Molecular regulation of microsomal triglyceride transfer protein gene, MTP : Functional genetic studies in relation to cardiovascular disease

Abstract: The microsomal triglyceride transfer protein, MTP, is expressed mainly in the liver, intestine and in the heart. It is crucial for the assembly and secretion of apob-containing lipoproteins, chylomicrons in the intestine and very-low density lipoproteins (VLDL) in the liver. MTP's role in the heart is not fully understood, but it is likely to provide an export system for excess triglycerides from the heart muscle. We hypothesized that functional polymorphisms in the MTP gene are likely to influence lipoprotein concentrations in plasma, and the lipid metabolism of the heart. In Paper I, we show that two promoter polymorphisms, -493G>T and -164T>C, and one missense polymorphism in exon 3, I>T128, are in almost complete linkage disequilibrium. Healthy 50-year old men who are homozygous for the three less common alleles have significantly lowered plasma total and LDL cholesterol levels, slightly higher body mass index (BMI) and waist circumference compared with carriers of the common alleles. In Paper II, the West of Scotland Coronary Prevention Study (WOSCOPS) was genotyped for the -493G>T polymorphism, and individuals homozygous for the less common -493T allele had significantly increased risk of coronary heart disease (CHD) and higher prevalence of minor ECG abnormalities at baseline. We also observed decreased expression of MTP mRNA in myocardial biopsies homozygous for the -493T allele. Haplotype analysis did not provide any additional information over and above the MTP -493G>T polymorphism. In Paper III, we evaluated the structural consequences of the I>T128 missense polymorphism. Circular dichroism (CD) analysis showed that the uncommon T128 variant is greatly destabilized. Limited proteolysis showed increased susceptibility to proteolysis in the T128 variant, and in a ligand-binding assay of MTP to LDL particles, also assayed by proteolysis, decreased stability of the T128-LDL complex was observed compared with the 1128-LDL complex. The results were interpreted based on a structural model of the Nterminal domain of MTP, where residue 128 is located in a surface-exposed position. These results indicate that the I>T128 polymorphism in exon 3 of the MTP gene confers a reduced local structural stability in the Nterminal MTP domain, leading to reduced binding of MTP to LDL particles. In Paper IV, the promoter polymorphisms -493G>T and -164r>C were evaluated in vitro, Transient transfection studies in HepG2 cells using minimal promoter constructs containing the less common -164C allele revealed a 30% decrease in transcriptional activity of MTP compared with the common T allele. Performing similar assays with longer constructs containing both the -493G>T and -164T>C polymorphisms showed that the less common -493T and -164C alleles confer a 30% decreased transcriptional activity. Electrophoretic mobility shift assays (EMSA) demonstrated differential binding between the -164 common T and less common C variants, and super shift analysis showed that SREBP-1 binds to the less common C variant, but not the common T variant. This thesis show that a polymorphism in the MTP gene is associated with increased risk of CHD unrelated to lipoprotein levels. In vitro studies of the less common T128 variant MTP protein show thermal destabilization, increased susceptibility to proteolysis and decreased binding of MTP to LDL particles compared with the common I variant. The less common -493T and -164C alleles show decreased transcriptional activity of the gene and differential binding of nuclear factors. Together the results suggest that these polymorphisms may contribute to an impairment of triglyceride export from the heart, which could in part explain the increase in risk of CHD observed in individuals homozygous for these less common alleles compared with individuals carrying the common alleles.