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MUTATION SCREENING OF AUTOSOMAL DOMINANT HYPERCHOLESTEROLEMIA IN GREECE
154
Poster Sessions PO33 Advances in clinical care and diagnostics
study. This suggests vascular disease can be studied at a population level long before conventional endpoints materialize. REFINE Reykjavik Study can therefore be used to potentially identify new risk factors for cardiovascular disease. PO33-558
MUTATION SCREENING OF AUTOSOMAL DOMINANT HYPERCHOLESTEROLEMIA IN GREECE
E. Laios, E. Koniari, E. Tsoutsou, N. Skouma, E. Drogari. 1st Department of Pediatrics, Unit of Metabolic Disease, Ag. Sophia Children’s Hospital, University of Athens, Athens, Greece Background and aims: The diagnosis of autosomal dominant hypercholesterolemia (ADH) in Greece has primarily been based on clinical characteristics. In fact, only a limited number of Greek patients have been screened for LDLR mutations. Over the past three years, in an attempt to enhance our limited knowledge of the molecular basis of ADH in Greece, we have screened over 140 index cases presenting with ADH. Methods: Genomic DNA was PCR-amplified followed by mutation detection based on restriction-fragment length polymorphism, NanoChip analysis or sequencing. Results: We have genotyped 70 ADH index cases. 69 were found to have a single point mutation in the LDLR gene (20 cases of the G1285A mutation, 17 of C858A, 24 of G1646A, 9 of T517C). One patient carried the R3500Q mutation of the ApoB100 gene. An additional 105 relatives of the index cases have also been diagnosed with the corresponding LDLR mutation. A mutation has yet to be identified in an additional 72 index cases which have been screened for 3 to 10 LDLR mutations and 1 to 3 ApoB100 mutations. Finally, 6 patients in which an LDLR mutation was not indentified upon sequencing, were sequenced across the entire PCSK9 coding region and the exon-intron boundaries; a PCSK9 mutation was not found. Conclusion: FH genetic screening and diagnosis is not presently in effect in Greece. To our knowledge, our data represents the most systematic effort to categorize ADH mutations in Greece in order to establish a targeted molecular screening strategy suitable for Greek ADH patients. PO33-559
THE NONLINEAR OPTICAL RESPONSE OF HUMAN NATIVE AND OXIDIZED LDL: A NEW METHOD TO QUANTIFY THEIR AMOUNT IN THE PLASMA
S.L. Gomez 3 , R.F. Turchiello 2 , M.C. Jurado 4 , P. Boschcov 5 , M. Gidlund 4 , A.M. Figueiredo Neto 1 . 1 Instituto de Fisica, Universidade de Sao Paulo, Brazil; 2 Instituto de Quimica Universidade de Sao Paulo, Brazil; 3 Departamento de Fisica, Universidade Estadual de Ponta Grossa, Parana, Brazil; 4 Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Brazil; 5 Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Brazil Oxidative modifications of low-density lipoproteins are implicated in the development of atherosclerosis, and the choice of a method for monitoring the changes in LDL is a fundamental issue. The detection of such modifications, especially in early stages, has drawbacks, like the low specificity of the TBARS assay. The nonlinear optical responses of human native (LDL) and oxidized (oxLDL) by Cu2+ low-density lipoprotein particles, were investigated by the Z-scan technique (ZS), in the millisecond time-scale regime, as a function of the concentration of particles. ZS is commonly used by condensed matter physicists who measure the nonlinear index of refraction of materials. LDL particles are separated from the human plasma by ultracentrifugation and are dispersed in water at known concentrations. The sample is placed between glass plates, in slab geometry. After that, sample is illuminated by a laser beam and the transmittance is measured trough an iris placed in the front of a detector. The ZS signals increase linearly with concentration of LDL in a broad range of concentrations (from 0.25 to 1.75 mg/mL). The oxLDL do not show any measurable nonlinear optical response at the same experimental. We show that the main contribution to the nonlinear response of LDL comes from the phospholid fraction of the particles. Our method allows the quickly determination of the concentration of LDL and oxLDL in a sample, provided that the total amount of native and oxLDL is known from usual blood exams. Support: FAPESP, CNPq-IMFCx. [Chem. Phys. Lipid, 132 (2004) 185].
PO33-560
DOES THE OXIDATIVE STRESS AND CEREBRAL ATHEROSCLEROSIS INITIATE BRAIN HYPOPERFUSION AND THE DEVELOPMENT OF ALZHEIMER DISEASE?
G. Aliev 1,2 , J. Liu 3 , M. Puchowicz 4 , K. Xu 4 , S.L. Siedlak 5 , M.E. Obrenovich 5 , J.C. Shenk 1,2 , M.A. Smith 5 , E. Gasimov 6 , J.C. LaManna 4 , B.N. Ames 3 , G. Perry 1,5 . 1 Department of Biology of The College of Sciences, University of Texas At San Antonio, San Antonio, TX, USA; 2 Electron Microscopy Research Center, University of Texas At San Antonio, San Antonio, TX, USA; 3 Children’s Hospital Oakland Research Institute, Oakland, CA, USA; 4 Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland, OH, USA; 5 Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA; 6 Departments of Histology and Embryology, Baku Medical University, Baku, Azerbaijan Mechanisms involved in the pathogenesis of athero- and arteriosclerosis also appear to be relevant to Alzheimer’s disease (AD). We studied blood flow and cellular/subcellular features of vascular lesions and mitochondria in brain vascular wall cells of human AD brain biopsies, human short postmortem brain tissues, two transgenic mouse lines overexpressing amyloid beta precursor protein (AßPP) and ApoE4 mice as a model that mimics AD. We also studied the same features in aged rats and ApoE4 mice given selective mitochondrial antioxidants (Lipoic Acid +ALCAIR). In situ hybridization, using mitochondrial DNA (mtDNA) probes for human and mouse wild type and 5kb deleted mtDNA, was performed in conjunction with immunocytochemistry using antibodies against the oxidative stress and mitochondrial markers. Significantly more mitochondrial abnormalities were present in microvessels where lesions occurred. In situ hybridization revealed positive mtDNA signals in damaged mitochondria from the vascular endothelium and in perivascular cells of atherosclerotic lesioned vessels in human AD and the animal models of AD. These mtDNA deletions were associated with increased amounts of immunoreactive Aβ, 8OHG and COX in the same cellular and subcellular compartments. The animals that received treatment with antioxidants showed an improvement in cognitive performance and an absence of structural alterations. We hypothesize that oxidative stress induced by athero- and arteriosclerosis is responsible for cerebral blood flow reduction and most likely can lead to blood brain barrier failure and breakage during the development of AD that can be diminished by using selective mitochondrial antioxidants. Supported by: NIH, Alzheimer’s Association and Philip Morris USA. PO33-561
USE OF MICROARRAYS AND IMMUNOHISTOCHEMISTRY TO INVESTIGATE ACCELERATED ATHEROSCLEROSIS IN HUMAN GRAFT CORONARY ARTERY DISEASE
A. Rousoulieres 1,2 , S. Collot-Teixeira 3,4 , L. Chalabreysse 5 , K. Morser 3,4 , C. McDermott-Roe 3,4 , S. Yilmaz 3 , M. Leleu 4 , F. De Lorenzo 4,6 , A. Guzman 7 , J.B. Michel 7 , L. Sebbag 2 , P. Boissonnat 2 , F. Thivolet-Bejui 5 , J.L. McGregor 3,4,8 . 1 EA 1582, Faculte de Medecine RTH Laennec, Lyon, France; 2 Department of Heart Transplantation, Hopital Cardiologique Louis Pradel, Lyon, France; 3 Cardiovascular Division, Kings College London, UK; 4 Thrombosis Research Institute, London, UK; 5 Department of Pathology, Hopital Cardiologique Louis Pradel, France; 6 Beta cell Diabetes Centre, Chelsea and Westminster Hospital NHS Foundation Trust, London, UK; 7 INSERM U698, Paris, France; 8 INSERM U689, Hopital Lariboisiere, Paris, France Background and aims: Accelerated coronary atherosclerosis in cardiac transplant recipients is a major cause of morbidity and mortality. Graft coronary artery disease (CAD) or chronic rejection (CR) is the major limiting factor for long term survival after heart transplantation (HT). To investigate pathways implicated in accelerated atherosclerosis in chronic arterial graft rejection a rat aortic allograft model was used in tandem with microarrays and immunohistochemistry (IHC). Methods: Rat abdominal aortae were isografted (5) or allografted (5) from Brown-Norway to Lewis rats and grafts were harvested (Intima and media) after day 8, 25 or 60. Total isolated RNA was then used in whole genome 2-color Agilent microarrays. Rosetta Luminator and BRB software’s were used for candidate genes selection and validation was performed as indicated below. Results: Differentially expressed genes between iso and allo at day 8, 25 and 60 were respectively 1829, 2582 and 1925 (fold changes>2 or<-2 and Pvalues<0.05). Some of the investigated genes were altered at the time of
77th Congress of the European Atherosclerosis Society, April 26–29, 2008, Istanbul, Turkey
Poster Sessions PO33 Advances in clinical care and diagnostics
study. This suggests vascular disease can be studied at a population level long before conventional endpoints materialize. REFINE Reykjavik Study can therefore be used to potentially identify new risk factors for cardiovascular disease. PO33-558
MUTATION SCREENING OF AUTOSOMAL DOMINANT HYPERCHOLESTEROLEMIA IN GREECE
E. Laios, E. Koniari, E. Tsoutsou, N. Skouma, E. Drogari. 1st Department of Pediatrics, Unit of Metabolic Disease, Ag. Sophia Children’s Hospital, University of Athens, Athens, Greece Background and aims: The diagnosis of autosomal dominant hypercholesterolemia (ADH) in Greece has primarily been based on clinical characteristics. In fact, only a limited number of Greek patients have been screened for LDLR mutations. Over the past three years, in an attempt to enhance our limited knowledge of the molecular basis of ADH in Greece, we have screened over 140 index cases presenting with ADH. Methods: Genomic DNA was PCR-amplified followed by mutation detection based on restriction-fragment length polymorphism, NanoChip analysis or sequencing. Results: We have genotyped 70 ADH index cases. 69 were found to have a single point mutation in the LDLR gene (20 cases of the G1285A mutation, 17 of C858A, 24 of G1646A, 9 of T517C). One patient carried the R3500Q mutation of the ApoB100 gene. An additional 105 relatives of the index cases have also been diagnosed with the corresponding LDLR mutation. A mutation has yet to be identified in an additional 72 index cases which have been screened for 3 to 10 LDLR mutations and 1 to 3 ApoB100 mutations. Finally, 6 patients in which an LDLR mutation was not indentified upon sequencing, were sequenced across the entire PCSK9 coding region and the exon-intron boundaries; a PCSK9 mutation was not found. Conclusion: FH genetic screening and diagnosis is not presently in effect in Greece. To our knowledge, our data represents the most systematic effort to categorize ADH mutations in Greece in order to establish a targeted molecular screening strategy suitable for Greek ADH patients. PO33-559
THE NONLINEAR OPTICAL RESPONSE OF HUMAN NATIVE AND OXIDIZED LDL: A NEW METHOD TO QUANTIFY THEIR AMOUNT IN THE PLASMA
S.L. Gomez 3 , R.F. Turchiello 2 , M.C. Jurado 4 , P. Boschcov 5 , M. Gidlund 4 , A.M. Figueiredo Neto 1 . 1 Instituto de Fisica, Universidade de Sao Paulo, Brazil; 2 Instituto de Quimica Universidade de Sao Paulo, Brazil; 3 Departamento de Fisica, Universidade Estadual de Ponta Grossa, Parana, Brazil; 4 Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Brazil; 5 Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Brazil Oxidative modifications of low-density lipoproteins are implicated in the development of atherosclerosis, and the choice of a method for monitoring the changes in LDL is a fundamental issue. The detection of such modifications, especially in early stages, has drawbacks, like the low specificity of the TBARS assay. The nonlinear optical responses of human native (LDL) and oxidized (oxLDL) by Cu2+ low-density lipoprotein particles, were investigated by the Z-scan technique (ZS), in the millisecond time-scale regime, as a function of the concentration of particles. ZS is commonly used by condensed matter physicists who measure the nonlinear index of refraction of materials. LDL particles are separated from the human plasma by ultracentrifugation and are dispersed in water at known concentrations. The sample is placed between glass plates, in slab geometry. After that, sample is illuminated by a laser beam and the transmittance is measured trough an iris placed in the front of a detector. The ZS signals increase linearly with concentration of LDL in a broad range of concentrations (from 0.25 to 1.75 mg/mL). The oxLDL do not show any measurable nonlinear optical response at the same experimental. We show that the main contribution to the nonlinear response of LDL comes from the phospholid fraction of the particles. Our method allows the quickly determination of the concentration of LDL and oxLDL in a sample, provided that the total amount of native and oxLDL is known from usual blood exams. Support: FAPESP, CNPq-IMFCx. [Chem. Phys. Lipid, 132 (2004) 185].
PO33-560
DOES THE OXIDATIVE STRESS AND CEREBRAL ATHEROSCLEROSIS INITIATE BRAIN HYPOPERFUSION AND THE DEVELOPMENT OF ALZHEIMER DISEASE?
G. Aliev 1,2 , J. Liu 3 , M. Puchowicz 4 , K. Xu 4 , S.L. Siedlak 5 , M.E. Obrenovich 5 , J.C. Shenk 1,2 , M.A. Smith 5 , E. Gasimov 6 , J.C. LaManna 4 , B.N. Ames 3 , G. Perry 1,5 . 1 Department of Biology of The College of Sciences, University of Texas At San Antonio, San Antonio, TX, USA; 2 Electron Microscopy Research Center, University of Texas At San Antonio, San Antonio, TX, USA; 3 Children’s Hospital Oakland Research Institute, Oakland, CA, USA; 4 Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland, OH, USA; 5 Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA; 6 Departments of Histology and Embryology, Baku Medical University, Baku, Azerbaijan Mechanisms involved in the pathogenesis of athero- and arteriosclerosis also appear to be relevant to Alzheimer’s disease (AD). We studied blood flow and cellular/subcellular features of vascular lesions and mitochondria in brain vascular wall cells of human AD brain biopsies, human short postmortem brain tissues, two transgenic mouse lines overexpressing amyloid beta precursor protein (AßPP) and ApoE4 mice as a model that mimics AD. We also studied the same features in aged rats and ApoE4 mice given selective mitochondrial antioxidants (Lipoic Acid +ALCAIR). In situ hybridization, using mitochondrial DNA (mtDNA) probes for human and mouse wild type and 5kb deleted mtDNA, was performed in conjunction with immunocytochemistry using antibodies against the oxidative stress and mitochondrial markers. Significantly more mitochondrial abnormalities were present in microvessels where lesions occurred. In situ hybridization revealed positive mtDNA signals in damaged mitochondria from the vascular endothelium and in perivascular cells of atherosclerotic lesioned vessels in human AD and the animal models of AD. These mtDNA deletions were associated with increased amounts of immunoreactive Aβ, 8OHG and COX in the same cellular and subcellular compartments. The animals that received treatment with antioxidants showed an improvement in cognitive performance and an absence of structural alterations. We hypothesize that oxidative stress induced by athero- and arteriosclerosis is responsible for cerebral blood flow reduction and most likely can lead to blood brain barrier failure and breakage during the development of AD that can be diminished by using selective mitochondrial antioxidants. Supported by: NIH, Alzheimer’s Association and Philip Morris USA. PO33-561
USE OF MICROARRAYS AND IMMUNOHISTOCHEMISTRY TO INVESTIGATE ACCELERATED ATHEROSCLEROSIS IN HUMAN GRAFT CORONARY ARTERY DISEASE
A. Rousoulieres 1,2 , S. Collot-Teixeira 3,4 , L. Chalabreysse 5 , K. Morser 3,4 , C. McDermott-Roe 3,4 , S. Yilmaz 3 , M. Leleu 4 , F. De Lorenzo 4,6 , A. Guzman 7 , J.B. Michel 7 , L. Sebbag 2 , P. Boissonnat 2 , F. Thivolet-Bejui 5 , J.L. McGregor 3,4,8 . 1 EA 1582, Faculte de Medecine RTH Laennec, Lyon, France; 2 Department of Heart Transplantation, Hopital Cardiologique Louis Pradel, Lyon, France; 3 Cardiovascular Division, Kings College London, UK; 4 Thrombosis Research Institute, London, UK; 5 Department of Pathology, Hopital Cardiologique Louis Pradel, France; 6 Beta cell Diabetes Centre, Chelsea and Westminster Hospital NHS Foundation Trust, London, UK; 7 INSERM U698, Paris, France; 8 INSERM U689, Hopital Lariboisiere, Paris, France Background and aims: Accelerated coronary atherosclerosis in cardiac transplant recipients is a major cause of morbidity and mortality. Graft coronary artery disease (CAD) or chronic rejection (CR) is the major limiting factor for long term survival after heart transplantation (HT). To investigate pathways implicated in accelerated atherosclerosis in chronic arterial graft rejection a rat aortic allograft model was used in tandem with microarrays and immunohistochemistry (IHC). Methods: Rat abdominal aortae were isografted (5) or allografted (5) from Brown-Norway to Lewis rats and grafts were harvested (Intima and media) after day 8, 25 or 60. Total isolated RNA was then used in whole genome 2-color Agilent microarrays. Rosetta Luminator and BRB software’s were used for candidate genes selection and validation was performed as indicated below. Results: Differentially expressed genes between iso and allo at day 8, 25 and 60 were respectively 1829, 2582 and 1925 (fold changes>2 or<-2 and Pvalues<0.05). Some of the investigated genes were altered at the time of
77th Congress of the European Atherosclerosis Society, April 26–29, 2008, Istanbul, Turkey