Functional analysis of the synonymous R385R mutation in the low-density lipoprotein receptor gene.
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.
Tveten
Kristian
K
Medical Genetics Laboratory, Department of Medical Genetics, Rikshospitalet University Hospital, Oslo, Norway.
Khoo
Kah-Lin
KL
Berge
Knut Erik
KE
Leren
Trond P
TP
Kulseth
Mari Ann
MA
D000293:Adolescent; D000328:Adult; D000483:Alleles; D017398:Alternative Splicing / Q000502:physiology*; D001483:Base Sequence; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D002648:Child; D005091:Exons; D005260:Female; D017930:Genes, Reporter; D004854:Herpesvirus 4, Human / Q000235:genetics; D006579:Heterozygote; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics; D007438:Introns; D008214:Lymphocytes / Q000378:metabolism / Q000821:virology; D008297:Male; D008875:Middle Aged; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D022821:RNA Splice Sites / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D022821:RNA Splice Sites; D012333:RNA, Messenger; D011973:Receptors, LDL