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Functional analysis of the synonymous R385R mutation in the low-density lipoprotein receptor gene.
- Source :
-
Genetic testing and molecular biomarkers [Genet Test Mol Biomarkers] 2009 Apr; Vol. 13 (2), pp. 243-8. - Publication Year :
- 2009
-
Abstract
- Familial hypercholesterolemia is caused by mutations in the low-density lipoprotein receptor (LDLR) gene. The synonymous mutation R385R has been shown to introduce a cryptic splice site in exon 9. The aims of this study were to establish to what extent the cryptic splice site is selected ahead of the normal splice site and to determine if the aberrant transcript is degraded by nonsense-mediated mRNA decay. The relative amount of the aberrant transcript was determined by real-time PCR and found to vary from 25% to 45% in heterozygous familial hypercholesterolemia individuals. Epstein-Barr virus-transformed lymphocytes were established from one heterozygous patient, and treatment of these cells with cycloheximide increased the amount of aberrant transcript, indicating that the aberrant transcripts are degraded by nonsense-mediated mRNA decay. Cloning of reverse transcriptase-PCR products from one of the heterozygous patients and introduction of the R385R mutation into a minigene reporter construct revealed an almost exclusive use of the cryptic splice site in the mutated allele. Thus, the synonymous mutation R385R converts the mutated allele to a null allele unable to produce functional mRNA.
- Subjects :
- Adolescent
Adult
Alleles
Base Sequence
Cell Line, Transformed
Cell Line, Tumor
Cell Transformation, Viral
Cells, Cultured
Child
Exons
Female
Genes, Reporter
Herpesvirus 4, Human genetics
Heterozygote
Humans
Hyperlipoproteinemia Type II genetics
Introns
Lymphocytes metabolism
Lymphocytes virology
Male
Middle Aged
Models, Genetic
Molecular Sequence Data
RNA Splice Sites genetics
RNA, Messenger analysis
RNA, Messenger metabolism
Transfection
Alternative Splicing physiology
Mutation
Receptors, LDL genetics
Receptors, LDL metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1945-0257
- Volume :
- 13
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Genetic testing and molecular biomarkers
- Publication Type :
- Academic Journal
- Accession number :
- 19371225
- Full Text :
- https://doi.org/10.1089/gtmb.2008.0125