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SUPPLEMENTAL DIGITAL CONTENT 1
Supplementary Methods
Setting
Selection of controls
PCR and sequencing methods
In vitro functional assessment of SLCO1B1 rs4149056 variant
Genome-Wide Association Screen: Subjects and selection of SNPs
Re-genotyping in cases and controls
Statistical analysis
Supplementary Results
Supplementary Table 1: Characteristics of rhabdomyolysis case subjects who did and did
not participate in the study
Supplementary Table 2: Primers and PCR conditions for CYP2C8 sequencing
Supplementary Table 3: Primers and PCR conditions for SLCO1B1 sequencing
Supplementary Table 4: Primers and PCR conditions for a) UGT1A1 and b) UGT1A3
sequencing
Supplementary Table 5: Characteristics of GWA screen study subjects
Supplementary Table 6. SNPS selected for genotyping by gene, type, platform and
outcome
Supplementary Table 7. SLCO1B1 tagSNP, FSNP and novel SNP analysis. Models
adjusted for age at statin use, sex, and race.
Supplementary Table 8. CYP2C8 tagSNP, FSNP and novel SNP analysis. Models
adjusted for age at statin use, sex, and race.
Supplementary Table 9. UGT1A1 and 1A3 tagSNP, FSNP and novel SNP analysis.
Models adjusted for age at statin use, sex, and race.
Supplementary Table 10. Distribution of genotype by type of statin among controls
Supplementary Figure 1. Rhabdomyolysis case subject recruitment
Supplementary References
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Supplementary Methods
Setting
The HVH study is a set of inter-related case-control studies at Group Health Cooperative,
Seattle. In the HVH study, the cases include members who have had an incident
myocardial infarction, stroke, venous thromboembolism, or an episode of atrial
fibrillation [1-3]. Controls are a stratified random sample of Group Health members,
matched to the myocardial infarction cases by age, sex, calendar year and hypertension
status. Available data from the HVH study included medical record reviews, telephone
interviews, medication information, and DNA from venous blood samples. The CHS is a
NHLBI-funded cohort study of risk factors for cardiovascular disease in adults aged 65
years and older in four US communities. In CHS, subjects were recruited from random
samples of Medicare eligibility lists in 4 US communities: Hagerstown MD, Pittsburgh
PA, Sacramento CA, or Winston-Salem NC [4]. Available data from the CHS study
included medical histories, risk factor information, medication data, and DNA from
venous blood samples.
Selection of controls
We attempted to frequency match HVH controls to the rhabdomyolysis cases by age and
sex at approximately a ratio of 2 to 1. However, there were too few young female statin
using controls. We instead selected additional young male statin users to try and reach the
desired case-control matching ratio within decade of age. This resulted in a sex inbalance
between cases and HVH controls.
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PCR and Sequencing Methods
DNA samples were PCR amplified using the primers listed in supplementary tables 2-4.
PCR primers were designed using Primer 3 (http://frodo.wi.mit.edu) and the amplicons
cover the exons, intron-exon boundaries, promoter and the UTR regions of CYP2C8,
SLCO1B1, UGT1A1 and UGT1A3 gene. There are 3 general PCR conditions used for
all the assays: the P601X35, Betaine P601X35, and Q561X35 conditions. The P601X35
protocol started with 4 ng of genomic DNA incubated in a 10 l reaction composed of 1
µl each of the Forward and Reverse primers (both at 1µM), 0.7 l of 50 mM MgCl2, 0.4
l of 2.5 mM dNTP, 0.15U of Taq polymerase, and 1 l of 10X Taq Buffer (Buffer and
enzyme are from Invitrogen Corporation, Carlsbad, CA) with cycling conditions of
denaturation at 95°C for 2 min followed by 35 cycles of 92°C for 10 sec, annealing at
60°C for 20 sec, and extension at 72°C for 1 min. At the end of the 35 cycles, the
reaction mixture was held at 72°C for 10 min before being cooled to 4°C until the next
step. With the Betaine P60X135 protocol, 4 ng of genomic DNA was incubated in a 10
l reaction composed of 2.5 µl each of the Forward and Reverse primers (both at 1 µM),
0.42 l of 50 mM MgCl2, 0.42 l of 5M Betaine (Sigma-Aldrich Corporation, St. Louis,
MO), 0.16 l of 2.5 mM dNTP, 0.15U of Taq polymerase, and 0.83 l of 10X Taq
Buffer, with the same cycling conditions as the P601X35 protocol. The Q561X35
protocol started with 8 ng of genomic DNA that was incubated in a 10 l reaction
composed of 2 µl each of the Forward and Reverse primers (both at 1M), 0.4 l of 2.5
mM dNTP, 0.3U of Qiagen Taq polymerase, 1 l of 10X Buffer, and 2 l of Q-mix
(Buffer, Q-Mix, and enzyme are from the Qiagen hot start kit (Qiagen, Inc., Valencia,
CA) with cycling conditions of denaturation at 95°C for 5 min followed by 35 cycles of
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94°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 1 min. At the end
of the 35 cycles, the reaction mixture was held at 72°C for 10 min before being cooled to
4°C until the next step.
The 10 l PCR product was purified by incubation with 0.4 l of PCR Clean-up Reagent
and 3.6 l of PCR Clean-Up Dilution Buffer (both from PerkinElmer Life Sciences, Inc.,
Boston, MA) at 37°C for 1 hour followed by enzyme inactivation at 90°C for 15 minutes.
The purified PCR product was sequenced using Applied Biosystems PRISM BigDye
terminator sequencing Version 3.1 on an ABI Prism 3730xl DNA analyzer (Applied
Biosystems, Inc., Foster City, CA). The 12 l sequencing reaction was composed of 2.5
l of purified PCR product, 4.5 l of 1 µM sequencing primer, 1 l BigDyeV3.1, 2 l of
5X buffer, and 2 l water. Cycling conditions were 96°C for 2 min, 25 cycles of 96°C
for 15 sec, 50°C for 1 sec, 60°C for 4 minutes. After sequencing, the DNA sequence files
were imported into and aligned with SEQUENCHER 4.6 (Gene Codes Corporation, Ann
Arbor, MI) for variant analysis.
In vitro functional assessment of SLCO1B1 variant (p.Val174Ala)
The SLCO1B1 reference cDNA was cloned from human liver tissue and inserted into the
pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) and subsequently inserted into
pcDNA5/FRT vector (Invitrogen). A plasmid containing the variant c.521T>C
(p.Val174Ala, rs4149056) was constructed by site-directed mutagenesis using a
QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the
manufacturer’s protocol. The following primer sequences for the site-directed
mutagenesis were designed using QuickChange Primer Design Program
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(http://www.stratagene.com/sdmdesigner/default.aspx) from Stratagene: 5’ACATGTGGATATATGCGTTCATGGGTAATAT-3’ and 3’TGTACACCTATATACGCAAGTACCCATTATA-5’. PCR for the site-directed
mutagenesis (SDM) was performed according to the following conditions: 30 sec at
95°C for denaturation/activation followed by 15 cycles of denaturation at 95°C for 30
sec, 1 min annealing at 55°C and extension at 68°C for 14.5 minutes. The SDM product
was digested by addition of 1 l of Dpn I enzyme and incubated for 1 hour at 37°C to
remove the methylated reference plasmid. The Dpn I digested product was transformed
via XL1-Blue Supercompetent cells from Strategene and each transformation reaction
was plated on LB-ampicillin (100 g/ml ampicillin) agar plates and incubated overnight
at 37°C. Colonies were selected and purified plasmid obtained by QIAfilter Plasmid
Midi Kit (QIAGEN, Inc., Valencia, CA) was sequenced using ABI PRISM BigDye
terminator sequencing Version 3.1 on an ABI Prism 3730xl DNA analyzer (Applied
Biosystems, Inc., Foster City, CA) to verify the insertion of the variant. After
sequencing, the DNA sequence files were imported into and aligned with
SEQUENCHER 4.8 (Gene Codes Corporation, Ann Arbor, MI) for variant analysis.
Human embryonic kidney epithelial Flp-In (HEK293/FRT) cells (Invitrogen) were stably
transfected with pcDNA5/FRT, pcDNA5/FRT/SLCO1B1 and pcDNA5/FRT/SLCO1B1c.521T>C vectors using FuGENE 6 transfection reagent (Roche Applied Sciences,
Mannheim, Germany). Briefly, on the day before transfection, 1.5 x 105 HEK293/FRT
cells were seeded in a BD multiwell 24-well plate (BD Biosciences Discover Labware,
Bedford, MA) and incubated for 24 hr in 250 l of Dulbecco’s Modified Eagle Medium
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4.5% glucose (DMEM-H-21; UCSF Cell Culture Facility, San Francisco, CA) and 10%
heat inactivated fetal bovine serum (UCSF Cell Culture Facility) growth media. The
next day cells were transfected with a DNA:FuGENE 6 complex containing 80 ng of
vector, 720 ng pOG44, a Flp-recombinase expression vector (Invitrogen), 2.4 l
FuGENE 6 and 17.6 l Opti-MEM (UCSF Cell Culture Facility). The cells were
incubated at 37°C, >95% relative humidity and 5% CO2 for 24 hr before they were split.
Selection media containing 150 g/ml of hygromycin (Invitrogen), 100 g/ml Zeocin
(Invitrogen), 1% penicillin and streptomycin (UCSF Cell Culture Facility) and 89%
DMEM-H21 was added 48 hr post transfection. Colonies were isolated and screened for
the expression of SLCO1B1. Briefly, RNA was isolated from each individual colony
using a RNeasy Plus Micro Kit (Qiagen) per the manufacturer’s protocol. The isolated
RNA was used to make cDNA via iScript cDNA Synthesis kit (Bio-Rad Laboratories,
Hercules, CA) per manufacturer’s protocol followed by quantitative PCR (qPCR).
The qPCR protocol started with 2 l of cDNA incubated in a 11 l total reaction
composed of 0.5 µl each of the Forward (5’-TCTTCTCTTGTTGGTTTTATTGACG-3’)
and Reverse primers (3’-TCCCATAATGAAACAACCGATTC-5’) (both at 1 µM), 5 l
of Power SYBR Green PCR Master Mix (Applied Biosystems) and 3 l of distilled
nuclease free water. The qPCR reaction was transferred to Applied Biosystems
Prism7900HT Real-Time PCR Systems (Applied Biosystems) with cycling conditions of
denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec denaturations,
annealing/extension at 60°C for 1 min for data collection. The qPCR data was analyzed
by 7900HT Version 2.3 Sequence Detection Systems (Applied Biosystems).
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Stably transfected HEK293/FRT cells expressing the empty vector, SLCO1B1 reference
and c.521T>C variant (Val174Ala) were plated onto poly-D-lysine-coated 24-well plates
(BD Biosciences Discover Labware). Cerivastatin (CER) cellular accumulation studies
were performed 24 hr post cell seeding. The accumulation study started with first
aspirating the media and washing the cells two times followed by 15 min incubation with
warm Krebs-Henseleit buffer (UCSF Cell Culture Facility). Following the removal of
the buffer, cells were incubated with 5 nM [3H]-cerivastatin (American Radiolabeled
Chemicals, St. Louis, MO) for a 5 min accumulation study at 37°C, >95% relative
humidity and 5% CO2 incubator. Accumulation was stopped by removing CER and
washing the cells three times with ice-cold Krebs-Henseleit buffer. The cells were lysed
by addition of 500 l/well of lysis buffer composed of 0.1 N NaOH and 0.1% sodium
dodecyl sulfate. The intracellular concentration of CER was measured via liquid
scintillation counting by transferring 400 l of the lysate to 2 ml Ecolite scintillation
fluid (MP Biomedicals, Irvine, CA) in a Mini-Scintillation vial (Denville Scientific,
Metuchen, NJ). The disintegration per minute (dpm) value for each sample, measured
by LS-6500 Multi-Purpose Scintillation Counter (Beckman Coulter, Brea, CA), were
normalized to the sample protein concentration measured using a BCA protein assay kit
(Pierce Biotechnology Inc, Rockford, IL).
Genome-Wide Association Screen
Study subjects of African descent were excluded from the GWA screen. Rhabdomyolysis
subjects with missing information on race were assumed to be of European descent. The
CHS GWA controls included CHS candidate gene controls who were not of African
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descent plus other CHS statin users who were not part of the candidate gene study but
had genome-wide data available (n=645).
Logistic regression models with robust standard errors were used to estimate the
association for each SNP. SNPs were modeled additively. Models were adjusted for age
and sex. 292,461 SNPs with minor allele frequencies ≥5% were analyzed. The genomic
control lambda was 1.029. The one expected false positive threshold was 1/292,461 or
3.42E-06. We used a less conservative threshold for re-genotyping due to concerns about
bias caused by chip, batch or center effects.
Re-genotyping in cases and controls
Ten of the 96 SNPs attempted on the Illumina platform failed. Nine samples with call
rates <95% after removing the failed SNPs were excluded. Excluding failed samples and
SNPs, the Illumina call rate was 99.92%. Thirteen of the 83 SNPs attempted failed
TaqMan and pyrosequencing efforts. An additional four SNPs with
TaqMan/pyrosequencing call rates <95% were considered failures. Fifteen subject
samples with TaqMan/pyrosequencing call rates <95% were excluded. Excluding failed
samples and SNPs, the TaqMan/pyrosequencing call rate was 99.58%. Among the nonfailed SNPs, several were monomorphic in the candidate gene subjects and were
excluded from the analysis. SNPs that were highly correlated with another SNP were also
dropped. A description of the SNPs we attempted to genotype and their outcome is
detailed in Supplementary Table 6.
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Statistical analysis
Six rhabdomyolysis cases with missing race information were assumed to be of European
descent. Case and control status was permuted 1000 times for each gene to obtain the
permutation p-values.
Supplementary Results
Complete results for the candidate genes SLCO1B1, CYP2C8, and the UGTs are
described in Supplementary Tables 7, 8 and 9. There was no association of UGT
tagSNPs, F-SNPs or novel SNPs with cerivastatin-associated rhabdomyolysis
(permutation p-value=0.523). Results remained null for the UGTs when analyses were
limited to definite cases, and subjects who didn’t use gemfibrozil.
Other investigators have reported variation in the genes underlying heritable metabolic
muscle diseases, such McArdles disease, carnitine palmitoyltransferase II deficiency, and
myoadenylate deaminase deficiency to be risk factors for statin-associated
rhabdomyolysis [5]. We only had GWA data for only 1 variant related to these diseases
(rs17602729, AMPD1) and it was not associated with rhabdomyolysis (p=0.831).
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Supplementary Table 1: Characteristics of rhabdomyolysis case subjects
who did and did not participate in the study
Rhabdomyolysis cases
Rhabdomyolysis subjects
included in the study
not included in the study*
N=185
N=770
Female, %
61.1
58.6
Age, mean (std. dev.)
63.5 (10.6)
66.4 (11.49)
Age, range
34-89
29-94
European descent, %
90.8
85.6
*Of the 770 subjects, 12 were missing information on sex, 206 were missing
information on age, and 603 were missing information on race.
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Supplementary Table 2: Primers and PCR conditions for CYP2C8 sequencing
Primer Sequence
Amplicon
Size (base
Name
Covers
P1
P2
pairs)
Amplicon 1
Pro,3'UTR
GAACCCCAATGGGTATCAGAA
CAGCTGTGAGTTCACTCC
967
Amplicon 2
Ex 1, 3'UTR
CACACACTAAATTAGCAGGGAGTG
TTCAGAGGGAGTATTTTGCTTTACAA
379
Amplicon 3
Ex 2, 3
GGCACATCACAGGCCATCTA
CCTACACCCTATGAACCAACACA
890
Amplicon 4
Ex 4
GGTCCCCAACTTTTCTCTTCC
ACCCCTTGCACTTCTGATGG
795
Amplicon 5
Ex 5
TTTCCCTTCAAAATGGACATGA
TGAAACCTTTCTTCTGTTCCACA
743
Amplicon 6
Ex 6
ATGAAGGCCATTGCCAGAAG
TGCTGGCTCTCCTTACCACA
846
Amplicon 7
Ex 7
TGGCTGGTTGTACTTCTGGAC
TGCATGAACATGTTAAGTCTTTCC
651
Amplicon 8
Ex 8
CTTCAAATGTGATTGGAAAGCTC
GGAGCTCTTGGGTGCCTTAG
623
Amplicon 9
Ex 9, 5'UTR
ATGGAAACTCAAAATGGCAAAA
TCCTCACCCTCTTCTCCTTTG
1000
Total bases sequenced>>>
6894
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Supplementary Table 3: Primers and PCR conditions for SLCO1B1 sequencing
Primer Sequence
Amplicon
Size (base
Name
Covers
P1
P2
pairs)
Amplicon 1
Pro
TTTTGCTGCAACCATATCAACA
CCTTACTTTTGGGAATGGCTTTT
730
Amplicon 2
Pro + Ex1
CCAGGTGGTATCTCCAGTCTCC
GCTCTGTGTCTTCCACACGC
598
Amplicon 3
5'UTR, Ex 2
CAGCATTGACCTAGCAGAGTGG
TTCCTAAATATGTCGTGATCAATCC
586
Amplicon 4
Ex 3
ACCAAATTAGAAATGATGCTTTATCAG
AAGTATGACATGGCGTTAGTTTGC
677
Amplicon 5
Ex 4
TGTCTTGGACTCTATTTGCATCC
CACTTAGTGGGTATCTTCTCAAAAGG
348
Amplicon 6
Ex 5
TTTACCCATCACATCTCTTAAAACACA
CTGTGTTGTTAATGGGCGAAC
468
Amplicon 7
Ex 6, Ex 7
GAGTCCATTAGACCCTTTTCCTTT
TCAACATCCAAGCCACCATC
1017
Amplicon 8
Ex 8
TTCATACCATTATTTCCCTGAACC
TTTGAACTCTCCCAGTTTAGACCA
634
Amplicon 9
Ex 9
AAGCTGTGAACAGCCTGTGG
TGAGTCTTGATTTCAAAGTCCATAAA
686
Amplicon 10
Ex 10
TTGCAGGGACATGGATGAAG
TCTAAGCCTTACTTTTCCCATTCA
930
Amplicon 11
Ex 11
TTTCCAGAAATAATCCAGTGACATC
GGCTTTAGTCAAATGAGGTGCTT
494
Amplicon 12
Ex 12
TTCCTCCTCAGGGCATGTCT
TTGTCAGTCTGTGTCTTCAGATTCTT
834
Amplicon 13
Ex 13
TGATGCATTGAATAAATAAGGGAATAA
CGAATTCTCCTTTAGGTCCATCA
513
Amplicon 15
Ex 14
TCCCATATTAACCAACATAACTTCCA
GACATGAGGAGAGTTTTGGAAACA
779
Amplicon 16
Ex 15, 3'UTR
TTTCTATGGCTTCATAACCCTATTACA
CTGAATCAATGCAATGCTGTTT
465
12
13
Amplicon 17
Ex 15, 3'UTR
TGGGGCAGATAGTGAAACACA
13
GGGCCCACTGGAAACTTAAC
688
Total bases sequenced>>>
10447
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Supplementary Table 4: Primers and PCR conditions for a) UGT1A1 and b) UGT1A3 sequencing
a)
Primer Sequence
Amplicon
Size (base
Name
Covers
P1
P2
pairs)
Pro, 5'UTR,
Amplicon 1
Ex 1
GCCTTCTGTTTAATTTCTGGAAAAG
GGGAACAGCCAGACAAAAGC
765
Amplicon 2
Ex 1
GATTCTTTCCTGCAGCGTGT
TGCCAAAGACAGACTCAAACC
822
Amplicon 3
Ex 2
CTGGATTTTGCATCTCAAGGA
GGCAGGGAAAAGCCAAATCT
483
Amplicon 4
Ex3, 4
CACGTAGTGCATACACCCTTGTAA
TGAAACAACGCTATTAAATGCTACG
898
Amplicon 5
Ex 5, 3'UTR
AAGTTTGGAAAATCTGGTAGTCTTC
TTTAAAGCACTCTGGGGCTG
542
Amplicon 6
Ex 5, 3'UTR
TGGCTACCGGAAATGCTTGGGGA
TGCATGCACACGCAATGAAGGCG
946
Total bases sequenced>>>
4456
b)
Primer Sequence
Name
Covers
Amplicon 1
Pro
P1
GCTGTGCCTGCTACATTTGC
P2
AGACTGGAGCCTTCGGCATT
14
Amplicon
Size (bp)
827
15
Amplicon 2
Ex 1-1
GGAGGGCACTCTGTCTTCCA
TCAGGGCCTCATTATGTAGTAGCTC
544
Amplicon 3
Ex 1-2
CAGTGGTCCTCACCCCAGAG
TGACAAGGAGAAGCAGAAATGAA
822
Total bases sequenced>>>
15
2193
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Supplementary Table 5: Characteristics of genome-wide association screen study subjects
Rhabdomyolysis
Cardiovascular Health
Cases
Study Controls
N=175
N=645
Female, %
60.6
66.8
Age (mean, std)
63.6 (10.3)
77.1 (5.0)
Age (range)
34-89
65-91
European descent*, %
94.9
99.7
Statin user, %
100.0
100.0
Gemfibrozil user, %
65.1
0.2
Location
US, Canada
CA, MD, PA, NC
Genotyping lab
Fred Hutchinson
Cedar-Sinai
Cancer Research Center
Chip
Illumina 370 CNV quad
Illumina 370 CNV duo
*Subjects of African descent were excluded from the genome-wide association screen
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Supplementary Table 6. SNPS* selected for genotyping by gene, type, platform and outcome
Gene
Class
Platform
SNP
CYP2C8
tag
illumina
rs1058932
CYP2C8
tag
illumina
rs11572079
CYP2C8
tag
illumina
rs11572093
CYP2C8
tag
illumina
rs11572105
CYP2C8
tag
illumina
rs11572126
CYP2C8
tag
illumina
rs11572155
CYP2C8
tag
illumina
rs11572172
CYP2C8
tag
illumina
rs11572177
CYP2C8
tag
illumina
rs1891071
CYP2C8
tag
illumina
rs1934953
CYP2C8
tag
illumina
rs1934956
CYP2C8
tag
illumina
rs3752988
CYP2C8
tag
illumina
rs7909236
CYP2C8
tag
illumina
rs7912549
CYP2C8
tag
illumina
rs41286886
Status
failed illumina, successfully genotyped on taqman
dropped, in high LD with another SNP
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18
CYP2C8
tag
illumina
rs4548799
monomorphic
CYP2C8
tag
illumina
rs11572133
dropped, in high LD with another SNP
CYP2C8
tag
taqman
rs11572127
dropped, in high LD with another SNP
CYP2C8
tag
taqman
rs3216029
failed taqman
CYP2C8
fsnp
illumina
rs10509681
CYP2C8
fsnp
illumina
rs1058930
CYP2C8
fsnp
illumina
rs11572102
CYP2C8
fsnp
taqman
rs11572080
CYP2C8
fsnp
taqman
rs11572103
CYP2C8
fsnp
taqman
rs17851796
CYP2C8
novel
illumina
ss107123045
CYP2C8
novel
illumina
ss179319936
CYP2C8
novel
illumina
ss179319937
CYP2C8
novel
illumina
ss179319939
CYP2C8
novel
illumina
ss179319940
CYP2C8
novel
taqman
ss86217920
CYP2C8
novel
taqman
ss86217921
CYP2C8
novel
taqman
ss86217922
failed illumina, successfully genotyped on taqman
monomorphic
dropped, in high LD with another SNP
monomorphic
failed taqman
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19
CYP2C8
novel
taqman
ss179319938
CYP2C8
novel
taqman
ss86217923
CYP2C8
novel
taqman
ss86217924
failed taqman
CYP2C8
novel
taqman
ss86217925
failed taqman
SLCO1B1
tag
illumina
rs11045790
SLCO1B1
tag
illumina
rs11045813
dropped, in high LD with another SNP
SLCO1B1
tag
illumina
rs11045884
dropped, in high LD with another SNP
SLCO1B1
tag
illumina
rs11045889
SLCO1B1
tag
illumina
rs11045891
SLCO1B1
tag
illumina
rs12372157
SLCO1B1
tag
illumina
rs12427008
SLCO1B1
tag
illumina
rs12812795
SLCO1B1
tag
illumina
rs1463565
SLCO1B1
tag
illumina
rs16923519
SLCO1B1
tag
illumina
rs2010668
SLCO1B1
tag
illumina
rs2291074
SLCO1B1
tag
illumina
rs2291077
SLCO1B1
tag
illumina
rs2417955
failed illumina, successfully genotyped on taqman
dropped, in high LD with another SNP
19
20
SLCO1B1
tag
illumina
rs3829306
failed illumina
SLCO1B1
tag
illumina
rs3829307
failed illumina
SLCO1B1
tag
illumina
rs4149026
failed illumina, successfully genotyped on taqman
SLCO1B1
tag
illumina
rs4149028
dropped, in high LD with another SNP
SLCO1B1
tag
illumina
rs4149034
SLCO1B1
tag
illumina
rs4149035
SLCO1B1
tag
illumina
rs4149058
SLCO1B1
tag
illumina
rs4149081
SLCO1B1
tag
illumina
rs7139376
SLCO1B1
tag
illumina
rs7296796
SLCO1B1
tag
illumina
rs7969341
SLCO1B1
tag
illumina
rs976754
SLCO1B1
tag
illumina
rs991262
SLCO1B1
tag
taqman
rs11045805
SLCO1B1
tag
taqman
rs11045834
SLCO1B1
tag
taqman
rs12371604
SLCO1B1
tag
taqman
rs34671512
SLCO1B1
tag
taqman
rs35733645
dropped, in high LD with another SNP
failed taqman
20
21
SLCO1B1
tag
taqman
rs4149015
SLCO1B1
tag
taqman
rs4149030
SLCO1B1
tag
taqman
rs4149033
SLCO1B1
tag
taqman
rs4149057
SLCO1B1
tag
taqman
rs4149061
SLCO1B1
tag
taqman
rs4149066
failed taqman, low call rate
SLCO1B1
tag
taqman
rs4149071
failed taqman
SLCO1B1
tag
taqman
rs7138177
SLCO1B1
tag
taqman
rs7489119
SLCO1B1
tag
taqman
rs7955751
SLCO1B1
fsnp
illumina
rs11045853
SLCO1B1
fsnp
illumina
rs2306282
SLCO1B1
fsnp
taqman
rs11045819
SLCO1B1
fsnp
taqman
rs11557087
SLCO1B1
fsnp
taqman
rs2306283
SLCO1B1
fsnp
taqman
rs4149056
SLCO1B1
fsnp
taqman
rs4603354
SLCO1B1
novel
illumina
ss86217929
monomorphic
monomorphic
monomorphic
monomorphic
21
22
SLCO1B1
novel
illumina
ss107123049
SLCO1B1
novel
illumina
ss86217934
SLCO1B1
novel
illumina
ss179319947
SLCO1B1
novel
illumina
ss179319948
monomorphic
SLCO1B1
novel
taqman
ss86217938
failed taqman
SLCO1B1
novel
taqman
ss86217926
SLCO1B1
novel
taqman
ss86217927
SLCO1B1
novel
taqman
ss107123046
SLCO1B1
novel
taqman
ss86217928
SLCO1B1
novel
taqman
ss107123047
SLCO1B1
novel
taqman
ss86217930
SLCO1B1
novel
taqman
ss179319944
SLCO1B1
novel
taqman
ss86217931
SLCO1B1
novel
taqman
ss86217932
SLCO1B1
novel
taqman
ss86217933
SLCO1B1
novel
taqman
ss179319945
monomorphic
SLCO1B1
novel
taqman
ss179319946
failed taqman
SLCO1B1
novel
taqman
ss86217935
monomorphic
monomorphic
failed taqman
22
23
SLCO1B1
novel
taqman
ss86217936
SLCO1B1
novel
taqman
ss86217937
UGT
tag
illumina
rs1018124
UGT
tag
illumina
rs1042640
UGT
tag
illumina
rs10929303
UGT
tag
illumina
rs11563251
UGT
tag
illumina
rs17862880
UGT
tag
illumina
rs17868337
UGT
tag
illumina
rs17868341
dropped, in high LD with another SNP
UGT
tag
illumina
rs1983023
failed illumina
UGT
tag
illumina
rs2003569
UGT
tag
illumina
rs2302538
UGT
tag
illumina
rs28899468
UGT
tag
illumina
rs35665780
UGT
tag
illumina
rs4148326
UGT
tag
illumina
rs4148328
UGT
tag
illumina
rs45449995
dropped, in high LD with another SNP
UGT
tag
illumina
rs4663971
dropped, in high LD with another SNP
dropped, in high LD with another SNP
failed illumina, also failed taqman, low call rate
dropped, in high LD with another SNP
23
24
UGT
tag
illumina
rs6708136
UGT
tag
illumina
rs6717546
UGT
tag
illumina
rs6747843
failed illumina
UGT
tag
illumina
rs7574296
failed illumina
UGT
tag
illumina
rs8330
UGT
tag
illumina
rs887829
UGT
tag
illumina
rs929596
dropped, in high LD with another SNP
UGT
tag
illumina
rs10929302
dropped, in high LD with another SNP
UGT
tag
illumina
rs4124874
dropped, in high LD with another SNP
UGT
tag
illumina
rs10179091
UGT
tag
illumina
rs4663968
dropped, in high LD with another SNP
UGT
tag
illumina
rs6714634
dropped, in high LD with another SNP
UGT
tag
taqman
UGT1A1*28
failed taqman, low call rate
UGT
tag
taqman
rs12471326
UGT
tag
taqman
rs28946889
failed taqman
UGT
tag
taqman
rs33979061
monomorphic
UGT
tag
taqman
rs35765465
failed taqman
UGT
tag
taqman
rs4148329
dropped, in high LD with another SNP
24
25
UGT
fsnp
illumina
rs1042709
UGT
fsnp
illumina
rs17862867
UGT
fsnp
illumina
rs17868336
UGT
fsnp
illumina
rs28898619
UGT
fsnp
illumina
rs4148323
UGT
fsnp
taqman
rs13406898
UGT
fsnp
taqman
rs17851756
monomorphic
UGT
fsnp
taqman
rs17862869
failed taqman, low call rate
UGT
fsnp
taqman
rs28898617
UGT
fsnp
taqman
rs28898618
UGT
fsnp
taqman
rs28898620
monomorphic
UGT
fsnp
taqman
rs28900376
monomorphic
UGT
fsnp
taqman
rs28934877
monomorphic
UGT
fsnp
taqman
rs3821242
failed taqman
UGT
fsnp
taqman
rs6431625
UGT
novel
illumina
ss86217916
UGT
novel
illumina
ss86217908
UGT
novel
illumina
ss86217910
monomorphic
dropped, in high LD with another SNP
monomorphic
25
26
UGT
novel
illumina
ss86217917
UGT
novel
taqman
ss179319949
UGT
novel
taqman
ss86217909
UGT
novel
taqman
ss86217911
failed taqman
UGT
novel
taqman
ss86217912
monomorphic
UGT
novel
taqman
ss86217913
UGT
novel
taqman
ss86217915
UGT
novel
taqman
ss86217918
UGT
novel
taqman
ss86217919
Intergenic
GWA
taqman
rs10049478
Intergenic
GWA
taqman
rs1519414
Intergenic
GWA
taqman
rs6703753
Intergenic
GWA
taqman
rs7556683
NTN1
GWA
taqman
rs1880646
RYR2
GWA
taqman
rs2819742
*Single nucleotide polymorphism
26
27
Supplementary Table 7. SLCO1B1 tagSNP*, FSNP and novel SNP analysis. Models adjusted for age at statin use,
sex, and race.
Minor Allele
Minor Allele
Frequency
Frequency
Odds ratio (95%
(Controls)
(Cases)
confidence interval)
N=732
N=185
Modeled Allele
p-value
TagSNPs
rs11045805
# copies C allele
0.170
0.160
0.89 ( 0.64 to 1.23)
0.48
rs11045834
# copies A allele
0.310
0.240
0.74 ( 0.57 to 0.97)
0.03
rs12371604
# copies G allele
0.240
0.310
1.47 ( 1.12 to 1.92)
0.005
rs12427008
# copies C allele
0.068
0.052
0.73 ( 0.44 to 1.23)
0.24
rs34671512
# copies G allele
0.053
0.041
0.80 ( 0.44 to 1.45)
0.47
rs4149015
# copies A allele
0.056
0.044
0.75 ( 0.42 to 1.32)
0.31
rs4149026
# copies C allele
0.290
0.250
0.80 ( 0.60 to 1.07)
0.13
rs4149030
# copies A allele
0.490
0.480
1.06 ( 0.82 to 1.36)
0.65
rs4149033
# copies A allele
0.210
0.270
1.41 ( 1.06 to 1.87)
0.02
rs4149057
# copies A allele
0.430
0.480
1.40 ( 1.11 to 1.78)
0.005
rs4149061
# copies C allele
0.470
0.480
1.09 ( 0.85 to 1.39)
0.50
rs7138177
# copies G allele
0.120
0.150
1.55 ( 1.11 to 2.16)
0.01
27
28
rs7489119
# copies A allele
0.040
0.037
0.88 ( 0.46 to 1.66)
0.69
rs11045790
# copies G allele
0.074
0.050
0.69 ( 0.41 to 1.18)
0.18
rs11045889
# copies A allele
0.340
0.390
1.22 ( 0.96 to 1.56)
0.10
rs11045891
# copies C allele
0.170
0.130
0.70 ( 0.49 to 0.99)
0.04
rs12372157
# copies C allele
0.410
0.440
1.12 ( 0.89 to 1.42)
0.32
rs12812795
# copies A allele
0.051
0.067
1.33 ( 0.83 to 2.16)
0.24
rs1463565
# copies C allele
0.420
0.410
0.86 ( 0.67 to 1.09)
0.22
rs16923519
# copies G allele
0.230
0.230
1.16 ( 0.87 to 1.55)
0.32
rs2010668
# copies A allele
0.063
0.086
1.75 ( 1.11 to 2.76)
0.02
rs2291074
# copies G allele
0.062
0.048
0.88 ( 0.51 to 1.52)
0.65
rs2417955
# copies T allele
0.410
0.390
1.11 ( 0.87 to 1.41)
0.42
rs4149034
# copies A allele
0.370
0.320
0.87 ( 0.67 to 1.14)
0.33
rs4149035
# copies A allele
0.380
0.400
1.17 ( 0.91 to 1.51)
0.23
rs4149058
# copies G allele
0.250
0.310
1.43 ( 1.09 to 1.86)
0.009
rs7139376
# copies A allele
0.110
0.120
1.11 ( 0.74 to 1.64)
0.62
rs7296796
# copies G allele
0.095
0.110
1.22 ( 0.82 to 1.82)
0.33
rs7969341
# copies G allele
0.180
0.260
1.61 ( 1.20 to 2.15)
0.001
rs976754
# copies G allele
0.073
0.055
0.75 ( 0.44 to 1.27)
0.28
28
29
rs991262
# copies A allele
0.065
0.061
0.70 ( 0.41 to 1.21)
0.20
rs2306283
# copies C allele
0.430
0.400
0.96 ( 0.75 to 1.24)
0.78
rs11045819
# copies G allele
0.150
0.120
0.71 ( 0.50 to 1.01)
0.06
rs4149056
# copies A allele
0.140
0.240
1.89 ( 1.40 to 2.56)
3.62E-05
FSNPs
Novel SNPs
≥ 1 copy of a
Collapsed
minor allele
0.059
0.087
2.37 (1.22 to 4.63)
0.01
ss86217927
# copies C allele
0.031
0.061
2.16 ( 1.25 to 3.72)
0.005
ss86217930
# copies – allele
0.400
0.430
1.17 ( 0.91 to 1.51)
0.22
Gene-wide permutation test result p=0.002
Results for one SLCO1B1 F-SNP (rs11045853) are not shown because the minor allele was only observed in 1 control subject of African descent.
*Single nucleotide polymorphism
29
30
Supplementary Table 8. CYP2C8 tagSNP*, FSNP and novel SNP analysis. Models adjusted for age at statin use,
sex, and race.
Minor allele frequency
Minor allele frequency
Odds ratio (95%
(Controls) N=732
(Cases) N=185
confidence interval)
Modeled Allele
p-value
TagSNPs
rs11572079
# copies C allele
0.010
0.008
0.65 ( 0.20 to 2.09)
0.48
rs1058932
# copies A allele
0.220
0.180
0.78 ( 0.56 to 1.07)
0.13
rs11572093
# copies A allele
0.280
0.340
1.33 ( 1.03 to 1.72)
0.03
rs11572105
# copies A allele
0.032
0.033
1.20 ( 0.64 to 2.25)
0.58
rs11572126
# copies A allele
0.130
0.120
0.87 ( 0.59 to 1.28)
0.48
rs11572155
# copies G allele
0.053
0.070
1.51 ( 0.91 to 2.50)
0.11
rs11572172
# copies C allele
0.075
0.047
0.69 ( 0.41 to 1.15)
0.16
rs11572177
# copies G allele
0.330
0.410
1.42 ( 1.11 to 1.82)
0.005
rs1891071
# copies G allele
0.360
0.350
1.01 ( 0.79 to 1.29)
0.95
rs1934953
# copies G allele
0.310
0.280
0.90 ( 0.70 to 1.17)
0.45
rs3752988
# copies G allele
0.360
0.300
0.75 ( 0.57 to 0.98)
0.03
rs41286886
# copies A allele
0.002
0.003
1.60 ( 0.17 to 15.42)
0.69
rs7909236
# copies A allele
0.210
0.190
0.82 ( 0.60 to 1.10)
0.19
rs7912549
# copies G allele
0.150
0.160
1.23 ( 0.89 to 1.70)
0.22
30
31
F-SNPs
rs1058930 (*4)
# copies G allele
0.054
0.041
0.67 ( 0.36 to 1.26)
0.22
rs11572080 (*3)
# copies T allele
0.097
0.087
0.73 ( 0.47 to 1.13)
0.16
rs11572103 (*2)
# copies T allele
0.013
0.006
0.68 ( 0.15 to 3.12)
0.62
rs10509681 (*3)
# copies G allele
0.100
0.081
0.64 ( 0.42 to 0.98)
0.04
0.018
0.038
2.06 (0.79 to 5.37)
0.14
Novel SNPs
≥ 1 copy of a
collapsed
minor allele
Gene-wide permutation test result p=0.07
Results for one CYP2C8 F-SNP, rs11572102, are not shown because the minor allele was only observed in 3 control subjects of African descent.
*Single nucleotide polymorphism
31
32
Supplementary Table 9. UGT1A1 and 1A3 tagSNP*, FSNP and novel SNP
analysis. Models adjusted for age at statin use, sex, and race.
Minor
Minor
allele
allele
frequency
frequency
(Controls)
(Cases)
Odds ratio (95%
N=732
N=185
confidence interval)
pvalue
TagSNPs
rs10179091
# copies G allele
0.460
0.450
1.03 ( 0.81 to 1.30)
0.84
rs1018124
# copies G allele
0.083
0.061
0.78 ( 0.48 to 1.27)
0.32
rs2003569
# copies A allele
0.150
0.120
0.77 ( 0.52 to 1.13)
0.18
rs35665780
# copies A allele
0.002
0.006
2.63 ( 0.40 to 17.48)
0.32
rs4148326
# copies G allele
0.460
0.440
1.02 ( 0.80 to 1.30)
0.85
rs4148328
# copies A allele
0.370
0.340
0.87 ( 0.68 to 1.12)
0.28
rs4148329
# copies G allele
0.450
0.460
1.11 ( 0.86 to 1.41)
0.42
rs887829
# copies A allele
0.320
0.330
1.13 ( 0.88 to 1.46)
0.35
rs17868337
# copies A allele
0.020
0.030
1.45 ( 0.69 to 3.04)
0.33
rs28899468
# copies G allele
0.032
0.042
1.61 ( 0.84 to 3.07)
0.15
rs1042640
# copies G allele
0.190
0.230
1.34 ( 0.99 to 1.80)
0.06
rs11563251
# copies A allele
0.150
0.120
0.86 ( 0.59 to 1.26)
0.45
rs2302538
# copies G allele
0.160
0.150
0.95 ( 0.67 to 1.34)
0.77
rs6717546
# copies A allele
0.380
0.410
1.17 ( 0.90 to 1.51)
0.24
rs8330
# copies C allele
0.230
0.270
1.33 ( 1.00 to 1.77)
0.05
FSNPS
≥ 1 copy of a minor
collapsed
allele
0.018
0.011
1.23 (0.23 to 6.62)
0.81
rs17868336
# copies G allele
0.040
0.039
0.96 ( 0.50 to 1.83)
0.89
32
33
rs17862867
# copies A allele
0.099
0.089
0.92 ( 0.59 to 1.41)
0.69
rs28898618
# copies C allele
0.039
0.038
0.93 ( 0.49 to 1.79)
0.83
rs6431625
# copies G allele
0.360
0.360
1.12 ( 0.89 to 1.42)
0.34
Novel SNPs
≥ 1 copy of a minor
collapsed
allele
0.010
0.032
2.87 (0.87 to 9.42)
0.08
ss86217909
# copies A allele
0.055
0.052
0.90 ( 0.51 to 1.57)
0.70
ss179319949
# copies A allele
0.004
0.003
1.19 ( 0.15 to 9.17)
0.87
Gene-wide permutation test result p=0.52
*Single nucleotide polymorphism
33
34
Supplementary Table 10. Distribution of genotype by type of statin among controls
Number of
Overall
Simvastatin*
Lovastatin
Pravastatin
Fluvastatin
Atorvastatina
Cerivastatin
minor alleles
N=721
N=313
N=254
N=71
N=21
N=59
N=4
%
%
%
%
%
%
%
0
74
74
75
73
81
71
75
1
24
25
22
27
19
25
25
2
2
1
3
0
0
3
0
0
38
36
41
42
33
39
75
1
47
52
43
48
48
46
25
2
14
12
17
10
19
15
0
rs4149056,
SLCO1B1
rs2819742
RYR2
*One Cardiovascular Health Study control reported being on both atorvastatin and simvastatin.
Chi square p-value for rs4149056=0.855
Chi square p-value for rs2819742=0.478
34
35
Supplementary Figure1. Rhabdomyolysis case subject recruitment
Plaintiffs with
rhabdomyolysis who
had settled their cases
N=955
Contacted by attorney
N=918 (96%)
Screened out by attorney
N=37
Released contact
information to study
staff
N=291 (30%)
Did not release contact
information to study staff
N=627 (321 refusals, 304 no response, 2
Consented to
participate in study
N=221 (23%)
Did not consent to participate
in study
N=70
Eligible
N=215 (23%)
Ineligible
N=6
Usable DNA sample
N=186 (19%)
No / unusable DNA sample
N=29 (27 subjects were deceased, 2
unknown)
subjects without DNA)
Included in candidate
gene study
N=185 (19%) (excluded 1
that failed genotyping)
Included in GWA
N=175 (excluded 8 subjects of
African descent, 2 with low DNA,
1 that failed genotyping)
35
36
Supplementary References
1. Psaty BM, Heckbert SR, Koepsell TD, Siscovick DS, Raghunathan TE, Weiss NS, et
al. The risk of myocardial infarction associated with antihypertensive drug therapies.
Jama. 1995;274:620-625.
2. Heckbert SR, Wiggins KL, Glazer NL, Dublin S, Psaty BM, Smith NL, et al.
Antihypertensive treatment with ACE inhibitors or beta-blockers and risk of incident
atrial fibrillation in a general hypertensive population. Am J Hypertens. 2009;22:538544.
3. Smith NL, Hindorff LA, Heckbert SR, Lemaitre RN, Marciante KD, Rice K, et al.
Association of genetic variations with nonfatal venous thrombosis in postmenopausal
women. Jama. 2007;297:489-498.
4. Fried LP, Borhani NO, Enright P, Furberg CD, Gardin JM, Kronmal RA, et al. The
Cardiovascular Health Study: design and rationale. Ann Epidemiol. 1991;1:263-276.
5. Vladutiu GD, Simmons Z, Isackson PJ, Tarnopolsky M, Peltier WL, Barboi AC, et al.
Genetic risk factors associated with lipid-lowering drug-induced myopathies. Muscle
Nerve. 2006;34:153-162.
36