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Mutations in the gene encoding sterol-regulatory element-binding protein-2 in hypercholesterolaemic subjects
Posters 6. Genetics and Atherosclerosis two groups. Among conventional CV risk factors, tot-C. and LDL-C. levels (p < 0.001 both) and BMI (p c 0.001) were significantly higher in the case group than in control subjects. We did not find any significant association between genetic polymorphisms and MI in elderly subjects. These results needs to be confirmed by larger studies on older subjects
M
ASSOCIATION OF THE LDLR GENE POLYMORPHISMS WITH LDLR mRNA EXPRESSION IN MONONUCLEAR CELLS AND RESPONSE TO ATORVASTATIN THERAPY IN SUBJECTS WITH HETEROZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA
L.A. Salazar’*3, M.H. Hirata’, N. Forti*, J. Diamen$, SD. Giannini*, R.D.C. Hirata’ . ‘Faculty of Pharmaceutical Sciences: ‘Heart Institute
(InCor), University of Sao”Paula, Sao Paulo, SP, Brazil; 3Faculty of Medicine, Unioersity of La Fnmtera, Temuco, Chile Recently studies in Brazil have demonstrated the association between polymorphisms at the LDL receptor (LDLR) gene and high serum lipid concentrations, lower response to HMG-CoA reductase inhibitors (statins) and susceptibility to coronary artery disease. The present study investigated the possible influence of three genetic polymorphisms (HincII - exon 12, AuaII - exon 13 and PuuII - intron 15) of the LDLR gene on LDLR mRNA expression and response to Atorvastatin therapy in subjects with heterozygous Familial Hypercholesterolemia (FH). Twenty-five unrelated patients (5 men and 20 women) with elevated LDL-cholesterol levels (6.3*1.6 mmol/L); aged 55&19 yr. entered our 4-week study. Patients with secondary forms of dyslipidemia and those with diabetes mellitus, hypothyroidism or obesity were excluded. other lipid lowering medications were discontinued for at least 1 month before the study. Subsequently, FH patients were treated with 10 mg of Atorvastatin per day. Blood samples were collected at weeks 0 and 4 for lipid profile analysis in serum and LDLR gene expression evaluation in peripheral blood mononuclear cells (PBMC) by RTPCR assay. The LDLR genotypes were determined by PCR-RFLP technique. At week 4, Atorvastatin significantly reduced TC, LDL-C and ApoB levels from baseline (by -25%, -31%, and -21%, respectively, P < 0.01) in 19 patients (Responders, RF). Gn the other hand, 6 patients presented less than 15% of reduction on LDL-C levels (No responders group, NR). Our data also have shown that Atorvastatin increase LDLR mRNA expression in responder patients when compared to baseline levels (P < 0.0001). LDLR mRNA expression in NR patients was lower in comparison with the RE at the weeks 0 and 4 (P < 0.0001). In addition, when the individuals were categorized by genotypes, those subjects carrying the A+A+IAuaII (N = 13) and P-P-IPuuII (N = 15) genotypes presented lower LDLR mRNA expression and lower response to Atorvastatin therapy (P < 0.05). This study shows an important association between the lipid-lowering response to Atorvastatin, LDLR mRNA expression in PBMC and polymorphisms at the LDLR gene in subjects with FH from Brazil. Financial Support: FAPESP-Brazil
M
69
these mutations in a key regulator of the cholesterol metabolism are associated with elevated plasma cholesterol concentrations and hence, suggest an involvement of SREBP-2 in the development of hypercholesterolaemia.
IP92
EVALUATION OF THE FUNCTIONALITY OF A NEW POLYMORPHISM IN THE CETP GENE PROMOTER ASSOCIATED WITH PLASMA HDL-C LEVELS
W. Le Goff, M. Guerin, V Nicaud, C. Dachet, M.J. Chapman, Insenn Unite 321 Hopital de la Pitie, 75651, Parts
J. Thillet.
Cholestetyl ester transfer protein (CETP) plays a key role in reverse cholesterol transport by mediating the transfer of cholesteryl ester (CE) from HDL to atherogenic ApoB-containing lipoproteins, including VLDL, IDL and LDL. We describe a new frequent polymorphism located in the human CETP gene promoter at position -971. This polymorphism is significantly associated with both plasma HDL-C levels and CETP concentration (p = 0.006 and p = 0.009, respectively) in the ECTIM study (CHD cases and controls). In addition the -97 lG/A polymorphism interacts significantly with the functional -629C/A site and the TaqIB polymorphism with respect to plasma HDL-C levels (p = 0.0014 and p = 0.012, respectively), but not with plasma CETP concentration. These results suggest-that the effect of the -971G/A uolvmornhism on both olasma CETP concentration and HDL-C levels are independent. Transient hansfection of HepG2 cells revealed that the -971G/A promoter polymorphism constitutes a non-functional marker. Our findings suggest the existence of as yet unidentified functional polymorphisms in the CETP gene promoter that could explain the association between specific polymorphisms of the CETP gone and both plasma HDL-C and CETP concentrations in both control subjects and CHD cases.
m
PARAOXONASE DISEASE
AND RISK OF CARDIOVASCULAR
HEART
C. GarcBs, M. Benavente, R. Rubio, E. Viturro, C. Maicas, D. Lopez, J. Romero, E. Martin, J. Farm, M. de Gya. Fundacidn Jimlnez Diaz,
Madrid, Spain Paraoxonase (PONl) is a HDL-associated enzyme that prevents ox&dative modification of LDL. The PONl gene has a polymorphic site at 192 position. PONl activity varies markedly among genotypes and the polymorphism has been inconsistently asocciated with cardiovascular heart disease (CHD). Objective: To analyse the relationship between PONl and CHD. Methods: We have determined PON genotypes by PCR amplification and AlwI restriction enzyme analysis, and measured the serum activity of paraoxon hydrolysis in 173 survivors of myocardial infarction (MI) admitted at the Coronary Care Unit at two hospitals of our area, and compare with the result of those determinations in a population-based control sample. Results: There are not significant differences in genotype frequencies between groups, but patients showed lower activities than controls in the whole group and within each genotype group.
MUTATIONS IN THE GENE ENCODING STEROLEEGUEATORY ELEMENT-BINDING PROTEIN-2 IN HYPERCROLESTEROLAEMIC SUBJECTS
PY. Muller, A.R. Miserez. Department of Clinical-Biological Sciences,
University of Basel; Division of Endocrinology, Diabetology, and Clinical Nuhition, Base1 University Clinics, Hebelshasse 20, CH-4031 Basel, Switzerland Human cells maintain their cholesterol homeostasis by the regulated cleavage of membrane-bound transcription factors, so-called sterol-regulatory element-binding proteins (SRFBPs). If cells are deprived of cholesterol, the SREBPs are released from the membranes of the endoplasmic reticulum by two proteolytic steps and are transported into the nucleus. The SRBBPs bind to specific nucleotide sequences in the promoters of the low-density lipoprotein receptor gene and of a variety of other key genes involved in cholesterol and triglyceride homeostasis. Given the central role of the SREBPs in the regulation of the cholesterol metabolism, we investigated whether hypercholesterolaemic subjects harbor specific sequence variations in crucial functional domains of SREBP-2. Exons 4 to 11, encoding the DNA-binding and the regulatory domains of SREBP-2, were analysed in a cohort of hypercholesterolaemic subjects. Three missense mutations were found in the CGGH-terminal regulatory domain and one missense mutation in the DNA-binding domain. In summary, this is the first report describing mutations in the human SRBBP-2 gene. Our results open the possibility that
Conclusions: These results confirm the hypothesis that genotypes do not predict CHD and support the convenience of PONl activity determination in cardiovascular disease
Study sponsored by Fundacidn Ramdn Areces. w
FAMILIAL DEFECTIVE POPULATION
apo B 100 IN A SOUTH EUROPEAN
J.T. Real, F.J. Chaves’, I. Ejarque, M.J. Catala, M.A. Priego, J.F. Ascaso, M.E. Armengod’, R. Cannena. Endocrtnology, Hospital Clinico,
Uniuersitat de Valencia; ‘FIB, Valencia. Spain Purpose of the Study: 1) To investigate the prevalence of familial defective apo B 100 (FDB) in a South European population of familial hypercholesterolemia (FH). 2) Study the lipid phenotype of FDB.
72nd EAS Congress
M
ASSOCIATION OF THE LDLR GENE POLYMORPHISMS WITH LDLR mRNA EXPRESSION IN MONONUCLEAR CELLS AND RESPONSE TO ATORVASTATIN THERAPY IN SUBJECTS WITH HETEROZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA
L.A. Salazar’*3, M.H. Hirata’, N. Forti*, J. Diamen$, SD. Giannini*, R.D.C. Hirata’ . ‘Faculty of Pharmaceutical Sciences: ‘Heart Institute
(InCor), University of Sao”Paula, Sao Paulo, SP, Brazil; 3Faculty of Medicine, Unioersity of La Fnmtera, Temuco, Chile Recently studies in Brazil have demonstrated the association between polymorphisms at the LDL receptor (LDLR) gene and high serum lipid concentrations, lower response to HMG-CoA reductase inhibitors (statins) and susceptibility to coronary artery disease. The present study investigated the possible influence of three genetic polymorphisms (HincII - exon 12, AuaII - exon 13 and PuuII - intron 15) of the LDLR gene on LDLR mRNA expression and response to Atorvastatin therapy in subjects with heterozygous Familial Hypercholesterolemia (FH). Twenty-five unrelated patients (5 men and 20 women) with elevated LDL-cholesterol levels (6.3*1.6 mmol/L); aged 55&19 yr. entered our 4-week study. Patients with secondary forms of dyslipidemia and those with diabetes mellitus, hypothyroidism or obesity were excluded. other lipid lowering medications were discontinued for at least 1 month before the study. Subsequently, FH patients were treated with 10 mg of Atorvastatin per day. Blood samples were collected at weeks 0 and 4 for lipid profile analysis in serum and LDLR gene expression evaluation in peripheral blood mononuclear cells (PBMC) by RTPCR assay. The LDLR genotypes were determined by PCR-RFLP technique. At week 4, Atorvastatin significantly reduced TC, LDL-C and ApoB levels from baseline (by -25%, -31%, and -21%, respectively, P < 0.01) in 19 patients (Responders, RF). Gn the other hand, 6 patients presented less than 15% of reduction on LDL-C levels (No responders group, NR). Our data also have shown that Atorvastatin increase LDLR mRNA expression in responder patients when compared to baseline levels (P < 0.0001). LDLR mRNA expression in NR patients was lower in comparison with the RE at the weeks 0 and 4 (P < 0.0001). In addition, when the individuals were categorized by genotypes, those subjects carrying the A+A+IAuaII (N = 13) and P-P-IPuuII (N = 15) genotypes presented lower LDLR mRNA expression and lower response to Atorvastatin therapy (P < 0.05). This study shows an important association between the lipid-lowering response to Atorvastatin, LDLR mRNA expression in PBMC and polymorphisms at the LDLR gene in subjects with FH from Brazil. Financial Support: FAPESP-Brazil
M
69
these mutations in a key regulator of the cholesterol metabolism are associated with elevated plasma cholesterol concentrations and hence, suggest an involvement of SREBP-2 in the development of hypercholesterolaemia.
IP92
EVALUATION OF THE FUNCTIONALITY OF A NEW POLYMORPHISM IN THE CETP GENE PROMOTER ASSOCIATED WITH PLASMA HDL-C LEVELS
W. Le Goff, M. Guerin, V Nicaud, C. Dachet, M.J. Chapman, Insenn Unite 321 Hopital de la Pitie, 75651, Parts
J. Thillet.
Cholestetyl ester transfer protein (CETP) plays a key role in reverse cholesterol transport by mediating the transfer of cholesteryl ester (CE) from HDL to atherogenic ApoB-containing lipoproteins, including VLDL, IDL and LDL. We describe a new frequent polymorphism located in the human CETP gene promoter at position -971. This polymorphism is significantly associated with both plasma HDL-C levels and CETP concentration (p = 0.006 and p = 0.009, respectively) in the ECTIM study (CHD cases and controls). In addition the -97 lG/A polymorphism interacts significantly with the functional -629C/A site and the TaqIB polymorphism with respect to plasma HDL-C levels (p = 0.0014 and p = 0.012, respectively), but not with plasma CETP concentration. These results suggest-that the effect of the -971G/A uolvmornhism on both olasma CETP concentration and HDL-C levels are independent. Transient hansfection of HepG2 cells revealed that the -971G/A promoter polymorphism constitutes a non-functional marker. Our findings suggest the existence of as yet unidentified functional polymorphisms in the CETP gene promoter that could explain the association between specific polymorphisms of the CETP gone and both plasma HDL-C and CETP concentrations in both control subjects and CHD cases.
m
PARAOXONASE DISEASE
AND RISK OF CARDIOVASCULAR
HEART
C. GarcBs, M. Benavente, R. Rubio, E. Viturro, C. Maicas, D. Lopez, J. Romero, E. Martin, J. Farm, M. de Gya. Fundacidn Jimlnez Diaz,
Madrid, Spain Paraoxonase (PONl) is a HDL-associated enzyme that prevents ox&dative modification of LDL. The PONl gene has a polymorphic site at 192 position. PONl activity varies markedly among genotypes and the polymorphism has been inconsistently asocciated with cardiovascular heart disease (CHD). Objective: To analyse the relationship between PONl and CHD. Methods: We have determined PON genotypes by PCR amplification and AlwI restriction enzyme analysis, and measured the serum activity of paraoxon hydrolysis in 173 survivors of myocardial infarction (MI) admitted at the Coronary Care Unit at two hospitals of our area, and compare with the result of those determinations in a population-based control sample. Results: There are not significant differences in genotype frequencies between groups, but patients showed lower activities than controls in the whole group and within each genotype group.
MUTATIONS IN THE GENE ENCODING STEROLEEGUEATORY ELEMENT-BINDING PROTEIN-2 IN HYPERCROLESTEROLAEMIC SUBJECTS
PY. Muller, A.R. Miserez. Department of Clinical-Biological Sciences,
University of Basel; Division of Endocrinology, Diabetology, and Clinical Nuhition, Base1 University Clinics, Hebelshasse 20, CH-4031 Basel, Switzerland Human cells maintain their cholesterol homeostasis by the regulated cleavage of membrane-bound transcription factors, so-called sterol-regulatory element-binding proteins (SRFBPs). If cells are deprived of cholesterol, the SREBPs are released from the membranes of the endoplasmic reticulum by two proteolytic steps and are transported into the nucleus. The SRBBPs bind to specific nucleotide sequences in the promoters of the low-density lipoprotein receptor gene and of a variety of other key genes involved in cholesterol and triglyceride homeostasis. Given the central role of the SREBPs in the regulation of the cholesterol metabolism, we investigated whether hypercholesterolaemic subjects harbor specific sequence variations in crucial functional domains of SREBP-2. Exons 4 to 11, encoding the DNA-binding and the regulatory domains of SREBP-2, were analysed in a cohort of hypercholesterolaemic subjects. Three missense mutations were found in the CGGH-terminal regulatory domain and one missense mutation in the DNA-binding domain. In summary, this is the first report describing mutations in the human SRBBP-2 gene. Our results open the possibility that
Conclusions: These results confirm the hypothesis that genotypes do not predict CHD and support the convenience of PONl activity determination in cardiovascular disease
Study sponsored by Fundacidn Ramdn Areces. w
FAMILIAL DEFECTIVE POPULATION
apo B 100 IN A SOUTH EUROPEAN
J.T. Real, F.J. Chaves’, I. Ejarque, M.J. Catala, M.A. Priego, J.F. Ascaso, M.E. Armengod’, R. Cannena. Endocrtnology, Hospital Clinico,
Uniuersitat de Valencia; ‘FIB, Valencia. Spain Purpose of the Study: 1) To investigate the prevalence of familial defective apo B 100 (FDB) in a South European population of familial hypercholesterolemia (FH). 2) Study the lipid phenotype of FDB.
72nd EAS Congress