- Email: [email protected]
Identification of novel cardiovascular susceptibility genes by mouse genetics and gene expression profiling
Thursday June 29, 2000: Poster Abstracts P:W30 Genetics of Lipoprotein Metabolism
266
controlled laboratory. Statistical analysis was performed using the ANOVA. Analysis was carded out in men and women together. Results: The effects of apoE and apoB polymorphisms were as expected. Gender differences have been observed, but no significant gene-gender interaction was detected. The carders of the apoe2 and apoB II alleles have the lowest, and the carriers of the apoe4 and apoB ID or DD alleles have the highest levels of plasma lipides (see table). Polymorphism apt E apt B
N
Apolipoprotein B (g/l)
LDLcholesterol (mmol/I)
+allele2 +allele2 3/3 3/3 +allele4 +allele4
22 14 80 100 18 21
1.02:t: 0.24 1.084- 0.38 1.13:t=0.26 1.204- 0.28 1.285:0.27 1.31 4- 0.26 0.002
2.75 5:1.03 3.095:1.12 3.09 5:0.87 3.47 4- 0.91 3.495:0.95 3.645:1.06 0.005
11 +alleleD I1 +alleleD 1I +alleleD p
Conclusions: In this sample, haplotype analysis of the apt E and apt B polymorphisms emphasizes the importance of combined and additive effect of more genes in plasma lipid parameters determination. i T h P 9 : W 3 0 [ Fluorescence based detection of the CETP TaqlB polymorphism: Identification of a new C775T polymorphism in intron 1 by melting curve analysis
Forty middle-aged men with dyslipidemia and established CAD, undergoing intensive lipid-lowering therapy, were selected for the study of the impact of the HL gene promoter polymorphism on changes in HL activity, LDL buoyancy and lipoprotein levels and its association with CAD regression. The change in coronary stenosis was assessed by repeat quantitative angiography, the HL polymorphism by PCR amplification, HL activity by the use of 14C-labeled substrate and LDL buoyancy by plasma density gradient ultracentrifugation. Subjects with the CC genotype had the greatest decrease in HL activity (p < 0.005 vs. TC and 7T by ANOVA) and the greatest improvement in LDL density (p < 0.005) and HDL2-C (p < 0.05) with therapy. These subjects had the greatest CAD angiographic improvement, with 95% of them experiencing regression of coronary stenosis, while 63% of TC and none of the TT patients had disease regression (X 2 = 16.43; p < 0.001). In patients with established CAD and dyslipidemia, the HL gene - 5 1 4 C--~T polymorphism predicts 16% of the change in coronary stenosis with lipid lowering treatment. This association appears to be accounted for by the modulating effect of this polymorphism on HL-associated steps in LDL and HDL metabolism. This study identifies a gene polymorphism that strongly influences the lipid and clinical response to lipid-lowering drugs. ) ThP11 :W30 J Use of SNP technology to define discase-bearing hapiotypes of the a p t AI-CIII-AIV gene region in
familial combined hyperlipidemia
D. Teupser, W. Rupprecht, J. Thiery, D. Seidel. Institute of Clinical
M. Groenendijk I , R.M. Cantor2, G.M. Dallinga-Thie I ./Department of
Chemistry, University Hospital LMU, Munich, Germany
Internal Medicine, University Medical Center Utrecht, the Netherlands; 2Department of Human Genetics, UCLA, Los Angeles, USA
Objective: Cholesteryl ester transfer protein (CETP) plays a crucial role in reverse cholesterol transport. It facilitates the transfer of cholesteryl esters from HDL to apoB-containing lipoproteins. The TaqlB polymorphism of the CETP gene has an influence on plasma CETP and HDL levels and the progression of CAD. The aim of the present study was to determine the performance of two new fluorescence based detection systems in the analysis of the TaqlB genotype in CAD patients. Methods: The TaqlB genotype was determined by RFLP analysis using TaqI. A total of 150 patients, 50 patients each carrying the B i l l , B1/2 and B2/2 genotypes were selected. Subsequently, the genotype was also analyzed by fluorescence based detection assays established in our laboratory with the SDS 7700 (TaqMan) and the LightCycler. DNA sequencing was performed with a ABI PRISM 377 sequencer. Results: The TaqlB genotypes obtained with the fluorescence assays corresponded to those of the RFLP analysis with the exception of three samples from heterozygous patients (B 1/2), that were misclassified as homozygous for the B2 allele with the TaqMan system. Melting curve analysis of these sampies in the LightCycler revealed a distinct melting pattern with an additional melting point at 59°C. The same melting point also occurred in samples from four patients homozygous for the B 1 allele. DNA-sequencing revealed a new, previously unknown C775T nucleotide exchange in intron 1 of the CETP gene just 8 basepairs apart from the TaqlB nucleotide exchange. Conclusion: Determination of the TaqlB polymorphism in the SDS 7700 resulted in unexpected misclassifications due to a previously unknown C775T polymorphism of the CETP gene. However, the new polymorphism could be detected with the LightCycler due to the occurrence of a different melting point. Ongoing studies indicate, that the C775T polymorphism may be an additional risk factor for the progression of CAD. I T h P 1 0 : W 3 0 [ A common hepatic lipase gene promoter variant
determines clinical response to intensive lipid lowering treatment A. Zambon 1, S.S. Deeb 2, B.G. Brown I , J.E. Hokanson 1, S. Bertocco 3 , J.D. Brunzell I ./Dept. of Medicine; 2Div. of Medical Genetics, University of
Washington, Seattle, USA; 3Dept. of Medicine, University of Padua, Italy Our understanding is limited as to why lipid and coronary artery disease (CAD) response to lipid-towering therapy varies among different subjects. The common - 5 1 4 C--* T promoter polymorphism of the hepatic lipase (HL) gene affects HL activity, The C allele is associated with higher HL activity and a more atherogenic lipid profile: more dense LDL particles and lower levels of HDL2-cholesterol. Intensive lipid-lowering therapy lowers HL activity, increases LDL and HDL buoyancy and promotes CAD regression. We tested the hypothesis that subjects who experience the greatest CAD regression from these favorable effects are those with the CC genotype, associated with a more atherogenic lipid profile.
In previous studies we have reported linkage and association in the ape AI-CIII-AIV gene region on chromosome 11 with Familial Combined Hyperlipidemia (FCHL). Using sequence analysis we identified five new SNP variants in this gene region, a C317T variant in intron 2 of the apoAl gene and four variants in the intergenic region between apt AI and apt CIII, T3213C, A3235C, T3287C and A5132C. Haplotypes consisting of 7 SNPs, including Mspl in promoter of apt AI gene and Sstl in non-coding region of ape CIII, were assigned using parsimony of inheritance in 33 FCHL families. The distribution of these haplotypes was significantly different between the probands (n = 33) and spouse controls (n = 230) (p = 0.0005). Three major haplotypes were observed in both groups. The haplotype with only wild type alleles was significantly less frequent in probands (0.46 vs 0.62, p = 0.013), whereas the haplotype with the M1, 317T, 3213T, 3235C, 3287C, 5232C and $2 alleles had a frequency of 15% in probands vs 4% in spouse controls (p = 0.0005). Also the third haplotype consisting of the M2, 317% 3213C, 3235A, 3287C, 5232C and SI alleles was significantly more frequent in probands (26% vs 11%, p = 0.001). The Mspl marker and T3213C variant were in complete linkage disequilibrium and are exclusively present on the same haplotype. Similarly the $2 allele and the A3235C variant reside on one haplotype. The C317% T3287C and A5132C markers occurred in both haplotypes. In conclusion, successful application of SNP technology provides evidence that two truly distinct haplotypes independently predispose to FCHL and they account together with the wild type for 90% of the occurring haplotypes in these FCHL families.
ThP12:W30 ] Identification of novel cardiovascular susceptibility genes by mouse genetics and gene expression profiling C.J.A. Moen 1, M.J.J. Gijbels I , A. Kreeft I , P.J.J. van Gorp I , A. Tholens I , M.C. Jong 2, L.M. Havekes2, R.R. Frants I , M.H. Hofker t . 1Leiden University
Medical Center; 2TNO-PG, Leiden, The Netherlands In order to identify new genes involved in the development of hyperlipidemia and atherosclerosis, we use transgcnic APOE3Leiden (E3L) mice. In contrast to non-transgenic mice, these E3L mice have high lipid levels due to a defect in the uptake of remnant lipoproteins from the plasma, allowing the analysis of genetic factors involved in VLDL metabolism. We use two different, complementary approaches: (l) genetic mapping: We tested female F1 hybrids and backcross mice between strain E3L and 7 inbred strains (CBA, C3H, NZB, FVB, 129, BALB/c, AKR) for their lipid levels and susceptibility to atherosclerosis to look for the presence of dominant and recessive modifiers, after feeding several diets. Notably, FVB carries modifiers that increase the levels of cholesterol as well as triglycerides on all diets and also influence the susceptibility to atherosclerosis. These modifiers are currently being mapped in (E3L x FVB) F2 crosses.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
Thursday June 29, 2000: Poster Abstracts P:W30 Genetics of Lipoprotein Metabolism (2) Gene expression profiling using microarraying to identify genes that are differentially regulated under different (genetic or dietary) conditions. Of special interest am genes that are differentially expressed a) during overexpression of APOE b) in E3L mice on different diets c) in {FVB x (FVB x E3L)} backcross mice with high lipid levels compared to backcross mice with low lipid levels. Initial experiments showed that among 26 differentially expressed genes, 5 were absent or downregulated in backcross mice with low cholesterol levels compared to backcross mice with nigh cholesterol levels, whereas 21 were upregulated. Combining the two genetic approaches will facilitate the identification of novel (candidate) genes and pathways involved in lipid metabolism and atherosclerosis.
ThPl 3:W30/ Identification of (dietary respouse) genes involved in lipid metabofism and susceptibility to atherosclerosis in mice J
Aria J. Kreeft 1, Corina J.A. Moen 1, Marten H. Hofker l, Rune R. Frants I , Louis M. Havekes 3 , Erno Vreugdenhil2 , Nicole A. Datson 2. Depts. of
I Human and Clinical Genetics; 2Medical Pharmacology, Leiden university Medical Centre; 3TNO-PG, Gaubius laboratory, Leiden, The Netherlands Serial Analysis of Gene Expression (SAGE) is used to identify new genes involved in lipid metabolism and susceptibility to atherosclerosis. As a model system we use APOE3Leiden (E3L) transgenic mice on a C57BI/6 background. These mice show a profound diet-induced hyperlipidemia and are susceptible to the cholesterol-raising factor cafestol. SAGE allows a qualitative and quantitative analysis of thousands of transcripts simultaneously. Currently the first liver expression profiles have been obtained from the E3L mice and of C57BI/6 on standard chow. Over 11,000 transcripts have been analyzed of both mice. These 11,000 transcripts represent 4000 different genes. Of these 4000 different genes, about 100 genes were differentially expressed. Fourty-one percent of these genes are significantly upregulated in E3L mice, while fourty-two percent are downregulated and seventeen percent are completely absent in E3L compared to C57BI/6. The differences in expression levels found are currently being validated using Northern blot analysis. High abundant genes are involved in several metabolic processes such as the synthesis of lipoprotein particles, glycolysis and gluconeogenesis and detoxification processes. Currently two more SAGE expression profiles are being generated to find genes responding to dietary factors e.g. E3L on moderate high fat diet and E3L on moderate high fat diet containing cafestol.
T h P 1 4 : W 3 0 ] Characterisation of apolipoprotein E sendal, an i
apolipoprotein E variant associated with lipoprotein glomerulopathy
M.M. Hoffmann, E. Panagiotou, H. Schamagl, W. Marz.
Albert-Ludwigs-University, Department of Clinical Chemistry Freiburg, Germany It has been well-known that nephrotic syndrome and chronic renal failure are associated with lipid and lipoprotein abnormalities. It has only recently become obvious that structural variants of apolipoprotein E may be responsible for the development of a new glomerolar disease, lipoprotein glomerulopathy (LPG). A major hallmark of this disease is the deposition of lipoproteins and apolipoproteins in the mesangium. In addition several patients show a lipid profile reminiscent of type III hyperlipoproteinemia. The aim of our study was to obtain a more precise insight into the molecular mechanisms linking apoE to the pathogenesis of LPG. Purification of apoE from a single patient's plasma is hardly feasible, especially because all carders of apoE Sendai are heterozygous for this variation. Thus, we introduced the Arg145->Pro exchange into the wild type apoE3 cDNA using site-specific-mutagenesis. ApoE2, apoE3 and apoE Sendai were expressed in the baculo virus system. LDL receptor binding was studied using recombinant apoE complexed with phospholipid particles and with VLDL from a patient with familiar apoE deficiency. We examined cellular binding, uptake and degradation of these lipoproteins in human skin fibroblasts. ApoE2 and apoE Sendai displayed clearly reduced receptor binding activities compared to apoE3. Heparin binding of apoE Sendai was lower compared to apoE3 but nigher than that of apoE2. Further studies will have to address the question in which way apoE Sendai lead to LPG. Taking into account that several carders of apo E Sendai without any LPG symptoms exist, it is likely that renal factors cooperate with apo E variants in the development of lipoprotein glomernlopathy.
267
1 T h P 1 5 : W 3 0 ] A373P/R451Q CETP mutations, ltDL cholesterol, and risk of ischemic heart disease. The Copenhagen city
heart study B. Agerholm-Larsen A. Tybjaerg-Hansen, P. Schnohr, R. Steffensen, B.G. Nordestgaard. University of Copenhagen, Denmark
Objective: We studied two common mutations in cholesteryl ester transfer protein (CETP) gene, A373P and R451Q; CETP mediates transfer of cholesteryl ester from high-density lipoprotein (HDL) in exchange for triglycerides in apolipoprotein B containing lipoproteins. Methods: We genotyped 8467 healthy women and men from the Danish general population, and 1636 Danish women and men with ischemic heart disease. Results: The prevalence of heterozygous carriers of 373P and 451Q was 0.10 and 0.07, and of homozygous carders 0.003 and 0.002, respectively. All carriers of the 451Q allele also carried the 373P allele. Plasma levels of HDL cholesterol in women (mean 4- SE in lnmol/L) were 1.74 4- 0.01 for non-carriers of 373P, 1.62 4- 0.02 for heterozygous carriers, and 1.38 4- 0.09 for homozygous carriers (ANOVA: P < 0.001). In men, the equivalent values were 1.40 4- 0.01, 1.26 4- 0.02, and 1.19 4- 0.09 (ANOVA: P < 0.001). When we adjusted for age alone, or a group of risk factors, CETP genotype did not predict risk of ischemic heart disease. When adjusting for a group of risk factors plus HDL cholesterol, women not treated with hormonal replacement therapy carrying the 373P allele had a decreased risk of ischemic heart disease (odds ratio: 0.64; 95%CI: 0.43--0.96), whereas the risk of ischemic heart disease in male or treated female carders was statistically unaffected. Conclusions: The A373P/R451Q polymorphism in CETP is associated with decreases in HDL cholesterol of 0.12--0.36 and 0.14-4).21 mmol/L in women and men, and possibly with a paradoxical 36% decrease in risk of ischemic heart disease in women.
ThP16:W30]Familial recessive hypercholesterolemia (FRH): Characterization of 10 homozygotes and 9 obligate heterozygotes G. Zuliani, A. Pacifico I , M. Ciccarese I , P. Pintus2, M. Arca 3 , A. Corsini4, Maioli I , R. Fellin. Department of Internal Medicine 11,
University of Ferrara; 1Department of Internal Medicine, University of Sassari; 2Brotzu Hospital, Cagliari; 3Department of Internal Medicine 11, University of Rome "La Sapienza"; 4Institute of Pharmacological Science, University of Milan, Italy Objective: We described an Italian family with several cases of hypercholesterolemia and/or sudden death. Probands had the clinical waits of homozygous familial hypercholesterolemia (FH), while parents were normal. The involvement of low density lipoprotein receptor (LDL-R) and apo B genes was excluded. LDL-R activity in fibroblasts, and LDL binding to LDL-receptor were normal in probands. We investigated the metabolic mechanisms underlying this new recessive disorder, and found a reduction in the in vivo LDL catabolism caused by a selective reduction in hepatic LDL uptake. We report 3 new Italian families, and describe the characteristics of 10 probands (5 M and 5 F), and 9 parents (5 M and 4 F) from 5 different pedigrees. Methods: Plasma lipids were measured by standardized methods. LDL-R activity was determined by evaluating 125I-LDL internalization/degradation in cultured fibroblasts. LDL binding to LDL-R was measured in vitro by competition experiments in cultured fibroblasts. Results: Probands (age: 13-47 yrs): all had tendinous/tuberous xanthomas, 4 had xanthelasmas. Coronary angiography was pathological in 6 out of 6. Lipoprotein parameters were (range, mg/dL): Tot Chol (450--642), LDL-C (372-592), HDL-C (28-58), Triglycerides (49-208), apo B (207-365), apo A-I (83-137). LDL-R activity in vitro was (range, % of normal): uptake (70-140), degradation (70-150). LDL binding activity in vitro was 84--100% of normal. Parents (age 51-83 yrs): 2 had type 2 diabetes, 2 had hypertension. Nobody had xanthomas or xanthelasmas. Lipoprotein parameters were (range, mg/dL): Tot Chol (172-237), LDL-C (103-153), HDL-C (40-70), Triglycerides (64-180), apo B (88-165), apo A-I (119-173). Conclusions: 1) all FRH homozygotes have the clinical and laboratory features of FH homozygotes despite having normal LDL-R activity in fibroblasts and normal LDL binding activity; 2) all parents (FRH obbligate heterozygotes) show normal phenotype, supporting the hypothesis of an autosomal recessive trasmission of this disease.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
266
controlled laboratory. Statistical analysis was performed using the ANOVA. Analysis was carded out in men and women together. Results: The effects of apoE and apoB polymorphisms were as expected. Gender differences have been observed, but no significant gene-gender interaction was detected. The carders of the apoe2 and apoB II alleles have the lowest, and the carriers of the apoe4 and apoB ID or DD alleles have the highest levels of plasma lipides (see table). Polymorphism apt E apt B
N
Apolipoprotein B (g/l)
LDLcholesterol (mmol/I)
+allele2 +allele2 3/3 3/3 +allele4 +allele4
22 14 80 100 18 21
1.02:t: 0.24 1.084- 0.38 1.13:t=0.26 1.204- 0.28 1.285:0.27 1.31 4- 0.26 0.002
2.75 5:1.03 3.095:1.12 3.09 5:0.87 3.47 4- 0.91 3.495:0.95 3.645:1.06 0.005
11 +alleleD I1 +alleleD 1I +alleleD p
Conclusions: In this sample, haplotype analysis of the apt E and apt B polymorphisms emphasizes the importance of combined and additive effect of more genes in plasma lipid parameters determination. i T h P 9 : W 3 0 [ Fluorescence based detection of the CETP TaqlB polymorphism: Identification of a new C775T polymorphism in intron 1 by melting curve analysis
Forty middle-aged men with dyslipidemia and established CAD, undergoing intensive lipid-lowering therapy, were selected for the study of the impact of the HL gene promoter polymorphism on changes in HL activity, LDL buoyancy and lipoprotein levels and its association with CAD regression. The change in coronary stenosis was assessed by repeat quantitative angiography, the HL polymorphism by PCR amplification, HL activity by the use of 14C-labeled substrate and LDL buoyancy by plasma density gradient ultracentrifugation. Subjects with the CC genotype had the greatest decrease in HL activity (p < 0.005 vs. TC and 7T by ANOVA) and the greatest improvement in LDL density (p < 0.005) and HDL2-C (p < 0.05) with therapy. These subjects had the greatest CAD angiographic improvement, with 95% of them experiencing regression of coronary stenosis, while 63% of TC and none of the TT patients had disease regression (X 2 = 16.43; p < 0.001). In patients with established CAD and dyslipidemia, the HL gene - 5 1 4 C--~T polymorphism predicts 16% of the change in coronary stenosis with lipid lowering treatment. This association appears to be accounted for by the modulating effect of this polymorphism on HL-associated steps in LDL and HDL metabolism. This study identifies a gene polymorphism that strongly influences the lipid and clinical response to lipid-lowering drugs. ) ThP11 :W30 J Use of SNP technology to define discase-bearing hapiotypes of the a p t AI-CIII-AIV gene region in
familial combined hyperlipidemia
D. Teupser, W. Rupprecht, J. Thiery, D. Seidel. Institute of Clinical
M. Groenendijk I , R.M. Cantor2, G.M. Dallinga-Thie I ./Department of
Chemistry, University Hospital LMU, Munich, Germany
Internal Medicine, University Medical Center Utrecht, the Netherlands; 2Department of Human Genetics, UCLA, Los Angeles, USA
Objective: Cholesteryl ester transfer protein (CETP) plays a crucial role in reverse cholesterol transport. It facilitates the transfer of cholesteryl esters from HDL to apoB-containing lipoproteins. The TaqlB polymorphism of the CETP gene has an influence on plasma CETP and HDL levels and the progression of CAD. The aim of the present study was to determine the performance of two new fluorescence based detection systems in the analysis of the TaqlB genotype in CAD patients. Methods: The TaqlB genotype was determined by RFLP analysis using TaqI. A total of 150 patients, 50 patients each carrying the B i l l , B1/2 and B2/2 genotypes were selected. Subsequently, the genotype was also analyzed by fluorescence based detection assays established in our laboratory with the SDS 7700 (TaqMan) and the LightCycler. DNA sequencing was performed with a ABI PRISM 377 sequencer. Results: The TaqlB genotypes obtained with the fluorescence assays corresponded to those of the RFLP analysis with the exception of three samples from heterozygous patients (B 1/2), that were misclassified as homozygous for the B2 allele with the TaqMan system. Melting curve analysis of these sampies in the LightCycler revealed a distinct melting pattern with an additional melting point at 59°C. The same melting point also occurred in samples from four patients homozygous for the B 1 allele. DNA-sequencing revealed a new, previously unknown C775T nucleotide exchange in intron 1 of the CETP gene just 8 basepairs apart from the TaqlB nucleotide exchange. Conclusion: Determination of the TaqlB polymorphism in the SDS 7700 resulted in unexpected misclassifications due to a previously unknown C775T polymorphism of the CETP gene. However, the new polymorphism could be detected with the LightCycler due to the occurrence of a different melting point. Ongoing studies indicate, that the C775T polymorphism may be an additional risk factor for the progression of CAD. I T h P 1 0 : W 3 0 [ A common hepatic lipase gene promoter variant
determines clinical response to intensive lipid lowering treatment A. Zambon 1, S.S. Deeb 2, B.G. Brown I , J.E. Hokanson 1, S. Bertocco 3 , J.D. Brunzell I ./Dept. of Medicine; 2Div. of Medical Genetics, University of
Washington, Seattle, USA; 3Dept. of Medicine, University of Padua, Italy Our understanding is limited as to why lipid and coronary artery disease (CAD) response to lipid-towering therapy varies among different subjects. The common - 5 1 4 C--* T promoter polymorphism of the hepatic lipase (HL) gene affects HL activity, The C allele is associated with higher HL activity and a more atherogenic lipid profile: more dense LDL particles and lower levels of HDL2-cholesterol. Intensive lipid-lowering therapy lowers HL activity, increases LDL and HDL buoyancy and promotes CAD regression. We tested the hypothesis that subjects who experience the greatest CAD regression from these favorable effects are those with the CC genotype, associated with a more atherogenic lipid profile.
In previous studies we have reported linkage and association in the ape AI-CIII-AIV gene region on chromosome 11 with Familial Combined Hyperlipidemia (FCHL). Using sequence analysis we identified five new SNP variants in this gene region, a C317T variant in intron 2 of the apoAl gene and four variants in the intergenic region between apt AI and apt CIII, T3213C, A3235C, T3287C and A5132C. Haplotypes consisting of 7 SNPs, including Mspl in promoter of apt AI gene and Sstl in non-coding region of ape CIII, were assigned using parsimony of inheritance in 33 FCHL families. The distribution of these haplotypes was significantly different between the probands (n = 33) and spouse controls (n = 230) (p = 0.0005). Three major haplotypes were observed in both groups. The haplotype with only wild type alleles was significantly less frequent in probands (0.46 vs 0.62, p = 0.013), whereas the haplotype with the M1, 317T, 3213T, 3235C, 3287C, 5232C and $2 alleles had a frequency of 15% in probands vs 4% in spouse controls (p = 0.0005). Also the third haplotype consisting of the M2, 317% 3213C, 3235A, 3287C, 5232C and SI alleles was significantly more frequent in probands (26% vs 11%, p = 0.001). The Mspl marker and T3213C variant were in complete linkage disequilibrium and are exclusively present on the same haplotype. Similarly the $2 allele and the A3235C variant reside on one haplotype. The C317% T3287C and A5132C markers occurred in both haplotypes. In conclusion, successful application of SNP technology provides evidence that two truly distinct haplotypes independently predispose to FCHL and they account together with the wild type for 90% of the occurring haplotypes in these FCHL families.
ThP12:W30 ] Identification of novel cardiovascular susceptibility genes by mouse genetics and gene expression profiling C.J.A. Moen 1, M.J.J. Gijbels I , A. Kreeft I , P.J.J. van Gorp I , A. Tholens I , M.C. Jong 2, L.M. Havekes2, R.R. Frants I , M.H. Hofker t . 1Leiden University
Medical Center; 2TNO-PG, Leiden, The Netherlands In order to identify new genes involved in the development of hyperlipidemia and atherosclerosis, we use transgcnic APOE3Leiden (E3L) mice. In contrast to non-transgenic mice, these E3L mice have high lipid levels due to a defect in the uptake of remnant lipoproteins from the plasma, allowing the analysis of genetic factors involved in VLDL metabolism. We use two different, complementary approaches: (l) genetic mapping: We tested female F1 hybrids and backcross mice between strain E3L and 7 inbred strains (CBA, C3H, NZB, FVB, 129, BALB/c, AKR) for their lipid levels and susceptibility to atherosclerosis to look for the presence of dominant and recessive modifiers, after feeding several diets. Notably, FVB carries modifiers that increase the levels of cholesterol as well as triglycerides on all diets and also influence the susceptibility to atherosclerosis. These modifiers are currently being mapped in (E3L x FVB) F2 crosses.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
Thursday June 29, 2000: Poster Abstracts P:W30 Genetics of Lipoprotein Metabolism (2) Gene expression profiling using microarraying to identify genes that are differentially regulated under different (genetic or dietary) conditions. Of special interest am genes that are differentially expressed a) during overexpression of APOE b) in E3L mice on different diets c) in {FVB x (FVB x E3L)} backcross mice with high lipid levels compared to backcross mice with low lipid levels. Initial experiments showed that among 26 differentially expressed genes, 5 were absent or downregulated in backcross mice with low cholesterol levels compared to backcross mice with nigh cholesterol levels, whereas 21 were upregulated. Combining the two genetic approaches will facilitate the identification of novel (candidate) genes and pathways involved in lipid metabolism and atherosclerosis.
ThPl 3:W30/ Identification of (dietary respouse) genes involved in lipid metabofism and susceptibility to atherosclerosis in mice J
Aria J. Kreeft 1, Corina J.A. Moen 1, Marten H. Hofker l, Rune R. Frants I , Louis M. Havekes 3 , Erno Vreugdenhil2 , Nicole A. Datson 2. Depts. of
I Human and Clinical Genetics; 2Medical Pharmacology, Leiden university Medical Centre; 3TNO-PG, Gaubius laboratory, Leiden, The Netherlands Serial Analysis of Gene Expression (SAGE) is used to identify new genes involved in lipid metabolism and susceptibility to atherosclerosis. As a model system we use APOE3Leiden (E3L) transgenic mice on a C57BI/6 background. These mice show a profound diet-induced hyperlipidemia and are susceptible to the cholesterol-raising factor cafestol. SAGE allows a qualitative and quantitative analysis of thousands of transcripts simultaneously. Currently the first liver expression profiles have been obtained from the E3L mice and of C57BI/6 on standard chow. Over 11,000 transcripts have been analyzed of both mice. These 11,000 transcripts represent 4000 different genes. Of these 4000 different genes, about 100 genes were differentially expressed. Fourty-one percent of these genes are significantly upregulated in E3L mice, while fourty-two percent are downregulated and seventeen percent are completely absent in E3L compared to C57BI/6. The differences in expression levels found are currently being validated using Northern blot analysis. High abundant genes are involved in several metabolic processes such as the synthesis of lipoprotein particles, glycolysis and gluconeogenesis and detoxification processes. Currently two more SAGE expression profiles are being generated to find genes responding to dietary factors e.g. E3L on moderate high fat diet and E3L on moderate high fat diet containing cafestol.
T h P 1 4 : W 3 0 ] Characterisation of apolipoprotein E sendal, an i
apolipoprotein E variant associated with lipoprotein glomerulopathy
M.M. Hoffmann, E. Panagiotou, H. Schamagl, W. Marz.
Albert-Ludwigs-University, Department of Clinical Chemistry Freiburg, Germany It has been well-known that nephrotic syndrome and chronic renal failure are associated with lipid and lipoprotein abnormalities. It has only recently become obvious that structural variants of apolipoprotein E may be responsible for the development of a new glomerolar disease, lipoprotein glomerulopathy (LPG). A major hallmark of this disease is the deposition of lipoproteins and apolipoproteins in the mesangium. In addition several patients show a lipid profile reminiscent of type III hyperlipoproteinemia. The aim of our study was to obtain a more precise insight into the molecular mechanisms linking apoE to the pathogenesis of LPG. Purification of apoE from a single patient's plasma is hardly feasible, especially because all carders of apoE Sendai are heterozygous for this variation. Thus, we introduced the Arg145->Pro exchange into the wild type apoE3 cDNA using site-specific-mutagenesis. ApoE2, apoE3 and apoE Sendai were expressed in the baculo virus system. LDL receptor binding was studied using recombinant apoE complexed with phospholipid particles and with VLDL from a patient with familiar apoE deficiency. We examined cellular binding, uptake and degradation of these lipoproteins in human skin fibroblasts. ApoE2 and apoE Sendai displayed clearly reduced receptor binding activities compared to apoE3. Heparin binding of apoE Sendai was lower compared to apoE3 but nigher than that of apoE2. Further studies will have to address the question in which way apoE Sendai lead to LPG. Taking into account that several carders of apo E Sendai without any LPG symptoms exist, it is likely that renal factors cooperate with apo E variants in the development of lipoprotein glomernlopathy.
267
1 T h P 1 5 : W 3 0 ] A373P/R451Q CETP mutations, ltDL cholesterol, and risk of ischemic heart disease. The Copenhagen city
heart study B. Agerholm-Larsen A. Tybjaerg-Hansen, P. Schnohr, R. Steffensen, B.G. Nordestgaard. University of Copenhagen, Denmark
Objective: We studied two common mutations in cholesteryl ester transfer protein (CETP) gene, A373P and R451Q; CETP mediates transfer of cholesteryl ester from high-density lipoprotein (HDL) in exchange for triglycerides in apolipoprotein B containing lipoproteins. Methods: We genotyped 8467 healthy women and men from the Danish general population, and 1636 Danish women and men with ischemic heart disease. Results: The prevalence of heterozygous carriers of 373P and 451Q was 0.10 and 0.07, and of homozygous carders 0.003 and 0.002, respectively. All carriers of the 451Q allele also carried the 373P allele. Plasma levels of HDL cholesterol in women (mean 4- SE in lnmol/L) were 1.74 4- 0.01 for non-carriers of 373P, 1.62 4- 0.02 for heterozygous carriers, and 1.38 4- 0.09 for homozygous carriers (ANOVA: P < 0.001). In men, the equivalent values were 1.40 4- 0.01, 1.26 4- 0.02, and 1.19 4- 0.09 (ANOVA: P < 0.001). When we adjusted for age alone, or a group of risk factors, CETP genotype did not predict risk of ischemic heart disease. When adjusting for a group of risk factors plus HDL cholesterol, women not treated with hormonal replacement therapy carrying the 373P allele had a decreased risk of ischemic heart disease (odds ratio: 0.64; 95%CI: 0.43--0.96), whereas the risk of ischemic heart disease in male or treated female carders was statistically unaffected. Conclusions: The A373P/R451Q polymorphism in CETP is associated with decreases in HDL cholesterol of 0.12--0.36 and 0.14-4).21 mmol/L in women and men, and possibly with a paradoxical 36% decrease in risk of ischemic heart disease in women.
ThP16:W30]Familial recessive hypercholesterolemia (FRH): Characterization of 10 homozygotes and 9 obligate heterozygotes G. Zuliani, A. Pacifico I , M. Ciccarese I , P. Pintus2, M. Arca 3 , A. Corsini4, Maioli I , R. Fellin. Department of Internal Medicine 11,
University of Ferrara; 1Department of Internal Medicine, University of Sassari; 2Brotzu Hospital, Cagliari; 3Department of Internal Medicine 11, University of Rome "La Sapienza"; 4Institute of Pharmacological Science, University of Milan, Italy Objective: We described an Italian family with several cases of hypercholesterolemia and/or sudden death. Probands had the clinical waits of homozygous familial hypercholesterolemia (FH), while parents were normal. The involvement of low density lipoprotein receptor (LDL-R) and apo B genes was excluded. LDL-R activity in fibroblasts, and LDL binding to LDL-receptor were normal in probands. We investigated the metabolic mechanisms underlying this new recessive disorder, and found a reduction in the in vivo LDL catabolism caused by a selective reduction in hepatic LDL uptake. We report 3 new Italian families, and describe the characteristics of 10 probands (5 M and 5 F), and 9 parents (5 M and 4 F) from 5 different pedigrees. Methods: Plasma lipids were measured by standardized methods. LDL-R activity was determined by evaluating 125I-LDL internalization/degradation in cultured fibroblasts. LDL binding to LDL-R was measured in vitro by competition experiments in cultured fibroblasts. Results: Probands (age: 13-47 yrs): all had tendinous/tuberous xanthomas, 4 had xanthelasmas. Coronary angiography was pathological in 6 out of 6. Lipoprotein parameters were (range, mg/dL): Tot Chol (450--642), LDL-C (372-592), HDL-C (28-58), Triglycerides (49-208), apo B (207-365), apo A-I (83-137). LDL-R activity in vitro was (range, % of normal): uptake (70-140), degradation (70-150). LDL binding activity in vitro was 84--100% of normal. Parents (age 51-83 yrs): 2 had type 2 diabetes, 2 had hypertension. Nobody had xanthomas or xanthelasmas. Lipoprotein parameters were (range, mg/dL): Tot Chol (172-237), LDL-C (103-153), HDL-C (40-70), Triglycerides (64-180), apo B (88-165), apo A-I (119-173). Conclusions: 1) all FRH homozygotes have the clinical and laboratory features of FH homozygotes despite having normal LDL-R activity in fibroblasts and normal LDL binding activity; 2) all parents (FRH obbligate heterozygotes) show normal phenotype, supporting the hypothesis of an autosomal recessive trasmission of this disease.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000