84 Atherosclerosis Supplements 11, no. 2 (2010) 17–108 length ABCG4 (2.0 kb) transfected cells. Since there was large discrepancy in the molecular w...

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Atherosclerosis Supplements 11, no. 2 (2010) 17–108

length ABCG4 (2.0 kb) transfected cells. Since there was large discrepancy in the molecular weight of ABCG4 protein as compared with previous report, we performed to analyze the isoform of ABCG4. The mRNAs were extracted from AD brain, and ABCG4 isoforms were cloned using rapid amplification of cDNA ends (RACE) technique. Here we report the cloning of a novel isoform of human ABCG4. 5 -RACE identified an upstream exon and 3 kinds of splicing, not deduced from known sequences of ABCG4. 3 -RACE also identified an exon not deduced from known sequences of ABCG4, and it was short-isoform of ABCG4 (sABCG4) which was only half size (1.1 kb) of full-length ABCG4. Examination of the expression of sABCG4 mRNA revealed that sABCG4 mRNA was also expressed in macrophage by RT-PCR. sABCG4 transfected cells expressed sABCG4 proteins with both soluble and membrane bound forms. The sABCG4 is possible to regulate HDL metabolism to interact with other lipid transporters, in addition soluble ABCG4 might be a novel marker for AD and cardiovascular diseases. P315 GENETIC VARIATION IN THE ABCA1-GENE ABROGATES THE PROTECTIVE EFFECT OF ADIPONECTIN ON CAROTID IMT C. Ciardi1 , A. Sandhofer1 , B. Iglseder2 , B. Paulweber3 , J. Patsch1 . 1 Department of Internal Medicine I, Medical University Innsbruck, Innsbruck, 2 Department of Geriatrics, 3 First Department of Internal Medicine, Paracelsus Private Medical University Salzburg, Salzburg, Austria Introduction: The ATP-binding cassette transporter A1 (ABCA1) facilitates amongst others the initial step of the reverse cholesterol transport by transporting cholesterol from macrophages to lipid-poor HDL precursors. Variants in the ABCA1-gene have been associated with a lower carotid intima media thickness (IMT). Adiponectin levels are correlated negatively with carotid IMT and positively with HDL-C. Recent in vitro data suggested an influence of adiponectin on ABCA1-mediated cholesterol efflux from macrophages. Objective: Our objective was to investigate the R219K polymorphism of the ABCA1 gene influences the atheroprotective effect of adiponectin. Design: 680 men randomly were selected from the SAPHIR-population. Methods: The R219K-variant was determined by RFLP-analysis, carotid IMT by high-resolution B-mode ultrasound and plasma adiponectin levels by ELISA. Results: In carriers of the K-variant (n = 334) adiponectin shows a negative correlation with carotid IMT (r = −0.137, p = 0.013), whereas in wild-type subjects (n = 346) homozygous for the R allele no correlation can be detected (r = −0.044, p = 0.42). In carriers this atheroprotective effect persisted after adjustment for age, blood pressure, LDL-cholesterol, C-reactive protein and insulin resistance (standardized Beta = −0.129, p = 0.009). Plasma adiponectin levels correlated positively with HDL-C in the whole study population (r = 0.337, p < 0.001), in wildtype subjects (r = 0.312, p < 0.001) and in carriers of the K-variant (r = 0.387, p < 0.001). However, adjustment for HDL-cholesterol abrogates the correlation of IMT and adiponectin in the whole population, wildtype subjects and carriers of the K-variant. Conclusion: Variation in the ABCA1-gene influences the atheroprotective effect of adiponectin, suggesting that adiponectin influences carotid IMT at least partly via ABCA1. P316 NEW SPLICING MUTATIONS OF MTP LEADING TO SEVERE ABETALIPOPROTEINEMIA E. Mas1,2 , V. Pons2 , C. Rolland2 , M. Nauze2 , M. Danjoux3 , G. Gaibelet2 , ´ A. Sassolas4 , E. Levy ´ 5 , F. Terce´ 2 , X. Collet2 . 1 Unite´ de Gastroenterologie, ´ ´ ´ ˆ Hepatologie et Nutrition − Departement de Pediatrie − Hopital des Enfants, 2 3 ´ ˆ Inserm U563; CPTP, IFR 150, Departement d’Anatomopatholgie, Hopital ´ Purpan Purpan, Toulouse, 4 Laboratoire des Dyslipidemies, Centre de Biologie 5 ´ ˆ de Nutrition et de Biochimie, Hopital Est, Lyon, France, Departements ´ Montreal, ´ QC, Canada Sainte-Justine et Universite´ de Montreal, Background: Abetalipoproteinemia (ABL) is a rare autosomal recessive disease characterized by very low plasma levels of cholesterol and triglycerides (TG) and the absence of apo B-containing lipoproteins. It is the consequence of microsomal triglyceride transfer protein (MTP) deficiency. Methods: We reported 2 new patients with a severe form of ABL. We determined MTP mutations and we analysed their functional involvement in MTP function. Results: These children have the intestinal and biochemical characteristics of ABL including duodenal fat storage and absence of chylomicrons and LDL lipoproteins. We confirmed that they are compound heterozygotes with 619 G>T and c.1237−28 A>G mutations. cDNA analysis revealed alternative splicing with deletion of exon 6 and 10, respectively. Deletion of exon 6 introduced a frame shift in the reading frame and a premature stop codon at position 234; the truncated protein was not translated. Although D10-MTP was normally present at the endoplasmic reticulum (ER), there was no transfer of TG using duodenal biopsies. Conclusion: Amino acids 413–448 are required for MTP TG transfer but their loss does not impair the localization of MTP at the ER.

Poster Presentations

P317 FUNCTION OF HDL MODIFYING PROTEINS IN THE HUMAN PLACENTA J. Saarela1 , J. Metso1 , R. Kaaja2 , W.J. Schneider3 , M. Jauhiainen1 . 1 Public Health Genomics Unit, National Institute for Health and Welfare, 2 Department of Obstetrics and Gynaecology, University of Helsinki, Helsinki, Finland, 3 Department of Medical Biochemistry, Medical University of Vienna/MFPL, Vienna, Austria Object of the study: Pre-eclampsia is a common disorder during pregnancy and a serious threat for both the mother and the baby. Furthermore, it predisposes both the mother and the baby later in life to cardiovascular diseases. In the pre-eclamptic placenta many changes already occur during early pregnancy and the placenta becomes greasy due to dysfunctional lipidmetabolism. The reason why pre-eclampsia develops is unknown and predictive markers are lacking. We aimed to decipher how HDL modifying proteins (e.g. PLTP, CETP, ABCA1, ABCG1, SRBI) participate in the transfer of lipids from the maternal to the embryonic circulation and whether any of them can be used as predictive markers of the disorder. Methods: Measurement of human placenta PLTP- and CETP-activity (radiometric methods), PLTP-mass (ELISA), Western-blotting, qRT-PCR, and lipid measurements to determine fattening of the placenta. As cell model, we are using human placenta derived BeWo-cells and aim to study regulation of LXR/RXR/FXR target genes and the effect of estrogen and progesterone in the context of HDL metabolism. Results: Our preliminary results show that in type-1-diabetes placenta PLTP activity is increased (39%) and in pre-eclampsia placenta reduced (37%) compared to control placenta. In addition, in diabetic placenta phospholipid levels are increased (55%). Properties of the BeWo-cell derived PLTP are similar to plasma PLTP. Conclusions: The majority (44%) of the umbilical cord cholesterol is carried by HDL-particles. PLTP, CETP, ABCA1, ABCG1, and SRBI are highly expressed in human placenta. The preliminary results suggest that HDL modifying factors are important in maternal-fetal lipid transport. P318 EZETIMIBE EFFECT ON SERUM apoB48 LEVELS IN FAMILIAL HYPERCHOLESTEROLEMIA AND TYPE 2 DIABETES M. Sudo, S. Takemitsu, Y. Sato, M. Nagao, T. Harada, Y. Nakajima, K. Tanimura, F. Okajima, S. Oikawa. Nippon Medical School, Tokyo, Japan Background and Aims: Chylomicron synthesis followed by the assembly with apoB48 will be increased in diabetic condition or insulin resistant state. Ezetimibe is a unique agent binding to NPC1L1 molecule of enterocyte plasma membrane and inhibits cholesterol absorption. On the other hand cholesterol absorption will be increased in diabetes or insulin resistant state through NPC1L1 function. The focus of the present study is on the changes of serum lipids and apoB48 levels after monotherapy with Ezetimibe for 3−4 months in hypercholesterolemia patients. Materials and Methods: Subjects were FH (n = 30, 57±11 years old, mean±SD), Type 2 diabetes (DM, n = 33, 58±11), nonDMnonFH (n = 22, 56±11). Serum lipids and apoB48 levels were studied before and after Ezetimibe monotherapy (10 mg/day) during 3−4 months. Results: Ezetimibe significantly decreased serum TC, LDL-C and non-HDL-C levels in each group. The %changes of LDL-C were higher in DM (−22%) compared to FH (14%) and nonDMnonFH (−15%). The significant changes of serum TG and apoB48 levels were observed in DM (−10% and 6%, respectively), but not in FH or nonDMnonFH. It was suggested that the decrease of apoB48 after treatment with Ezetimibe was related to TG reduction. Conclusion: Inhibition of cholesterol absorption by Ezetimibe will be more effective in DM compared to nonDM subjects. Ezetimibe effect on apoB48 will be affected through chylomicron metabolism. P319 RESEQUENCING THE LPL GENE IN PATIENTS WITH VARIOUS HYPERTRIGLYCERIDEMIAS TO DETERMINE THE RELATIVE IMPORTANCE OF RARE ALLELES AND COMMON VARIANTS D. Evans, J. Azar, F. Beil. Endokrinologie und Stoffwechsel, Med. Klinik III, UKE, Hamburg, Germany Common SNPs in the LPL gene have been shown to have a modest affect on triglyceride levels in a number of studies whereas resequencing studies in patients with severe hypertriglyceridemia (>10 mmol/l) have shown that rare mutations are frequent. It is the aim of this study to determine the role of rare mutations in the LPL gene in patients with moderate hypertriglyceridemia. The exons plus exon/intron boundaries of the LPL gene of three groups of patients were sequenced: Group A. 97 patients with severe hypertriglyceridemia defined as triglycerides above 10 mmol/l. Group B, 220 patients with triglycerides above the 95th percentile for age and sex but below 10 mmol/l. Group C 120 patients with APOE2/2 genotype. In addition to known SNPs, 18 rare variants were identified, 5 of which have been previously reported (V69L, 5×, G188E 3×, I194T 1×, I225T, 2× and c440–443delACTA 1×). New variants identified were: M-36L, R89W, Y94S, I196F, Y206D, Y262X, S266P, S277N, C278T, M301I and E347D. In group A 12 patients were carriers of at least one rare variant and