Genetic Variants of Proprotein
Convertase Subtilisin/Kexin
Type 9 (PCSK9)
Implications for Low-Density Lipoprotein Cholesterol
(LDL-C)
USA-145-100024(1)
© 2014 Amgen Inc. All rights reserved. Not for Reproduction.
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
16.7%
Others
14%
ApoB
2.3%
PCSK9
67%
LDLR
*Autosomal Dominant Hypercholesterolemia form of FH
1. van der Graaf A, et al. Circulation. 2011;123:1167-1173. 2. Seidah NG, et al. J Mol Med. 2007;85:685-696.
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
Decreased LDLR surface concentration
PCSK9 secretion
Lysosomal degradation
LDLR/PCSK9
routed to lysosome
LDL = low-density lipoprotein; LDLR = low-density lipoprotein receptor
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
3. Zhang DW, et al. J Biol Chem. 2007;282:18602-18612.
Regulation of PCSK9 is Dynamic
PCSK9 is produced primarily by
the liver, kidney, and intestine1
Plasma PCSK9
Upregulates PCSK9
• Cholesterol depletion2,3
• Cholestyramine2
• Sterol regulatory elementbinding protein 21,3,4
• Statins3,4
Downregulates PCSK9
• Dietary and cellular cholesterol4
• Long-term fasting1
• Bile acids3,4
1. Horton JD, et al. J Lipid Res. 2009;50:S172-S177. 2. Lopez D. Biochem Biophys Acta. 2008;1781:184191. 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.
4
Genetic Variants of PCSK9 Demonstrate its
Importance in Regulating LDL Levels
PCSK9 Gain of Function = Less LDLRs1
Lysosomal degradation of LDLR
Gain-of-function PCSK9
PCSK9 Loss of Function = More LDLRs1
Recycling of LDLR
Loss-of-function PCSK9
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
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.
PCSK9 GOF Mutations
Clinical Outcomes Associated With Genetic
Mutations for Gain of PCSK9 Function
PCSK9 Function1
FH-associated
physical
abnormalities1
1. Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders. Vol. 2. Oxford, UK: Atlas Medical Publishing Ltd.
2010:3-23.
7
Case Reports Highlight Hypercholesterolemia
Associated With PCSK9 GOF Mutations
F216L mutation1,2
R218S mutation3
French proband
Age: 49 years
French proband presented
with tendinous xanthoma and
arcus corneae
Age: 45 years
TC: 441 mg/dL
TC: 402 mg/dL
LDL-C: 356 mg/dL
LDL-C: 293 mg/dL
Tendon
xanthoma4
TC = total cholesterol
Reprinted from The Lancet, Vol. 362, Durrington P,
Copyright 2003, with permission from Elsevier.
1. Abifadel M, et al. Nat Genet. 2003;34:154-156. 2. Abifadel M, et al. In: Toth PP. The Year in Lipid Disorders.
8
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.
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
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
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.
Adapted from Ouguerram K, et al. Arterioscler Thromb Vasc Biol. 2004;24:1448-1453.
9
PCSK9 GOF Mutations Associated With FH*1
PCSK9
Genotype
Mutation
Type
Biochemical Phenotype
S127R
Missense
5x 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
Missense
Leads to decreased LDLR expression and
activity1
Elevated LDL-C1
R218S
Missense
Normal processing and secretion but loss of
PCSK9 enzymatic activity1
Tendon xanthomas, arcus
corneae4
Tendon xanthomas4
Missense
10‒25x higher affinity for LDLR; decreased
LDLR recycling and increased
degradation1,5
D374Y
Clinical/Biochemical
Phenotype
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
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.
10
PCSK9 LOF Mutations
Clinical Outcomes Associated With Genetic
Mutations for Loss of PCSK9 Function
PCSK9 Function
Reduced plasma
levels of TC
and LDL-C1,2, 3,4
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.
12
PCSK9 LOF Mutations Are Associated With
Decreased Plasma LDL-C Concentrations
Distribution of Plasma LDL-C in
Black Subjects (mg/dL)
30
10
10
0
0
0
50
100
PCSK9Y142X or
PCSK9C679X
(N = 85)
30
150
200
250
300
50th Percentile
0
10
10
0
0
100
150
200
250
300
81% of PCSK9Y142X and PCSK9C679X subjects
had mean plasma LDL-C below 50th percentile
Adapted from Cohen JC, et al. New Engl J Med. 2006;354:1264-1272.
100
150
200
250
300
250
300
50th Percentile
30
20
50
50
PCSK9R46L Allele
(N = 301)
20
0
50th Percentile
No PCSK9R46L
Allele
(N = 9,223)
20
Frequency (%)
Frequency (%)
30
50th Percentile
No Nonsense
Mutation
(N = 3,278)
20
Distribution of Plasma LDL-C in
White Subjects (mg/dL)
0
50
100
150
200
Moderate mean plasma LDL-C-lowering
effect in PCSK9R46L allele carriers
13
PCSK9 LOF Mutations and Variants
Associated With Hypocholesterolemia
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 is
not 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.
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.
14
2010:3-23.
PCSK9 LOF Compound Heterozygote
With No Detectable Circulating PCSK9
Paternal allele: PCSK9ΔR97
(disrupted processing/secretion)
LDL-C: 39 mg/dL
A
LDL-C (mg/dL):
Percentile
Maternal allele: PCSK9Y142X
(disrupted synthesis)
LDL-C: 49 mg/dL
1
2
PCSK9-Y142X
39
<1
B
49
<1
1
C
PCSK9-R97
2
77
21
3
14
<1
1
2
30
<1
3
37
<1
104
61
Compound heterozygote:
No immunodetectable circulating PCSK9
Mutation prevented autocatalytic cleavage and secretion of PCSK9
LDL-C: 14 mg/dL
Adapted from Zhao Z, et al. Am J Hum Genet. 2006;79:514-523.
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)
Normal
Heterozygous
C679C/C679C (CC)
Homozygous*
C679C/C679X (CX)
mean (SD)
C679X/C679X (XX)
mean (SD)
629
1
23
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)
n
Age (y)
*Total deficiency in PCSK9; no adverse clinical sequelae were reported in this individual.
†P < 0.005.
ǂP < 0.001.
Adapted from Hooper AJ, et al. Atherosclerosis. 2007;193:445-448.
16
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
PCSK9 Gain of Function
(GOF):2-5
Less LDLRs
More LDL-C
PCSK9 Loss of Function
(LOF):2,4
More LDLRs
Less LDL-C
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.17
Proc
Natl Acad Sci U S A. 2009;106:9546-9547.