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Paraoxonase-1 gene expression: The importance of the T(−107)C promoter polymorphism, and a role for transcription factors Sp1 and SREBP
96 ~-~
De Oya THE HYPOTRIGLYCERIDEMIC EFFECT OF PRAVASTATIN IS ASSOCIATED W I T H A DECREASE IN SERUM APO-E CONCENTRATION IN OUR HYPERLIPEMIC POPULATION
M. De Ova 1, I. Campo 2, E. Viturro 1, B. Cano 1, M. Benavente 1, R. Rubio 1, C. Garces 1. ~Fundacion Jimenez Diaz," 2 Centro de Salud de Las Rozas,
Madrid, Spain Apolipoprotein E (apo E) is a polymorphic protein existing in humans in three common isoforms: apo E2, apo E3, and apo E4, coded by three alleles: e2, ~3 and e4. It has been suggested that both the apo E concentration and apo E genotype play an important role in lipoprotein metabolism and thus, the apoE concentration could be related with cardiovascular disease. The aim of this study was to analyze the influence of pravastatin treatment (40 mg/day) on serum apoE levels in 24 patients with primary moderate hypercholesterolemia. Serum apoE concentrations were determined by inmunoturbidimetry. After a 4-week washout phase, treatment with pravastatin for 6 weeks, besides decreasing serum total cholesterol (26%, p<0.00l), LDL cholesterol (29%, p<0.001), apoB (27%, p<0.00l) and triglycerides levels (27%, p <0.001), significantly reduced serum apoE levels (28%, p<0.001). Decrease in apoE levels was correlated with decrease in triglycerides. We believe that the hypotriglyceridemic effect of pravastatin in our study is associated with the apoE concentration decrease that we found in our sample. Since pravastatin does not play a direct role in either the synthesis or the hydrolysis of triglycerides, we suggest this compound causes art increase of (B and E) receptors. As these receptors bind and internalize those lipoproteins containing apoB and apoE, which includes VLDL and remnants, plasma triglycerides decrease. This could be another potential mechanism to explain the cardiovascular benefits of pravastatin. Supported by a grant from Laboratorios Dr. Esteve, Spain. OF THE e3e4 APOE GENOTYPE DISPLAY LESS [i45]CARRIERS SUSCEPTIBILITY TO IN VITRO OXIDATION OF LDL M. De Oya 1, R. Rubio 1, H. Ortega2, M. Benavente 1, E. Viturro 1, B. Cano 1, D. Gomez-Coronado2, M.A. Lasuncion2, C. GarceQ.
]Fundacion Jimenez Diaz; 2Hospital Ramon y Cajal, Madrid, Spain Background: We have examined the relationship between apoE and low density lipoprotein (LDL) oxidation in vitro to address whether susceptibility to LDL oxidation varied among apoE genotypes and thereby might explain differences in susceptibility to suffer atfierosclerotic lesions of subjects of the different genotypes. Methods: The susceptibility of LDL to oxidative modification was determined as the production of conjugated dienes induced by copper. Oxidation experiments were performed in 45 healthy subjects (15 from each of the three most common apoE genotypes: ~2e3, e3~3 and ~3~4). Results: Significant differences in lag time and tl/2 were detected between LDL samples of subject with the ~2e3 genotype and the ~3~3 and e3~4 genotypes. By stepwise multiple regression analysis, e2 was selected as the only significant predictor of the lag time, explaining 10.7% of the variance of this parameter (p<0.05). Both the ~2 allele (17.3%, p<0.01) and the betacarotene content in LDL (16.5%, p<0.01) were selected as predictors of the tl/2, together explaining 28.5% of its variance. LDL beta-carotene content was selected as the only predictor of the maximal oxidation rate of LDL, being responsible for 18.2% (p<0.01) of the variance of this variable. Conclusions: LDL particles of those with the ~2~3 genotype are more resistant to oxidation than LDL particles of individuals with the ~3e3 or ~3e4 genotype. Our findings are consistent with the evidence of differences in the risk for the development of arteriosclerotic lesions among carriers of different ApoE genotypes. Supported by a grant from Laboratorios Dr. Esteve, Spain.
[•
PARAOXONASE-1 GENE EXPRESSION: THE IMPORTANCE OF THE T(-107)C PROMOTER POLYMORPHISM, AND A ROLE FOR TRANSCRIPTION FACTORS SP1 AND SREBP
attempted to identify potential mechanisms. Highly significant differences (p<0.0001) in serum mass and activity were observed as a function of promoter haplotype structure. These could be essentially attributed to the -107 (C/T) site with no significant contribution from the -824 or -907 sites. Studies of potential mechanisms centered on transcription factors Spl and sterol regulatory element binding proteins (SREBP). The -107 polymorphic position lies in the centre of a potential binding site for Spl. The T variant polymorphism disrupts this site. EMSA analysis revealed that Spl binds an 80bp PON1 promoter fragment containing the -107 site and has a lower affinity for the T v C containing fragment. Two regions with homology to SREBP sites are found adjacent to the Spl site surrounding -107. Spl is known to act as synergistic co-regulator of SREBPla and 2. SREBP la and 2 were found to bind to the PON1 promoter and increase expression up to 25 fold. In conclusion we identify the -107 polymorphic site as predominantly important in an in vivo context and by in vitro analysis offer a mechanistic explanation for its effect. ~-~
TWO NOVEL POLYMORPHISMS OF THE LDL-RECEPTOR GENE IN GREEK PATIENTS W I T H FAMILIAL HYPERCHOLESTEROLEMIA
G. DedoussiQ, C. Pitsavos2, J. Skoumas2, G. SokarP, M. Pmssa 1, C. Stefanadis 2, R Toutouzas2. JDepartment of Diet and Nutrition,
Harokopio University of Athens," 2Department of Cardiology, Hippocratio Hospital, Medical School of Athens, Greece Familial hypercholesterolemia (FH; MIM# 143890) is an autosomal dominant disorder caused by mutations in the low-density lipoprotein receptor gene (LDLR;MIM#143890, Genbank Accession Number: 000527) affecting approximately 1 in 500 individuals worldwide. So far, over 700 mutations have been reported for the LDLR gene and account for FH, database website www.ucl.ac.uk/fh.3. Multiple restriction fragment length polymorphisms (RFLPs), identified in the gene, can be used to study haplotype associations in families with FH. Two new restriction fragment length polymorphisms (RFLP) in exons 13 and 4 of the low-density lipoprotein receptor (LDLR) gene are described. They were detected from two patients with FH, by the denaturing graduing gel electrophoresis (DGGE) method and automated sequencinq. In exon 13 the codon for Asn 619 was converted from AAC to AAT. The presence of the mutant allele abolishes a HinclI restriction site. In exon 4 the codon for Cysl07 was converted from TGT to TGC. The presence of the mutant allele abolishes a Mae III restriction site. The noveI polymorphisms could be useful on the haplotype construction and in association studies of genotype to phenotype. [i4-~ FAMILIAL DEFECTIVE APOLIPOPROTEIN B-1011 VERSUS FAMILIAL HYPERCHOLESTEROLEMIA: AN ASSESSMENT OF RISK S.W. Fouchier1, E.I.G. Sijbrands 2, J.J.P. Kastelein 1, LC. Defesche I .
1Department of Vascular Medicine, Academic Medical Centre, Amsterdam; 2Department of Internal Medicine, Erasmus Medical Centre, Rotterdam, The Netherlands Background: Patients with Familial Hypercholesterolemia (FH) and Familial Defective apolipoprotein B (FDB) have severely increased LDLcholesterol levels that expose them to an increased risk for premature coronary disease (CAD). Previous data on FDB patients, however, were collected in patients referred to Lipid Clinics and were therefore subject to clinical sampling bias. Therefore, we assessed the clinical phenotype of FDB in a population free from selection on CAD in order to compare the atherosclerotic burden with that of heterozygous FH. Design: The study population was actively recruited in a large-scale screening program for inherited hypercholesterolemia, in which FH and FDB heterozygotes were diagnosed by standard molecular techniques. Findings: Patients with FH and FDB differed significantly from their unaffected relatives with regard to plasma total cholesterol (Table).
I. Leviev, S.R Deakin, M.-C. Brulhart, R.W. James. University Hospital,
Geneva, Switzerland Serum paraoxonase-1 (PON1) levels are primary determinants of the antioxidant and anti-inflammatory capacities of HDL and are implicated in protection from atherosclerosis. There is a wide spectrum of serum PON1 mass in man, to which promoter polymorphisms make important contributions. The present study analysed the relative importance of individual promoter polymorphisms (-107, -824, -907) to in vivo variations in serum PON1 and
FH FDB
n LDL (aft&mar0
95% CI
p
399/t048 5.65 t22/378 5.27
5.5I 5.79 <0.00I 4.95 5.64 <0.00I
LDL diff. 95% CI (aff/unaf0 2.29 t.78
p
2.I6 2.42 <0.00i t.56 2.00 <0.00i
affAmaff:affected versusunaffected; LDL:LDL-cholesterollevels hi lmnol/1;95% CI: 95% coiffi.dence hlterval;LDL diff. (aff/tmaf0:meandifferencehi LDL-cholesterollevelsbetweenaffected and tmaffected hldividuals.
73rd EAS Congress
De Oya THE HYPOTRIGLYCERIDEMIC EFFECT OF PRAVASTATIN IS ASSOCIATED W I T H A DECREASE IN SERUM APO-E CONCENTRATION IN OUR HYPERLIPEMIC POPULATION
M. De Ova 1, I. Campo 2, E. Viturro 1, B. Cano 1, M. Benavente 1, R. Rubio 1, C. Garces 1. ~Fundacion Jimenez Diaz," 2 Centro de Salud de Las Rozas,
Madrid, Spain Apolipoprotein E (apo E) is a polymorphic protein existing in humans in three common isoforms: apo E2, apo E3, and apo E4, coded by three alleles: e2, ~3 and e4. It has been suggested that both the apo E concentration and apo E genotype play an important role in lipoprotein metabolism and thus, the apoE concentration could be related with cardiovascular disease. The aim of this study was to analyze the influence of pravastatin treatment (40 mg/day) on serum apoE levels in 24 patients with primary moderate hypercholesterolemia. Serum apoE concentrations were determined by inmunoturbidimetry. After a 4-week washout phase, treatment with pravastatin for 6 weeks, besides decreasing serum total cholesterol (26%, p<0.00l), LDL cholesterol (29%, p<0.001), apoB (27%, p<0.00l) and triglycerides levels (27%, p <0.001), significantly reduced serum apoE levels (28%, p<0.001). Decrease in apoE levels was correlated with decrease in triglycerides. We believe that the hypotriglyceridemic effect of pravastatin in our study is associated with the apoE concentration decrease that we found in our sample. Since pravastatin does not play a direct role in either the synthesis or the hydrolysis of triglycerides, we suggest this compound causes art increase of (B and E) receptors. As these receptors bind and internalize those lipoproteins containing apoB and apoE, which includes VLDL and remnants, plasma triglycerides decrease. This could be another potential mechanism to explain the cardiovascular benefits of pravastatin. Supported by a grant from Laboratorios Dr. Esteve, Spain. OF THE e3e4 APOE GENOTYPE DISPLAY LESS [i45]CARRIERS SUSCEPTIBILITY TO IN VITRO OXIDATION OF LDL M. De Oya 1, R. Rubio 1, H. Ortega2, M. Benavente 1, E. Viturro 1, B. Cano 1, D. Gomez-Coronado2, M.A. Lasuncion2, C. GarceQ.
]Fundacion Jimenez Diaz; 2Hospital Ramon y Cajal, Madrid, Spain Background: We have examined the relationship between apoE and low density lipoprotein (LDL) oxidation in vitro to address whether susceptibility to LDL oxidation varied among apoE genotypes and thereby might explain differences in susceptibility to suffer atfierosclerotic lesions of subjects of the different genotypes. Methods: The susceptibility of LDL to oxidative modification was determined as the production of conjugated dienes induced by copper. Oxidation experiments were performed in 45 healthy subjects (15 from each of the three most common apoE genotypes: ~2e3, e3~3 and ~3~4). Results: Significant differences in lag time and tl/2 were detected between LDL samples of subject with the ~2e3 genotype and the ~3~3 and e3~4 genotypes. By stepwise multiple regression analysis, e2 was selected as the only significant predictor of the lag time, explaining 10.7% of the variance of this parameter (p<0.05). Both the ~2 allele (17.3%, p<0.01) and the betacarotene content in LDL (16.5%, p<0.01) were selected as predictors of the tl/2, together explaining 28.5% of its variance. LDL beta-carotene content was selected as the only predictor of the maximal oxidation rate of LDL, being responsible for 18.2% (p<0.01) of the variance of this variable. Conclusions: LDL particles of those with the ~2~3 genotype are more resistant to oxidation than LDL particles of individuals with the ~3e3 or ~3e4 genotype. Our findings are consistent with the evidence of differences in the risk for the development of arteriosclerotic lesions among carriers of different ApoE genotypes. Supported by a grant from Laboratorios Dr. Esteve, Spain.
[•
PARAOXONASE-1 GENE EXPRESSION: THE IMPORTANCE OF THE T(-107)C PROMOTER POLYMORPHISM, AND A ROLE FOR TRANSCRIPTION FACTORS SP1 AND SREBP
attempted to identify potential mechanisms. Highly significant differences (p<0.0001) in serum mass and activity were observed as a function of promoter haplotype structure. These could be essentially attributed to the -107 (C/T) site with no significant contribution from the -824 or -907 sites. Studies of potential mechanisms centered on transcription factors Spl and sterol regulatory element binding proteins (SREBP). The -107 polymorphic position lies in the centre of a potential binding site for Spl. The T variant polymorphism disrupts this site. EMSA analysis revealed that Spl binds an 80bp PON1 promoter fragment containing the -107 site and has a lower affinity for the T v C containing fragment. Two regions with homology to SREBP sites are found adjacent to the Spl site surrounding -107. Spl is known to act as synergistic co-regulator of SREBPla and 2. SREBP la and 2 were found to bind to the PON1 promoter and increase expression up to 25 fold. In conclusion we identify the -107 polymorphic site as predominantly important in an in vivo context and by in vitro analysis offer a mechanistic explanation for its effect. ~-~
TWO NOVEL POLYMORPHISMS OF THE LDL-RECEPTOR GENE IN GREEK PATIENTS W I T H FAMILIAL HYPERCHOLESTEROLEMIA
G. DedoussiQ, C. Pitsavos2, J. Skoumas2, G. SokarP, M. Pmssa 1, C. Stefanadis 2, R Toutouzas2. JDepartment of Diet and Nutrition,
Harokopio University of Athens," 2Department of Cardiology, Hippocratio Hospital, Medical School of Athens, Greece Familial hypercholesterolemia (FH; MIM# 143890) is an autosomal dominant disorder caused by mutations in the low-density lipoprotein receptor gene (LDLR;MIM#143890, Genbank Accession Number: 000527) affecting approximately 1 in 500 individuals worldwide. So far, over 700 mutations have been reported for the LDLR gene and account for FH, database website www.ucl.ac.uk/fh.3. Multiple restriction fragment length polymorphisms (RFLPs), identified in the gene, can be used to study haplotype associations in families with FH. Two new restriction fragment length polymorphisms (RFLP) in exons 13 and 4 of the low-density lipoprotein receptor (LDLR) gene are described. They were detected from two patients with FH, by the denaturing graduing gel electrophoresis (DGGE) method and automated sequencinq. In exon 13 the codon for Asn 619 was converted from AAC to AAT. The presence of the mutant allele abolishes a HinclI restriction site. In exon 4 the codon for Cysl07 was converted from TGT to TGC. The presence of the mutant allele abolishes a Mae III restriction site. The noveI polymorphisms could be useful on the haplotype construction and in association studies of genotype to phenotype. [i4-~ FAMILIAL DEFECTIVE APOLIPOPROTEIN B-1011 VERSUS FAMILIAL HYPERCHOLESTEROLEMIA: AN ASSESSMENT OF RISK S.W. Fouchier1, E.I.G. Sijbrands 2, J.J.P. Kastelein 1, LC. Defesche I .
1Department of Vascular Medicine, Academic Medical Centre, Amsterdam; 2Department of Internal Medicine, Erasmus Medical Centre, Rotterdam, The Netherlands Background: Patients with Familial Hypercholesterolemia (FH) and Familial Defective apolipoprotein B (FDB) have severely increased LDLcholesterol levels that expose them to an increased risk for premature coronary disease (CAD). Previous data on FDB patients, however, were collected in patients referred to Lipid Clinics and were therefore subject to clinical sampling bias. Therefore, we assessed the clinical phenotype of FDB in a population free from selection on CAD in order to compare the atherosclerotic burden with that of heterozygous FH. Design: The study population was actively recruited in a large-scale screening program for inherited hypercholesterolemia, in which FH and FDB heterozygotes were diagnosed by standard molecular techniques. Findings: Patients with FH and FDB differed significantly from their unaffected relatives with regard to plasma total cholesterol (Table).
I. Leviev, S.R Deakin, M.-C. Brulhart, R.W. James. University Hospital,
Geneva, Switzerland Serum paraoxonase-1 (PON1) levels are primary determinants of the antioxidant and anti-inflammatory capacities of HDL and are implicated in protection from atherosclerosis. There is a wide spectrum of serum PON1 mass in man, to which promoter polymorphisms make important contributions. The present study analysed the relative importance of individual promoter polymorphisms (-107, -824, -907) to in vivo variations in serum PON1 and
FH FDB
n LDL (aft&mar0
95% CI
p
399/t048 5.65 t22/378 5.27
5.5I 5.79 <0.00I 4.95 5.64 <0.00I
LDL diff. 95% CI (aff/unaf0 2.29 t.78
p
2.I6 2.42 <0.00i t.56 2.00 <0.00i
affAmaff:affected versusunaffected; LDL:LDL-cholesterollevels hi lmnol/1;95% CI: 95% coiffi.dence hlterval;LDL diff. (aff/tmaf0:meandifferencehi LDL-cholesterollevelsbetweenaffected and tmaffected hldividuals.
73rd EAS Congress