The A/G polymorphism in the -78 position of the apolipoprotein A-I promoter does not have a direct effect on transcriptional efficiency.

A promoter polymorphism A/G at position 78 bp upstream of the transcription initiation site characterizes the human apolipoprotein A-I gene. Some studies correlated the higher Apo A-I levels or increased Apo A-I transcription efficiency with the A allele, while other studies did not confirm these results. We have investigated the in vitro effects of this transition on the transcriptional efficiency of ApoAI gene by creating two sets of identical constructs with the whole Apo A-I promoter, carrying the A or the G, linked to the complete ApoAI gene. The relative activity of the two promoter alleles was determined through a quantitative RT-PCR system after transient tranfections of human HepG2 cell line in basal state and after stimulation with retinoic acid or 17beta-estradiol. Our results exclude differences in promoter activity linked to the A or G promoter alleles either in basal or in stimulated conditions. The data suggest that the A/G polymorphism does not directly affect the transcriptional efficiency of ApoAI gene, although it may be in linkage disequilibrium with other regulatory sequences and the combination of these elements may explain the contradictory results of the ApoAI gene expression.

Functional characterization of the novel mutation IVS 8 (-11delC) (-14T>A) in the intron 8 of the glucocerebrosidase gene of two Italian siblings with Gaucher disease type I.

Gaucher disease, the most common glycolipid storage disease, can be caused by a large variety of mutations. We report here the identification and characterization of a novel mutation in the human glucocerebrosidase gene, IVS 8 (-11delC) (-14T>A), in two siblings with Gaucher disease type I which occurs within the 3' end of intron 8. Both siblings were compound heterozygotes for the IVS 8 (-11delC) (-14T>A) mutation and for the c.626 G>C (R170P) substitution within exon 6. No mRNA species carrying the IVS 8 (-11delC) (-14T>A) mutation were detected by RT-PCR analysis of the RNA extracted from the patients' fibroblasts. To study the possible effects of the IVS 8 (-11delC) (-14T>A) sequence alteration on the splicing of the proximal exon 9, we have established an in vitro system generating a minigene carrying the genomic region of human glucocerebrosidase spanning from exon 8 to exon 10. Transfections into the human Hep3B cell line of the wild-type construct resulted in the expression of mRNA with the glucocerebrosidase exons correctly spliced. On the contrary, transfections of the construct carrying the IVS 8 (-11delC) (-14T>A) mutation resulted in the expression of mRNA with an 11-bp insertion located between the end of exon 8 and the beginning of exon 9. These results indicated that the 5243T>A substitution created a new 3' splice site 11 bp upstream of the wild-type one, leading to the incorporation into the mRNA of these extra 11 bases. Moreover, the new 3' splice site created by this 5243T>A transversion was preferred over the wild-type one in 100% of cases. The in vitro studies suggest that, in the patients, the 11-bp inclusion causes a shift in the reading frame with the generation of a stop codon after codon 388 which undergoes early degradation.

Polymorphism of the apolipoprotein E gene and early carotid atherosclerosis defined by ultrasonography in asymptomatic adults.

Clinical and autoptical studies have suggested a predisposing role of the allele E4 of apolipoprotein E (apoE) in the development of atherosclerosis and cardiovascular disease. To investigate the possible contribution of apoE allele polymorphism to the carotid intima-media thickness (IMT) as assessed by ultrasound, we studied 260 asymptomatic nondiabetic subjects (121 men, 139 women; mean +/- SD age, 53 +/- 7 years), randomly selected from the population register of the inhabitants of Trieste, Italy. B-mode ultrasound was used to quantify the maximum IMT at 12 sites on the near and far wall of the common, bifurcation, and internal carotid arteries. ApoE genotypes were determined from amplified apoE sequences by restriction isotyping. The frequencies of E2, E3, and E4 alleles were 0.073, 0.827, and 0.100, respectively. As expected, subjects with E4 allele had the highest levels of total serum cholesterol and LDL cholesterol, subjects with E2 allele had the lowest levels, and those with E3 genotype had intermediate levels. The echographic measurements of carotid IMT showed increasing values from E2 to E4 carriers. After adjustment for total and LDL cholesterol serum levels, triglycerides, ratio of LDL to HDL cholesterol, age, sex, and body mass index, ANCOVA showed that the common carotid IMT was significantly greater (P = .029) in subjects with E4 allele compared with E3 carriers. Our data confirm the influence of apoE4 on cholesterol levels and clearly show that apoE genotype affects carotid atherosclerosis in its early stages in middle-aged asymptomatic subjects.