Pharmacogenetics of Statin Therapies

Daniel I. Chasman, Ph.D.Division of Preventive Medicine

Brigham and Women’s Hospital

Johanna and Ralph DeStefano Personalized Health Care

Conference OSU Medical CenterColumbus, OH

Oct 6, 2011

Disclosure

Funding for this research provided by

AstraZenecaCelera

Background and research questions

• BackgroundThere is large inter-individual response to statin therapy as

measured by LDL-C reduction, a strong predictor of risk reduction. Some of this variation may be correlated with genetic variation.

• Research questionsWhat genes, in the entire genome, carry common genetic

variation associated with LDL-C lowering on statin therapy?

What are the magnitudes of these effects?

Are there interactions involving these gene variants?

To what extent do the genetic effects explain variation in inter-individual statin response?

Some previous genetic analyses of LDL-C lowering with statin treatment

• Candidate gene analysis1

HMGCR – target of statin therapy

APOE – major apolipoprotein of VLDL, IDL, chylomicrons

LDLR – LDL receptor

ABCG5/8 – sterol transporters

CYP7A1 – cytochrome P450 family metabolizing enzyme

ABCG2 – transporter in liver, kidney• Genome-wide association studies (GWAS)2

CLMN association in GWAS of PRINCE, CAP, and TNT (pravastatin, simvastatin, atorvastatin)

GRIK4 association in GWAS of TNT (atorvastatin)

SLCO1B1 association myopathy GWAS of SEARCH (simvastatin)

References1JAMA. 2004 291:2821, ATVB 2010 30:1485, Circ. 2008 117:1537; Athero. 2004 175:287; Am J Cardi. 2004 93:104. Athero. 2001 158:183. Circulation Cardiovascular genetics 2010 doi: 10.1161. 2PLoS One. 2010 5:e9763 , N Engl J Med. 2008 ;359:789, Circ Cardio Genet. 2009 2:173.

CYP’s

LDLRAPOEABCG5/8

HMGCR

degradation

hepatocyte

inhibition of cholesterolsynthesis

hepatocyte

effects on cholesteroltransport

hepatocytevascular system

peripheral tissues

Known pharmacologic pathways for statin therapy

uptake

intestinehepatocyte

excretion

hepatocyterenal cells

temporal sequence of statin pharmacology

ABCB1ABCG2

SLCO1B1

Genome-wide association study (GWAS)

• Focus on single nucleotide polymorphisms (SNPs), the most prevalent form of genetic variation in people

• SNPs typically have two alleles, the major allele (≥50% in the population) and the minor allele (<50%)

• In a single experiment, examine all common SNPs at once. For 1% allele frequency, approx. 1 million SNPs.

• Require very stringent significance, e.g. p < 5 x 10-8

• Test for association of the minor allele with LDL-C response among individuals taking statin

Population with genome-wide data from JUPITER

• JUPITER trial enrolled 17,802 participants with LDL-C < 130mg/dL and C-reactive protein (CRP) ≥ 2mg/L for primary prevention with random allocation to rosuvastatin (20 mg/day). Treatment highly effective in this population1

• Genotyping on the Illumina Omni 1M Quad platform by Illumina• 8,782 of the 12,649 JUPITER participants with consent and

genotype had verified European ancestry• Compliance limits sample to 6,934• SNPs excluded when failing Hardy-Weinberg equilibrium test

at P < 10-6, with the exception of rs7412 at APOE (E2 v. E3)• 820,411 SNPs pass QC with minor allele frequency > 1%

1N Engl J Med. 2008 359:2195.

Clinical characteristics of study sample(all European ancestry)

characteristic placebo statin p*

3414 3520

age (yrs) 66.0 (60.0-71.0) 66.0 (60.0-71.0) 0.52

sex (N (%) female) 1086 (31.8) 1112 (31.6) 0.86

BMI (kg/m^2) 28.7 (25.6-32.1) 28.7 (25.8-32.1) 0.76

hypertension (N (%)) 1886 (55.2) 1985 (56.4) 0.35

smoking_cigs (N (%)) 443 (13.0) 460 (13.1) 0.94

LDL-C (mg/dL) 110.0 ( 97.0-120.0) 110.0 ( 96.0-120.0) 0.16

HDL-C (mg/dL) 50.0 (41.0-61.0) 49.0 (41.0-60.0) 0.40

triglycerides (mg/dL) 115.5 (84.0-163.0) 117.0 (85.0-167.0) 0.10

Δ LDL-C (mg/dL) 3.0 (-15.0-7.0) -54.0 (41.0-66.0)

Δ HDL-C (mg/dL) 1.0 (-5.0-3.0) 3.0 (-8.0-1.0)

Δ triglycerides (mg/dL) 0 (-24-25) -18.5 (-3.0-50.0)

Defining LDL-C response to statin therapy

Absolute LDL-C response:

LDL-C at 12 months – LDL-C baseline

Fractional (%) LDL-C response:

LDL-C at 12 months – LDL-C baseline = absolute ΔLDL-C

LDL-C baseline LDL-C baseline

Statistical power: JUPITER sample with genome-wide genetic information is the largest to date with a single statin administered at a single dose

ΔLDL-C (mg/dL) Δ fr. LDL-C (%)

MAF p=0.05 5x10-8 p=0.05 5x10-8

0.05 3.8 8.5 3.7 8.3

0.1 2.8 6.2 2.7 6.0

0.2 2.1 4.7 2.0 4.5

0.5 1.7 3.7 1.6 3.6

Genome-wide association of baseline LDL-C

~820K SNPs

Genome-wide association ofLDL-C lowering with rosuvastatin

< Absolute LDL-C reduction

Fractional LDL-C reduction >

Genome-wide association of LDL-C lowering with placebo

< Absolute LDL-C reduction

Fractional LDL-C reduction >

Magnitude of effects: best SNP at each locus

    absolute LDL-C reduction (mg/dL) fractional LDL-C reduction (%)

chr. pos. gene SNP MAF effect (se) p SNP MAF effect (se) p*

1p32.3 PCSK9 rs17111584 0.05 4.3 (1.4) 5.8E-04 rs11591147 0.03 -4.5 (1.7) 3.1E-04

4q22.1 ABCG2 rs2199936 # 0.11 -5.2 (0.9) 2.1E-12 rs1481012# 0.11 -5.1 (0.9) 1.7E-15

6q26 LPA rs10455872 0.05 6.2 (1.3) 3.5E-09 rs10455872 0.05 6.8 (1.2) 5.0E-15

19q13.32 APOE rs71352238 0.10 4.2 (1.0) 2.9E-04 rs7412 0.15 -5.1 (0.8) 5.8E-19

    baseline LDL-C (mg/dL)

chr. pos. gene SNP MAF effect (se) p

1p32.3 PCSK9 rs11591147 0.03 -5.0 (0.8) 4.7E-11

4q22.1 ABCG2 N.S.

6q26 LPA N.S.

19q13.32 APOE rs7412 0.15 -6.1 (0.4) 1.6E-53

LDL-C lowering

Baseline LDL-C

# high LD

Distribution of effect by genotype

Total genetic effect: proportion of variance explained at genome-wide loci

“●” indicates locus with genome-wide association (p<5x10-8)

For comparison, age, BMI, sex, smoking status, region explain:3.5% of absolute LDL-C response3.7% of fractional LDL-C response

Genes from genome-wide analysis• PCSK9 (chr. 1)

Serine protease with functions in LDLR protein degradation• ABCG2 (chr. 4)

Widely-expressed (hepatic, renal, elsewhere) transporter studied for multi-drug resistance phenotype in chemotherapy (as BCRP). Variation also associated with plasma urate levels. Effects observed in candidate analysis of LDL-C lowering with rosuvastatin*.

• LPA (chr. 6)Apolipoprotein(a) component of Lp(a). Plasma Lp(a) levels almost entirely

determined by genetic variation at LPA. LDL-C includes contribution from cholesterol in Lp(a) particles.

• APOE (chr. 19)Major apolipoprotein component of VLDL, IDL, chylomicrons.

*Circ Cardiovasc Genet. 2010 Jun 1;3(3):276-85.

Validation

No replication, but …• Genome-wide standard of significance (p<5x10-8) imposed• All loci previously recognized in genetics of statin response literature• Winner’s curse probably not a strong influence on effect estimates• Associations not merely due to individuals with extreme LDL-C since

such individuals were excluded by the trial design• No effects at all in placebo

Sub-genome-wide significant loci(5x10-8<P<5x10-6)

absolute LDL-C reduction(mg/dL)

fractional LDL-C reduction(%)

chr. SNP pos maf beta (se)* p* beta (se)* p* genes

2q21.3 rs6730157 135623558 0.35 3.4 (0.6) 3.0E-05 3.7 (0.6) 8.2E-07 RAB3GAP1

6p22.3 rs6924995 16269404 0.21 4.1 (0.7) 5.3E-07 3.8 (2.9) 1.4E-06 IDOL (MYLIP)

6q23.1 rs7769153 131298057 0.03 8.9 (1.9) 1.1E-04 10.3 (1.8) 1.7E-07 EPB41L2

9q22.1 rs1875620 90729879 0.45 2.8 (0.6) 7.2E-07 2.2 (0.6) 3.7E-04 C9orf47, S1PR3, SHC3

19p12 rs931608 22405962 0.12 4.2 (0.9) 2.7E-07 3.6 (0.9) 7.9E-07 LOC342994, ZNF98

IDOL (inducible degrader of LDL receptor)

• IDOL (originally named MYLIP)

Sterol responsive ubiquitin-mediated pathway for post-transcriptional regulation (degradation?) of the LDL receptor1

Regulated by LXRRecently associated with baseline LDL-C2

Candidate therapeutic target for “statin-like” regulation ofLDL-C levels mediated through the LDL receptor

• EPB41LD

Unknown function but shares band 4.1 homology with IDOL

1Science 2009 325:100-1042PLoS Genetics 2009 5:e1000730. Nature 2010 466:707-13.

Candidate associations          absolute LDL-C reduction (mg/dL)   fractional LDL-C reduction (%)

chr gene SNP pos maf effect* (se) p*   effect* (se) p*

5 HMGCR rs17244841 74678611 0.05 1.9 (2.0) 5.00E-01 1.8 (2.0) 5.70E-01

rs17238540 74691254 0.02 1.7 (2.0) 6.20E-01 1.6 (2.0) 8.30E-01

rs12916 74692295 0.39 1.1 (0.6) 3.10E-01 1 (0.6) 3.90E-01

rs6989121 74717529 0.20 1.2 (0.7) 9.7e-02 (9.5e-01) 0.99 (0.7) 1.3e-01 (9.8e-01)

rs104744332 74652599 0.33 1.0 (0.6) 2.6e-01 (1e+00) 1.1 (0.6) 9.5e-02 (9.4e-01)

12 SLCO1B1 rs4149056 21222816 0.16 2.7 (0.8) 1.70E-04 2.6 (0.8) 7.70E-05

rs4363657 21259989 0.17 2.8 (0.8) 1.80E-04 2.8 (0.7) 4.00E-05

rs123172681,2 21243808 0.16 3.2 (0.8) 2.9e-05 (3.8e-03) 3.2 (0.8) 4.1e-06 (5.3e-04)

19 LDLR rs6511720 11063306 0.15 -1.7 (0.8) 1.50E-01 -2.6 (0.8) 4.60E-03

rs688 11088602 0.43 -0.58 (0.6) 5.50E-01 -0.4 (0.6) 9.50E-01

rs1433099 11103658 0.27 0.023 (0.7) 5.90E-01 0.28 (0.6) 6.30E-01

rs116721231 11055823 0.05 4.4 (1.0) 6.6e-04 (2.4e-02) 4 (1) 5.9e-03 (2.0e-01)

    rs116684772 11056030 0.24 -1.9 (0.7) 9.2e-03 (2.9e-01)   -2.1 (0.7) 1.8e-03 (6.4e-02)

No associations at GRIK4, CLMN, APOB, CYP3A5, CYP2C9

1,2locus-wide best SNP for absolute (1) or fractional (2) LDL-C reduction

Interaction analysis

• No interaction among lead SNPs at genome-wide loci• No interaction between lead SNPs and other SNPs across

genome• No interaction with sex• No evidence for conditional associations within top loci• However, evidence for PCSK9 X LDLR interaction with

fractional LDL-C reduction (pint=0.002)

rs11668477 (LDLR)

rs11591147 (PCSK9) non-carrier carrier (~15%)

non-carrier -51.6% -52.0

carrier (~6%) -51.5 -57.0

CYP’s

HMGCR

degradation

hepatocyte

inhibition of cholesterolsynthesis

hepatocyte

effects on cholesteroltransport

hepatocytevascular system

peripheral tissues

APOEPCSK9

LPALDLRIDOL

APOB

Influence of common genetic variation on rosuvastatin therapy in JUPITER

uptake

intestinehepatocyte

SLCO1B1

excretion

hepatocyterenal cells

ABCG2

temporal sequence of statin pharmacology

Predicting LDL-C reductionGenetic score: sum of inherited “risk alleles”

absolute LDL-C response fractional (%) LDL-C response

Effects of genetic score

 beta

(95% CI) R2 OR > median

absolute ΔLDL-C-5.0 (mg/dL)(-6.06- -3.93) 2.3 (%)

1.54(1.41-1.69)

fractional ΔLDL-C-5.5 (%)

(-6.57- -4.5) 3.11.93

(1.75-2.12)

Estimates per unit of score, i.e. per inherited allele

Another candidate (KIF6)

KIF6 gene non-synonymous variant (rs20455, MAF=34.7%). Minor allele (719Arg) has greater CV risk and greater response to atorvastatin (CARE, WOSCOP).

No effect observed in JUPITER. See: Ridker et al. Circ Cardiovasc Genet. 2011 Apr 14. Lack of association may be related to differences between rosuvastatin and other statins

Summary

• In JUPITER, three (3) loci genome-wide significant association for LDL-C reduction with random rosuvastatin (20mg/dL) allocation: ABCG2, LPA, APOE

• An additional locus (PCSK9) for LDL-C reduction arises from genome-wide association with baseline LDL-C.

• Per allele, the lead SNPs are associated with a -5.2 mg/dL (ABCG2) and a +6.2 mg/dL (LPA) change in absolute LDL-C; a -5.1 mg/dL change in fractional LDL-C change (APOE)

• In total, 2.8% and 6.7% of the variance explained by four loci in absolute and fractional LDL-C reduction respectively

• A sub-genome-wide association at IDOL is consistent with current understanding of LDL receptor regulation

• Additional candidate analysis supports a role for variation in SLCO1B1 and LDLR

• A genetic risk score reveals dependence of median LDL-C response on genetics but only explains a small proportion of the variance

• No interaction effects with rosuvastatin observed for KIF6 variant

Collaborators and support

• BWHPaul M Ridker, MD, MPHAudrey Chu, PhDFranco Guilianini, PhDJean MacFadyen, BS

• AstraZenecaFredrik Nyberg, MD, PhD, MPHBryan Barratt, PhD

• SupportAstraZeneca

Celera