nature publishing group
Genotype in the Cytochrome
P450 3A4 Gene, a Rapid Metabolizer
of Sex Steroids, Is Associated With Low Bone
Mineral DensityYS Kang1, SY Park1, CH Yim1, HS Kwak1, P Gajendrarao2, N Krishnamoorthy2, S-C Yun3,
KW Lee2 and KO Han1
Osteoporosis is influenced by genetic factors. The interindividual variability in the activity of CYP3A, the metabolic
enzyme of sex hormones, may result from genetic polymorphisms. In a study of 2,178 women of ages 40–79 years,
the presence of the CYP3A4*18 variant was found to be significantly associated with low bone mass. In vitro functional
analyses indicate that CYP3A4*18 is a gain-of-function mutation in sex steroid metabolism, resulting in rapid oxidation
of estrogens and testosterone; in vivo pharmacokinetics using midazolam (MDZ) verify the altered activity of the
CYP3A4*18, showing lower metabolic turnover in the mutant than in the wild type. Molecular modeling reveals the
structural changes in the substrate recognition sites of CYP3A4*18 that can cause changes in enzymatic activity and that
potentially account for the difference between the catalytic activities of estrogen and MDZ, depending on the genotype.
The results indicate that a genetic variation in the CYP3A4 gene—as a gain-of-function mutation in the metabolism of
certain CYP3A substrates, including sex steroids—may predispose individuals to osteoporosis.
Osteoporosis is a multifactorial disease with a strong genetic endogenous compounds, but genetic factors are also among the
component. Genetic factors influence bone mass, bone size, most plausible mechanisms.
bone quality, and bone turnover, and they may modulate the
The CYP3A activity of the adult human liver is the sum
risk of osteoporosis.1 Many candidate genes have thus far been activity of at least two CYP3A family members: CYP3A4 and
suggested, but none has yet been supported strongly and consist-
CYP3A5. To date, approximately 40 allelic variants in the
ently by subsequent studies.
gene have been reported as showing marked ethnic
The members of the cytochrome P450 3A (CYP3A) subfamily differences in allele frequencies.6,7 CYP3A5, the second-most
are the major enzymes in the nicotinamide adenine dinucleotide important CYP3A protein in the liver, has characteristic poly-
phosphate-oxidase–dependent oxidative metabolism of vari-
morphic expression caused by genetic variation; certain genetic
ous endogenous and exogenous compounds, including sex variations, such as CYP3A5*3
, give rise to an
hormones. A wide interindividual variability in the expres-
aberrantly spliced mRNA with a premature stop codon, which
sion and catalytic activity of CYP3A has been reported in the produces a nonfunctioning protein.8,9
general population.2 The interindividual variation, exceeding
We therefore hypothesized that genetic variations of CYP3A
30-fold in some populations, may influence the circulating lev-
proteins, the important metabolizing enzymes of estrogen,
els of endogenous sex steroids and thereby mediate the risk of might be among the major determinants in the development
certain estrogen-associated diseases such as osteoporosis.3–5 of osteoporosis. To identify the candidate genetic variations in
This variation is, at least partly, caused by multiple environ-
gene, we sequenced the entire coding region and
mental factors, including induction by drugs, chemicals, and performed detailed structural and functional studies, including
The first three authors contributed equally to this work.
1Department of Internal Medicine, Cheil General Hospital and Women's Healthcare Center, Kwandong University College of Medicine, Seoul, Korea;
2Division of Applied Life Sciences (BK21 Program), Environmental Biotechnology, National Core Research Center, Gyeongsang National University, Jinju, Korea;
3Department of Preventive Medicine, University of Ulsan College of Medicine, Seoul, Korea. Correspondence: KO
Received 19 May 2008; accepted 11 September 2008; advance online publication 19 November 2008.
VOLUME 85 NUMBER 3 MARCH 2009 www.nature.com/cpt
table 1 Clinical characteristics and bone density data of the subjects (n = 2,178)
Age at menopause (years)
Spine BMD (g/cm2)
Values are means ± SD.
BMD, bone mineral density; BMI, body mass index; YSM, years since menopause.
value adjusting for age, BMI, and CYP3A5
value adjusting for age, BMI, and CYP3A4
both in vitro
and in vivo
analyses for candidate genotypes. We
also assessed the CYP3A5*3
al eles that are known to contribute
to the reduction of CYP3A5 activity. We analyzed the associa-
tion of CYP3A
genotypes with bone mineral density (BMD) in
CYP3A4 and CYP3A5 genotyping analysis
A screening search for base changes in all 13 exons in the
gene in 225 Koreans identified two mutations: a T →
Lumbar spine BMD (g/cm
C point mutation (L293P) at codon 293, named CYP3A4*18
and a silent mutation (L295L) at codon 295. Among 2,178
Korean women of ages ranging from 40 to 79 years, CYP3A4*18
was detected in 53 women, with an allelic frequency of 1.2%:
2,125 were wild type (WT), 52 were heterozygotes, and 1 was a
Years since menopause
homozygote. For CYP3A5
, the genotyping revealed that 62.7%
of the subjects were the CYP3A5*3/*3
genotype and the allelic
frequency of CYP3A5*3
was 79.5%. Significant linkage disequi-
librium was shown between the two genotypes in our study
ʹ = 0.80, P <
0.001), as presented in the previous data.10
CYP3A4 polymorphism and BMD
The clinical characteristics of the study groups in relation to
genotypes are listed The groups
were all balanced with regard to clinical variables. Subjects with
genotype were significantly associated with low
lumbar spine BMD after adjusting for age, body mass index
(BMI), and CYP3A5
= 0.014). However, in analy-
ses of BMD according to CYP3A5
genotype, no BMD difference Figure 1
The association of CYP3A4
genotype with lumbar and femur bone
was observed in women with deficient CYP3A5 activity as com-
mineral density (BMD). (a
) Relationship of lumbar spine BMD to years since
menopause (YSM), relative to CYP3A4
genotype, in 2,178 women of ages
pared to those with whole CYP3A5 activity, thereby indicating 40–79 years. The slope relating to the CYP3A4*18
genotype did not differ from
that CYP3A5 has no significant influence on bone metabolism that of the CYP3A4*1
(wild type) genotype (P
= 0.437). YSM values
Subsequent analyses to assess the differences between <6 months were arbitrarily set to YSM = 0. (b
) Comparison of femur BMD by
genotypes, adjusted for age, years since menopause, CYP3A4
genotype in 1,353 women in whom femur BMD measurements were
BMI, or CYP3A5
genotype, did not elucidate any significant inter-
performed using a QDR-2000. BMD comparisons were adjusted for age, body mass index, and CYP3A5
action effects between the CYP3A4
genotype and each covariate.
The relationship between lumbar spine BMD and years since subjects. Their BMD was measured by dual-energy X-ray absorp-
menopause in subjects with the CYP3A4
genotype is shown in tiometry using the QDR-2000 Adjusted BMD for
No significant differences in slope were found between the total hip was significantly lower in CYP3A4*18
genotypes. To assess the femoral BMD, we analyzed 1,353 in CYP3A4*1
= 0.027). We also observed a tendency
ClInICAl PhArMACOlOgY & TherAPeuTICs
VOLUME 85 NUMBER 3 MARCH 2009
toward association of CYP3A4*18
with a low adjusted BMD at netic tests: metabolic turnover for MDZ was significantly lower
the femoral neck, trochanter, and Ward's triangle; however, this in CYP3A4*18 than in CYP3A4 WT.
tendency did not reach statistical significance.
Molecular modeling construction for the CYP3A4
In vitro functional analysis of CYP3A4 enzyme activity
To compare CYP3A4 enzyme activity for metabolism of sex hor-
To investigate the structural behavior of the proteins, molecu-
mones in vitro
, we obtained both types of recombinant enzymes lar modeling studies and molecular dynamic simulation were
using CYP3A4*1 or CYP3A4*18 prepared in a baculovirus system. carried out, for the WT and CYP3A4*18 proteins. As shown
Western blotting and CO spectra indicated that both were present both proteins have centrally located hemes; how-
in the form of their holoproteins (data not shown). The levels of ever, there is a structural difference between the WT and
the individual metabolites (16α-OHE1, 2-OHE1, and 4-OHE1) in
the oxidations of estrone (E1) tended to be higher with the mutant
enzyme, CYP3A4*18, than with the CYP3A4*1 enzyme (
The oxidative metabolite of testosterone, 6β-hydroxytestosterone,
was present in significantly higher quantities by the CYP3A4*18
enzyme These results suggest that CYP3A4*18 has
a higher catalytic efficiency for both estrogen and testosterone
metabolism than the CYP3A4 WT enzyme does.
In vivo pharmacogenetics for CYP3A4 polymorphism
Midazolam concentration (ng/ml)
Midazolam (MDZ) is the drug that is most widely used as an
probe for phenotyping CYP3A activity.11,12 We carried out
a pharmacokinetic test to compare MDZ metabolic activity in 13
subjects and 26 CYP3A4*1+
subjects, each matched
for age, sex, BMI, and CYP3A5
concentration–time plot revealed that the mean plasma concen-
trations of MDZ after a 7.5 mg oral administration of MDZ tended
to be lower in subjects with the CYP3A5*3
genotype, but that this
difference was not statistical y significant (data not shown). On
the other hand, plasma concentrations were significantly higher
subjects. The CYP3A4*18
+ subjects showed
diminished MDZ clearance and increased MDZ area under the
plasma concentration curve as compared to CYP3A4*1
Midazolam AUC (ng·h/ml)
= 0.029, P
< 0.001, respectively), thereby suggesting that
is associated with decreased catalytic activity for
MDZ. To confirm the contradictory data resulting from the MDZ
pharmacokinetic tests, we tested the in vitro
MDZ kinetics with
recombinant enzymes The maximum reaction veloc-
ity value for 1-OH MDZ in the case of CYP3A4*1 was greater
than the maximum reaction velocity value for CYP3A4*18. These
results were consistent with the results from in vivo
table 2 estrone and testosterone hydroxylase activity with
Metabolite formation rate (min
Figure 2 In vivo
pharmacokinetics and in vitro
enzyme kinetics to assess the
differences in midazolam (MDZ) oxidation relative to the CYP3A4
) The mean MDZ plasma concentration and (b
) MDZ clearance and area under
the plasma concentration curve after a 7.5-mg oral dose of MDZ indicated that
CYP3A4*18 is associated with decreased metabolism of MDZ as compared
to CYP3A4*1. *P
< 0.05, P
values by linear mixed-effects model. (c
characteristics of MDZ biotransformation in vitro
. Kinetics of 1-hydroxy (OH)
Values are means ± SD of three experiments. Concentration is expressed as ng/ml.
MDZ was determined using recombinant CYP3A4*1 and CYP3A4*18. Assays
Substrate concentrations were as follows: estrone 200 μmol/l and testosterone 250 μmol/l.
were performed at substrate concentrations between 10 and 250 μmol/l for MDZ. Data were analyzed using the Michaelis–Menten equation.
6β-OHT, 6β-hydroxytestosterone; E1, estrone.
VOLUME 85 NUMBER 3 MARCH 2009 www.nature.com/cpt
occupied by negative (E374) and polar (S119) residues.
Although a more refined study using molecular dynamic sim-
ulation would be necessary to confirm this issue, our results
showed that the docking mode of MDZ bonding to the active
site is different from that of testosterone, suggesting compat-
ibility with our enzyme kinetic studies.
This study shows that the CYP3A4*18
variant of the CYP3A4
gene is associated with low BMD in Korean women, causing the
conformational changes in the SRS regions of the mutant protein
that lead to change in enzymatic activity.
The major CYP3A family members expressed in the adult
human liver, CYP3A4 and CYP3A5, were targeted in our study
as candidate genes for osteoporosis. Large race-related differ-
ences in the CYP3A4
gene have been reported. For instance,
(M445T) and CYP3A4*17
(F189S) are found only
in Caucasians, CYP3A4*15
(R162Q) is found only in Africans,
(D174H) is found in both Caucasians and
Africans.14,15 We have therefore screened all the exons of the
gene in Koreans and identified two single-nucleotide
polymorphisms: L293P and the silent mutation, L295L, which
is a novel single-nucleotide polymorphism detected first in
our study. CYP3A4*18
has been identified only in some Asian
populations, including Japanese and Chinese people, but not
in Caucasians or African Americans. The certain mutations
Molecular dynamic simulation and molecular docking results for
reported in Chinese or Japanese people, such as CYP3A4*3
CYP3A4*1 (wild type) and CYP3A4*18. (a
) Secondary structural details of
CYP3A4*1 (illustration in gray) where the heme is shown as ruby-colored
(P218R), and CYP3A4*6
(A17776), were not identified in our
) Snapshots taken from the simulations at 2 ns, CYP3A4*1 (orange)
screening.10,16 These findings might be explained by the pres-
superimposed with CYP3A4*18 (green) (RMSD = 2.13Å). Comparison of the
ence of ethnic differences even among Asians.
secondary structural changes on helix I between CYP3A4*1 (lower box) and CYP3A4*18 (upper box) are presented in an enlarged view in the small boxes.
CYP3A4*18 has shown a significantly higher turnover
Docking mode of (c
) midazolam (blue) and (d
) testosterone (aqua green) in
activity for both testosterone and insecticide chlorpyrifos
the CYP3A4*1 active site cavity. Residues are represented as gray sticks.
.14 However, the functional significance of CYP3A4*18
in drug metabolism or disease pathogenesis has not been clari-
CYP3A4*18 proteins. Codon 293 is located at the start of the fied in vivo
. The data we present in this study suggest, for the
highly conserved helix I, which has been known to play an first time in the field of osteoporosis genetics, that gene muta-
essential role in substrate specificity 12 The most tion possibly contributes to disease vulnerability, and that the
important change in the L293P secondary structural elements CYP3A4*18
genotype is one of the genetic risk factors for low
was observed in the conserved helix I: a long, straight α helix bone mass. For CYP3A5, the second CYP3A family member in
in the WT was modified into two small α helices separated by a the adult human liver, we focused on the CYP3A5*3
short loop in the final conformation of CYP3A4*18 because the CYP3A5*3/*3
homozygote is common in Koreans17
small boxes). This change reduces stability, and the consequent and is not able to make functioning CYP3A5 by alternative splic-
fluctuation on the separated helices could lead to changes in ing. No BMD difference was observed between women with
the other substrate recognition sites (SRSs) through SRS4 of whole CYP3A5 activity and those with deficient CYP3A5 activ-
helix I. SRS3 and SRS1 were closer to SRS4 in the CYP3A4*18 ity, thereby indicating that CYP3A5 has no significant influence
than in the WT protein. Furthermore, the conformational on bone metabolism.
shifts on the G and F helices observed in CYP3A4*18 could
The molecular modeling strongly supports our hypoth-
affect all the SRS regions except SRS6. To uncover the pos-
esis, showing the significant secondary structural change in
sible conformation of substrate–enzyme binding in the active CYP3A4*18. Surprisingly, change of a single amino acid, L293P,
site, molecular docking was carried out using GOLD, version at the beginning of helix I has an influence on the overall pro-
3.1.1 (CCDC Software Ltd., Cambridge, UK), with the MDZ tein structure and leads to the modification of the arrangement
and testosterone structures The active of SRS regions, the important sites for substrate recognition,
site pocket is mostly hydrophobic with neutral residues. and substrate access to the active site. Our in vitro
and in vivo
One side of the substrate is brokered by nonpolar residues pharmacokinetic studies using a conventional probe drug,
(I301, F304, A305, I369, and A370), and the other side is MDZ, also indicate that CYP3A4*18
is a functional mutation.
ClInICAl PhArMACOlOgY & TherAPeuTICs
VOLUME 85 NUMBER 3 MARCH 2009
In our in vivo
pharmacokinetic study, subjects with CYP3A4*18
The genotype-specific differences in BMD were shown first
exhibited lower enzyme activity in MDZ hydroxylation as com-
in our study involving the CYP3A4*18 protein. The results of
pared to the CYP3A4*1
group matched for the CYP3A5
an additional search for the kinetics of metabolism and con-
type. This result was confirmed by our in vitro
enzyme kinetic struction of enzyme structure suggest a possible mechanism to
assay with different doses of MDZ. The data obtained from both explain the effect of CYP3A4*18 on bone mass. This finding
and in vivo
experiments with MDZ are contradictory suggests that the CYP3A4
polymorphism may be a predictor
to ours and to previous data using sex steroids as substrates. for osteoporosis in some Asian populations. Genetic markers in
Molecular docking studies with the WT structure showed that estrogen metabolism could be clinically useful for identifying
the pattern of MDZ bonding to the active site is different from subjects at risk for osteoporosis and also for estrogen-related
that of testosterone. Therefore, structural changes and variable diseases such as breast cancer.
docking patterns detected in our modeling studies suggest that
the conformational change in CYP3A4*18 may lead to the MethoDs
alteration of metabolic activity, depending on substrate types. subjects and measurement of BMD.
The subjects in our study were a
CYP3A4*18, therefore, seems to be a two-faced mutation in hospital-based series of 2,178 healthy women of ethnic Korean back-
metabolic activity: it acts as a rapid metabolizer of sex steroids, ground who had visited the hospital for a general checkup between
1994 and 2004. The protocol for the study was approved by the Chell
but, on the other hand, it is a poor metabolizer of some drugs, General Hospital Institutional Review Board, and informed con-
such as MDZ.
sent was obtained from the participants. We enrolled women aged
CYP3A4 is thought to be responsible for the phase I metabo-
40–79 years, and those who had a history of chronic medical dis-
lism of numerous structural y diverse exogenous and endogenous ease or were taking medications that could affect bone and calcium
molecules, including steroids, fatty acids, prostaglandins, and metabolism were excluded from the study. Each patient was clini-
cally examined, and routine biochemical tests were performed to
lipid-soluble vitamins.18 As the critical role of estrogen in the exclude underlying diseases.
maintenance of skeletal health has been well demonstrated, and
BMD at the lumbar spine (L2–L4) was measured by dual-energy
the degree of estrogen exposure is a determining factor for bone X-ray absorptiometry using a QDR-2000 (Hologic, Bedford, MA), or
mass,19,20 we evaluated whether CYP3A4*18 could affect estro-
XR-36 (Norland, Fort Atkinson, WI). The precision errors (the coeffi-
gen metabolism. Estrogens are metabolized to a large number of cients of variation for the in vivo
BMD measurements) were 0.65 and
0.7%, respectively. The scores measured by XR-36 were converted to
oxidated (2-, 4-, 6α-, 6β-, 12β-, 15α-, 16α-, and 16β-hydroxylated) those of QDR-2000 in accordance with the conversion equation used
metabolites, mainly by CYP3A enzymes.21,22 CYP3A4 has a cat-
in a previous study25: QDR-2000 = (0.876 × XR-36) + 0.124. Data were
alytic activity predominantly for the 2-hydroxylation (which is obtained in 1,353 women for BMD at the proximal femur, measured by
devoid of estrogen activity) rather than for the 4-hydroxylation dual-energy X-ray absorptiometry using the QDR-2000. The precision
of estrogen.23,24 In our study, CYP3A4 had high catalytic activity error was 1.2%.
for the oxidation of both estrogen and testosterone. Although it DnA genotyping in CYP3A genes.
To determine the presence of
is difficult to obtain statistically significant changes in estrogen genetic variations in the CYP3A4
gene, the 596 bp 5′-upstream and
metabolism by CYP3A4*18 due to the smal and multiple peaks all 13 exons were amplified and sequenced in DNA from 225 ran-
of metabolites, the oxidation of major metabolites of estrone by domly selected, unrelated Koreans. The identified single-nucleotide
CYP3A4*18 had a tendency to be greater than that associated polymorphism, CYP3A4*18
(L293P), was detected by PCR and
with the WT protein. As previous data show,14 the production the restriction fragment length polymorphism method, using for-
ward primer (5′-TGATGCCCTACATTGAT CTGA-3′) and reverse
of 6β-hydroxytestosterone by CYP3A4*18 from the metabolism primer (5′-GTGGTGAGGAGGCATTTTTG-3′) and restriction
of testosterone was significantly higher than that produced by enzyme Msp
I (NEB, Beverly, MA). On the basis of the published
variant alleles, we developed specific PCR–restriction
Based on the data from our experiments, the plausible expla-
fragment length polymorphism tests for CYP3A5*3
nation for low BMD in CYP3A4*18
+ women might be that a The sequences of the primers are as follows: forward primer
(5′-TGGCATAGGAGATACCCACG-3′) and reverse primer (5′-GT
gain-of-function mutation for sex steroids on codon 293 in the GGTCCAAACAGGGAAGAAATA-3′).
gene results in the rapid metabolic clearance of sex hor-
mones, including estrogens, leading to a relative sex-hormone CYP3A4 enzyme activity in vitro.
The human CYP3A4*1
deficiency and consequent rapid bone turnover. To confirm our the vector pUV1 was a generous gift from Dr Gonzales (National
hypothesis more clearly, it would be helpful to assess the cir-
Cancer Institute, National Institutes of Health). A mutant contain-
culating estrogen concentrations, estrogen metabolites, or the ing CYP3A4*18
was made using the GeneEditor in vitro
skeletal responsiveness (such as bone turnover and BMD) to the mutagenesis kit (Promega, Madison, WI). Recombinant CYP3A4*1
hormone therapy, in relation to the various genotypes in a large and CYP3A4*18 were expressed using a baculovirus expression sys-
tem purchased from Clontech Laboratories (Mountain View, CA).
population. Our study involving a less common genotype found For western blot analysis, sodium dodecyl sulfate–polyacrylamide gel
only in Asians might have limitations in its clinical implications, electrophoresis was used to separate the recombinant proteins, after
but our pharmacogenetic approach focusing on metabolizing which the proteins were transferred onto nitrocellulose membranes.
enzymes was able to identify one of the oligogenic determinants The membranes were incubated with anti-CYP3A4 primary anti-
in osteoporosis. Searching for and grouping of oligogenic deter-
body for 1 h at room temperature. An enhanced chemiluminescent
kit (Pierce, Rockford, IL) was used for immunodetection. Enzyme
minants in serial metabolic pathways of candidate molecules can content was monitored by means of the reduced CO spectrum, using a
be a powerful tool to predict future osteoporosis.
DW-2000 Spectrophotometer. Protein concentration was determined
VOLUME 85 NUMBER 3 MARCH 2009 www.nature.com/cpt
by the Bradford method.26 Next, we compared the catalytic activities
(A080016), and the MOST/KOSEF for the Environmental Biotechnology
of CYP3A4*1 and CYP3A4*18 for estrone, testosterone, and MDZ in
National Core Research Center (R15-2003-012-02001-0).
accordance with previously described methods.14 Metabolites were
analyzed using a liquid chromatographic–tandem mass spectrometric
ConFliCt oF inteRest
system (API 2000; MDS Sciex, Concord, Ontario, Canada). Formation
The authors declared no conflict of interest.
data for 1-hydroxy (OH)-MDZ for MDZ were fitted to a Michaelis–
Menten model. All experiments were performed in triplicate.
2008 American Society for Clinical Pharmacology and Therapeutics
In vivo pharmacokinetics using MDZ as a probe.
1. Ralston, S.H. & de Crombrugghe, B. Genetic regulation of bone mass and
with the CYP3A4*18
allele and 26 normal healthy volunteers matched
susceptibility to osteoporosis. Genes Dev
, 2492–2506 (2006).
for age (±2 years), sex, and CYP3A5
genotype were enrol ed in the
2. Daly, A.K., Cholerton, S., Gregory, W. & Idle, J.R. Metabolic polymorphisms.
pharmacokinetic study using MDZ as a phenotypic probe
, 129–160 (1993).
drug. After an overnight fast, all subjects received a single 7.5-mg oral
3. Westlind, A., Löfberg, L., Tindberg, N., Andersson, T.B. & Sundberg, M.
dose of MDZ (time 0). Blood samples were obtained before the drug
Interindividual differences in hepatic expression of CYP3A4: relationship
was administered and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, and 24 h afterward.
to genetic polymorphism in the 5′-upstream regulatory region. Biochem.
Pharmacokinetic parameters included MDZ concentration in plasma,
Biophys. Res. Commun
, 201–205 (1999).
area under the plasma concentration curve, and total clearance.
4. Rogers, J.F., Rocci, M.L. Jr., Haughey, D.B. & Bertino, J.S. Jr. An evaluation of
the suitability of intravenous midazolam as an in vivo marker for hepatic
Weight-normalized oral MDZ clearance was calculated by dividing
cytochrome P4503A activity. Clin. Pharmacol. Ther
, 153–158 (2003).
total clearance by body weight (in kilograms).
5. Wilkinson, G.R. Drug metabolism and variability among patients in drug
response. N. Engl. J. Med
, 2211–2221 (2005).
Protein structure preparation for molecular modeling and molecular
6. Human Cytochrome P450 (CYP) Allele Nomenclature Committee
Molecular modeling studies were used for
<http://www.imm.ki.se/CYPalleles/> (2006). Accessed 27 June 2006.
investigating the structure of WT CYT3A4 and the effects of its
7. Burk, O. & Wojnowski, L. Cytochrome P450 3A and their regulation. Naunyn
mutation (CYP3A4*18) on structural stability. For this study, the
Schmiedebergs Arch. Pharmacol
, 105–124 (2004).
three-dimensional coordinates of the CYP3A4 protein were obtained
8. Kuehl, P. et al
. Sequence diversity in CYP3A promoters and characterization of
from the Protein Data Bank (PDB ID: 1TQN).27 The X-ray crystal
the genetic basis of polymorphic CYP3A5 expression. Nat. Genet
structure of the WT protein was resolved, and the missing part was
recovered using the HOMOLOGY module in the INSIGHTII pro-
9. Floyd, M.D. et al
. Genotype-phenotype associations for common CYP3A4
and CYP3A5 variants in the basal and induced metabolism of midazolam in
gram (Insight II, version 2005.3L; Accelrys., San Diego, CA). The
European- and African-American men and women. Pharmacogenetics 13
CYP3A4*18 mutation model was constructed by the INSIGHTII
10. Fukushima-Uesaka, H. et al
. Haplotypes of CYP3A4 and their close linkage with
The molecular dynamic simulations were performed using
CYP3A5 haplotypes in a Japanese population. Hum. Mutat
, 100 (2004).
GROMACS Simulation Software, version 3.3.1, (a web-based software
11. Watkins, P.B. Noninvasive tests of CYP3A enzymes. Pharmacogenetics 4
available at http://www.gromacs.org/)28 to study protein structural
behavior in the polar environment. To compare the binding confor-
12. Streetman, D.S., Bertino, J.S. Jr. & Nafziger, A.N. Phenotyping of drug-
mation of MDZ and testosterone, the study of the molecular docking
metabolizing enzymes in adults: a review of in-vivo cytochrome P450
of the substrates to the active site of the protein was carried out using
phenotyping probes. Pharmacogenetics 10
, 187–216 (2000).
13. Khan, K.K., He, Y.Q., Domanski, T.L. & Halpert, J.R. Midazolam oxidation by
GOLD, version 3.1.1. (CCDC Software Ltd., Cambridge, UK)29
cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding sites and of the metabolic pathway that leads to enzyme inactivation.
The data were presented as mean values ± SD,
, 495–506 (2002).
and compared using Student's unpaired t-
or Mann–Whitney U
14. Dai, D. et al
. Identification of variants of CYP3A4 and characterization of their
and one-way analysis of variance or Kruskal–Wallis test as appro-
abilities to metabolize testosterone and chlorpyrifos. J. Pharmacol. Exp. Ther
priate. Multiple linear regression analysis was used to adjust BMD
, 825–831 (2001).
for confounding factors such as age, years since menopause, BMI,
15. Lamba, J.K. et al
. Common allelic variants of cytochrome P4503A4 and
genotype. The interactions between genotype and
their prevalence in different populations. Pharmacogenetics 12
several covariates, such as age, years since menopause, BMI, and
16. Hsieh, K.P. et al
. Novel mutations of CYP3A4 in Chinese. Drug Metab. Dispos
genotype, were examined using linear regression analysis.
Comparison of MDZ clearance and area under the plasma concentra-
17. Park, S.Y., Kang, Y.S., Jeong, M.S., Yoon, H.K. & Han, K.O. Frequencies of CYP3A5
tion curve between the CYP3A4*18 and CYP3A4*1 proteins was per-
genotypes and haplotypes in a Korean population. J. Clin. Pharm. Ther
formed using a linear mixed-effects model. Underlying assumptions
regarding linear regression and mixed-effects models were checked by
18. Handschin, C. & Meyer, U.A. Induction of drug metabolism: the role of nuclear
residual plot, normal probability plot of the residuals, and the absolute
receptors. Pharmacol. Rev
, 649–673 (2003).
residual plot, and no relevant violations were found. A P
19. Leelawattana, R. et al
. The oxidative metabolism of estradiol conditions
was considered to indicate a significant difference. Statistical analysis
postmenopausal bone density and bone loss. J. Bone Miner. Res
was performed using commercially available software, SPSS 11.0 for
20. Nguyen, T.V., Jones, G., Sambrook, P.N., White, C.P., Kelly, P.J. & Eisman, J.A.
Windows (SPSS, Chicago, IL) and the Statistical Analysis System pro-
Effects of estrogen exposure and reproductive factors on bone mineral
gram, version 9.1 (SAS Institute, Cary, NC).
density and osteoporotic fractures. J. Clin. Endocrinol. Metab
Linkage analysis between the CYP3A4
was performed using SNPstats, a web-based software available at
21. Lee, A.J., Kosh, J.W., Conney, A.H. & Zhu, B.T. Characterization of the
http://bioinfo.iconcologia.net/SNPstats. Linkage disequilibrium results
NADPH-dependent metabolism of 17beta-estradiol to multiple
are presented as D′ and P
values. We defined a D′ ≥ 0.70 as high linkage
metabolites by human liver microsomes and selectively expressed human
disequilibrium and a P
value <0.05 as a significant value (e.g., the calcu-
cytochrome P450 3A4 and 3A5. J. Pharmacol. Exp. Ther
lated D′ value is significant).30
22. Lee, A.J., Mills, L.H., Kosh, J.W., Conney, A.H. & Zhu, B.T. NADPH-dependent
metabolism of estrone by human liver microsomes. J. Pharmacol. Exp. Ther
, 838–849 (2002).
This study was supported by grants from the Korea Healthcare technology
23. Lee, A.J., Cai, M.X., Thomas, P.E., Conney, A.H. & Zhu, B.T. Characterization
project, Ministry for Health, Welfare and Family Affairs, Republic of Korea
of the oxidative metabolites of 17beta-estradiol and estrone formed by 15
ClInICAl PhArMACOlOgY & TherAPeuTICs
VOLUME 85 NUMBER 3 MARCH 2009
selectively expressed human cytochrome p450 isoforms. Endocrinology 144
27. Yano, J.K., Wester, M.R., Schoch, G.A., Griffin, K.J., Stout, C.D. & Johnson, E.F. The
structure of human microsomal cytochrome P450 3A4 determined by X-ray
24. Tsuchiya, Y., Nakajima, M. & Yokoi, T. Cytochrome P450-mediated
crystallography to 2.05-A resolution. J. Biol. Chem
, 38091–38094 (2004).
metabolism of estrogens and its regulation in human. Cancer Lett
28. Van Der Spoel, D., Lindahl, E., Hess, B., Groenhof, G., Mark, A.E. &
Berendsen, H.J. GROMACS: fast, flexible, and free. Comput. Chem
25. Kim, S.W., Lim, C.H., Han, K.O., Jung, H.Y., Min, H.K. & Han, I.K. Standardization
of dual energy X-ray absorptionmetry (DXA) in spinal BMD of Korean
29. Jones, G., Willett, P., Glen, R.C., Leach, A.R. & Taylor, R. Development and
women and phantom. 10th International Congress of Endocrinology,
validation of a genetic algorithm for flexible docking. J. Mol. Biol
San Francisco, CA 1996. 372.
26. Bradford, M.M. A rapid and sensitive method for the quantitation of
30. Bento, J.L. et al
. Genetic analysis of the GLUT10 glucose transporter (SLC2A10)
microgram quantities of protein utilizing the principle of protein-dye binding.
polymorphisms in Caucasian American type 2 diabetes. BMC Med. Genet
, 248–254 (1976).
VOLUME 85 NUMBER 3 MARCH 2009 www.nature.com/cpt
Australasian Society for Immunology Incorporated PP 341403100035 ISSN 1442-8725 Infection Immunity and Immunogenetics Unit, Pathology and Laboratory Medicine, University of Western Australia Can a HIV patient who once progressed to AIDS ever regain a normal immune system on antiretroviral therapy (ART)? Why do some HIV patients beginning ART have an uneventful immune recovery, whilst others develop immune restoration disease? Are the effects of CMV similar in HIV patients, transplant recipients and healthy aging? Why is HCV disease more severe in HIV patients and what determines how HCV patients respond to therapy?
PROGETTO UNIVA 2013 Journal Club Pietro Gareri, MD, PhD Geriatra ASP Catanzaro Lamezia Terme 3 Luglio 2013 Drug-induced parkinsonism (DIP) was recognized in the early 1950s as a commoncomplication of antipsychotic therapy; initially considered to be present in 4 - 40%of patients treated with the first neuroleptics