Molecular regulation of microsomal triglyceride transfer protein gene, MTP : functional genetic studies in relation to cardiovascular disease
Author: Ledmyr, Helena
Date: 2004-12-17
Location: Föreläsningssalen, Haga Forum
Time: 10.00
Department: Institutionen för medicin / Department of Medicine
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.
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.
List of papers:
I. Ledmyr H, Karpe F, Lundahl B, McKinnon M, Skoglund-Andersson C, Ehrenborg E (2002). "Variants of the microsomal triglyceride transfer protein gene are associated with plasma cholesterol levels and body mass index." J Lipid Res 43(1): 51-8
Pubmed
II. Ledmyr H, McMahon AD, Ehrenborg E, Nielsen LB, Neville M, Lithell H, MacFarlane PW, Packard CJ, Karpe F; WOSCOPS executive (2004). "The microsomal triglyceride transfer protein gene-493T variant lowers cholesterol but increases the risk of coronary heart disease. " Circulation 109(19): 2279-84. Epub 2004 May 10
Pubmed
III. Ledmyr H, Andresen C, Ottosson L, Sunnerhagen M, Ehrenborg E (2004). "The I/T128 missense polymorphism in the microsomal triglyceride transfer protein gene influences stability and ligand binding of the protein." (Manuscript)
IV. Ledmyr H, Karpe F, Murphy C, Gafvels M, Ehrenborg E (2004). "A genetic promoter variant at position - 164 in the microsomal triglyceride transfer protein gene influences activity and binding of transcriptional factors." (Manuscript)
I. Ledmyr H, Karpe F, Lundahl B, McKinnon M, Skoglund-Andersson C, Ehrenborg E (2002). "Variants of the microsomal triglyceride transfer protein gene are associated with plasma cholesterol levels and body mass index." J Lipid Res 43(1): 51-8
Pubmed
II. Ledmyr H, McMahon AD, Ehrenborg E, Nielsen LB, Neville M, Lithell H, MacFarlane PW, Packard CJ, Karpe F; WOSCOPS executive (2004). "The microsomal triglyceride transfer protein gene-493T variant lowers cholesterol but increases the risk of coronary heart disease. " Circulation 109(19): 2279-84. Epub 2004 May 10
Pubmed
III. Ledmyr H, Andresen C, Ottosson L, Sunnerhagen M, Ehrenborg E (2004). "The I/T128 missense polymorphism in the microsomal triglyceride transfer protein gene influences stability and ligand binding of the protein." (Manuscript)
IV. Ledmyr H, Karpe F, Murphy C, Gafvels M, Ehrenborg E (2004). "A genetic promoter variant at position - 164 in the microsomal triglyceride transfer protein gene influences activity and binding of transcriptional factors." (Manuscript)
Issue date: 2004-11-26
Publication year: 2004
ISBN: 91-7140-142-3
Statistics
Total Visits
Views | |
---|---|
Molecular ...(legacy) | 189 |
Molecular ... | 110 |
Total Visits Per Month
September 2023 | October 2023 | November 2023 | December 2023 | January 2024 | February 2024 | March 2024 | |
---|---|---|---|---|---|---|---|
Molecular ... | 1 | 2 | 0 | 0 | 2 | 1 | 0 |
Top country views
Views | |
---|---|
United States | 59 |
Sweden | 44 |
Germany | 40 |
China | 36 |
Hong Kong | 12 |
South Korea | 9 |
Ireland | 7 |
Finland | 6 |
Russia | 5 |
Ukraine | 5 |
Top cities views
Views | |
---|---|
Kiez | 15 |
Central District | 9 |
Seoul | 9 |
Sunnyvale | 9 |
Shenzhen | 8 |
Beijing | 6 |
Dublin | 6 |
São Paulo | 4 |
Ashburn | 3 |
Ballerup | 3 |