- Email: [email protected]
[32] IDENTIFICATION OF DELETIONS IN LDLR GENE BY MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION ANALYSIS
S8
Nutrition, Metabolism & Cardiovascular Diseases (2009) S1–S32
29 THE CHOLESTEROL ESTERIFICATION PROCESS IS IMPAIRED IN PATIENTS WITH CHRONIC KIDNEY DISEASE P. Conca, S. Pileggi, S. Penco, G. Busnach, G. Franceschini, L. Calabresi. Center Grossi Paoletti, Dept. Pharmacological Sciences, University of Milano, Italy E-mail: [email protected] Background: Chronic kidney disease (CKD) is associated with profound alterations of plasma lipid/lipoproteins profile, characterized by a reduction in high-density lipoprotein (HDL) cholesterol levels and an increase in plasma triglyceride levels. Objective of this study was to investigate the cholesterol esterification process in patients with CKD. Methods: Plasma lipid profile, the cholesterol esterification process, and HDL subpopulations were evaluated in 143 patients with end-stage renal disease (ESRD) and 40 patients with CKD stages IV to V, in comparison with a group of control subjects. Results: Patients with ESRD exhibited a significant reduction of plasma HDL, mainly due to a reduction in LpA-I:A-II particles. The unesterified/total cholesterol ratio was significantly increased, while cholesterol esterification rate, LCAT activity, and plasma LCAT concentration were reduced. The percentage of pre-beta HDL was greater in patients with ESRD compared to controls, while HDL subpopulation distribution was unchanged. No mutations in the LCAT gene were detected in any of the analyzed subjects. Patients affected by CKD stages IV to V showed an intermediate phenotype. Conclusions: Chronic kidney disease is associated with a compromised cholesterol esterification process, likely due to a reduced LCAT concentration in plasma; this alteration, more evident in patients with ESRD, appears to worsen with the progression of the disease. 30 METABOLIC SYNDROME: CLINICAL GOVERNANCE IN THE GENERAL PRACTITIONER M. Coronelli, M. Framarin, I. Coronelli. Asl 312 Pavia, Italy E-mail: [email protected] Introduction: For general practitioners, the Clinical Governance, offers the opportunity to better themselves through activities aimed to assess the effectiveness and appropriateness of the services provided. The Clinical governance can find application in medicine of territory in respect of any chronic and the general practitioner has all the characteristics to play a major role in the diagnosis and treatment of metabolic syndrome. The first step to do is the assessment of individual risk in the medium and long term. Metabolic syndrome is combatted,first,by empowering the patient and encouraged to change their lifestyle. The removal of individual risk factors (inactivity physical, obesity and high fat diet)was addressed with the help of a psychological service business in our medical group. Materials and methods: The date were collected on all accessess to our clinic, interval of time between 01.01.2008 31.07.2008. All patient,except those who present themselves for reasons not related morbidity, were included. The criteria of AHA were applied to identify patients with metabolic syndrome. In the period of time included in the study were presented at the clinic 976 patients equal to 59.48% of the total assisted in this clinic. The selection has resulted in identifying 755 patients equal to 77.36% of the total (55.69 female and 43.31 male). Applying to these 755 persons the criteria of AHA were selected who could be labeled as suffering from metabolic syndrome. It follows a cohort of 123 patients (16.26%) divided into 77 female (62.60%) and 46 male (37.40) all with metabolic syndrome. Were than taken into account all the possible associations of three citeria and it was evaluated its frequency in campions. Results: Analyzing the population shows that most are subject over 65 years old with a small minority under 40 years old. From analysis of values of BMI of the patients selected it appears that almost all of overweight patients (86.99%) and almost 70% is obese; only a small percentage is severely obese. For the purpose of diagnosis is yet more interesting to consider waist circumference.Again there is marked tendency to obesity visceral. Were also assessed blood pressure, the triglicerides, the HDL cholesterol and blood sugar and has been investigated smoking habits, because predictor of cardiovascular risk. It was also evaluated the pharmacological management of the patient. Conclusions: This study shows that it’s possible, in a general practice clinic,apply the most recent diagnostic criteria for metabolic syndrome thus identifying pearl strictly and universally accepted nearly all of the subjects with metabolic syndrome. It was shown that psycological counselling is useful in improving the lifestyle of those patients with poor motivations. 31 FAMILIAL COMBINED HYPERLIPIDEMIA AND CAROTID IMT G. Covetti2 , A. Iannuzzi2 , M. Gentile1 , P. Pauciullo1 , G. Iannuzzo1 , G. Marotta1 , A. Bresciani1 , S. Panico1 , P. Rubba1 . 1 Dip Med Clin e Sperim, Univ. Federico II, 2 AORN Cardarelli, Napoli, Italy E-mail: [email protected] Background: Familial combined hyperlipidemia (FCHL) is the most common genetic lipid disorder. In patients or families ascertained for premature coronary artery disease, FCHL is identified as the most common familial dyslipidemia. Recently we demonstrated that vascular reactivity was impaired in the arteries of patients with FCHL. Measurement of carotid intima-media thickness (IMT) is an independent marker of both prevalent and incident cardio-vascular disease (CVD). IMT has been investigated in small cohorts of
hyperlipidemic FCHL subjects by comparison with healthy controls and was not always demonstrated to be more enlarged in FCHL than in control populations. Aim of our study was to perform carotid sonography for evaluation of IMT in a sample of FCHL patients and to compare the findings with those of healthy subjects. Materials and Methods: We studied 54 (34 M, 20 F) hyperlipidemic adults with a clinical diagnosis of FCHL consecutively evaluated at our Lipid Clinic. During the same period, we also evaluated 144 healthy control subjects (72 M, 72 F). All subjects were assessed for standard cardiovascular risk factors. B-mode ultrasound imaging of the right and left carotid arteries was performed in hyperlipidemic and control subjects using a standardized imaging protocol for the IMT measurements. Results: As expected from study design, FCHL patients showed a higher number and potency of cardiovascular risk factors than healthy controls (Cholesterol 266±10.0 vs 200±3.1 mg/dl, p < 0.001; Triglycerides 245±32 vs 97±4.0 mg/dL, p < 0.001; HDL-C 41±2.0 vs 49±1.1 mg/dL, p < 0.001; apoB 138±4.9 vs 97±1.8 mg/dL, p < 0.001; SBP 137±3.0 vs 118±1.3 mmHg, p < 0.001); with the exception of BMI (26.7±0.51 vs 25.4±0.32 kg/m2 , p = 0.07). In a univariate analysis carotid IMT was significantly higher in FCHL patients (0.65±0.01 vs 0.59±0.01 mm, p < 0.001) compared with healthy controls. FCHL patients were older (50±1.5 vs 45±1.0 years, p < 0.01) and in a multivariate analysis, after adjustment for age and gender, there was still a statistically significant difference between the two groups (p < 0.05). Conclusions: The present study shows that hyperlipidemic FCHL patients have increased carotid IMT compared with healthy controls, after adjustment for age and gender. The increased carotid IMT observed in FCHL patients is in line with their enhanced risk of CVD 32 IDENTIFICATION OF DELETIONS IN LDLR GENE BY MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION ANALYSIS M.N. D’Agostino, M. Romano, M.D. Di Taranto, G. Marotta, M. Gentile, P. Rubba, G. Fortunato. CEINGE S.C.a r.l. Biotecnologie Avanzate, Napoli, Italia e Dip. di Biochimica e Biotecnologie Mediche, Italy E-mail: [email protected] Introduction: Autosomal dominant hypercholesterolemias are due to defects in the LDL receptor (LDLR) gene, in its ligand the apolipoprotein B-100 (APOB) gene or to gain-of-function mutations in the proprotein convertase subtilisin kexin type 9 gene (PCSK9) (1). LDLR mutations account for most cases of familial hypercholesterolemia (FH). Materials and Methods: We enrolled 56 unrelated patients from Southern Italy with a clinical diagnosis of FH. The mutation screening was performed by direct sequencing of the LDLR gene (promoter and the 18 exons). In the patients without detectable mutations at sequence analysis, we carried out an multiplex ligation-dependent probe amplification (MLPA) analysis to search for large rearrangements. Copy number quantification of the 18 exons of the LDLR gene was performed by the SALSA MLPA P062B kit (MRC-Holland) according to the supplier’s instructions. To obtain gene dosage quotients, the results were compared with three control samples analysed at the same time using the Coffalyser data analysis tool (MRC-Holland) and confirmed with Long-PCR. Results and Conclusions: Direct sequencing of the promoter and encoding regions of the LDLR gene revealed mutations in 40/56 patients (mutation rate 71.4%). In the 16 patients without detectable mutations at sequence analysis, we carried out an MLPA analysis to search for large rearrangements and found gross deletions in 3/16 subjects. Using these two methods, we identified mutations in 43/56 subjects (mutation rate, 76.8%). This result supports the concept that genetic screening in FH disease should include the detection of large rearrangements. 33 PPARg2 PRO12ALA POLYMORPHISM AFFECTS ADIPOSE TISSUE DISTRIBUTION AND INTERACTS WITH ACE I/D POLIMORPHISM ON THE MODULATION OF BODY WEIGHT E. Dalla Nora, C. Marcello, F. Di Vece, J.M. Sanz, C. Bosi, C. Bolognesi, R. Fellin, A. Passaro. Dept. Clinical and Exper. Medicine, Sect. Int. Med., Geront. and Geriatrics, University of Ferrara, Italy E-mail: [email protected] Introduction: Metabolic Syndrome is believed to be attributable to the combined effect of genetic and environmental factors. Adipose tissue related genes, PPARg and ACE are good candidates. Objective: Investigate the possible association of PPARg2Pro12Ala and ACE I/D polymorphisms with Metabolic Syndrome components and whether there is an interaction between these two genetic variants. Materials and methods: 364 patients, consecutively referred to our outpatients clinic, were enrolled. Waist circumference, blood pressure and BMI were recorded and body composition estimated by impedance analysis. Metabolic Syndrome was diagnosed according to NCEP-ATPIII criteria. A blood sample was obtained for glucose, insulin, lipid profile determination and DNA isolation for Pro12Ala PPARg 2 and ACE I/D genotyping. Results: Carriers of a PPARg2 Ala allele (XA) show a significantly higher BMI and fat mass but a significantly lower systolic blood pressure. No difference in waist circumference and other metabolic features were detected. Pro12Ala is an independent predictor of waist circumference. Subjects carrying different ACE polymorphism did not differ in terms of BMI, waist, blood pressure, glucose and insulin levels and lipid profile. Metabolic syndrome prevalence
Nutrition, Metabolism & Cardiovascular Diseases (2009) S1–S32
29 THE CHOLESTEROL ESTERIFICATION PROCESS IS IMPAIRED IN PATIENTS WITH CHRONIC KIDNEY DISEASE P. Conca, S. Pileggi, S. Penco, G. Busnach, G. Franceschini, L. Calabresi. Center Grossi Paoletti, Dept. Pharmacological Sciences, University of Milano, Italy E-mail: [email protected] Background: Chronic kidney disease (CKD) is associated with profound alterations of plasma lipid/lipoproteins profile, characterized by a reduction in high-density lipoprotein (HDL) cholesterol levels and an increase in plasma triglyceride levels. Objective of this study was to investigate the cholesterol esterification process in patients with CKD. Methods: Plasma lipid profile, the cholesterol esterification process, and HDL subpopulations were evaluated in 143 patients with end-stage renal disease (ESRD) and 40 patients with CKD stages IV to V, in comparison with a group of control subjects. Results: Patients with ESRD exhibited a significant reduction of plasma HDL, mainly due to a reduction in LpA-I:A-II particles. The unesterified/total cholesterol ratio was significantly increased, while cholesterol esterification rate, LCAT activity, and plasma LCAT concentration were reduced. The percentage of pre-beta HDL was greater in patients with ESRD compared to controls, while HDL subpopulation distribution was unchanged. No mutations in the LCAT gene were detected in any of the analyzed subjects. Patients affected by CKD stages IV to V showed an intermediate phenotype. Conclusions: Chronic kidney disease is associated with a compromised cholesterol esterification process, likely due to a reduced LCAT concentration in plasma; this alteration, more evident in patients with ESRD, appears to worsen with the progression of the disease. 30 METABOLIC SYNDROME: CLINICAL GOVERNANCE IN THE GENERAL PRACTITIONER M. Coronelli, M. Framarin, I. Coronelli. Asl 312 Pavia, Italy E-mail: [email protected] Introduction: For general practitioners, the Clinical Governance, offers the opportunity to better themselves through activities aimed to assess the effectiveness and appropriateness of the services provided. The Clinical governance can find application in medicine of territory in respect of any chronic and the general practitioner has all the characteristics to play a major role in the diagnosis and treatment of metabolic syndrome. The first step to do is the assessment of individual risk in the medium and long term. Metabolic syndrome is combatted,first,by empowering the patient and encouraged to change their lifestyle. The removal of individual risk factors (inactivity physical, obesity and high fat diet)was addressed with the help of a psychological service business in our medical group. Materials and methods: The date were collected on all accessess to our clinic, interval of time between 01.01.2008 31.07.2008. All patient,except those who present themselves for reasons not related morbidity, were included. The criteria of AHA were applied to identify patients with metabolic syndrome. In the period of time included in the study were presented at the clinic 976 patients equal to 59.48% of the total assisted in this clinic. The selection has resulted in identifying 755 patients equal to 77.36% of the total (55.69 female and 43.31 male). Applying to these 755 persons the criteria of AHA were selected who could be labeled as suffering from metabolic syndrome. It follows a cohort of 123 patients (16.26%) divided into 77 female (62.60%) and 46 male (37.40) all with metabolic syndrome. Were than taken into account all the possible associations of three citeria and it was evaluated its frequency in campions. Results: Analyzing the population shows that most are subject over 65 years old with a small minority under 40 years old. From analysis of values of BMI of the patients selected it appears that almost all of overweight patients (86.99%) and almost 70% is obese; only a small percentage is severely obese. For the purpose of diagnosis is yet more interesting to consider waist circumference.Again there is marked tendency to obesity visceral. Were also assessed blood pressure, the triglicerides, the HDL cholesterol and blood sugar and has been investigated smoking habits, because predictor of cardiovascular risk. It was also evaluated the pharmacological management of the patient. Conclusions: This study shows that it’s possible, in a general practice clinic,apply the most recent diagnostic criteria for metabolic syndrome thus identifying pearl strictly and universally accepted nearly all of the subjects with metabolic syndrome. It was shown that psycological counselling is useful in improving the lifestyle of those patients with poor motivations. 31 FAMILIAL COMBINED HYPERLIPIDEMIA AND CAROTID IMT G. Covetti2 , A. Iannuzzi2 , M. Gentile1 , P. Pauciullo1 , G. Iannuzzo1 , G. Marotta1 , A. Bresciani1 , S. Panico1 , P. Rubba1 . 1 Dip Med Clin e Sperim, Univ. Federico II, 2 AORN Cardarelli, Napoli, Italy E-mail: [email protected] Background: Familial combined hyperlipidemia (FCHL) is the most common genetic lipid disorder. In patients or families ascertained for premature coronary artery disease, FCHL is identified as the most common familial dyslipidemia. Recently we demonstrated that vascular reactivity was impaired in the arteries of patients with FCHL. Measurement of carotid intima-media thickness (IMT) is an independent marker of both prevalent and incident cardio-vascular disease (CVD). IMT has been investigated in small cohorts of
hyperlipidemic FCHL subjects by comparison with healthy controls and was not always demonstrated to be more enlarged in FCHL than in control populations. Aim of our study was to perform carotid sonography for evaluation of IMT in a sample of FCHL patients and to compare the findings with those of healthy subjects. Materials and Methods: We studied 54 (34 M, 20 F) hyperlipidemic adults with a clinical diagnosis of FCHL consecutively evaluated at our Lipid Clinic. During the same period, we also evaluated 144 healthy control subjects (72 M, 72 F). All subjects were assessed for standard cardiovascular risk factors. B-mode ultrasound imaging of the right and left carotid arteries was performed in hyperlipidemic and control subjects using a standardized imaging protocol for the IMT measurements. Results: As expected from study design, FCHL patients showed a higher number and potency of cardiovascular risk factors than healthy controls (Cholesterol 266±10.0 vs 200±3.1 mg/dl, p < 0.001; Triglycerides 245±32 vs 97±4.0 mg/dL, p < 0.001; HDL-C 41±2.0 vs 49±1.1 mg/dL, p < 0.001; apoB 138±4.9 vs 97±1.8 mg/dL, p < 0.001; SBP 137±3.0 vs 118±1.3 mmHg, p < 0.001); with the exception of BMI (26.7±0.51 vs 25.4±0.32 kg/m2 , p = 0.07). In a univariate analysis carotid IMT was significantly higher in FCHL patients (0.65±0.01 vs 0.59±0.01 mm, p < 0.001) compared with healthy controls. FCHL patients were older (50±1.5 vs 45±1.0 years, p < 0.01) and in a multivariate analysis, after adjustment for age and gender, there was still a statistically significant difference between the two groups (p < 0.05). Conclusions: The present study shows that hyperlipidemic FCHL patients have increased carotid IMT compared with healthy controls, after adjustment for age and gender. The increased carotid IMT observed in FCHL patients is in line with their enhanced risk of CVD 32 IDENTIFICATION OF DELETIONS IN LDLR GENE BY MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION ANALYSIS M.N. D’Agostino, M. Romano, M.D. Di Taranto, G. Marotta, M. Gentile, P. Rubba, G. Fortunato. CEINGE S.C.a r.l. Biotecnologie Avanzate, Napoli, Italia e Dip. di Biochimica e Biotecnologie Mediche, Italy E-mail: [email protected] Introduction: Autosomal dominant hypercholesterolemias are due to defects in the LDL receptor (LDLR) gene, in its ligand the apolipoprotein B-100 (APOB) gene or to gain-of-function mutations in the proprotein convertase subtilisin kexin type 9 gene (PCSK9) (1). LDLR mutations account for most cases of familial hypercholesterolemia (FH). Materials and Methods: We enrolled 56 unrelated patients from Southern Italy with a clinical diagnosis of FH. The mutation screening was performed by direct sequencing of the LDLR gene (promoter and the 18 exons). In the patients without detectable mutations at sequence analysis, we carried out an multiplex ligation-dependent probe amplification (MLPA) analysis to search for large rearrangements. Copy number quantification of the 18 exons of the LDLR gene was performed by the SALSA MLPA P062B kit (MRC-Holland) according to the supplier’s instructions. To obtain gene dosage quotients, the results were compared with three control samples analysed at the same time using the Coffalyser data analysis tool (MRC-Holland) and confirmed with Long-PCR. Results and Conclusions: Direct sequencing of the promoter and encoding regions of the LDLR gene revealed mutations in 40/56 patients (mutation rate 71.4%). In the 16 patients without detectable mutations at sequence analysis, we carried out an MLPA analysis to search for large rearrangements and found gross deletions in 3/16 subjects. Using these two methods, we identified mutations in 43/56 subjects (mutation rate, 76.8%). This result supports the concept that genetic screening in FH disease should include the detection of large rearrangements. 33 PPARg2 PRO12ALA POLYMORPHISM AFFECTS ADIPOSE TISSUE DISTRIBUTION AND INTERACTS WITH ACE I/D POLIMORPHISM ON THE MODULATION OF BODY WEIGHT E. Dalla Nora, C. Marcello, F. Di Vece, J.M. Sanz, C. Bosi, C. Bolognesi, R. Fellin, A. Passaro. Dept. Clinical and Exper. Medicine, Sect. Int. Med., Geront. and Geriatrics, University of Ferrara, Italy E-mail: [email protected] Introduction: Metabolic Syndrome is believed to be attributable to the combined effect of genetic and environmental factors. Adipose tissue related genes, PPARg and ACE are good candidates. Objective: Investigate the possible association of PPARg2Pro12Ala and ACE I/D polymorphisms with Metabolic Syndrome components and whether there is an interaction between these two genetic variants. Materials and methods: 364 patients, consecutively referred to our outpatients clinic, were enrolled. Waist circumference, blood pressure and BMI were recorded and body composition estimated by impedance analysis. Metabolic Syndrome was diagnosed according to NCEP-ATPIII criteria. A blood sample was obtained for glucose, insulin, lipid profile determination and DNA isolation for Pro12Ala PPARg 2 and ACE I/D genotyping. Results: Carriers of a PPARg2 Ala allele (XA) show a significantly higher BMI and fat mass but a significantly lower systolic blood pressure. No difference in waist circumference and other metabolic features were detected. Pro12Ala is an independent predictor of waist circumference. Subjects carrying different ACE polymorphism did not differ in terms of BMI, waist, blood pressure, glucose and insulin levels and lipid profile. Metabolic syndrome prevalence