Human Genome

Human Genome; Arranged on multiple

chromosomes; twenty three pairs of
chromosomes;
■ Twenty two pairs (autosomes).
■ One pair (sex chromosome) (xx) (female) or
(xy) (male).

Humans have 23 pairs of chromosome in

every cell (except mature red blood cells..);
Gametes or sex cells (sperm and eggs) have
half the normal complement of chromosomes.
 Human Genome

Genome

Chromosome

Gene Gene
 Human genomes
I

Nuclear genome I Mitochondrial genome 16.5 Kb I

3.2Gb
I
25% 75'4 I I
Genes and gene Extragenic 2rRNA 22tRNA 13 polypeptKle
related DNA DNA genes genes encod111g genes
I
I
1o,i, 90% 1m 40%
Coding and Non-coding Unique low copy Repetmve DNA I
regulatory DNA DNA number DNA

I l I I
Pseudogenes I Gene lntrons Interspersed Tandem
lragmenlS untranslated repeats repeats
sequences, etc
 Gene Structure

■ Most of the genes consist of; short

coding sequences or exons are
interrupted by a longer intervening
noncoding sequence or introns;
although a few genes in the human
genome have no introns.
exon intron exon intron exon
untranslated region {UTR) coding sequence {CDS) intron
DNA Organization
 The Central Dogma of Molecular Biology

О DNA molecules serve as templates for

either complementary DNA strands
during the process of replication or
complementary RNA during the
process of transcription.

О RNA molecules serve as a template for

ordering amino acids by ribosomes
The Central Dogma of Molecular Biology

Replication mRNA
Translation
DNA (protein synthesis)
/
� ;:::! '>fA.lh. - "OC

/
Transcription Ribosome
(RNA synthesis)
\
Protein
 Application of Molecular Biology

О Research
О Diagnosis
О Transplantation
О Paternity
О Forensic analysis
О Gene therapy
 Variability Among Patients
Idea: Use Molecular Marl<ers to
Make Better Treatment Decisions
+ Gene expression
Imaging

Family history-- Information �

.>" integration
Clinical data

Was the outcome

predicted accurately? Outcome
�----�

� Real I/
Outcome
:w
Genomics, Bioinformatics & Medicine
http://biochem158
.stanford.edu/

Pharmacogenomics
http://biochem158.stanford.edu/Drug-Development.html
Doug Brutlag 2011
The Proposition of the Usual
Dose

“The dose makes the

poison”
Paracelsus
(1493-1541)

The art of healing comes from nature,

not from the physician.
Therefore the physician must start
from nature, with an open mind.
The Problem of Variability

“Variability is the law of life, and

as no two faces are the same,
so no two bodies are alike,
and no two individuals react
alike,
and behave alike under the
abnormal conditions which we
Sir William
know as disease.”
Osler
(1849-1919)
•

Courtesy of Felix W. Frueh Ur Stl FDA

Doug B u ag 2011
 Paradox of Modern Drug
Development
Clinical trials provide evidence of
efficacy and safety at usual doses
in populations

Physicians treat individual

patients who can vary widely in
their response to drug therapy
 How Can The Causes of
Variability be Unraveled?

Pharmacogenomics: systemic genomic

analysis in populations of treated
subjects to identify variants that predict
drug response including the occurrence
of adverse reactions
Pharmacogenomics Strategy
Applied to the Practice of
Medicine

GCCCGCCTC

GCCCA CCTC
From McLeod and Evans, Ann Rev of Pharmacol and Toxicol, 2001:
 Personalized Medicine

• Medicine is personal:
o We are all different.
o Some of our differences translate into how we react to
drugs as individuals.
o This is why personalized medicine is important to
everyone.
• Why does someone need twice the standard dose to
be effective?
• Why does this drug work for you but not me?
• Why do I have side-effects and you don’t?
• Why do some people get cancer and others don’t?
• Why is anecdotal information irrelevant to your
own health and treatment?

Doug Brutlag 2011

 Is Medicine a Science or an Art?

If it were not for the great variability among

individuals, medicine might well be a science,
not an art.
o Sir William Osler, Physician 1892
o Johns Hopkins School of Medicine
o Johns Hopkins Hospital
o Father of modern medicine

Doug Brutlag 2011

 The Goal of Personalized Medicine

• The Right Dose of

• The Right Drug for
• The Right Indication for
• The Right Patient at
• The Right Time.

Courtesy FelixDoug
W.rBFutlrueh
ag 2011
 Pharmacogenetics & Pharmacogenomics

• Pharmacogenetics: The role of genetics in

drug responses.
o F. Vogel. 1959

• Pharmacogenomics: The science that allows

us to predict a response to drugs based on
an individuals genetic makeup.
o Felix Frueh, Associate Director of Genomics, FDA

Courtesy FelixDoug
W.rBFutlrueh
ag 2011
 Pharmacogenetics & Pharmacogenomics
http://www.ph
armgkb.org/

• Pharmacogenetics: study of individual gene-drug

interactions, usually one or two genes that have
dominant effect on a drug response (SIMPLE
relationship)

• Pharmacogenomics: study of genomic influence on

drug response, often using high-throughput data
(sequencing, SNP chip, expression, proteomics -
COMPLEX interactions)

o PharmGKB Website:
http://www.pharmgkb.org/

Doug B u ag 2011
Courtesy of Michelle Whirlr-Ctl arillo
 Purine Analogs:
A Case Study in Pharmacogenetics
• 6-mercaptopurine, 6-thioguanine, azathioprine

• Used to treat lymphoblastic leukemia, autoimmune disease,

inflammatory bowel disease, after transplant

• Interferes with nucleic acid synthesis

• Therapeutic index limited by myelosuppression

(treatment limited by immune suppression side effect)

6-mercaptopurine 6-thioguanine azathioprine

Doug Brutlag 2011
 Pharmacogenetics: A Case Study
Pharmacogenetics: A Case Study
lndividuob respond diffo.-.ntty to tho Tho diversity in rotpon$0S is A"or o timpto blood tffl,
antl-l+ukllffll3 drug 6-tnercaptoputi.,.. du. to valil,tk>nt In tho Q-'O lndlvlduolt c:.an bo gl- d-
for 11n enzyme c:.all,ecl TPMT, or or medication ti/lat ere tailored
thlopurlna methyltramforasa. to their g-llc p,ofile.

Mostpeople
metal>ol:r:. .,,. drug
qulel(ly. Do- Med
to be hoQh onough 10
bfft 14IUI<...... 8f>d

ov..... -lolbol•ff
atowty and � iow«
d,vg'"°
aoaea to a¥00d liOxlc uo.
effect• Of ll'oe <1111\).

A amd poniOn Of�

n'MIW>oloie lhOdn'9 so
poorly that Its of'lecls �
can be &atlll. Dose for
All e,o.tn,
51ow�lzor
fn'MT def'lclen1)

Doug B u ag 2011
 Courtesy of Michelle Whirlr-Ctl arillo

Pharmacogenetics: A Case Study

Pharmacogenetics: A Case Study
lndividuob respond diffo.-.ntty to tho Tho diversity in rotpon$0S is
antl-l+ukllffll3 drug 6-tnercaptoputi.,.. du. to valil,tk>nt In tho Q-'O
for 11n enzyme c:.all,ecl TPMT, or
thlopurlna methyltramforasa.
A"or o timpto blood tffl,
lndlvlduolt c:.an bo gl- d-
or medication ti/lat ere tailored
to their g-llc p,ofile.

Mostpeople
metal>ol:r:. .,,. drug
qulel(ly. Do- Med
to be hoQh onough 10
bfft 14IUI<...... 8f>d

ov..... -lolbol•ff
atowty and � iow«
d,vg'"°
aoaea to a¥00d liOxlc uo.
effect• Of ll'oe <1111\).

A amd poniOn Of�

n'MIW>oloie lhOdn'9 so
poorly that Its of'lecls �
can be &atlll. Dose for All e,o.tn,
51ow�lzor
fn'MT def'lclen1)

Doug B u ag 2011
 Courtesy of Michelle Whirlr-Ctl arillo

Pharmacogenetics: A Case Study

Pharmacogenetics: A Case Study
lndividuob respond diffo.-.ntty to tho Tho diversity in rotpon$0S is
antl-l+ukllffll3 drug 6-tnercaptoputi.,.. du. to valil,tk>nt In tho Q-'O
for 11n enzyme c:.all,ecl TPMT, or
thlopurlna methyltramforasa.
A"or o timpto blood tffl,
lndlvlduolt c:.an bo gl- d-
or medication ti/lat ere tailored
to their g-llc p,ofile.

Mostpeople
metal>ol:r:. .,,. drug
qulel(ly. Do- Med
to be hoQh onough 10
bfft 14IUI<...... 8f>d

ov..... -lolbol•ff
atowty and � iow«
d,vg'"°
aoaea to a¥00d liOxlc uo.
effect• Of ll'oe <1111\).

A amd poniOn Of�

n'MIW>oloie lhOdn'9 so
poorly that Its of'lecls �
can be &atlll. Dose for All e,o.tn,
51ow�lzor
fn'MT def'lclen1)

Doug B u ag 2011
Courtesy of Michelle Whirlr-Ctl arillo
 Metabolism of 6-MP

AZATHIOPRINE

OXIDIZED XO TPMT 6-METHYl

6-MERCAPTOPURINE 1111

•
METABOLITES MERCAPTOPURINE

! HPRT

!
IMP DEHYOAOGENASE
GMP SYNTHETASE

6·THIOGUANINE
NUCLEOTIDES
Doug B u ag 2011
L Wang and R Weinshilboum, Oncogene 25, 1629-1638
r(2tl006)
 Thiopurine S-methyl Transferase Activity
and Personalized Dosage
Genotype/Phenotype Drug Dose
TPMT Alleles
Conventional Dosing
*1 -oomtotmD-
ATG
*2�
ATO 38C
02
*3A� uu,. ro . TPMT-
Deficiency

•
... r·· ,._.....
04eoA A711lC
*3C�
ATO A718G

10
!
Individualized Dosing
II 500

i II
I. ·2. "3A. '3C

10 20
TPMi activity
Eichelbaum et al., Annu. Rev. Med. 2006.57:11r9t-l
137.

Doug B u ag 2011
 Warfarin: Significant Problems for Rats!
Warfarin
0

•
• taters
WAR FARI
BAIT
PATE STEINER
TOUTES PHARMCIES
rr DROCiUERIES

Doug Brutlag 2011

 Warfarin: Significant Problems for Humans!

• Ranks #1 in total mentions of deaths for drugs

causing adverse events (from death certificates)

• Ranks among the top drugs associated hospital

emergency room visits for bleeding

• Overall frequency of major bleeding range from 2%

to 16% (versus 0.1% for most drugs)

• Minor bleeding event rates in randomized control

trials of new anticoagulants has been as high as 29%
per year.

Doug Brutlag 2011

 Warfarin: Significant Problems for Humans!

• Case Report July 2, 2008

o Company director dies of brain
hemorrhage after heading a football
o Consultant neurosurgeon told the
inquest the warfarin effect was
probably the cause of the death
o It can happen to anyone!

• Other Warfarin Patients

• Case Report July 2, 2008
o Joseph Stalin

Doug Brutlag 2011

 Why Maintaining Warfarin
Therapeutic Range is Critical
Wa arm reatmen
Relationship between I NR control and outcomes
Incidence rate of stroke and major bleeding (per 1 DO-person years)

Increased risk of Target Increased risk or

tnrornboernbonc Therapeutic bleeding
Range
1orti----------- - - - ev e n

-•
ts

80

60

40

20

0
<2.0 2.0-3.0 3.1-3.9 4.0-4.9 >5

..
N Engl -J t.le<I 1995;333.5-10•
Doug B u ag 2011
J European Atrial Fibrillation Trial Study Group, N Engl J Med 1995;333r :t5l
-10.
 Finding Doses to Maintain Therapeutic
Anticoagulation is Largely Trial and Error
25
Approved Dose Range
."c..:.' 1111
•
20
.r.s. ..
( 1 )
Dose AdjJJ.stments

.. 115 No (5 mg)= 16%

Yes(< 5 mg)= 51%
0
Yes (> 5 mg) = 33%
'-
(1) 110
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E ::J 5
z
0
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T""" T""" N N ("I') co � � LO LO CD CD I'- CX)

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LO
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Weekly Dose
� (!j� Doug Brutlag 2011
Warfarin Levels Depend on Two
Enzymes – CYP2C9 & VKORC1

I@ © ! Doug Brutlag 2011

 Genetic Analysis Permits

• More rapid determination of stable

therapeutic dose.
• Better prediction of dose than clinical
methods alone.
• Applicable to the 70-75% of patients not in
controled anticoagulation centers.
• Reduces between 4,500 and 22,000 serious
bleeding events annually.
• Genetic testing now required by FDA

Doug Brutlag 2011

 What are Targeted Drugs?

• Often, drugs are only effective in specific

“sub-populations” (responders).
• Early identification of responders can have a
dramatic effect of treatment success.
• Treatment of non-responders puts these
individuals at unnecessary risk of adverse
events, while providing no benefit.
• Personalized Medicine allows the
identification of responders and non-
responders for targeted therapies.
• This is happening today!
Doug Brutlag 2011
Doug Brutlag 2011
 Personalized Drugs

• Herceptin (breast cancer, target: Her2/neu)

• Erbitux (colorectal cancer, target: EGFR)
• Tarceva (lung cancer, target: EGFR)
• Strattera (attention-deficit/hyperactivity
disorder, Metabolism: P4502D6)
• 6-MP (leukemia, Metabolism: TPMT)
• Antivirals (i.e. resistance based on form of HIV)

• etc. and the list is growing rapidly ...

Doug Brutlag 2011

 FDA Requires Genetic Tests
for Certain Therapies
List of FDA Required or Recommended Biomarker
Tests in Drug Labels

User Prevalence
(%)

•
13
Biomarker Test Drug Example (ns36.l million)
CYP2C9 Recommended Warfarin 2.0896
EGFR Required Cetuximab 0.0001
G6PD deficiency Recommended Dapsone 0.025i
G6PD deficiency Recommended Rasburicase 0.0000
HER2/neu
overexpression Required Trastuzumab 0.0003
TPMT variants Recommended Azathioprine 0.1168
TPMT variants Recommended Mercaptopurine 0.0541
TPMT variants Recommended Thioguanine 0.0012
UGTlAl variants Recommended Lrinotecan 0.0002
Urea cycle
enzyme deficiency Recommended Valproic acid 0.48
Total 2.768
CYP = cytochrome P-t,o; EGFR = human epidermal growth factor receptor; G6PD = glucose-e-
phosphate dehydrogenase; HER2/neu =
human epidennal growth factor receptor 2; TPMT =
thlopurlne S-melhyltransferase.

Courtesy of Michelle Whirlr-Ctl arillo

Doug B u ag 2011
Roche Chip for Cytochrome P450
Genes: CYPC19 and CYP2D6

Doug B u ag 2011
Xie and Frueh, Pharmacogenomics steps toward Personalized Medicine, Personalized Medicine 2005, 2, 3 2 5
r t l
-337
 Treatment of
Pulmonary Tuberculosis
• Approximately 20％ of the pat ents
treated with Rifampicin and INH
develop hepatotoxicity.
• Metabolic enzyme of INH is N T2.
• Rifampicin strongly induces many
drug metabolizing enzymes.
40
Metabolic pathways of isoniazid
CONHN Rifampicin
NAT2 H2
Acetylation N Enzym
CONHNHCOC Isoniazid einductio
n
H3 Acetyl (INH) Hydrolysis

N isoniazid COO
Hydrolysis
H
N Acetylation
NH2NHCOC
AcHe3tylhydrazine NAT2 NH2NH
(AcHz)
D2iacetylh
NAT2 ydrazine Isonicotinyl glycine
(NHCOCH3)
 2
H ydrazine
(Hz)
Hepatotoxi
c
41
Mutation of Nacetyltransferase 2

A→C 759 C→T

434
T→C G→A A→C G→A 803 A→G
111 191 341 499 845 A→

* ** * * * * * ****
*
190 282 481 590 857
C→T C→T C→ G→A G→A
Ｔ

ＳＮ
42
Frequency of the NAT2 genotypes in patients

Genotyp n %
e RA type (40.3%)
NAT2*4 / *4 4 40.3
6
NAT2*4 / *5 4 3.5
NAT2*4
/ *6 3 29.8 IA type (46.5%)
NAT2*4
/ *7 4 13.2
/ 1
NAT2*5 *5
/
NAT2*5 *6 5
SA type (13.2%)
NAT2*5 / *7 0 0.0
NAT2*6 / *6 7 6.1
NAT2*6 / *7 4 3.5
NAT2*7 / *7 2 1.8
43
4 0
Incidence of INHRifampicin (RFP) induced
hepatotoxicity and NAT2 genotype

Total
(n=114)

RA
type
(n=
IA4
6)
*
*
type
(n =
S A *
53) 4 6 8 10 P<0.01
type
(n=15)
0 2
0 In0cidenc0e (%) 0 0
normal hepatotoxicit
 yof norma4l 4
　　　　 and 2 x before administration.
Trough plasma concentration of INH and
hydrazine metabolites in relation to
Plasma concentration (μM)
NAT2 gene polymorphisms

* * * R (n=29
8 * * * )(n=31

AI )(n=
6 7)
AS
A
*p < 0.0 vs RA &
ImA ean± D
4

0
INH AcHz Hz
45
 Serum aminotransferase (
Case in SA type
Case 1（
f ema
l e 69yea
r )s
35
0
300

25 drug
0 ASmonitoring
20 TALT
0
15
0
10
0
NAT2*6/*6 (SA type)
5
0
0
0 1 2 3 40 50 6 7 8 90
INH Da
0 0 0 0 0
(mg/ y
40 10 20
da y ) 0
R F P 0 発疹のため 0 投与中
(mg/day) 3 止
検出菌数 0 0 0
 2 1 ± 46
（蛍光
+ +
 Plasma INH concentrationtime profile

(μg/
observed in patient of case1
INH concentration
7
6
400mg/
mL)

5 d20a0y
Reduce mg/
4 d day
3

0
0 5 1 1
5 47
 Cases in RA type
Case 5 (male 62years)

INH conc. (μg/mL)

: NAT2*4/*4 (RA type)
3

Poor Response 2
Case 5
Mean of RA
INH 400mg/day (po) type
+RFP+SM 1 (400 mg/day)

0 5 1 1
INH 400mg/day(po)　 Time after adminis r0ation (hr)
　+200mg/day(inhalation) 5
Cure +RFP+SM
48
 INHRFP induced hepatotoxicity
INH＋RF
P
slow acetylator　　intermediate 　rapid acetylator
acetylator
(SA type)　 (IA type) (RA type)

Liver
Toxicit Continue
y Increase dose
Change
regimen
Cure
Delaye d 49

RFP:rifampicin
 Plasma INH concentrationtime profile
INH concentration (μg/

Patients (n )：114
1
SparseRplasma (n )：
0
278
(1 – 4Apoints/person)
1 ty
Dose: INH 200
p e
mg a t steady
0. state
1 IA
0. Same dose
01 but different ty
pe
0.0 plasma concentrations
ra Time after
1 1 0
01 0 5 administ
t S Therapeutic range
i A
Minimal inhibitory
o tyconcentration 50

(MIC)
n

(
h
r
)
5
 Simulation of dose adjustment of INH
based on NAT2 genotype (preliminary)
Presen
INH conc. (μg/

t Simulated
Cs
3. msa
mL)

0 x

mi
0.2
n

0 1 2 H 0 1 2 h
2 4 R 2 4 r
All RAtype tSyApe
20ty0pmesg x 2 IA type
/day
500 mg x 2 /day
250 mg x 2 /day
100 mg x 2 /day 51
Clinical trial for genotype based chemotherapy
against pulmonary tuberculosis

◆ Multicenter prospective randomized clinical trial

◆ Rationalized dosing of isoniazid based on NAT2
gene polymorphism
◆ Safety, Efficacy, Pharmacoeconomics

Patients with pulmonary tuberculosis

NAT2
NAT2 genotyping Gene
chip

RAtype IAtype SAtype

52
 Modern Rendition of
Paracelsus

“The dose makes the

poison, but differently for
genetically different
individuals.”
Paracelsus
(1493-1541)
 Application of Molecular Biology

О Research
О Diagnosis
О Transplantation
О Paternity
О Forensic analysis
О Gene therapy
Application of Molecular Biology
Everywhere

THANK YOU