Pharmacogenomics

PHARMACOGENOMICS
PRESENTED BY : ANAND SAGAR TIWARI
M.PHARM (FIRST SEMESTER)
DEPT. OF PHARMACOLOGY
GUIDED BY : Mrs. ARATI MALPANI
M.PHARMA(Ph.D)

Overview
Basic concepts
Gene mapping and cloning of disease gene
Genetic variation and its role in health pharmacology
Polymorphisms affecting drug metabolism
Genetic variation in drug transporters
Genetic variation in G protein coupled receptors
Application of Proteomics, Genomics, Metabolomics ,
Functionomics & Nutrigenomics .

Basic concepts :
• Gene : A gene is the basic physical and functional unit of
heredity. Genes are made up of DNA wherein they act as a
instruction to make molecules called proteins. All genes
don’t code for proteins. The Human Genome Project
estimated that humans have between 20,000 – 25,000
genes.
• Every person has two copies of each gene one inherited from
each parent. Though genes are same in most of the people, a
small number of them are slightly different.
• Alleles are forms of the same gene with small differences in
their sequence of DNA bases.

Human Genome Project
It was an ambitious international project that started bin 1990s
molecular biologists to understand the genomes of humans and other
organisms.
It is designed to identify and locate 35,000-40,000 genes present on
23 pairs of chromosome contained in the nucleus of the cell of
humans.
USA took lead later to be joined in by 14 other countries namely
Australia , Canada, Brazil, Denmark, France, Germany , Israel, Italy ,
Japan, Netherlands, Russia, Sweden and UK.
It was believed that this project will give insight into the fundamental
mechanisms of life and lead to an era of molecular medicine with
new ways to prevent, diagnose and treat a disease.

Goals of HGP:
To determine the complete nucleotide sequence of DNA of each
chromosome.
To construct the detailed genetic and physical maps of nearly 3 billion
base pairs.
Detailed study of information about the structure , organization and
function of human DNA.
To diagnose genetic disease like cancer, cardiovascular disease ,
autoimmunity and mental illness.
To develop efficient technology for the identification of genes.
To identify ethical, legal and social implications that may arise from
the project.

Achievements:
Identification of nearly 30,000-35,000 genes.
Identification of 3164.7 million nitrogen bases in the human genome.
Study of size and number bases of genes. The average gene consists
of 3000 bases but the size vary greatly.
Establishment of the fact that almost all nucleotide bases are exactly
the same in all people (99.9 %).
Identification of chromosome I with 2968 genes and Y chromosome
with only 231 genes.
Identification of 1.4 million locations in human genome where single
base DNA difference occurs.

Genetic code :
• The three nucleotide(triplet) base sequences in m RNA that act as a
code words fore amino acids in protein constitute the genetic code or
simply CODONS. The genetic code may be regarded as a dictionary of
nucleotide bases (A,G,C &U) that determine the sequence of amino
acid in proteins.
• These four bases produce 64 different combinations of three base
codons.
• 3 CODONS viz. UAA, UAG, UGA don’t code for amino acids thus
known as termination codons or non-sense codons .These CODONS
are named as amber , ochre and opal codons.
• Initiation codons are AUG(methionine) and GUG.

World of -OMICS :
PROTEOMICS
GENOMICS
NUTRIGENOMICS
METABOLOMICS
FUNCTIONOMICS
The suffix –OMICS itself means the field of study in
Biology. At the same time the suffix –OME is used to address
the objects of study of such fields as PROTEOME , GENOME
etc.

High Throughput Screening
• High Throughput screening (HTS) is a method for scientific
experimentation especially used in drug discovery and relevant to the
fields of biology and chemistry.
• Using robotics, data processing control software , liquid handling
devices and sensitive detectors it allows a researcher to quickly
conduct millions of chemical, genetic or pharmacological tests.
• Through this tests one can easily and rapidly identify active
compounds, antibodies or genes that modulate a particular bio-
molecular pathway.
• Thus it helps to know the starting point for drug design and for
understanding the non-interaction of a particular location.

Pharmacogenomics
Pharmacogenomics is the study of the role of the genome in drug
response.
Its name(pharmaco + genomics) reflects in combining of
Pharmacology and Genomics.
Thus it analyzes how the genetic makeup of an individual affects
his/her response to the drugs.
It deals with the influence of acquired and inherited genetic variation
on drug response in patients by correlating gene expression or single
nucleotide polymorphisms with pharmacokinetics and
pharmacodynamics.
Through the utilization of pharmacogenomics it is hoped that
pharmaceutical drug treatments can deviate from what is dubbed as
“ONE DOSE FITS ALL”.

History :
It was recognized by Pythagoras around 510 B.C. when he made a
connection between the dangers of fava bean ingestion with
hemolytic anemia and oxidative stress.This was later validated to the
deficiency of G6PD deficiency.
In 1956 reports of prolonged paralysis and fatal reactions linked to
genetic variation in patients who lacked butyryl-cholinesterase
following administration of succinylcholine injection during
anesthesia.
In the late 1960s twin studies supported the influence of genetic
involvement in drug metabolism with identical twins sharing
remarkable similarities to drug response compared to fraternal twins,
The term pharmacogenomics first began in 1990s.

Gene mapping:
Gene mapping is the process of determining the order of and relative
distance between genetic markers on a chromosome based on the
pattern of inheritance.
Gene mapping describes the method used to identify the locus of a
gene and the distance in between the genes . The essence of all
genome mapping is to place the collection of molecular markers onto
their respective positions on the genome.
The first step of building a genetic map are the development if
genetic markers and a mapping population. The closer the markers
are on the chromosome the more likely they are to be passed on to
the next generation together.
The quality of genetic map depends on the number of genetic
markers and the size of the mapping population.

• Molecular markers are the fragments of DNA that are
associated with a certain location within the genome.
Molecular markers are used in biotechnology to identify a
particular sequence of DNA in a pool of unknown DNA.
e.g. RFLP (Restriction Fragment Length Polymorphism);AFLP(Amlified
Fragment Length Polymorphism).

Types:
• There are two different types of Mapping
oGenetic mapping
oPhysical mapping
Both maps are collection of genetic markers and gene loci.
Genetic maps distances are based on the genetic linkage information
while physical maps use actual physical distances usually measured in
no of base pairs.
Physical map could be a more accurate representation of
genome.

Uses of gene mapping
• The great use of genetic mapping is that it can identify the relative
positions of genes based solely on their phenotypic effect.
• Identification of genes is usually the first step in understanding the
genome of a species ; mapping of the gene is usually the first step of
the identification of the gene .
• Gene mapping is usually the starting point of many downstream
studies.
• The process to identify a genetic element that is responsible for a
disease is also referred to as mapping.

Gene cloning
Molecular cloning is a set of experimental methods in molecular
biology that are used to assemble recombinant DNA molecules and to
direct their replication in the host cells.
Molecular cloning generally uses DNA sequences from two different
organisms: the species that is the source of the DNA to be cloned and
the species that will serve as the living host for replication of the DNA.
This will generate a population of organisms in which r-DNA
molecules are replicated along with the host DNA . Because they
contain foreign DNA molecules they can be also called as Transgenic
or Genetically Modified Organisms

Requirements
In standard molecular cloning experiments the cloning of any DNA
fragment essentially involves seven steps:
Choice of host organism and cloning vector
Preparation of vector DNA
Preparation of DNA to be cloned
Creation of r-DNA
Introduction of r-DNA into host organism
Selection of organism containing r-DNA
Screening for clones with desired DNA inserts and biological
properties.

Genetic variation
• Genetic variation is the difference in DNA among individuals. There
are multiple sources of genetic variation including mutation and
genetic recombination.
• Identification of genetic variation can be carried out by the variation
in the phenotypic characters in either quantitative traits or discrete
traits.
• Genetic variation can be studied by the variation in the order of the
bases in the nucleotide of genes.

Genetic variation contd…..
oPolymorphisms : Variation between individuals in a population
oSubstitution : Fixed variation in between individuals of species.
Factors affecting genetic variation are :
i. Mutations
ii. Gene flow
iii. Genetic drift
iv. Natural selection
v. Speciation
vi. Reproduction

Mutations
• These are spontaneous and permanent change in the genetic
makeup of an individual . This change may produce an
alteration in the character.
• Gene mutation is also called point mutation.
• Due to mutation in the gene one of the amino acids
glutamine gets replaced by valine , resulting in Sickle cell
anemia.
• Again mutation in chromosome can be leading to either
Turner’s syndrome or Klinefelter’s syndrome.

Gene flow
• Gene flow is defined as the transfer of genes between two
interbreeding population which differ genetically.
• Gene flow is mainly brought about by migration in
hybridization.
• When there is a random removal of alleles from a donor
population and introduced into a recipient population the
allele frequency of both the population is effected.
• This change in gene frequency leads to increased genetic
variation and causes evolutionary change.

Genetic drift
• It is the random change in gene frequency in a small
population purely by chance.
• Genetic drift may lead to the fixation or loss of certain
genes irrespective of their adaptive value.
• Genetic drift is also called FOUNDER EFFECT.

Natural selection
• It is the process of selection of better adapted individuals
with useful variations by nature.
• The fittest or better adapted individuals survive , reproduce
and contribute their genes to the next generation while the
less adapted are rejected by nature.
• Thus natural selection is the process of differential
reproduction that leads to differential contribution of
genotypes to the gene pool of the next generation.

Speciation
• It is the process of formation of new species.
• During this process organisms with a favorable
variations are allowed to reproduce by nature at a
faster rate than the others.
• Due to the cumulative effect of this process new
structures are formed which in turns leads to the
formation of new species.

What is g-protein ?
• Also known as guanine nucleotide binding proteins.
• Family of protein that acts as a molecular switch inside the
cell.
• Activity regulated by factors that controls their ability to bind
and hydrolyze GTP into GDP.
• When they are bound to GTP, they are “on” and when they
are bound to GDP they are “off”. G-Protein belongs to the
larger group of enzymes called GTPase.
• There are two classes of G Proteins
i. Monomeric small GTPase.
ii. Heterotrimeric G- Protein complexes (alpha,beta and
gamma subunit).

G Protein coupled receptor
• 7 trans membrane helices connected by alternating cytosolic extra
cellular loop.
• C terminal : Inside the cell
• N terminal : Extra cellular region
• Extra cellular portion has unique messenger binding site
• Cytosolic loop allow receptor to interact with G Protein
• The eventual effect of agonist induced activation is a change in the
relative orientations of the TM helices(likened to a twisting motion)
leading to a wider intercellular surface and revelation of residues of
the intracellular helices and TM domains crucial to signal transduction
function (i.e. G Protein coupling).
• In case of inverse agonists or antagonists binding to different sites the
effect must be prevention of TM helix orientation.

Genetic variation in GPCR
1. G protein coupled receptor is the main molecular target for
drug action which are estimated to be having around 600
GPCR genes in the human genome.
2. Confounding factors in assessing the therapeutic relevance
of variant GPCR alleles include
a) Interaction of a single drug with multiple closely related
receptors.
b) Poorly defined binding pockets to accommodate drug
ligands in different orientations or at alternative receptor
domains
c) Possibility of multiple receptor conformation
d) Multiple signaling pathways engaged by a single receptor.

Causes of gentic variation
1. Sequence variations of the human genome
2. Structure and function of GPCR
3. GPCR coupling to g proteins and other signaling
pathways
4. GPCR binding pockets
5. Spontaneous GPCR signaling
6. Multiple receptor conformations with distinct
functions.

Causes contd……
1. Sequence variation introduces variability in the genetic makeup.
Suspected to play an important role in diseases and variable
response in drug therapy . SNPs account for 80% of sequence
variations.
2. Molecular architecture might permit the prediction of functionally
relevant domains where sequence variations are more likely to
alter receptor function . Normally TM domains are highly
conserved, the loops are variable in sequence and length &b the C
& N tails represent the most diverse element.
3. Exchange of single amino acid residues can lead to constitutive
receptor activation . Considerable no. of human polymorphisms
enhance signaling or even the receptor constitutively causing
serious genetic disorders.

Causes contd……..
4. GPCRs are flexible structures and may accommodate ligands
in various ways. It exists in multiple conformations. Discrete
signaling pathways are triggered by discrete conformational
states of GPCR.
5. GPCR thought to be coupling to heterotrimeric G Proteins
composed of alpha, beta & gamma subunits. It displays
considerable heterogenecity with a predicted no. of 27
different alpha, 5 beta and 13 gamma subunits . However
proteins like kinases, arrestin and phosphatases modulate
receptor functions at different do mains that are possible
targets for polymorphic effects.

I. Impaired or Enhanced agonist signaling efficacy :
Several inactivating sequence variants of peptide receptors
have been associated with congenital disorders. E.g. A
point mutation causing truncation of Thyrotropin
stimulating hormone receptor leads to. Leydig’s cell
hyperplasia.
II. V2 vasopressin receptors : A number of mutations in
the gene encoding the V2 vasopressin receptor leads to
functionally inactive receptor protein that are causative for
nephrogenic diabetes insipidus. This a clear indication that
receptor activity depends on intact signaling pathways.

I. Thromboxane A2 Receptor : This receptor performs an
essential role in hemostasis by inducing platelet
aggregation . An R60L amino acid substitution in the first
cytoplasmic loop of TBAX2 receptor causes a dominantly
inherited bleeding disorder characterized by defective
platelet response to TBXA2. This leads to decreased
agonist induced second messenger formation.
II. P2Y12ADP Receptor : This receptor sub-type is shown to
be the target for anti thrombotic drugs such as ticlodipine
& clopidogrel. 2-nucleotide deletion in a region mapping
to the end of TMD6 is associated with a rare bleeding
disorder.

Importance of genetic variation
i. Genetic variation is important because it allows individuals
with a given species to adapt to their environment.
ii. We all are born with adaptation if don’t naturally have
these differences in our DNA we can’t obtain them.
iii. If we are different because of genetic variation and we are
able to adapt this is known as Survival of Natural Selection.
iv. Adaptation make an organism different and if it occurs
over a long enough period of time we get new species.

ROLE OF GENETIC VARIATION IN HEALTH
Gene therapy equipment
Gene addition therapies
Gene repairing(editing) therapies
Vectors for gene transfer

Polymorphisms
• The existence together of many forms of DNA sequences at a
locus within the population.
• A discontinuous genetic variation that results in different
forms or types of individuals among the members of single
species.
• Genetic polymorphisms of drug metabolizing enzyme give
rise to distinct subgroups in the population that differ in
their ability to perform certain drug transformation
reactions.
• Polymorphisms are generated by mutations in the gene for
these enzymes which cause decreased , increased or absent
enzyme expression or activity by multiple molecular

Contd….
1. Genetic differences in drug metabolism are the result of
genetically based variation in alleles for genes that code for
enzymes responsible for the metabolism of drugs.
2. In polymorphisms the genes contain abnormal base pairs
or multiples or abnormal alleles leading to altered enzyme
function.
3. Difference in enzyme activity occur at different rates
according to racial group.

Single nucleotide polymorphisms
1. Single change in one allele of a gene responsible for a
variety of metabolic processes including enzymatic
metabolism.
2. The combination of alleles encoding the gene determines
the activity and effectiveness of the enzyme.
3. The overall function of the enzyme is the phenotype of
enzyme function.
(Phenotype : The observable physical or biochemical
characteristics determined by both genetic makeup and
environmental issues.)

Drug metabolism
• The metabolism of drugs and other xenobiotics into more hydrophilic
metabolites is essential for their elimination from the body as well as
for the termination of their biological effect.
• Drug metabolism reactions are classified as either Phase-1
functionalization reaction or Phase 2 biosynthetic reactions (
Conjugation reaction).
• Enzyme system involved with the biotransformation of drugs is
located in the liver.
• These biotransformation reactions are carried out by CYPs
(CYTOCHROME P 450 isoforms) and by a variety of transferases.
• Phase 1 Reactions : Oxidation , Reduction, Hydrolysis.
• Phase 2 Reactions : Acetylation,Glucouronidation, Sulfation,
Methylation

Drug metabolising enzymes
CYTOCHROME P450
VKORC 1
TPMT

VKORC 1
• The Vitamin K Epoxide Reductase Complex subunit 1 is
responsible for the pharmacodynamics of Warfarin.
• Along with CYP2C9 are useful for the identification of risk of
bleeding during warfarin administration.
• Warfarin works by inhibiting VKOR which is encoded by the
VKORC 1 gene.

TPMT
Thiopurine methyltransferase catalyzes the S-methylation of
thiopurines thus regulating the balance between cytotoxic
thioguanine nucleotide and inactive metabolites in
hematopoietic cells.
TPMT is highly involved in 6-MP metabolism .
Excessive levels of 6-MP can cause myelosuppression and
myelotoxicity.
Myelosuppression: A condition in which bone marrow activity
is reduced resulting in fewer red blood cells ,WBC and
Platelets.It is a side effect of some cancer treatments.]

Cytochrome P450
The most prevalent drug metabolizing enzymes are the Cytochrome
P450 enzyme.
The term was coined by Omura and Sato in 1962 to describe the
membrane bound , heme containing protein characterized by 450 nm
spectral peak when complexed with Carbon monoxide.
Human CYP families consist of 57 genes, 18 families and 44
subfamilies.
CYPs are arranged into these families and subfamilies based on the
basis of similarities identified by the amino acid sequence.

Nomenclature
• In case of cytochromes nomenclature enzymes that share
35-40 % identity are assigned to the same family by an
Arabic numeral and those that share 55-70 % make up of a
particular subfamily with a designated letter e.g. CYP2D6
refers to family 2, subfamily D and gene number 6.

CYPs Contd….
• Commonly treated CYPs include –CYP2D6, CYP2C19,CYP2C9,CYP3A4
and CYP3A5.
DRUG METABOLISED BY ENZYMES:
CYP2C9 : Tolbutamide , Ibuprofen, Diclofenac etc.
CYP2C19 : Diazepam, Omeprazole, Propranolol.
CYP3A4 : Erythromycin, cyclosporine etc.
CYP2D6 : Metoprolol , Codeine etc.

Mutations in metabolizing enzymes
Polymorphisms affect drug interactions by altering the
affect of inhibitors and inducers on the enzyme . It results in
an exaggerated effect or minimal effect on the substrate.
Inhibitor : An enzyme inhibitor is the one which binds to an
enzyme and decreases their activity.
Inducer : An enzyme inducer is a type of drug that
increases the metabolic activity of an enzyme either by
binding to the enzyme either by binding to it and activating it
or by increasing the expression of the gene coding for the
enzyme.

Mutations Contd……
Poor Metabolizers : Two defective alleles (e.g. CYP2D6*4/*5 and
CYP2D6*4/*4) or combination of alleles including one resulting in no
enzymes (e.g.CYP2DG*5 and CYP2D6*4 deletion).
Intermediate Metabolizers : These may be heterozygous having
only one wild type allele and one defective allele.
Normal Metabolizers: Carry wild type allele (e.g. CYP2D6*1/*3).
Wild type alleles encode genes for normal enzyme function.
 Extensive Metabolizers : Carry one wild type and one amplified
gene.( Ex. CYP2D6*1/*2, CYP2D6*A/1 and CYP2D6*1A/*5).
Ultrarapid Metabolizers : Carry two or more copies of amplified
gene. (e.g. CYP2D6*2/*3)

CYP2C9
• Enzyme in humans is encoded by CYP2C9 gene.
• Enzyme being highly polymorphic genetic polymorphism
does exist . More than 50 SNPs have been described in the
regulatory and coding regions of the CYP2C9 gene with some
of them associated with reduced enzyme activity compared
with wild type.
• For CYP2C9 substrates such as Warfarin and Phenytoin,
diminished metabolic capacity because of genetic
polymorphisms or drug-drug interactions can lead to toxicity
at normal therapeutic doses.

CYP2C19
It is a member of Cytochrome P450 mixed function oxidase
system.
It is involved in the metabolism of xenobiotics, including
many proton pump inhibitors and anti-epileptics.
It is a liver enzyme that acts on at least 10% drugs in current
use notably the antiplatelet agent Clopidogrel (Plavin) . Also
drugs that treat pain associated with ulcers such as
omperazole, antiseizure drugs such as mepheytoin , the
antimalarial Proguanil and anxiolytic diazepam

Polymorphisms
• Genetic polymorphism do exist for CYP2C19 expression with
approx. 3-5% of European and 15-20% of Asian population
being poor metabolizers with no CYP2C19 function.
• In patients with reduced gene activity Clopidogrel may not
be metabolized to its active form and therefore may not
achieve the required pharmacological activity in the body.
• Polymorphisms can be observed in CYP2C19*2, CYP2C19*3
and CYP2C19*17 genes.
• Clopidogrel being administered as a prodrug it depends on
the action of enzyme in the body to observe the desired
pharmacological effect.

CYP2C19 and Diazepam
I. Diazepam is demethylated by CYP2C19.
II. Plasma half life is longer in individuals who are
homozygous for the effective CYP2C19*2 allele compared
to those who are homozygous for the wild type allele.
III. Half life of desmethyldiazepam is also longer in CYP2C19
poor metabolizers.
IV. Diazepam induced toxicity may occur as a result of slower
metabolism – requires careful dosing in Asian population.

CYP2D6
In humans this enzyme is encoded by the CYP2D6 gene.
Primarily expressed in liver with expressions in areas of CNS including
the Substantia nigara.
Member of the CYP450 mixed function oxidase system and one of
the most important enzymes involved in the metabolism of
xenobiotics in the body.
Responsible for the metabolism and elimination of approx. 25% of
clinically used drugs via the addition or removal of certain functional
groups specifically hydroxylation, demethylation and dealkylation.
Also activates some prodrugs. Moreover metabolizes endogenous
substance such as 5-HT, neurosteroids and both m-tyramine and p-
tyramine which are metabolized into dopamine in the brain and liver.

Polymorphisms
• Variation exists in the efficiency and amount of CYP2D6
enzymes produced between individuals. Hence certain will
eliminate quickly while for others it is slow(poor
metabolizers).
• Due to genetic polymorphism in it shows the largest
phenotypical variability accounting for normal, reduced and
non-existent CYP2D6 function in subjects.
• The genetic basis of CYP2D6 mediated metabolic variability is
the CYP2D6 allele located on chromosome 22.
• These with certain allelic variation will show increased or
decreased or normal or no CYP2D6 function.

CYP3A4
• It is an important enzyme in the body mainly found in the
liver and in the intestine.
• Oxidizes small foreign organic molecules such as toxins or
drugs so that they can be removed from the body.
• Of the total metabolism carried out by CYP450 about 60% is
carried out by CYP3A4.
• Metabolizes substances like acetaminophen, codeine,
diazepam , steroids and certain carcinogens.
• At the same time many substances are bioactivated by
CYP3A4 to form their active compounds and many proteins
being toxicated into their toxic forms.

Genetic Variability
Over 28 SNPs have been reported none of them have
significant inter-individual variability in-vivo.
Alleles which have been reported to have minimal function
compared to wild type include CYP3A4*6 and CYP3A4*17.
Both of these lead to a decreased catalytic activity with
certain ligands including testosterone and nifedipin.

Genetic variation in drug transporters
• Introduction:
 Drug transporters are membrane bound proteins involved in the
uptake or efflux of drugs by several tissues such as the intestine , liver,
kidneys and brain.
 They can have a significant impact on the pharmacokinetics of
endogenous and exogenous compounds.
 Drug transporters along with drug metabolizing enzymes are major
determinants of drug disposition and are known to alter the response
to many commonly used drugs.
 For delivering efficacious medicine with the right dose for each
patient it is important to understand the contribution of genetic
variants for drug transporters.

Types of transporters
• Transporters can be divided into two families :
Uptake Transporters or Solute linked family [SLC family]
Efflux Transporters or ATP binding cassette,[ABC family].
ABC family are expressed in cells of several organs
such as liver , small intestine and kidney ,where they influence
the pharmacokinetics of drugs. Transporter proteins have
wide substrate specificity and therefore influence the
pharmacokinetics of the drugs.

Solute linked carrier transporters
• The solute carrier group of membrane transport proteins
include 400 members organized into 65 families.
• Solutes are carried are extremely diverse and include both
charged and uncharged organic molecules as well as
inorganic ions and the gas ammonia.
• It contains a number of hydrophobic transmembrane alpha
helices connected to each other by hydrophilic intra and
extra cellular loops.
• SLC group includes example of transport proteins that are
facilitative transporters ,secondary active transporters,
primary active transporters , ion channels and aquaporins.

Nomenclature
• Names of SLC family have the following format
i. SLCnXm where;
ii. SLC is the root name( solute carrier )
iii. n is an integer representing a family (e.g. 1-52)
iv. X = single letter (A,B,C…) denoting a subfamily
v. M= an integer representing an individual family member.
example : SLC1A1 is the first isoform of subfamily of SLC
family 1.

ATP binding cassette transporter
 The ABC are a transport system superfamily that is one of the largest
and possibly one of the oldest gene families.
 Consists of multiple subunits one or two of which are transmembrane
subunits and one or two of which are membrane associated with AAA
ATPases.
 The ATPase subunits utilize the energy of ATP binding and hydrolysis
to provide the energy needed for the translocation of substrates
across membranes either for uptake or for export of the substrate.
 ABC uptake porters take up a large variety of primary and secondary
metabolites. Some of them are involved in tumor resistance , cystic
fibrosis and a range of other inherited human disease.

• ABC genes are essential for many processes in the cell and
mutations in human genes cause or contribute to several
human genetic diseases.
• Many of them have been characterized and shown to be
casually related diseases present in humans as Cystic fibrosis,
Adrenoleukodystrophy , Stargardt disease, Ataxia and
Hyperinsulimenic hypoglycemia.
• Most of the eukaryotic ABC transporters are effluxers some
are not directly involved in transporting substance.
• Human ABC transporters are involved in several diseases that
arise from polymorphisms in ABC genes and rarely due to
complete loss of function of single ABC proteins.

P- Glycoprotein
• P –glycoprotein also known as multidrug resistance protein 1 or
cluster of differentiation 243 is an important protein of the ell
membrane that pumps many foreign substances out of the cells.
• It is an ATP dependent efflux pump.
• Discovered in 1971 by Victor Ling .
• The glycoprotein is encoded by ABCB1 gene.
• It transports various substrates across the cell membrane including
drugs like Colchicine, Quinidine, Tyrosine kinase inhibitors(Gefitinib,
Sunitinib).

ABC transporters subfamilies
• There are 49 known ABC transporters present in humans which are
classified into 7 families by Human Genome Organization.
1) ABCA ---- Family contains some of the largest transporters. 5 of
them are located in a cluster in the 17q24 chromosome.
Responsible for the transportation of cholesterol and lipids. e.g.
ABCA1,ABCA12.
2) ABCB ---- Consists of four full and seven half transporters . Some
are located in the BBB, liver , mitochondria which transport
peptides and bile. e.g. ABCB5
3) ABCC ---- Consists of 12 full transporters. Used in ion transport, cell
surface receptors, toxin secretion. Includes CFTR protein which
causes cystic fibrosis when deficient. e.g. ABCC6

Subfamilies contd…..
4) ABCD ---- Consists of 4 half transporters. All are used in
peroxisomes. e.g. ABCD1.
5) ABCE/ABCEF ---- Consist of 1 ABCE & 3 ABCF proteins .
These are not actually transporters but merely ATP binding
domains that were divided from the ABC family , but without
the transmembrane domains. These proteins regulate protein
synthesis or expression. e.g. ASBCE1, ABCF1,ABCF2.
6) ABCG ---- Consists of 6 reverse half transporters with the
NBF at the NH3+ end and the TM at the COO- end . Transports
lipids, diverse drug substrates, bile cholesterol and other
steroids. e.g.ABCG2, ABCG1.

Genetic variation
1. ABCA1 group is implicated in the development of genetic
diseases . e.g. In reverse Tangier’s disease the ABCA1
protein is mutated.
2. Also the ABCA4 maps to a region of chromosome p21 that
contains the gene for Stargardt’s disease. In rod
photoreceptors the gene is highly expressed and is
mutated in Stargardt’s disease.
3. For ABCB group cells that overexpress this protein exhibit
multi-drug resistance.
4. ABCE group is composed of genes that have ATP binding
domains that are closely related to other ABC transporters
but these genes don’t encode for transmembrane
domains.

5) ABCG genes have orientation opposite to that of other ABC genes.
The ABCG2 gene was discovered in cell lines selected for high level
resistance for Mitoxantrone and no expression of ABCB1 or ABCC1 or
ABCG2 can export Anthrocycline anticancer drugs as well as Topotecan
as substrates.
6) Mutation in ABCD1 is responsible for Adrenoleukodystrophy . The
cells of ALD patient feature accumulation of unbranched saturated fatty
acids.
7) ABCC are also known as multidrug resistance proteins[MRPs].
i. Disease like cystic fibrosis can be observed when there is genetic
mutation in CFTR( cystic fibrosis transmembrane regulator) a part
of this subfamily.
ii. Mutations in Sulphonylurea, a part of this subfamily receptors are a
potential cause of Neonatal diabetes mellitus.

Cystic fibrosis
i. A genetic disorder that affects mostly the lungs but also
the pancreas, liver, kidneys and intestine.
ii. It is inherited in autosomal recessive manner and caused
due to the presence of mutations in both the copies of the
gene for the CFTR Protein.
iii. Those with a single working copy are carriers and
otherwise mostly healthy.
iv. CFTR is involved in the production of sweat, digestive
fluids and mucus.
v. Most common among North European and affects one out
of every 3000 newborns.

Adrenoleukodystrophy
i. This a disease linked to the X chromosome( X- Linked recessive
inheritance). It is a result of fatty acid buildup caused by the
relevant enzymes not functioning properly, which then causes
damage to the myelin sheath of the nerves, resulting in seizures
and hyperactivity.
ii. It is caused by mutations in the ABCD1 gene located on the X
chromosome that codes for ALD a peroxisomal membrane
transporter protein.
iii. Males with an ABCD1 mutation are hemizygous as they have only a
single X chromosome.
iv. Female carriers will avoid severe manifestations but later may be
symptomatic.
v. Detection of mutation identifies an individual who is affected there
is no genotype-phenotype correlation.

Stargardt disease
i. It is the most common inherited single-gene retinal
disease.
ii. It usually has an autosomal recessive inheritance caused
by mutations in the ABCA4 gene.
iii. Symptoms commonly include wavy vision, blind spots,
blurriness,, loss of depth perception.
iv. The carrier frequency in the general population of ABCA4
alleles is 5-10% . Different combinations of ABCA4 genes
will result in widely different age of onset and retinal
pathology.

-Omics world explored
• Genomics: Genomics is that field of biology that focuses on
the structure, function , evolution ,mapping and editing of
genes.
• The field also involves the study of intragenomic (within the
genome ) phenomena such as epistasis(effect of one gene on
another), pleiotropy ( one gene affecting more than one
trait) , heterosis ( hybrid vigor ) and other interactions in
between loci and alleles within the genome.

Genomics :
In contrast to genetics which refers to the study of
individual genes and their roles in inheritance.
Genomics aims at the collective characterization and
quantification of all of an organism’s genes, their
interrelations and influence on an organism.
Genomics also involves the sequencing and analysis of
genomes through high throughput DNA sequencing and
Bioinformatics to analyze the function of each genome.

Applications
Genomics has provided applications in many fields namely;
I. Genomic medicine: Next generation genomic techniques has
allowed researchers to drastically understand the genetic bases of
drug response and disease.
II. Synthetic biology and bioengineering
III. Conservation genomics: Conservationist can use the
information gathered by genome sequencing in order to
better evaluate genetic factors key to species conservation
such as the genetic diversity of a population or whether an
individual is heterozygous for a recessive disorder.

Proteomics
• The word was coined in the year 1997.
• Proteomics is defined as the large scale study of proteins. As
known proteins are vital parts of living organisms with many
functions.
• Proteome is the entire set of proteins.
• Proteome gets modified as an individual undergoes stresses
or with time or distinct requirements.
• Involves the overall exploration of Proteomes from the
overall level of protein composition, structure and activity.

Types of Proteomics
i. Interaction proteomics: Protein-protein association
ii. Expression proteomics : Protein quantification
Level of any protein in cell at any given time can be controlled
by :
1. Rate of transcription of the gene.
2. The efficiency of the translation of m-RNA into protein.
3. The rate of degradation of protein into cell.

Applications:
1. Mining
2. Protein expression profiling: identification of proteins in a sample as
a function of a particular state of cell.
3. Protein network mapping : To determine protein interaction with
each other in living organism.
4. Mapping of protein modifications: Task to identify how and where
proteins are modified post-translationally.
5. Helps to study drug MoA , Disease Biomarkers ,Epigenetics, Spatial
localization etc.

Nutrigenomics
Nutrigenomics or nutrition genomics is a science studying
the relationship between human genome ,nutrition and
health.
People in this field work towards developing an
understanding of how the whole body responds to a food via
systems biology.
[ Systems biology is the computational and mathematical
analysis and modeling of complex biological system. The
Human Genome Project is an example of applied systems
thinking in biology which has led to new collaborative ways of
working on problems in the biological field of genetics.]

• The four basic tenets of Nutrigenomics are :
Improper diets are risk factor for disease.
Dietary chemicals alter gene expression or change
gene structure.
The degree to which diet influences the balance
between healthy and disease states may depend on
individuals genetic make up.
Some diet regulated genes are likely to play a role in
the onset , incidence, progression or severity of
chronic disease.

Advantages
• Increased focus on a healthy diet and lifestyle.
• Increased awareness of risk of certain conditions.
• Improved health quantity of life.
• Focus on prevention of diseases.
• Decrease morbidity and premature mortality.
• Reduced healthcare concepts.
• Better understanding of the mechanisms involved in
the disease suspectibility.

Disadvantages
• Attention is drawn away from other modifiable risk
factors.
• Focus only on specific nutrients and foods.
• Increased costs associated with personalized diets
and designer foods.
• Sometimes plagued with misleading claims.

• Specific dietary profiles can modulate the delicate balance
between health and disease acting either directly or
indirectly or gene expression.
• Personalized diets which takes into account individual
genotype represents the ultimate goal of NUTRIGENOMICS.
• Certain foods like cauliflower, broccoli contain chemicals that
actually tell our gene to direct biosynthesis of these
enzymes.
• In individuals who lack the required gene or possess gene
which give unclear instruction for making an enzyme that
metabolites amino acid – Phenylalanine leading to amino
acid build up causing brain damage.

Applications
• Nutritional science originally emerged as a field that studied
individuals lacking certain nutrients and the subsequent effects such
as scurvy which results from lack of Vitamin C.
• Obesity is another aspect studied. It’s genetic variation that
individuals respond to diet differently. By exploring the interaction
between dietary pattern and genetic factors one can suggest the field
that could prevent or reduce obesity.
Ex. Prader-Willi Syndrome [Characterized by insatiable appetite where
the paternal copy of the chromosome is erroneously deleted and the
maternal loci is inactivated by over methylation.]
 The APO B SNP rs5125356 is another diet related variation ; the A/G
heterozygous genotype was found to have association with obesity in
terms of BMI and waist circumference.

Future aspects
• In the coming years NUTRIOGENOMICS has the promise to be the
most interesting and exciting fields for cutting edge research.
• The different –OMICS discipline offer a variety of powerful ways to
understand what is going inside the cells in response to nutrients and
to see how these responses differ from person to person.
• Over the past decade a lot of study has been carried out regarding
which antioxidant is found in which food and at which level.
• It all started in 1920 when Goldberger showed that he could cure
pellagra by simply adding Baker’s yeast to the diet . After another 10
years it was concluded that it was Niacin in bakers yeast that cured it.

Functionomics
• Functionomics or functional genomics is a field of molecular
biology that attempts to describe gene functions and
interactions.
• Functinomics make use of the vast data generated by
proteomic and transcriptomic projects .
• Functional genomics focuses on the dynamic aspects such as
gene transcription, translation, regulation of gene expression
and protein-protein interactions.
• A major challenge remains to integrate functionomics with
genomics and metabolomics data and mainly their
integration with their physiological and pathophysiological
functional interpretation.

Applications
i. The main goal of functional genomics is to understand the function
of genes or proteins eventually all components of genome.
ii. It involves the study of mutation and polymorphism (SNP) as swell
as the measurement of molecular activities.
iii. Functionomics is concerned with Gene Interaction mapping.
iv. To study the DNA/protein interactions. Protein formed by the
translation of m-RNA play a major role in regulating gene
expression and to understand that it is necessary to identify DNA
sequence that interact with.
v. Required for DNA accessibility assays . These assays include Chip
sequencing, CUT& RUN sequencing and Calling cards .

-OMICS chain
• DNA ----------- GENOMICS
• |
• RNA------------ TRANSCRIPTOMICS
• |
• PROTEINS ---------PROTEOMICS
• |
• BIOCHEMICAL ---------- METABOLOMICS
• |
• BIOLOGICAL PHENOTYPE

Metabolomics
• Definition: Metabolomics is the scientific study of chemical
processes involving metabolites , the small molecule
substrates, intermediates and products of metabolism.
• The metabolome represents the complete set of
metabolites in a biological cell , tissue or an organ or an
organism.
• Thus metabolomics provides a direct functional readout of
the physiological state of an organism.

Do you know ??
• Genome can tell what could happen.
• |
• Transcriptome can tell what appears to be happening
• |
• Proteome can tell what makes it happen
• |
•Metabolome can tell what has happened and what is
happening.

How it started ?
• The first metabolite database called METLIN for searching
fragmentation from tandem mass spectrometry experiments
was developed by the Sizudak Lab at the SCRIPPS Research
Institute in 2005.
• In January 2007 scientists at the University of Alberta and
the University of Calgary the first draft of the Human
Metabolome.
• Thus concluded that each type of cell and tissue has a
unique metabolic fingerprint that can elucidate organ or
tissue specific information.

Metabolites
Metabolites are substrates , intermediates and products of
metabolism.
Within the context of metabolomics a metabolite is usually
defined as any molecule less than 1.5 kDa in size.
Exometabolomics or Metabolomic Fingerprinting is the
study of extracellular metabolites. It uses many techniques
from other subfields of metabolomics with application in
biofuel development, bioprocessing, determining drug
mechanism of action and studying intercellular interactions.

Applications:
1. Pharmacology and pre-clinical drug trials
2. New born screening
3. Toxicology
4. Transplant monitoring
5. Tools for functional genomics
6. Applied in novel therapeutics and detection of various forms of
cancer.
Metabologenomics is a novel approach to integrate metabolomics and
genomics data by correlating microbial exported metabolites with
predicted biosynthetic genes.
Fluoxomics is the further development of metabolomics which helps to
determine the reactions and can trace metabolites in a biological
system over time.

Basic workflow :
Sample collection,treatment , processing-------- Separation
technique[GC,HPLC,CE] ------- Detection Technique( NMR, MS)--------
Data analysis using multivariate analysis[ PCA( Principle Component
Analysis)]---------- Validation followed by clinical application --------
Sample collection, treatment and processing.
Human Metabolome Project: On 23rd January ,2007, Human
Metabolome Project led by Dr.David Wishart of the University
of Alberta, Canada completed first draft of Human
Metabolome consisting of database of approx. 2500
metabolites.

Problems and challenges :
i. Metabolites are having wide range of molecular weight
and large variation in concentration.
ii. Metabolome is much more dynamic than proteome and
genome, thus more time sensitive.
iii. Number of metabolites existing are far less than the no of
transcripts.
iv. Not all metabolites can be identified.

Future aspects:
• If Pathognomonic metabolic profiles of various diseases can be
identified and validated in various body fluids metabolomics may save
time , cost and effort in obtaining definitive diagnosis in situations
where no other test can provide answer.
• Can be adopted as a minimally invasive tool.
• Improved sensitivity will also be positive using cryogenecially cooled
NMR probes known as Cryoprobes.
• Analysis of metabolites in known stress response pathways by
molecular profiling helps to response to environmental or genetic
perturbations. Thus a multi-metabolite profile obtained from high
resolution MS(Mass Spectroscopy) offers potential for identification
of robust biomarkers to predict radiation toxicity of organs.

Pharamcogrenomics [Applications]
• Improved drug safety and reduced ADRs.
• Tailor treatments to meet patients unique genetic pre-
disposition, identifying optimal dosing.
• Improved drug discovery targeted to human disease
• Improved proof of principle for efficacy trials
• May be applied to the areas of Pain management,
Cardiology, Oncology, Psychiatry etc.
• In Cancer treatment pharmacogenomics tests are used to
identify which patients are most likely to respond to certain
types of drugs.

Challenges
• Although there appears to be a general acceptance of the basic
tenets of Pharmacogenomics amongst physicians and healthcare
professionals there are some challenges to be addressed.
i. Limitation on how to apply the test into clinical practice and
treatment.
ii. Lack of availability of test.
iii. Understanding and interpretation of evidence based research.
iv. Ethical, Logical and Social issue.
v. No sufficient evidence to validate the cost-effectiveness and
cost-consequence of the test.

Future aspects
I. Computational advances have enabled cheaper and faster
sequencing .Research has focused more on the genomic
mining and –OMIC technologies.
II. Also as the cost decreases the development of
personalized drug therapies will increase.
III. Technology now allows for the genetic analysis of
hundreds of target genes involved in medication and
response in less than 24 hours for under 1000
dollars(1000$).

Pharmacogenomics in India
• The current definition of PRECISION MEDICINE – Understanding
disease at a deeper level in order to develop more targeted therapy
clearly requires the armament of pharmacogenomics to succeed.
 Some of them are Neo-adjuvant chemotherapy[NACT] or research
on P-glycoproteins.
• NACT is usually provided to patients with locally advanced breast
cancers.
• P-gp an ATP dependent efflux pump is used as a part of NACT for the
management of cancer.
• ABCB1 gene codes for the P-gp.
• Case study in 48 patients showed that those with high expression of
P-gp had 3.6 times more chance of having poor response to NACT.

Conclusion:
•Though the concept of Pharmacogenomics is new it
has the potential in it to be adopted as the pioneer to
provide Tailor-made therapy to the patients thereby
bringing the concept of PRECISION MEDICINE a reality.

Reference
• Internet
• Rang and Dale molecular Pharmacology
• Genetic variation in human G protein coupled receptors:
Implications for drug therapy; Wolfgang Sadee;Elen Hoeg;
Julie Lucas and Darwin Wang.

Pharmacogenomics

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