title: T-->G or T-->A mutation introduced in the branchpoint consensus sequence of intron 4 of lecithin:cholesterol acyltransferase (LCAT) gene: intron retention causing LCAT deficiency.
abstract: Previous mutations associated with lecithin:cholesteryl acyltransferase (LCAT) deficiency syndromes have been identified in the coding regions of the LCAT gene. However, recently, an intron mutation was found in a family in which three sisters presented with fish-eye disease (FED). The probands were shown to be heterozygotes for a mutation in intron 4. The respective T-->C nucleotide substitution, 22 bases upstream of the 3'-splice site, causes a null allele as the result of complete intron retention. Since the natural mutation occurs in a putative branchpoint consensus sequence of the intron, it was hypothesized that the point mutation may disrupt the splicing of the pre-mRNA. To further study the functional significance of the above thymine residue in the branchpoint sequence, we introduced other nucleotides at this position, i.e., LCAT Int-4 MUT-1 (T-->G) and LCAT Int-4 MUT-2 (T-->A). After stable transfection of the mutated pNUT-LCAT minigenes into BHK cells, we could detect neither LCAT activity nor LCAT protein in the culture medium of the pNUT-LCAT Int-4 MUT-1 and pNUT-LCAT Int-4 MUT-2 cell lines, as was previously described for the natural mutation. To determine the effects of the introduced mutations on pre-mRNA splicing, total RNA from transfected BHK cells was used for RT-PCR analysis. All BHK cell lines were shown to transcribe the integrated LCAT minigenes. However, the sizes of these LCAT messengers indicated that intron 4 was retained in the pNUT-LCAT Int-4 MUT-1 and pNUT-LCAT Int-4 MUT-2 cell lines. Subsequent sequence analysis of the RT-PCR products demonstrated that the unspliced intronic sequences contained the introduced mutations. In conclusion, the observed retention of intron 4 of the LCAT gene is the result of the specific loss of a thymine residue two bases upstream of the branchpoint adenosine residue in the putative branchpoint consensus sequence. The results confirm that a single base change in the branchpoint consensus sequence of an intron can cause human disease although this sequence is poorly conserved in mammals.
authors.affiliation: Atherosclerosis Specialty Laboratory, Department of Pathology and Laboratory Medicine, St. Paul's Hospital and University of British Columbia, 1081 Burrard Street, Vancouver, British Columbia, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Li
authors.forename: J A
authors.initials: JA
authors.lastname: Kuivenhoven
authors.forename: A F
authors.initials: AF
authors.lastname: Ayyobi
authors.forename: P H
authors.initials: PH
authors.lastname: Pritchard
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016384:Consensus Sequence; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005124:Eye Abnormalities / Q000201:enzymology / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006579:Heterozygote; D006801:Humans; D007438:Introns; D007863:Lecithin Cholesterol Acyltransferase Deficiency / Q000201:enzymology* / Q000235:genetics*; D008954:Models, Biological; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D014162:Transfection
chemical_list: D012322:RNA Precursors; D004247:DNA; D007862:Phosphatidylcholine-Sterol O-Acyltransferase