- Email: [email protected]
Fluorescence-based sscp analysis of the LDL receptor gene by capillary electrophoresis
71st EAS Meeting Abstracts acceptedfor presentation in the abstract hook
186
and of those 182 (78.5%) presented abnormal hormone profile. There was a strong association between UA with sedentary lifestyle (57%); AMI with sedentary lifestyle and arterial hypertension (73%); Non Q wave Ml with sedentary lifestyle and dyslipidaemia (85%). Conclusion: Our study showed a strong association of ischemic heart disease in women with sedentary lifestyle, arterial hypertension, dyslipidaemia and a family history of CAD. What was striking was the low percentage of patients under HRT which might reinforce the importance of estrogen therapy to prevent cardiovascular disease in high risk women population. ATORVASTATIN (LIPITOR) IN THE TREATMENT OF PATIENTS WITH HYPERCHOLESTEROLEMIA S. Delaroudis A. Slavakis, M. Tzoiti, A. Kyroudi, C. Balaris, J. Sarris, M. Giannoulis, M. Kita, D. Sofianou, H. Hatzikonstantinou, A. Avramides.
Lipid Clinic of Endocrinology Dept, Microbiology Lab, Biochemistr3, Lab. Hippocration Hospital, Thessaloniki. Greece Introduction: Increased plasma cholesterol, lipoprotein and apolipoprotein levels play a significant role in the development and progression of atherosclerotic disease. Many trials have shown that reducing plasma cholesterol levels (especially LDL) can lead to reduction in coronary artery disease. HMG-CoA reductase inhibitors (statins) have been shown to be effective in lowering LDL levels in patients with hypercholesterolemia. Several studies have indicated that atorvastatin, a recently synthesised statin, is safe and effective in hypercholesterolemic patients. Objective: To evaluate lipid parameter responses to 10 mg atorvastatin given daily to patients with primary hypercholesterolemia. Methods and Patients: Seventeen patients (3 men, 14 women) with elevated total and LDL cholesterol, aged 60.9=t=7.4 yr. entered our 16 week study. Participants were recruited at the Outpatient Lipid Clinic o f Endocrinology Dept_ Patients with secondary hypercholesterolemia were excluded. Other lipid lowering medications were discontinued for at least 2 months before study. The patients, who were counselled to follow hypolipidemic diet > 3 months before the baseline phase. Subsequently they were placed on a 10 mg atorvatatin tablet per day. Each patient's plasma lipid profile [total, HDL, LDL cholesterol, triglycerides, ApoAl, ApoB and Lp(a)] was determined at weeks 0, 2, 8 & 16. Results: Atorvastatin, at week 16, significantly lowered total and LDLcholesterol levels from baseline (by -29.3% and -38.5% respectively, p < 0.001) and also caused reduction in TGL (-18.9%, NS), ApoB (-17.9% NS), ApoAl (-1.7% NS) and mild increase in HDL cholesterol (+5.2% NS) and Lp(a) (+2.6% NS) (see table, "p < 0.001). (mean +sd)
0 week
2 week
16 week
(96).4 (0-16
week) CHOL
328+55.7
246.2+53.11.)
231.9+20.7(°1
-29.3% (°1
TG L
176.4 + 86.6
1362+:53.1
150.8:k55.5
- 18.9'/,
HDL
49.7+: 10.8
51.3+: 10.7
52.3=1=11.9
+5.2%
LDL
243.1 :i:57.3
167.6+55.2(°~
149.4+16.9I ' )
-38.5% I*l
ApoAI
156.4+22.5
153.8-t- 19.7
- t.7%
ApoB
153. I +48.2
125.7+:37.5
- 17.9°/*
Lp(a)
30.4+:24.7
3 | .2::k:25.7
+2.6%
replacement of the gel, are controlled by parameter input to the software system. The DNA samples are placed into the tray and analyzed sample by sample; the data of the analysis were displayed on the monitor and could be saved as a data file. Therefore this system opens new horizons in SSCP analysis and documentation. However, up to now only a few trials exist for the use of capillary electrophoresis in SSCP analysis. In the present studies, we adapted SSCP analysis to capillary electrophoresis for analyzing mutation in the LDL receptor gene. Mutations in the LDL receptor gene cause a marked elevation o f plasma cholesterol and the clinical phenotype of familial hypercholesterolemia. Worldwide, there are now more than 350 different mutations described in the gene. Most of the mutations are single nucleotide substitutions, small insertions and deletions. The high frequency of familial hypercholesterolemia in the population, approximately I in 500, the kind of mutations and their heterogeneous localization in the gene make genetic screening techniques for unknown mutations very helpful. To analyze the promoter and all 18 exons of the LDL receptor gene, 20 different amplification reactions were necessary. For each PCR, the forward and reverse primers were 5' fluorescent-labelled with FAM and HEX, respectively. To test the sensitivity of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. With identical electrophoresis conditions (13 KV, 30°C, 30 min) 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities which could be amplified by increasing the electrophoresis temperature. The high sensitivity, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis well-suited for clinical applications. EFFECTS OF QUINAPRIL IN PATIENTS WITH METABOLIC SYNDROME B. Zonis, N. Volkova, V. Martirosov. Regional cardiodispensao;
Rostov-on-Don, Russia Angiotensine converting enzyme inhibitors are most suitable in metabolic syndrome treatment. Quinapril in this raw may appear effective due to its primary effects on tissue reunin-angiotensine system. In our research effects of quinapril therapy (an average daily dose of 10-20 rag) on blood pressure and indices of lipoproteidegram in 19 patients (10 women and 9 men) with metabolic syndrome have been investigated. Obesity was diagnosed in all the patients (the body weight index - 31.5+0.98 kg/m 2) primarily it was of abdominal type (women's waist/hip ratio - 0.89+0.022, men's - 1.0 t ±0.019)~ This allowed to suggest the presence of insulin resistance in the patients examined. All the patients were observed to have a significant increase (P ~ 0.001) of LDL and triglycerides concentrations with a decreased HDL content. The arterial hypertension was characterized as mild and moderate. By the end of the therapy first month a considerable decrease o f the systolic and diastolic blood pressure was recorded, and after a three-month continuous treatment its normalization was recorded. The apparent hypotensive effect was accompanied by a considerable decrease of concentration of LDL (P < 0.001) and triglycerides (P < 0.02) in blood plasma by the therapy third month, the lipoproteide HDL content being somewhat higher (P < 0.02). The concentration of glucose, uric acid, sodium and potassium in blood plasma was not significantly changed. Thus, the findings show a high efficacy of quinapril in patients with metabolic syndrome. At the same time, not only the apparent antihypertensive effect of the drug was observed, hut also the definite positive effect on the lipid metabolism.
Conclusions: This study suggests that atorvastatin is a very effective therapy in the management of primary hypercholesterolemia, with rapid onset of action, since 85% of total cholesterol reduction and 80% of LDL cholesterol reduction from baseline occurred within the first 2 weeks of treatment.
SAME EXTENT OF CORONARY CALCIFICATION IN HETEROZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA CAUSED BY A RECEPTOR DEFECTIVE AND A RECEPTOR NEGATIVE MUTATION IN DANISH PATIENTS
FLUORESCENCE-BASED SSCP ANALYSIS OF THE LDL RECEPTOR GENE BY CAPILLARY ELECTROPHORESIS
J.U. Brorholt-Petersen, T. Christiansen t , H.K. Jansen, J.M. Jensen, B. Raungaard, N. Gregersen2, O. Faergeman. Department of Internal
J. Geisel, T. Walz, M. Bodis, W. Herrmann. Department of Clinical
Chemistry. University of the Saarland, D-66421 Homburg/Saar, Germany Analysis of single strand conformation polymorphisms (SSCP) is widely used to screen for unknown mutations because of its simplicity and sensitivity. However, for a routine genetic test, SSCP analysis in non-denaturing gels is too complicated and time consuming, especially for analysis of small DNA samples numbers. The combination o f SSCP analysis with capillary electrophoresis offers, for the fast time, a high degree of automation. In the ABI 310 Genetic Analyzer all electrophoresis procedures, including the
Medicine and Cardiology, Aarhus Amtssygehus University Hospital, Aarhus; IDepartment of Radiology, Aarhus dmtssygehus University Hospital, Aarhus; 2Research Unitfor Molecular Medicine. Skejby University Hospital, Aarhus, Denmark Patients with homozygous familial hypercholesterolemia caused by receptor negative mutations have higher concentrations of LDL-cholesterol in plasma than patients homozygous for receptor defective mutations. Receptor defective mutations in homozygous form are often considered to he milder than receptor negative mutations in terms of clinical course o f atherosclerotic disease. In contrast, it is uncertain whether the degree
71st EAS Congress and Satellite Symposia
186
and of those 182 (78.5%) presented abnormal hormone profile. There was a strong association between UA with sedentary lifestyle (57%); AMI with sedentary lifestyle and arterial hypertension (73%); Non Q wave Ml with sedentary lifestyle and dyslipidaemia (85%). Conclusion: Our study showed a strong association of ischemic heart disease in women with sedentary lifestyle, arterial hypertension, dyslipidaemia and a family history of CAD. What was striking was the low percentage of patients under HRT which might reinforce the importance of estrogen therapy to prevent cardiovascular disease in high risk women population. ATORVASTATIN (LIPITOR) IN THE TREATMENT OF PATIENTS WITH HYPERCHOLESTEROLEMIA S. Delaroudis A. Slavakis, M. Tzoiti, A. Kyroudi, C. Balaris, J. Sarris, M. Giannoulis, M. Kita, D. Sofianou, H. Hatzikonstantinou, A. Avramides.
Lipid Clinic of Endocrinology Dept, Microbiology Lab, Biochemistr3, Lab. Hippocration Hospital, Thessaloniki. Greece Introduction: Increased plasma cholesterol, lipoprotein and apolipoprotein levels play a significant role in the development and progression of atherosclerotic disease. Many trials have shown that reducing plasma cholesterol levels (especially LDL) can lead to reduction in coronary artery disease. HMG-CoA reductase inhibitors (statins) have been shown to be effective in lowering LDL levels in patients with hypercholesterolemia. Several studies have indicated that atorvastatin, a recently synthesised statin, is safe and effective in hypercholesterolemic patients. Objective: To evaluate lipid parameter responses to 10 mg atorvastatin given daily to patients with primary hypercholesterolemia. Methods and Patients: Seventeen patients (3 men, 14 women) with elevated total and LDL cholesterol, aged 60.9=t=7.4 yr. entered our 16 week study. Participants were recruited at the Outpatient Lipid Clinic o f Endocrinology Dept_ Patients with secondary hypercholesterolemia were excluded. Other lipid lowering medications were discontinued for at least 2 months before study. The patients, who were counselled to follow hypolipidemic diet > 3 months before the baseline phase. Subsequently they were placed on a 10 mg atorvatatin tablet per day. Each patient's plasma lipid profile [total, HDL, LDL cholesterol, triglycerides, ApoAl, ApoB and Lp(a)] was determined at weeks 0, 2, 8 & 16. Results: Atorvastatin, at week 16, significantly lowered total and LDLcholesterol levels from baseline (by -29.3% and -38.5% respectively, p < 0.001) and also caused reduction in TGL (-18.9%, NS), ApoB (-17.9% NS), ApoAl (-1.7% NS) and mild increase in HDL cholesterol (+5.2% NS) and Lp(a) (+2.6% NS) (see table, "p < 0.001). (mean +sd)
0 week
2 week
16 week
(96).4 (0-16
week) CHOL
328+55.7
246.2+53.11.)
231.9+20.7(°1
-29.3% (°1
TG L
176.4 + 86.6
1362+:53.1
150.8:k55.5
- 18.9'/,
HDL
49.7+: 10.8
51.3+: 10.7
52.3=1=11.9
+5.2%
LDL
243.1 :i:57.3
167.6+55.2(°~
149.4+16.9I ' )
-38.5% I*l
ApoAI
156.4+22.5
153.8-t- 19.7
- t.7%
ApoB
153. I +48.2
125.7+:37.5
- 17.9°/*
Lp(a)
30.4+:24.7
3 | .2::k:25.7
+2.6%
replacement of the gel, are controlled by parameter input to the software system. The DNA samples are placed into the tray and analyzed sample by sample; the data of the analysis were displayed on the monitor and could be saved as a data file. Therefore this system opens new horizons in SSCP analysis and documentation. However, up to now only a few trials exist for the use of capillary electrophoresis in SSCP analysis. In the present studies, we adapted SSCP analysis to capillary electrophoresis for analyzing mutation in the LDL receptor gene. Mutations in the LDL receptor gene cause a marked elevation o f plasma cholesterol and the clinical phenotype of familial hypercholesterolemia. Worldwide, there are now more than 350 different mutations described in the gene. Most of the mutations are single nucleotide substitutions, small insertions and deletions. The high frequency of familial hypercholesterolemia in the population, approximately I in 500, the kind of mutations and their heterogeneous localization in the gene make genetic screening techniques for unknown mutations very helpful. To analyze the promoter and all 18 exons of the LDL receptor gene, 20 different amplification reactions were necessary. For each PCR, the forward and reverse primers were 5' fluorescent-labelled with FAM and HEX, respectively. To test the sensitivity of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. With identical electrophoresis conditions (13 KV, 30°C, 30 min) 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities which could be amplified by increasing the electrophoresis temperature. The high sensitivity, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis well-suited for clinical applications. EFFECTS OF QUINAPRIL IN PATIENTS WITH METABOLIC SYNDROME B. Zonis, N. Volkova, V. Martirosov. Regional cardiodispensao;
Rostov-on-Don, Russia Angiotensine converting enzyme inhibitors are most suitable in metabolic syndrome treatment. Quinapril in this raw may appear effective due to its primary effects on tissue reunin-angiotensine system. In our research effects of quinapril therapy (an average daily dose of 10-20 rag) on blood pressure and indices of lipoproteidegram in 19 patients (10 women and 9 men) with metabolic syndrome have been investigated. Obesity was diagnosed in all the patients (the body weight index - 31.5+0.98 kg/m 2) primarily it was of abdominal type (women's waist/hip ratio - 0.89+0.022, men's - 1.0 t ±0.019)~ This allowed to suggest the presence of insulin resistance in the patients examined. All the patients were observed to have a significant increase (P ~ 0.001) of LDL and triglycerides concentrations with a decreased HDL content. The arterial hypertension was characterized as mild and moderate. By the end of the therapy first month a considerable decrease o f the systolic and diastolic blood pressure was recorded, and after a three-month continuous treatment its normalization was recorded. The apparent hypotensive effect was accompanied by a considerable decrease of concentration of LDL (P < 0.001) and triglycerides (P < 0.02) in blood plasma by the therapy third month, the lipoproteide HDL content being somewhat higher (P < 0.02). The concentration of glucose, uric acid, sodium and potassium in blood plasma was not significantly changed. Thus, the findings show a high efficacy of quinapril in patients with metabolic syndrome. At the same time, not only the apparent antihypertensive effect of the drug was observed, hut also the definite positive effect on the lipid metabolism.
Conclusions: This study suggests that atorvastatin is a very effective therapy in the management of primary hypercholesterolemia, with rapid onset of action, since 85% of total cholesterol reduction and 80% of LDL cholesterol reduction from baseline occurred within the first 2 weeks of treatment.
SAME EXTENT OF CORONARY CALCIFICATION IN HETEROZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA CAUSED BY A RECEPTOR DEFECTIVE AND A RECEPTOR NEGATIVE MUTATION IN DANISH PATIENTS
FLUORESCENCE-BASED SSCP ANALYSIS OF THE LDL RECEPTOR GENE BY CAPILLARY ELECTROPHORESIS
J.U. Brorholt-Petersen, T. Christiansen t , H.K. Jansen, J.M. Jensen, B. Raungaard, N. Gregersen2, O. Faergeman. Department of Internal
J. Geisel, T. Walz, M. Bodis, W. Herrmann. Department of Clinical
Chemistry. University of the Saarland, D-66421 Homburg/Saar, Germany Analysis of single strand conformation polymorphisms (SSCP) is widely used to screen for unknown mutations because of its simplicity and sensitivity. However, for a routine genetic test, SSCP analysis in non-denaturing gels is too complicated and time consuming, especially for analysis of small DNA samples numbers. The combination o f SSCP analysis with capillary electrophoresis offers, for the fast time, a high degree of automation. In the ABI 310 Genetic Analyzer all electrophoresis procedures, including the
Medicine and Cardiology, Aarhus Amtssygehus University Hospital, Aarhus; IDepartment of Radiology, Aarhus dmtssygehus University Hospital, Aarhus; 2Research Unitfor Molecular Medicine. Skejby University Hospital, Aarhus, Denmark Patients with homozygous familial hypercholesterolemia caused by receptor negative mutations have higher concentrations of LDL-cholesterol in plasma than patients homozygous for receptor defective mutations. Receptor defective mutations in homozygous form are often considered to he milder than receptor negative mutations in terms of clinical course o f atherosclerotic disease. In contrast, it is uncertain whether the degree
71st EAS Congress and Satellite Symposia