LIPOPROTEINE ATEROGENE E ANTI-ATEROGENE ATEROGENE Sebastiano Calandra Dipartimento di Scienze Biomediche Università di Modena e Reggio Emilia
Incidence Rate/1000 200-150 - 100-50 - Women 0 Men <25 25-34 35-44 45-54 55-64 65-74 >75 HDL cholesterol level (mg/dl) Incidence of CHD in the Framingham Heart Study
CAD Relative Risk 1.00 3.21 Low Total Cholesterol (<212mg/dl) 2.41 3.78 High Total Cholesterol (>212mg/dl) Low HDL-C (<47 mg/dl) High HDL-C (>47 mg/dl) Physicians Health Study
128 CAD events (rate/1000/6 yr) 24 94 46 Low HDL-C (<35 mg/dl) High HDL-C (>35 mg/dl) Low Triglycerides (<200 mg/dl) High Triglycerides (>200 mg/dl) PROCAM Study
LOW HDL-CHOLESTEROL (HYPOALPHALIPOPROTEINEMIA) Plasma HDL-cholesterol levels (HDLc) below the 5-10 th percentile in the population. 28-33 mg/dl in males 33-39 mg/dl in females Most frequent lipoprotein abnormality found in patients with premature coronary artery disease
ANTI-ATHEROGENIC PROPERTIES OF HDL
HETEROGENEITY OF PLASMA HDL
REVERSE CHOLESTEROL TRANSPORT AND INTRAVASCULAR HDL REMODELLING Bile Macrophage SR-BI PL A-I TG LCAT A-I PL ABCA1 Liver Kidney
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
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
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
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
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
PRODUCTION OF APO A-I BY LIVER AND INTESTINE Liver Intestine A-I A-I A-II HDL HDL
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 95 62 y HDLc 89 AI 190 72 y HDLc 74 AI 157 66 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 147 4 y HDLc 46 AI 105 HDLc, Apo AI: mg/dl
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 129 57 y HDLc 75 AI 165 50 y HDLc 35 AI 79 53 y HDLc 40 AI 146 16 y HDLc 20 AI 70 28 y HDLc 53 AI 144 25 y HDLc 27 AI 75
Family 4 From Tuscany (Lucca) MI 84 y HDLc 24 Apo AI 69 78 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 62 54 y HDLc 25 Apo AI 76 51 y HDLc 4 Apo AI <5 50 y HDLc 3 Apo AI 6 48 y HDLc 23 Apo AI 74 47 y HDLc 4 Apo AI <5 44 y HDLc 4 Apo AI <5 39 y HDLc 39 Apo AI 115 31 y HDLc 35 Apo AI 131 33 y HDLc 54 Apo AI 133 26 y HDLc 29 Apo AI 85 20 y HDLc 18 Apo AI 57 15 y HDLc 23 Apo AI 76 12 y HDLc 68 Apo AI 152 22 y HDLc 21 Apo AI 69 19 y HDLc 28 Apo AI 81 26 y HDLc 39 Apo AI 109 22 y HDLc 56 Apo AI 127 21 y HDLc 45 Apo AI 123 17 y HDLc 25 Apo AI 79 HDLc mg/dl Apo AI mg/dl
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± 29.9 37.1 ± 19.6 44.5 ± 17.8 NS BMI (Kg/m 2 ) 26.6 ± 7.6 24.6 ± 4.0 25.0 ± 4.0 NS Tc (mg/dl) 177 ± 39 184 ± 48 192 ± 32 NS LDLc (mg/dl) 126 ± 24 142 ± 46 121 ± 28 NS HDLc (mg/dl) 34 ± 9 22 ± 5 54 ± 13 <0.0001 Tg (mg/dl) 90 ± 58 90 ± 51 91 ± 35 NS Apo AI (mg/dl) 89 ±17 67 ±15 138 ±18 <0.0001 Apo B (mg/dl) 86 ±23 92 ±27 84 ±19 NS
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 143-165 aa region.! Limited number of carriers of mutant apo A-I does not allow definite conclusions on CHD risk.
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
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
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
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
REVERSE CHOLESTEROL TRANSPORT AND INTRAVASCULAR HDL REMODELLING Bile Macrophage SR-BI PL A-I TG LCAT A-I PL ABCA1 Liver Kidney
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
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
Angina BP 56 y 66 y 53 y HDL 45 A-I 104 41 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
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 ± 29.9 36.9 ± 19.4 53.2 ± 24.4 43.3 ± 20.0 BMI (kg/m2) 26.6 ± 7.6 24.6 ± 4.0 22.4 ± 1.6 24.0 ± 3.5 TC (mmol/l) 4.42 ± 1.01 4.59 ± 1.30 4.30 ± 1.28 4.99 ± 0.85 LDL-C (mmol/l) 3.13 ± 0.70 3.56 ± 1.21 2.92 ± 1.22 3.06 ± 0.72 HDL-C (mmol/l) 0.89 ± 0.12 0.56 ± 0.14 0.73 ± 0.19 1.40 ± 0.31 TG (mmol/l) 1.01 ± 0.68 1.02 ± 0.59 1.47 ± 0.59 1.19 ± 0.58 Apo A-I (mg/dl) 89.0 ± 9.1 65.8 ± 15.1 86.9 ± 16.8 141.3 ± 18.0 Apo B (mg/dl) 83.5 ± 23.1 89.4 ± 27.4 82.2 ± 25.0 80.0 ± 15.7
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.
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
REVERSE CHOLESTEROL TRANSPORT AND INTRAVASCULAR HDL REMODELLING Bile Macrophage SR-BI PL A-I TG LCAT A-I PL ABCA1 Liver Kidney
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) 5 3 1 2 3 4 5 6 c.321c>a (Y83X) c.343 T>C (S91P) c.209 T>A (V46E) g.968-994 del (X26) c.141-145 del (X76) c.893 C>T (T274I) c.892 A>G (T274A) c.803 G>A (R244H) c.997 G>A (V309M)
Comparison between Italian FED and FLD FED FLD P N. 3M, 1F 10M Age (years) 25.2±8.8 40.6±14.8 Tc (mmol/l) 2.27±0.74 4.66±1.52 0.01 LDLc (mmol/l) 1.41±0.56 2.92±1.50 0.05 HDLc (mmol/l) 0.24±0.08 0.27±0.12 NS Fc/Tc 0.625±0.065 0.920±0.044 0.004 Triglyc. (mmol/l) 1.42±0.54 4.22±2.11 0.04 Apo A-I (mg/dl) 51.2±20.7 37.5±11.5 NS R (nmol/ml/h) 40.5±4.9 0 (n.v. 30-60) LCAT act. (nmol/ml/h) 0 0 NS (n.v. 25-55) LCAT mass (µg/ml) (n.v. 3.1-6.7) 2.18±0.74 1.07±0.64 0.008 Values are mean±sd.
Comparison between Heterozygous Carriers of Italian LCAT mutations and Unaffected Relatives Heterozygotes Normal relatives P N. 20M, 19F 13M, 10F Age (years) 44.0 ± 22.7 40.3 ± 24.3 NS BMI (kg/m 2 ) 24.5 ± 3.9 23.1 ± 3.8 NS Tc (mg/dl) 161.2 ± 37.5 190.2 ± 32.1 0.003 LDLc (mg/dl) 100.5 ± 29.8 117.5 ± 28.6 0.03 HDLc (mg/dl) 35.6 ± 8.5 54.1 ± 9.3 0.0001 Fc/Tc 0.31 ± 0.08 0.25 ± 0.04 0.0001 Triglyc. (mg/dl) 124.6 ± 55.3 108.8 ± 51.7 NS Apo A-I (mg/dl) 106.6 ± 18.5 142.9 ± 14.8 0.0001 Apo A-II (mg/dl) 29.5 ± 5.9 34.6 ± 5.3 0.008 Apo B (mg/dl) 79.0 ± 19.8 84.2 ± 14.8 NS LCAT act. (nmol/ml/h) 26.7 ± 15.4 35.2 ± 12.3 NS LCAT mass (µg/ml) 3.72 ± 1.46 4.41 ± 0.70 0.03 Values (mean±sd) are adjusted for gender, age and BMI.
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)
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.
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
HDL and Reverse Cholesterol Transport Bile Liver SR-BI A-I Mature HDL LCAT A-I Nascent ABCA1 HDL Macrophage
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
ABCA1-mediated cholesterol efflux Lipid free Apo A-I apoa-i C Nascent HDL C ABCA1 C ABCA1 ABCA1 ER/Golgi
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