CYP3A4 promoter variant is an

increased activity allele

Rachel Botbyl
Erin Schuetz Lab
Pharmaceutical Sciences Department
About me
 Cytochrome P450 (CYP) enzymes play
an important role in drug metabolism

Acts on a wide variety of commonly used

drugs, including many chemotherapeutic
CYP
agents

Abundant in the liver and small intestine

Drug X X---OH
 CYP3A4 is the dominant CYP isoform

CYP2C9/19
CYP2D6 CYP1A2
CYP2B6

CYP2C8

CYP2E1

CYP3A4/5

Studying pharmacogenetics of CYP3A4 is important

 CYP3A4 coding variants have been shown to
affect drug metabolism

CYP3A4 activity varies up to 100-fold between individuals

Genetic differences have been identified as a factor

3A4*1B 3A4*22 3A4*1G

 CYP3A4 activity is higher in
populations of African descent

Previous studies have indicated that populations of

African descent clear 3A4-metabolized drugs faster

Since 3A4*22 is a low-activity allele found primarily in

Caucasians, so we wanted to look for an allele with
increased activity
higher allele frequency in African Americans
promoter variation more likely to drive higher
expression

Hypothesis: there are additional genetic

factors which explain CYP3A4 variation
 rs140702888 is a 14 base pair deletion at -2.934
Kb of CYP3A4 promoter

3A4WTseq(1>156)caagctTGAGAGCACAGTATTGAGAGCACAGtagacac
3A414bpDELseq(1>142)caagctTGAGAGCACAGtagacac
 Promoter variant is correlated with higher
drug clearance in African American cohort
Midazolam clearance

Midazolam clearance
Higher level of drug clearance
suggests increased activity
Dr. Stephen D. Hall
Drug Disposition, Eli Lilly and Co.,
Indianapolis
 Increased 3A4 activity decreases
clinical efficacy of drugs

Active Inactive

SV SV--OH

WT 3A4
Reductions in LDL Cholesterol

SV SV---OH

VAR 3A4 Reductions in LDL Cholesterol

 Increased 3A4 activity decreases
clinical efficacy of drugs
Simvastatin blood concentration

LDL blood concentration

p=0.35 (additive)

WT VAR WT VAR
WT and VAR elements were cloned
into pGL4.23 vector
 Is there a difference in basal luciferase
promoter activity in VAR compared to
WT?

WT TGAGAGCACAG TAT TGAGAGCACAG LUC

14bpdeletion
VAR TGAGAGCACAG LUC
 Promoter variant had a higher activity compared to WT
indicating deletion allele is an increased activity allele

LS180 HepG2
1.6
1.8 **
Fold Change over pGL4.23

Fold Change over pGL4.23

1.4
1.6
1.4 1.2

1.2 1

1 0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0 0

WT VAR WT VAR

**p 0.005
 Promoter variant is lacking GTF2
binding site
Matrix Common to
Family p-value Match Total #sequences TempSeq_LO5z_vH3

O$VTBP 0.14 2 1 2

V$DICE 0.05 1 1 1

V$FKHD 0.10 1 1 1

V$RBP2 0.02 1 1 1

V$RU49 0.04 2 1 2

V$ZF13 0.06 1 1 1

TempSeq_X8CP4Be
_
Matrix Match Common to
Family p-value Total #sequences
DEL allele does not have DICE so co- O$VTBP 0.14 2 1 2
repressors can not bind and hence V$FKHD 0.10 1 1 1
V$RBP2 0.02 1 1 1
could lead to increase in 3A4 V$RU49 0.04 2 1 2
transcription/Activity
 Does GTF2 bind to 14 base pair
deletion sequence and suppress?

GTF2

14bp deletion LUC

 GTF2 acts as a repressor when
14bp sequence is present

2.5
LS180 HepG2
1.6
Fold Change over pGL 4.23

Fold Change over pGL 4.23

1.4
2
1.2

1.5 1

0.8 **
1
** 0.6

0.5 0.4

0.2
0 0
Control GTF2 Control GTF2

**p 0.005
 Does GTF2 suppress luciferase
activity in WT but not in VAR?

GTF2

WT TGAGAGCACAG TAT TGAGAGCACAG LUC

GTF2

14bpdeletion
VAR TGAGAGCACAG LUC
 In Summary

Current Conclusions
The promoter variant is a high activity allele and is associated with
increased drug clearance
GTF2 binds to the 14-bp sequence which is absent in the variant
GTF2 plays an important role in suppressing CYP3A4 expression

Future Directions
Explore the effect of GTF2 in the context of the CYP3A4 promoter

Long Term Goals

Use knowledge of variations in 3A4 activity to better predict and
control dose response in patients
 Acknowledgements
Schuetz Lab
Erin Schuetz, PhD
Amarjit Chaudhry, PhD
Kazuto Yasuda, PhD
Cynthia Cline
Samit Ganguly

POE Program
Suzanne Gronemeyer, PhD
James Marmion
Jenna Bencini
POE Class of 2016