genetic variants of proprotein convertase subtilisin/kexin type 9 (pcsk9) implications for...
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Genetic Variants of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)
Implications for Low-Density Lipoprotein Cholesterol (LDL-C)
© 2014 Amgen Inc. All rights reserved. Not for Reproduction. USA-145-100024(1)
PCSK9 Mutations Are Involved in Familial Hypercholesterolemia
• Familial hypercholesterolemia (FH) characterized by1:– Severely elevated LDL-C levels
• Mutations of three genes are primarily responsible for FH*2
1. van der Graaf A, et al. Circulation. 2011;123:1167-1173. 2. Seidah NG, et al. J Mol Med. 2007;85:685-696.
*Autosomal Dominant Hypercholesterolemia form of FH
67%LDLR
16.7%Others
2.3%PCSK9
14%ApoB
2
PCSK9 is a Key Regulator of LDLR Recycling
• PCSK9 mediates degradation of the LDLR by interacting with the extracellular domain and targeting the receptor for degradation1
1. Horton JD, et al. J Lipid Res. 2009;50:S172-S177. 2. Qian YW, et al. J Lipid Res. 2007;48:1488-1498. 3. Zhang DW, et al. J Biol Chem. 2007;282:18602-18612.
LDL = low-density lipoprotein; LDLR = low-density lipoprotein receptor
LDLR/PCSK9 routed to lysosome
Lysosomal degradation
PCSK9 secretion
Decreased LDLR surface concentration
3
Regulation of PCSK9 is Dynamic
1. Horton JD, et al. J Lipid Res. 2009;50:S172-S177. 2. Lopez D. Biochem Biophys Acta. 2008;1781:184-191. 3. Abifadel M, et al. Hum Mutat. 2009;30:supplementary information. 4. Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders. Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010:3-23.
PCSK9 is produced primarily by the liver, kidney, and intestine1
• Dietary and cellular cholesterol4
• Long-term fasting1
• Bile acids3,4
Plasma PCSK9Downregulates PCSK9
Upregulates PCSK9
• Cholesterol depletion2,3
• Cholestyramine2
• Sterol regulatory element-binding protein 21,3,4
• Statins3,4
4
Genetic Variants of PCSK9 Demonstrate its Importance in Regulating LDL Levels
PCSK9 Gain of Function = Less LDLRs1 PCSK9 Loss of Function = More LDLRs1
1. Steinberg D, et al. PNAS. 2009;106:9546-9547. 2. Cohen JC, et al. N Engl J Med. 2006;354:1264-1272. 3. Benn M, et al. J Am Coll Cardiol. 2010;55:2833-2842.
Mutations in the human PCSK9 gene that lead to a loss of PCSK9 function are found in 1% to 3% of the representative populations2,3
Lysosomal degradation of LDLR
Gain-of-function PCSK9 Loss-of-function PCSK9
Recycling of LDLR
PCSK9 GOF Mutations
Clinical Outcomes Associated With Genetic Mutations for Gain of PCSK9 Function
FH-associated physical
abnormalities1
PCSK9 Function1
1. Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders. Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010:3-23.
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Case Reports Highlight Hypercholesterolemia Associated With PCSK9 GOF Mutations
1. Abifadel M, et al. Nat Genet. 2003;34:154-156. 2. Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders. Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010:3-23. 3. Abifadel M, et al. Hum Mutat. 2009;30:520-529. 4. Durrington P. Lancet. 2003;362:717-731.
F216L mutation1,2
French proband
Age: 49 years
TC: 441 mg/dL
LDL-C: 356 mg/dL
R218S mutation3
TC: 402 mg/dL
LDL-C: 293 mg/dL
French proband presented with tendinous xanthoma and
arcus corneae
Age: 45 years
Tendon xanthoma4
TC = total cholesterol
Reprinted from The Lancet, Vol. 362, Durrington P, Copyright 2003, with permission from Elsevier.
8
GOF Mutations Are Associated With Increased ApoB-Containing Lipoproteins in Humans
– PCSK9S127R subjects had a 3-fold increase in apoB-100 vs normal controls, with a 5-fold increase in LDL• Increases in TC concentration of LDL were also observed
Adapted from Ouguerram K, et al. Arterioscler Thromb Vasc Biol. 2004;24:1448-1453.
Subject Lipoprotein Composition (mg/dL)
LDL‡
Subject Genotype TC TG ApoB
S1 PCSK9S127R 241.0 18.0 115.4
S2 PCSK9S127R 301.0 24.0 107.0
FH* subjects 345.0 37.0 217.0
SD 40.0 7.0 22.0
Controls 70.0 12.5 39.2
SD 16.7 3.2 5.0
*Similar trends for increases in TC concentration and apoB-100 were seen in VLDL and IDL but not HDL
9
IDL = intermediate-density lipoprotein; VLDL = very-low-density lipoprotein; HDL = high-density lipoprotein; S1 = subject 1; S2 = subject 2; SD = standard deviation*FH subjects carrying heterozygous LDL-R mutation.
PCSK9 GOF Mutations Associated With FH*1
1. Lopez D. Biochem Biophys Acta. 2008;1781:184-191. 2. Horton JD, et al. J Lipid Res. 2009;50:S172-S177. 3. Abifadel M, et al. Nat Genet. 2003;34:154-156. 4. Abifadel M, et al. Hum Mutat. 2009;30:520-529. 5. Cunningham D, et al. Nat Struct Mol Biol. 2007;14:413-419.
PCSK9 Genotype
Mutation Type
Biochemical Phenotype Clinical/Biochemical Phenotype
S127R Missense5x higher affinity for LDLR; decreased LDLR expression and activity; may interfere with trafficking of LDLR to the cell surface1,2
Cholesterol levels in 90th percentile; tendon xanthomas3
D129G MissenseLeads to decreased LDLR expression and activity1
Elevated LDL-C1
R218S MissenseNormal processing and secretion but loss of PCSK9 enzymatic activity1
Tendon xanthomas, arcus corneae4
D374Y Missense10‒25x higher affinity for LDLR; decreased LDLR recycling and increased degradation1,5
Tendon xanthomas4
Please refer to Lopez et al (2008) and Abifadel et al (2009) for comprehensive lists of PCSK9 mutations and variants.
*Autosomal Dominant Hypercholesterolemia form of FH
10
PCSK9 LOF Mutations
Clinical Outcomes Associated With Genetic Mutations for Loss of PCSK9 Function
1. Abifadel M, et al. Hum Mutat. 2009;30:520-529. 2. Abifadel M, et al. Hum Mutat. 2009;30:supplementary information. 3. Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders. Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010:3-23. 4. Benn M, et al. J Am Coll Cardiol. 2010;55:2833:2842.
Reduced plasma levels of TC
and LDL-C1,2, 3,4
PCSK9 Function
12
PCSK9 LOF Mutations Are Associated With Decreased Plasma LDL-C Concentrations
81% of PCSK9Y142X and PCSK9C679X subjects had mean plasma LDL-C below 50th
percentile
Distribution of Plasma LDL-C in Black Subjects (mg/dL)
Distribution of Plasma LDL-C in White Subjects (mg/dL)
Moderate mean plasma LDL-C-lowering effect in PCSK9R46L allele carriers
30
20
10
0
0 50 100 150 200 250 300
No NonsenseMutation
(N = 3,278)
50th Percentile
Freq
uenc
y (%
)
30
20
10
0
0 50 100 150 200 250 300
PCSK9Y142X or PCSK9C679X(N = 85)
50th Percentile
30
20
10
0
0 50 100 150 200 250 300
No PCSK9R46LAllele
(N = 9,223)
50th Percentile
30
20
10
0
0 50 100 150 200 250 300
PCSK9R46L Allele(N = 301)
50th PercentileFreq
uenc
y (%
)
Adapted from Cohen JC, et al. New Engl J Med. 2006;354:1264-1272. 13
PCSK9 LOF Mutations and Variants Associated With Hypocholesterolemia
1. Lopez D. Biochem Biophys Acta. 2008;1781:184-191. 2. Benn M, et al. J Am Coll Cardiol. 2010;55:2833-2842. 3. Cunningham D, et al. Nat Struct Mol Biol. 2007;14:413-419. 4. Mayne J, et al. Clin Chem. 2011;57:1415-1423. 5. Abifadel M, et al. Hum Mutat. 2009;30:520-529. 6. Zhao Z, et al. Am J Hum Genet. 2006;79:514-523. 7. Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders. Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010:3-23.
PCSK9 Genotype Mutation Type Biochemical Phenotype Clinical/Biochemical Phenotype
R46L Missense Polymorphism
No effect on processing or secretion1
11%‒16% reduction in LDL-C2
G106R Missense Defective protein that isnot secreted1
Reduced LDL-C1
Y142X Nonsense Disrupted protein synthesis resulting in no detectable protein3
40% reduction in LDL-C1
Q152H Missense Defective autocatalytic cleavage and secretion4
48% decrease in LDL-C; 79% decrease in plasma PCSK94
L253F Missense Poorly cleaved and secreted1 30% reduction in LDL-C3,5
A443T Missense Polymorphism
Normally cleaved and secreted; higher susceptibility to cleavage1
Modest (2%) reduction in LDL-C6
Q554E Missense Poorly cleaved and secreted1 Reduced LDL-C7
C679X Nonsense Disrupted protein folding; impaired protein secretion1
40% reduction in LDL-C1
Please refer to Lopez et al (2008) and Abifadel et al (2009) for comprehensive lists of PCSK9 mutations and variants.
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PCSK9 LOF Compound Heterozygote With No Detectable Circulating PCSK9
Adapted from Zhao Z, et al. Am J Hum Genet. 2006;79:514-523.
Paternal allele: PCSK9ΔR97
(disrupted processing/secretion)LDL-C: 39 mg/dL
Maternal allele: PCSK9Y142X
(disrupted synthesis)LDL-C: 49 mg/dL
Compound heterozygote: No immunodetectable circulating PCSK9Mutation prevented autocatalytic cleavage and secretion of PCSK9LDL-C: 14 mg/dL
PCSK9-Y142X
PCSK9-R97LDL-C (mg/dL):Percentile
39< 1
49<1
7721
14< 1
30< 1
37< 1
104 61
1 2
1 2 3
1 2 3
A
B
C
15
PCSK9C679X-Associated Cholesterol Lowering in a Population With Low LDL-C– A total of 653 young black women from Zimbabwe, a population in which
basal LDL-C is already low, were examined
– C679X mutation occurred in 3.7% of subjects (24 out of 653)• One homozygous PCSK9C679X/C679X subject was identified • PCSK9C679X was associated with a 27% reduction in LDL-C
(carriers [heterozygotes] versus noncarriers)
Adapted from Hooper AJ, et al. Atherosclerosis. 2007;193:445-448.
*Total deficiency in PCSK9; no adverse clinical sequelae were reported in this individual.†P < 0.005.ǂP < 0.001.
NormalC679C/C679C (CC)
mean (SD)Homozygous*
C679X/C679X (XX)
HeterozygousC679C/C679X (CX)
mean (SD)
n 629 1 23
Age (y) 24 (5) 21 25 (5)
Cholesterol (mmol/L) 3.6 (0.7) 2.2 3.1 (0.7)†
Triglyceride (mmol/L) 0.7 (0.3) 0.8 0.6 (0.2)
LDL-C (mmol/L) 2.2 (0.7) 0.4 1.6 (0.3)ǂ
HDL-C (mmol/L) 1.2 (0.4) 1.4 1.2 (0.4)
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Summary
–Genetic variants resulting in changes in PCSK9 function provide evidence for the role of PCSK9 in regulating LDLR for cholesterol homeostasis1
• PCSK9 genetic mutations are associated with LDL variances
1. Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders. Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd. 2010:3-23. 2. Horton JD, et al. J Lipid Res. 2009;50:S172-S177. 3. Lakoski SG, et al. J Clin Endocrinol Metab. 2009;94:2537-2543. 4. Abifadel M, et al. Hum Mutat. 2009;30:520-529. 5. Steinberg D, et al. Proc Natl Acad Sci U S A. 2009;106:9546-9547.
PCSK9 Gain of Function (GOF):2-5
Less LDLRsMore LDL-C
PCSK9 Loss of Function (LOF):2,4
More LDLRsLess LDL-C
17