Pharmacogenomic assessment of carboxylesterases 1 and 2
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Transcript of Pharmacogenomic assessment of carboxylesterases 1 and 2
www.elsevier.com/locate/ygeno
Genomics 84 (20
Pharmacogenomic assessment of carboxylesterases 1 and 2
Sharon Marsha,b, Ming Xiaob,c, Jinsheng Yua,b, Ranjeet Ahluwaliaa,b, Matthew Mintonb,c,
Robert R. Freimutha,b, Pui-Yan Kwokb,c, Howard L. McLeoda,b,*
aDepartment of Medicine, Washington University School of Medicine and the Siteman Cancer Center, St Louis, MO, USAbCREATE Pharmacogenetic Research Network, St Louis, MO, USA
cCardiovascular Research Institute, Department of Dermatology, University of California, San Francisco, CA, USA
Received 9 July 2004; accepted 15 July 2004
Available online 14 August 2004
Abstract
Human carboxylesterases 1 and 2 (CES1 and CES2) catalyze the hydrolysis of many exogenous compounds. Alterations in
carboxylesterase sequences could lead to variability in both the inactivation of drugs and the activation of prodrugs. We resequenced CES1
and CES2 in multiple populations (n = 120) to identify single-nucleotide polymorphisms and confirmed the novel SNPs in healthy European
and African individuals (n = 190). Sixteen SNPs were found in CES1 (1 per 300 bp) and 11 in CES2 (1 per 630 bp) in at least one population.
Allele frequencies and estimated haplotype frequencies varied significantly between African and European populations. No association
between SNPs in CES1 or CES2 was found with respect to RNA expression in normal colonic mucosa; however, an intronic SNP (IVS10-
88) in CES2 was associated with reduced CES2 mRNA expression in colorectal tumors. Functional analysis of the novel polymorphisms
described in this study is now warranted to identify putative roles in drug metabolism.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Carboxylesterase; Irinotecan; Pharmacogenomics; Pharmacogenetics; Single nucleotide polymorphism.
Introduction
Carboxylesterase enzymes are found across animal
species and play an important role in the hydrolysis of drugs
and xenobiotics. Drugs, including heroin, cocaine, salicy-
lates, steroids, and anticancer agents such as irinotecan, are
substrates of carboxylesterases [1,2]. Human tissues have a
range of carboxylesterases, including placenta, brain (CES3
and hBr1) and liver (CES1; OMIM 114835 and CES2;
OMIM 605278) forms [3].
CES1 and CES2 catalyze the hydrolysis of the
anticancer drug irinotecan (CPT-11) to its active form
SN-38 [4–7]. It has been demonstrated that CES2 has a
0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygeno.2004.07.008
* Corresponding author. Washington University School of Medicine,
Campus Box 8069, 660 S. Euclid Ave., St. Louis, MO 63110. Fax: +1 314
362 3764.
E-mail address: [email protected] (H.L. McLeod).
12.5-to 26-fold higher affinity for CPT-11 compared to
CES1 [4,8]. Although originally identified as liver
carboxylesterases, both are expressed (CES2 at high
levels) in gastrointestinal tissue [9,10], a main target for
CPT-11 therapy. Carboxylesterase 1 is also active in the
metabolism of several clinical and elicit drugs including
cocaine, heroin, and lidocaine [1]. Variations in drug-
metabolizing enzyme genes can contribute to adverse
drug reaction and increased sensitivity/resistance to drug
treatment [11].
The majority of variations at the DNA level (over 90%)
are in the form of single-nucleotide polymorphisms (SNPs).
Alterations in carboxylesterase sequences could lead to
variability in drug metabolism between patients; yet there is
little information available on the genetic variation in these
genes. The genomic structure of the CES1 gene has been
ascertained; the gene lies on chromosome 16q13–q22.1,
contains 14 exons and spans about 30 kb [12]. The CES2
gene is also located on 16q13–q22.1, contains 12 exons, and
04) 661–668
S. Marsh et al. / Genomics 84 (2004) 661–668662
spans about 9 kb (http://www.ensembl.org/). CES2 lies
approximately 11.1 cM downstream from CES1. The close
proximity and homology (73% coding region homology) of
these genes imply an ancestral gene duplication event at this
chromosomal region.
To better characterize the extent and influence of genetic
variation in CES1 and CES2 on gene expression, single
nucleotide polymorphism discovery in these genes was
performed by resequencing of 120 individuals from three
populations (Caucasian American, African American, and
Asian American). In addition, SNP information for CES1
Table 1
Source and allele frequencies of SNPs in (a) CES1 and (b) CES2
SNP Location Source Validation
European African
p q p q
a. CES1
5VUTR�469C N Aa Resequencing 0.80 0.20 0.43 0.57
5VUTR�40G N A Resequencing 0.85 0.15 0.71 0.29
562G N C (G188R)a Resequencing 0.83 0.17 0.48 0.52
601G N A (A201T)a Resequencing/
dbSNP rs2307243
0.99 0.01 1 0
IVS2 + 72T N C Resequencing/
dbSNP rs3848300
0.86 0.14 0.83 0.17
IVS4 + 10C N Ga Resequencing/
dbSNP rs2307244
0.48 0.52 0.79 0.21
IVS4 + 66G N Ta Resequencing/
dbSNP rs2307236
0.48 0.52 0.72 0.28
IVS8 + 105C N T Resequencing 0.78 0.22 0.79 0.21
IVS8 + 128A N C Resequencing/
dbSNP rs2302719
0.91 0.09 0.83 0.17
IVS10 + 36C N Ta Resequencing 1 0 0.98 0.02
IVS10 + 99C N Ta Resequencing 1 0 0.99 0.01
IVS12�135A N G Resequencing 0.98 0.02 0.98 0.02
IVS12�159G N Aa Resequencing 0.70 0.30 0.91 0.09
3VUTR + 1970A N G Resequencing 0.96 0.04 0.99 0.01
3VUTR + 2480A N G Resequencing 0.64 0.36 0.61 0.39
3VUTR + 2845C N Ta Resequencing 0.63 0.37 0.22 0.78
b. CES2
5VUTR�363C N Ga Resequencing 0.88 0.12 0.67 0.33
1175A N T (E392V) CGAP ID 1518410 1 0 1 0
1647C N T (L459L)a Resequencing/
CGAP ID 887566
0.99 0.01 0.96 0.04
IVS1�57T N C Resequencing 1 0 1 0
IVS2�152T N C Resequencing/
dbSNP rs2303218
1 0 1 0
IVS5�146C N T Resequencing 1 0 1 0
IVS5�4A N T Resequencing 1 0 1 0
IVS7 + 48G N A Resequencing 1 0 1 0
IVS10�108G N A Resequencing/
dbSNP rs2241409
1 0 1 0
IVS10�88C N Ta Resequencing/
dbSNP rs3893757
0.81 0.19 0.55 0.45
IVS11 + 160G N Aa dbSNP rs3848286 0.98 0.02 1 0
3VUTR + 2068C N A dbSNP rs1058925 1 0 1 0
3VUTR + 3071G N Aa Resequencing 0.82 0.18 0.20 0.8
a statistically significant differences in allele frequencies between Europeans andb Predicted allele frequencies from resequencing.c NI, not identified.
and CES2 was mined from publicly available databases
(CGAP-GAI, dbSNP, and JSNP). These databases list SNPs
identified in human genes by resequencing or computational
mining efforts [13].
SNP validation and haplotype inference both of novel
resequencing and of existing database SNPs were per-
formed in 95 European and 95 African healthy unrelated
individuals. In addition, the genotype-expression relation-
ship was determined for CES1 and CES2 in paired normal
and tumor samples from 52 patients with colorectal
cancer.
Resequencing
European Americanb Asian Americanb African Americanb
p q p q p q
0.8 0.2 1 0 0.80 0.20
0.54 0.46 0.71 0.29 0.77 0.23
0.71 0.29 0.89 0.11 0.64 0.34
0.76 0.24 0.92 0.08 0.71 0.29
0.96 0.04 0.99 0.01 0.95 0.05
0.56 0.44 0.45 0.65 0.70 0.30
0.80 0.20 0.62 0.38 0.41 0.59
0.65 0.35 0.81 0.19 0.79 0.31
0.91 0.09 0.90 0.01 0.87 0.13
1 0 1 0 0.90 0.10
1 0 1 0 0.95 0.05
0.60 0.49 0.36 0.64 0.76 0.24
0.54 0.46 0.88 0.12 0.67 0.33
0.95 0.05 0.83 0.17 1 0
0.65 0.35 0.48 0.52 0.70 0.30
0.93 0.07 0.86 0.14 1 0
0.99 0.01 1 0 0.99 0.01
NIc NI NI NI NI NI
0.99 0.01 1 0 1 0
1 0 0.98 0.02 1 0
1 0 0.80 0.20 1 0
1 0 0.99 0.01 1 0
1 0 1 0 0.90 0.10
1 0 1 0 0.99 0.01
0.94 0.06 0.94 0.06 0.74 0.26
0.99 0.01 0.99 0.01 0.96 0.04
NI NI NI NI NI NI
NI NI NI NI NI NI
0.90 0.10 0.89 0.11 0.53 0.47
Africans ( pb0.001).
S. Marsh et al. / Genomics 84 (2004) 661–668 663
Results
CES1 SNP discovery and validation
SNPs in CES1 were identified by resequencing approx-
imately 10.5 kb in 120 individuals (average of 1 SNP per 330
bp). Two of the 16 SNPs observed were nonsynonymous
cSNPs, 2 SNPs were found in the 5V-untranslated region, and12 were intronic SNPs. Two SNPs (IVS10+36 and
IVS10+99) were unique to the African population in this
study and the nonsynonymous SNPA201Twas unique to the
European population (Table 1a). All 16 SNPs identified in
resequencing were validated in a distinct sample set from
European and African blood donors. Nine of the 16 SNPs had
statistically significant differences in allele frequency
between European and African populations (Table 1a).
Fig. 1. Frequencies of (A) CES1 and (B) CES2 rare alleles in European and Afric
flanking region; all other SNPs lie within the amino acid coding region.
CES2 SNP discovery and validation
Ten SNPs in CES2 were identified by resequencing
approximately 10.5 kb in 120 individuals (average of 1
SNP per 630 bp). In addition, 3 SNPs were mined from
publicly available SNP databases. Of the 13 SNPs
identified in CES2, 1 synonymous cSNP (L549L) was
confirmed in the validation populations at a low frequency
(European q =0.01; African q =0.04); no nonsynonymous
cSNPs were identified in the validation populations. One
SNP (-363 CNG) was located in the 5V-untranslated region
of CES2 and one (+3071 C N T) was located in the 3V-untranslated region. Four SNPs were unique to the
resequencing study and not found in any public SNP
database, and 8/13 SNPs were found to be monomorphic
in the validation (African and European) populations in
an populations. IVS, intronic region; –, 5VUTR/flanking region; +, 3VUTR/
S. Marsh et al. / Genomics 84 (2004) 661–668664
this study (Table 1b; Fig. 1B). Five SNPs identified by
resequencing which were monomorphic in the validation
populations in this study were originally found in
American Asians and/or African Americans but not
American Caucasians (Table 1b). One nonsynonymous
cSNP was mined from CGAP (ID 1518410) and one
3VUTR SNP from dbSNP (rs1058925) but these were
monomorphic in the validation populations in this study,
and were not found by resequencing. One intronic SNP
found by resequencing (6% in American Caucasians) and
dbSNP (rs2241409) was found to be monomorphic in the
validation populations (IVS10-108GNA). Of the five SNPs
that were confirmed in the validation populations, one
(IVS12+160 CNT) was unique to the European population
(q=0.02). All 5 confirmed SNPs had statistically signifi-
cantly different allele frequencies between European and
African populations (pb0.001) (Table 1b).
CES1 and CES2 haplotypes
Inferred haplotypes in both populations for CES1 and
CES2 were generated using the Polymorphism Haplotype
and Analysis Suite [14]. In CES1, 7 common haplotypes
(=5% in at least one population) were identified (Table 2a;
Fig. 2A). Haplotype frequencies differed between the
populations (Table 2b; Fig. 2A), which is expected due to
the significant differences in allele frequencies between
European and African populations (Table 1a).
Six unique haplotypes were identified in CES2, 1 unique
to the European and 2 unique to the African population (Fig.
2B). As with CES1, haplotype frequencies differed dramat-
ically between the populations. Significant (pb0.001) link-
age is present between CES2 SNPs –363 CNG and +3071
GNA (|DV| = 1; pb0.001) and IVS10-88 CNT and +3071
GNA (|DV| = 0.9; pb0.001) in the European population but
not the African population.
CES1 and CES2 in colorectal cancer
CES1 and CES2 SNPs were evaluated in normal and
tumor samples from 52 colorectal cancer patients. No
significant difference in allele frequencies was observed
between the colorectal cancer patients and the European
validation samples ( pN0.05; data not shown). Genotype
differences were not observed between the normal and the
tumor tissue for any SNP in either gene, supporting
previously published data that chromosome 16q is not a
genomic region frequently lost in colon cancer [15]. A range
of RNA expression for carboxylesterase 1 and 2 was seen in
both the normal [2.5–73.0 (median 20.3) units for CES1,
38.0–1781.6 (median 599.1) units for CES2] and tumor
[1.0–33.1 (median 4.0) units for CES1, 21.0–1319.0
(median 228.5) units for CES2] colon samples. Analysis
using the Wilcoxon matched-pairs test showed a statistically
significant difference between normal and tumor tissues for
both CES1 (median tumor:normal ratio 0.24) and CES2
(median tumor:normal ratio 0.34; pb0.001 in both cases).
No significant correlation between RNA expression and
polymorphisms in CES1 was observed in either normal or
tumor tissue. Similarly, no correlation was seen between any
polymorphism in CES2 and normal tissue RNA expression.
However, the intronic SNP IVS10-88 C N T in CES2 was
associated with reduced RNA expression in the colon tumor
tissue (p = 0.02; Fig. 3).
Discussion
Carboxylesterases 1 and 2 are responsible for the
metabolism of the chemotherapy drug CPT-11 to its active
form, SN-38. Interindividual variability of CES2 activity
may be particularly important, as this is the most active
human carboxylesterase for irinotecan metabolism. This
study identified and validated polymorphisms in both the
CES1 and the CES2 genes using a novel resequencing
method and in silico mining. In European samples,
resequencing of pooled DNA identified only 1 apparent
false positive out of 26 predicted SNPs (3%), demonstrating
its utility as a high-throughput SNP identification method.
This compares to a 30% false positive rate from in silico
mining for SNPs in CES2. The public databases also only
contained 10/27 (37%) SNPs identified by resequencing.
This can partly be a factor of limited database information
on the original population used to identify the polymor-
phism. The lack of overlap among publicly available SNP
databases [13], and the high false positive rate from in silico
mining, is still a cause of concern for pharmacogenetics
research.
CES2 appears to have very little genetic variation, with
the majority of SNPs occurring in intronic regions. There
was a SNP found every 630 bp, almost half the frequency
of CES1 (1 SNP per 330 bp), on average much lower than
that observed by most recent projects. For example, in
human membrane transporter genes, SNPs were found on
average one every 141 bp [16]. The ratio of nonsynon-
ymous to synonymous SNPs in a gene is considered to be a
useful measure of the fraction of deleterious alleles per gene
and consequently the level of conservation of a particular
gene. The ratio observed for CES2 (0 nonsynonymous:1
synonymous) is lower than that observed for CES1 (2
nonsynonymous:0 synonymous) and other drug metabolism
or transporter genes (0.2–0.5) [16–18]. However, the low
SNP frequency in CES2 makes this comparison of unclear
value.
Differences in haplotype structure and frequency were
observed between African and European populations for
both genes (Figs. 2A and B). The strong linkage between
SNPs in the European population within both CES1 and
CES2 and the significant differences in allele frequencies
between the populations explain the low degree of
haplotype variation seen in this study. The striking differ-
ence between African and European haplotypes in these
Table 2
Structure and frequency of haplotypes found in z5% of at least one population for (a) CES1 and (b) CES1 in European and African populations
(a) CES1
5VUTR�469C N A
5VUTR�40G N A
562G N C
(G188R)
601G N A
(A201T)
IVS2 +
72T N C
IVS4 +
10C N G
IVS4 +
66G N T
IVS8 +
105C N T
IVS8 +
128A N C
IVS10 +
36C N T
IVS10 +
99C N T
IVS12�135A N G
IVS12�159G N A
3VUTR +
1970A N G
3VUTR +
2480A N G
3VUTR +
2845C N T
%
European
%
African
Hap1 A A C G T C G C A C C A G A G T b5% 20%
Hap2 A A C G T C G C C C C A G A A T b5% 10%
Hap3 A A G G T C T C A C C A G A G T 2% 10%
Hap4 C G C G T C T C A C C A A A G T 15% b5%
Hap5 C G C G T C T C A C C A A A A C 52% b5%
Hap6 A G G G C G G C A C C A G A G T 8% b5%
Hap7 C A C G T C T C A C C A A A A C 8% b5%
(b) CES2
5VUTR�363C N G
1647C N T
(L459L)
IVS11 +
160G N A
IVS10�88C N T
3VUTR +
3V071G N A
%
European
%
African
Hap1 C C G C G 80% 24%
Hap2 C C G T A 8% 19%
Hap3 G C G T A 6% 0%
Hap4 C C G T G 2% 20%
Hap5 G C G T G 0% 18%
Hap6 G C G C G 0% 13%
S.Marsh
etal./Genomics
84(2004)661–668
665
Fig. 2. Estimated frequencies of common haplotypes in European and African populations in (A) CES1 and (B) CES2. Haplotype numbers are as listed in
Table 2.
S. Marsh et al. / Genomics 84 (2004) 661–668666
genes raises interesting issues for the field of pharmacoge-
netics. With emphasis being placed on resequencing whole
genes and identifying btag SNPsQ [19,20], care should be
taken that data for one population are not used to represent
another. For example, the –363 SNP in CES2 may be
informative for the CES2+3071 SNP in a European
population (|DV| = 1), but genotyping at this position alone
Fig. 3. Colon tumor CES2 RNA expression demonstrating significantly
lower expression in individuals IVS10-88 homozygous T/T.
would miss information in the African population where
these SNPs are not in linkage disequilibrium.
The 29 SNPs evaluated in this study did not have
predictive value for RNA expression in human tissues.
Recently three distinct promoter regions have been identi-
fied for CES2 [21]. Variable RNA expression is influenced
by promoter usage, which may explain some of the variation
in CES2 RNA expression seen in this study. In this study
SNPs were not found in these three promoter regions,
implying that polymorphisms in the upstream region of
CES2 may not be key players in promoter choice and
subsequent CES2 expression. Functional analysis of the
polymorphisms identified here in CES1 and CES2 would
help to elucidate any role in carboxylesterase protein
expression and catalytic activity.
Materials and methods
Patients and samples
Genomic DNA was extracted from whole blood of 95
European and 95 African healthy volunteers as previously
described [22,23]. DNA and RNA samples from tumor and
S. Marsh et al. / Genomics 84 (2004) 661–668 667
paired normal colon tissues from 52 Dukes’ C colorectal
cancer patients (29 male; 23 female, age range 33–102
years; median 68 years) were prepared by the Siteman
Cancer Center Tissue Precurement Core. Written informed
consent was obtained from all patients to bank tumor
tissue and to perform genomic analysis. This study was
approved by the Washington University Human Subjects
Committee.
In silico SNP mining
SNPs in CES1 and CES2 were identified from dbSNP
(http://www.ncbi.nlm.nih.gov/SNP/index.html) and CGAP-
GAI (http://lpgws.nci.nih.gov/perl/snpbr).
Resequencing
Human genomic sequence for CES1 and CES2 were
retrieved fromUCSCGolden path (http://genome.ucsc.edu/).
Repeat regions were masked with RepeatMasker (Smit, AFA
and Green, P; http://www.ftp.genome.washington.edu/RM/
RepeatMasker.html) and primers for each exonic region
selected using a modified Primer3 program [24] (see
supplemental material). PCR product sizes ranged from
0.3 to 1 kb and included exons plus 100 bp of flanking
intronic sequences. In addition, 1 kb of 5V-and 3V-untrans-lated regions were resequenced for both genes. Resequenc-
ing was carried out using DNA samples of TSC DNA panel
(http://snp.cshl.org/allele_frequency_project/panels.shtml),
which were obtained from the Coriell Institutes (http://
coriell.umdnj.edu/ccr/ccrsumm.html) of 40 African Ameri-
can, 40 Asian American, and 40 Caucasian American
samples. Samples were resequenced in 8 pools of 5 samples
per population; one sample was resequenced individually
and used a reference sequence. Dye terminator sequencing
was used and samples were analyzed on an ABI 3700
capillary sequencer (ABI, Foster City, CA), Electrochroma-
tograms were aligned and analyzed using the Sequencher
(GeneCode, Ann Arbor, MI) and Mutation Surveyor
software (SoftGenetics, LLC. State College, PA).
SNP validation
SNPs in the CES1 and CES2 genes from the resequenc-
ing study, and SNPs mined using the publicly available
SNP databases, were validated in human genomic DNA
from 95 European, 95 African individuals, and 52 color-
ectal cancer patients. PCR primers were designed within
intronic regions where possible to reduce coamplification
of homologous sequence, using Primer Express version 1.5
(ABI, Foster City, CA), and the Pyrosequencing primers
were designed using the Pyrosequencing SNP Primer
Design Version 1.01 software (http://www.pyrosequencing.
com). Unique localization of the PCR primers was verified
using NCBI Blast (http://www.ncbi.nlm.nih.gov/blast/) (for
PCR primers and conditions, see supplemental material).
PCR was carried out using Amplitaq Gold PCR master mix
(ABI, Foster City, CA), 5 pmol of each primer (IDT,
Coralville, IA), and 1 ng DNA. Pyrosequencing was carried
out as previously described [25] using the Pyrosequencing
PSQ hs96A instrument and software (Pyrosequencing,
Uppsala, Sweden). CES2 SNP L549L was assayed using
PCR-RFLP (for PCR primers and enzyme information, see
supplemental material). The genotype was called variant if it
differed from the Refseq consensus sequence at the single-
nucleotide polymorphism position (http://www.ncbi.nlm.
nih.gov/LocusLink/refseq.html).
Quantitative real-time RT-PCR
Tissue total RNA was isolated from the colon tumor or
adjacent normal colon mucosa with the TRIzol RNA
isolation kit (Invitrogen, Carlsbad, CA), and was reverse-
transcribed into cDNA using Superscript II reverse tran-
scriptase (Invitrogen). Real-time PCR was carried out in a
10-Al reactionmix containing 2 Al of cDNA (10 ng/Al), 5 Al of2�TaqMan universal PCR master mix (Applied Biosystem,
Foster City, CA), and 3 Al of primer and probe mix (400 nM
each forward and reverse primers and 200 nM TaqMan probe
(IDT, Caralville, IA)). Primer sequences are as follows: for
CES1, forward 5V-TGAGTTTCAGTACCGTCCAAGCT-3V,reverse 5V-CTCATCCCCGTGGTCTCCTA-3V, probe 5V-CTCATCAGACATGAAACCCAAGACGGTG-3V; for
CES2, forward 5V-AATCCCAGCTATTGGGAAGGA-3V,reverse 5V-CTGGCTGGTCGGTCTCAAAC-3V, probe 5V-TGGCCTCAAGCCATCCTCCCATCT-3V. All real-time
PCR assays were performed in triplicate on an ABI PRISM
7700 Sequence Detector System (ABI, Foster City, CA) with
the following program: 508C for 2 min to activate uracil
N-glycosylase enzyme, 958C for 10 min to denature uracil
N-glycosylase and activate DNA polymerase, and 40 cycles
at 958C for 20 s and at 608C for 1 min. The sequence
detection program calculates a threshold cycle number (CT)
at which the reporter fluorescence generated by cleavage of
the probe is statistically greater than that of the background
signal [26]. In this study, relative expression level was
calculated using the modified comparative CT method as
previously described [26,27]; i.e., the relative expression
level of an individual target gene was normalized to the
reference gene (amyloid g precursor protein) and to one of
the 104 colon tumor and normal RNA samples that had the
maximum CT value (i.e., the lowest expression level, called
calibrator sample or 1X sample) in any target gene [28]. The
formula for relative gene expression was utilized as
previously described [27,28].
Statistical analysis
Genotype-frequency analysis of Hardy-Weinberg equili-
brium and haplotype analysis was carried out using the
Polymorphism and Haplotype Analysis Suite (http://www.
ilya.wustl.edu/~pgrn/) [14]. Allele frequency differences
S. Marsh et al. / Genomics 84 (2004) 661–668668
between populations were analyzed using the m2 test. RNA
expression statistical analyses were performed using STA-
TISTICA from StatSoft, Inc. (Tulsa, OK). Significance of
the difference of relative expression level between paired
tumor and normal samples was evaluated by the Wilcoxon
matched-pairs test. The significance level was set at pb0.05.
Correlation between variables was observed with Spearman
rank correlation.
Acknowledgments
The authors thank Christi Ralph for technical support and
Derek Van Booven for informatics assistance. This work
was supported by the Siteman Cancer Center
(P30CA091842), NIH (R21 CA102461-01), and the NIH
Pharmacogenetics Research Network (U01 GM63340);
http://pharmacogenetics.wustl.edu. All data have been
deposited into http://pharmgkb.org.
Appendix A
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.ygeno.
2004.07.008.
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