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
| Autor: | Jan Albert Kuivenhoven, Amir F. Ayyobi, Min Li, P. Haydn Pritchard |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Heterozygote
Sequence analysis RNA Splicing Biophysics Gene Expression Biology Transfection medicine.disease_cause Models Biological Polymerase Chain Reaction Biochemistry Cell Line Phosphatidylcholine-Sterol O-Acyltransferase Endocrinology Lecithin Cholesterol Acyltransferase Deficiency Cricetinae Consensus Sequence RNA Precursors medicine Consensus sequence Animals Humans Point Mutation Eye Abnormalities Gene Genetics Mutation Base Sequence Point mutation Lecithin Acyltransferase Deficiency Intron DNA Molecular biology Introns RNA splicing Female |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism. 1391:256-264 |
| ISSN: | 0005-2760 |
| DOI: | 10.1016/s0005-2760(97)00198-7 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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