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.

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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

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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

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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

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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

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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

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