such mechanisms include altered mRNA splicing ef ciency by disruption of exonic splicing enhancer or splicing silencer sites 17 and altered splicing through activation of cryptic splicing sites. [18][19][20] C1qA -Gly70 GGA disruption in the exon 2 could generate errors in the splicing mechanism and create a new acceptor splicing site, with frame-changing followed by production of an aberrant mRNA. A nonsense mutation-mediated decay of abnormal mRNA resulting from splicing errors would further explain a de ciency in protein production or its total absence.…”

Overexpression of Vacuolar ATPase 16-kDa Subunit in 10T1/2 Fibroblasts Enhances Invasion with Concomitant Induction of Matrix Metalloproteinase-2

“…DNA sequence analysis was made using the ABI PRISMTM 310 Genetical Analyzer, as described (14). The RT-PCR products were sequenced and confirmed.…”