LIPOPROTEINE ATEROGENE E ANTI-ATEROGENE ATEROGENE

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1 LIPOPROTEINE ATEROGENE E ANTI-ATEROGENE ATEROGENE Sebastiano Calandra Dipartimento di Scienze Biomediche Università di Modena e Reggio Emilia

2 Incidence Rate/ Women 0 Men < >75 HDL cholesterol level (mg/dl) Incidence of CHD in the Framingham Heart Study

3 CAD Relative Risk Low Total Cholesterol (<212mg/dl) High Total Cholesterol (>212mg/dl) Low HDL-C (<47 mg/dl) High HDL-C (>47 mg/dl) Physicians Health Study

4 128 CAD events (rate/1000/6 yr) Low HDL-C (<35 mg/dl) High HDL-C (>35 mg/dl) Low Triglycerides (<200 mg/dl) High Triglycerides (>200 mg/dl) PROCAM Study

5 LOW HDL-CHOLESTEROL (HYPOALPHALIPOPROTEINEMIA) Plasma HDL-cholesterol levels (HDLc) below the 5-10 th percentile in the population mg/dl in males mg/dl in females Most frequent lipoprotein abnormality found in patients with premature coronary artery disease

6 ANTI-ATHEROGENIC PROPERTIES OF HDL

7 HETEROGENEITY OF PLASMA HDL

8 REVERSE CHOLESTEROL TRANSPORT AND INTRAVASCULAR HDL REMODELLING Bile Macrophage SR-BI PL A-I TG LCAT A-I PL ABCA1 Liver Kidney

9 REVERSE CHOLESTEROL TRANSPORT AND INTRAVASCULAR HDL REMODELLING Bile Liver LDLR SR-BI A-I TP LCAT A-I Macrophage ABCA1 B TG VLDL/LDL Kidney

10 liver LDL-R LRP Apo B Apo E VLDL IDL LDL LPL PL Apo s FFA Cubilin kidney SR-B1 TG TP Bile acids Large HDL HL HDL2 LCAT SR-B1 adrenal PLTP HDL3 TGRL LPL Apo AII Apo AI LCAT Apo AI PL ABCA1 pre-ßhdl peripheral cells

11 CANDIDATE GENES IN PLASMA HDL DEFICIENCY (Hypoalphalipoproteinemia) CANDIDATE GENE Apo A-I Apo A-II ABCA1 LCAT LPL LPDL * PLTP * ABCC6 NPC1/NPC2 GBA FUNCTION IN HDL METABOLISM HDL structure, LCAT activation HDL structure HDL formation/ metabolism HDL remodelling HDL formation HDL formation HDL remodelling Unknown Unknown Unknown * No defects yet identfied in humans

12 CRITERIA FOR SELECTION OF PRIMARY HYPOALPHALIPOPROTEINEMIA HDL-C <5th percentile for age and gender Absence of secondary lipid disorders High likelihood of inherited low HDL-C (primary low HDL-C in at least one first-degree family member) 100 PROBANDS/FAMILIES UNDER INVESTIGATION

13 CANDIDATE GENES IN PLASMA HDL DEFICIENCY (Hypoalphalipoproteinemia) CANDIDATE GENE Apo A-I Apo A-II ABCA1 LCAT LPL LPDL PLTP ABCC6 NPC1/NPC2 GBA FUNCTION IN HDL METABOLISM HDL structure, LCAT activation HDL structure HDL formation/ metabolism HDL remodelling HDL formation HDL formation HDL remodelling Unknown Unknown Unknown

14 PRODUCTION OF APO A-I BY LIVER AND INTESTINE Liver Intestine A-I A-I A-II HDL HDL

15 Family 1 From Piedmont c.85 del C > 11X MI at 56 y 99 y From Venetia Julia Leukaemia at 69 y 60 y HDLc 42 AI y HDLc 89 AI y HDLc 74 AI y HDLc 38 AI 99 Gx, px, 3V-CAD, 4BP Gx, px 37 y HDLc 8 AI 0 32 y HDLc 7 AI 0 36 y HDLc 68 AI y HDLc 46 AI 105 HDLc, Apo AI: mg/dl

16 Family 2 From Piedmont (Turin) c.494 T>G (L141R) PVD MI MI MI MI MI CA-ATS AO-ATS 26 y HDLc 9 42 y HDLc 51 AI y HDLc 75 AI y HDLc 35 AI y HDLc 40 AI y HDLc 20 AI y HDLc 53 AI y HDLc 27 AI 75

17 Family 4 From Tuscany (Lucca) MI 84 y HDLc 24 Apo AI y HDLc 22 Apo AI 47 c.85 del C > 11X c.494 T>G (L141R) CAD CAD CAD MI BP CAD 57 y HDLc 13 Apo AI y HDLc 25 Apo AI y HDLc 4 Apo AI <5 50 y HDLc 3 Apo AI 6 48 y HDLc 23 Apo AI y HDLc 4 Apo AI <5 44 y HDLc 4 Apo AI <5 39 y HDLc 39 Apo AI y HDLc 35 Apo AI y HDLc 54 Apo AI y HDLc 29 Apo AI y HDLc 18 Apo AI y HDLc 23 Apo AI y HDLc 68 Apo AI y HDLc 21 Apo AI y HDLc 28 Apo AI y HDLc 39 Apo AI y HDLc 56 Apo AI y HDLc 45 Apo AI y HDLc 25 Apo AI 79 HDLc mg/dl Apo AI mg/dl

18 LIPID PROFILE IN CARRIERS OF APO AI MUTATIONS AND IN UNAFFECTED RELATIVES Mutation c.85 del C L141R Unaffected P* N. 3M, 4F 4M, 7F 11M, 11F Age (years) 43.4± ± ± 17.8 NS BMI (Kg/m 2 ) 26.6 ± ± ± 4.0 NS Tc (mg/dl) 177 ± ± ± 32 NS LDLc (mg/dl) 126 ± ± ± 28 NS HDLc (mg/dl) 34 ± 9 22 ± 5 54 ± 13 < Tg (mg/dl) 90 ± ± ± 35 NS Apo AI (mg/dl) 89 ±17 67 ± ±18 < Apo B (mg/dl) 86 ±23 92 ±27 84 ±19 NS

19 CONCLUSIONS (I)! The presence of two mutant alleles of A-I gene is the cause of severe HDL and apo A-I deficiency and appears to be associated with pcad at least in some kindred.! Heterozygotes for asynthetic apo A-I mutations have higher HDL and apo A-I levels than heterozygotes for missense apo A-I mutations located in aa region.! Limited number of carriers of mutant apo A-I does not allow definite conclusions on CHD risk.

20 APO A-I MUTATION L178P AND CHD n. 54 carriers of mutant apo A-I L178P have: " reduced apo A-I (- 50%) " reduced HDL-C (- 63%) " increased carotid IMT " increased CHD risk (20 fold) Kastelein et al. 2004

21 MUTANT APO A-I AND CHD 1. Associated with increased CHD risk L141R (?), L159P (?), L178P 2. No effect on CHD risk L144R 3. Associated with CHD protection R173C (A-I Milano) Main variables to be considered: a) Site of mutation in apo A-I b) Type of mutation (asynthetic vs. missense) c) Type of amino acid substitution d) Genotype (homozygous vs. heterozygous) e) Plasma level of apo A-I and HDL-C f) Subclasses of plasma HDL

22 CANDIDATE GENES IN PLASMA HDL DEFICIENCY (Hypoalphalipoproteinemia) CANDIDATE GENE Apo A-I Apo A-II ABCA1 LCAT LPL LPDL PLTP ABCC6 NPC1/NPC2 GBA FUNCTION IN HDL METABOLISM HDL structure, LCAT activation HDL structure HDL formation/ metabolism HDL remodelling HDL formation HDL formation HDL remodelling Unknown Unknown Unknown

23 Intracellular Extracellular synthesis recycling from LPs droplet PL PL PL ABCA1 PL PL PL Lipid-poor Apo AI PL+ PL+ * * * * * * * * * * * * * * PL recycling LCAT PL nascent HDL -rich HDL LDL VLDL * * * * TP TgTg Tg Tg Tg Tg PL

24 REVERSE CHOLESTEROL TRANSPORT AND INTRAVASCULAR HDL REMODELLING Bile Macrophage SR-BI PL A-I TG LCAT A-I PL ABCA1 Liver Kidney

25 E89 W685 L739 L745 F794 L821 Hydrophobic region P1370 V1655 L1740 S1764 M1822 N1852 V70 I703 V717 W767 F773 E843 F1348 I1677 I1718 F1786 N1800 R1873 NH2 Y46 A NBD-1 G933-T940 B A B V2261 COOH NBD-2 G1946-S1953

26 GENETIC DEFECTS OF ABCA1 TRANSPORTER (Italian series) PHENOTYPE Analphalipoproteinemia (recessive) Severe hypoalphalipoproteinemia (recessive) (Tangier Disease) Hypoalphalipoproteinemia (co-dominant) (Familial HDL Deficiency) N 5 6 N of Mutations 12

27 Angina BP 56 y 66 y 53 y HDL 45 A-I y 39 y 37 y 33 y HDL 46 A-I 141 HDL 30 A-I 93 HDL 29 A-I 85 HDL 38 A-I 89 ABCA1 (c.5703 A>C; R1901S) ABCA1 (del. C (A); H160>FsX173) 16 y 10 y HDL 7 HDL 38 A-I 12 A-I 103

28 CARRIERS OF APO A-I AND ABCA1 GENE MUTATIONS c.85delc Apo A-I L141R Apo A-I ABCA1 mutations Unaffected N subjects 3 M, 4 F 4 M, 7 F 6 M, 3 F 24 M, 31 F Age (years) 43.4 ± ± ± ± 20.0 BMI (kg/m2) 26.6 ± ± ± ± 3.5 TC (mmol/l) 4.42 ± ± ± ± 0.85 LDL-C (mmol/l) 3.13 ± ± ± ± 0.72 HDL-C (mmol/l) 0.89 ± ± ± ± 0.31 TG (mmol/l) 1.01 ± ± ± ± 0.58 Apo A-I (mg/dl) 89.0 ± ± ± ± 18.0 Apo B (mg/dl) 83.5 ± ± ± ± 15.7

29 CONCLUSIONS (II)! The presence of two mutant alleles of ABCA1 gene is associated with a severe HDL deficiency and pchd (in adults).! Heterozygotes for ABCA1 gene mutations are identified primarily for the presence of pchd (selection bias).! ABCA1 mutations may co-exist with mutations in other genes involved in lipoprotein metabolism.

30 CANDIDATE GENES IN PLASMA HDL DEFICIENCY (Hypoalphalipoproteinemia) CANDIDATE GENE Apo A-I Apo A-II ABCA1 LCAT LPL LPDL PLTP ABCC6 NPC1/NPC2 GBA FUNCTION IN HDL METABOLISM HDL structure, LCAT activation HDL structure HDL formation/ metabolism HDL remodelling HDL formation HDL formation HDL remodelling Unknown Unknown Unknown

31 REVERSE CHOLESTEROL TRANSPORT AND INTRAVASCULAR HDL REMODELLING Bile Macrophage SR-BI PL A-I TG LCAT A-I PL ABCA1 Liver Kidney

32 c.35 C>T (T-13M) c.31 del G (X-14) c.511 C>T (R147W) c.490 C>T (R140C) c.493 G>A (A141T) c.694 T>A (S208T) c.614 G>A (S181N) LCAT gene (16q 22.1) c.726 G>C (K218N) c.321c>a (Y83X) c.343 T>C (S91P) c.209 T>A (V46E) g del (X26) c del (X76) c.893 C>T (T274I) c.892 A>G (T274A) c.803 G>A (R244H) c.997 G>A (V309M)

33 Comparison between Italian FED and FLD FED FLD P N. 3M, 1F 10M Age (years) 25.2± ±14.8 Tc (mmol/l) 2.27± ± LDLc (mmol/l) 1.41± ± HDLc (mmol/l) 0.24± ±0.12 NS Fc/Tc 0.625± ± Triglyc. (mmol/l) 1.42± ± Apo A-I (mg/dl) 51.2± ±11.5 NS R (nmol/ml/h) 40.5±4.9 0 (n.v ) LCAT act. (nmol/ml/h) 0 0 NS (n.v ) LCAT mass (µg/ml) (n.v ) 2.18± ± Values are mean±sd.

34 Comparison between Heterozygous Carriers of Italian LCAT mutations and Unaffected Relatives Heterozygotes Normal relatives P N. 20M, 19F 13M, 10F Age (years) 44.0 ± ± 24.3 NS BMI (kg/m 2 ) 24.5 ± ± 3.8 NS Tc (mg/dl) ± ± LDLc (mg/dl) ± ± HDLc (mg/dl) 35.6 ± ± Fc/Tc 0.31 ± ± Triglyc. (mg/dl) ± ± 51.7 NS Apo A-I (mg/dl) ± ± Apo A-II (mg/dl) 29.5 ± ± Apo B (mg/dl) 79.0 ± ± 14.8 NS LCAT act. (nmol/ml/h) 26.7 ± ± 12.3 NS LCAT mass (µg/ml) 3.72 ± ± Values (mean±sd) are adjusted for gender, age and BMI.

35 CHD AND PRE-CLINICAL ATHEROSCLEROSIS IN SUBJECTS WITH LCAT DEFECTS # HOMOZYGOTES Familial LCAT deficiency Fish eye disease No evidence of CHD or pre-clinical atherosclerosis # HETEROZYGOTES No clinical phenotype No evidence of pre-clinical atherosclerosis (average IMT and maximum IMT lower that in controls)

36 UNANSWERED QUESTIONS We need to understand more about:! Factors which regulate the concentration and subpopulation of HDL.! HDL function in vivo.! Relationship between HDL concentration and cardioprotective function.! Subpopulations of HDL and cardioprotection.! HDL as target for therapy recommended HDL level.

37 HDL WORKING GROUP GENETICS MOLECULAR PATHOLOGY Averna M. (Palermo) Bertolini S. (Genova) Calandra S. (Modena) Cortese C. (Roma) BIOCHEMISTRY LL BIOLOLOGY Bellotti V. (Pavia) Bernini F. (Parma) Calabresi L. (Milano) Franceschini G. (Milano) VASCULAR STUDIES Baldassarre D. (Milano) Mannarino E. (Perugia) CASE FINDING FAMILY STUDIES Italian Lipid Clinic Network

38 HDL and Reverse Cholesterol Transport Bile Liver SR-BI A-I Mature HDL LCAT A-I Nascent ABCA1 HDL Macrophage

39 CANDIDATE GENES, PLASMA HDL AND CARDIOVASCULAR DISEASE Variations in plasma HDL level are at least 50% genetically determined: At least 15 genes involved Rare mutations of major candidate genes Many gene polymorphisms Interactions of a few major candidate genes

40 ABCA1-mediated cholesterol efflux Lipid free Apo A-I apoa-i C Nascent HDL C ABCA1 C ABCA1 ABCA1 ER/Golgi

41 Angina 59 y, 1V-CAD, PTCA, CA-ATS LDLc Tg HDLc Apo AI Apo B Apo E 62 y 105 mg/dl 194 mg/dl 28 mg/dl 102 mg/dl 92 mg/dl ε3ε3 ABCA1 Exon 49 c.6588 G>C Q2196H

High density lipoprotein metabolism

High density lipoprotein metabolism Lipoprotein classes and atherosclerosis Chylomicrons, VLDL, and their catabolic remnants Pro-atherogenic LDL HDL Anti-atherogenic Plasma lipid transport Liver VLDL FC