1. Genomics, personalized medicine
and pharmacogenomics
Genomics and personalized medicine are changing the field of pharmacogenomics by two ways:
by optimizing drug therapies and
by reducing adverse drug reactions.
In the near future, personalized medicine will allow physicians to predict which diseases you will develop, which therapeutics will work for you, and which drug dosages are appropriate.
6.1.PHRM-521.

2. Definition
Today, the phrase personalized medicine is used to describe the application of information from a patient’s unique genetic profile in order to select effective treatments that have minimal side-effects and to detect disease susceptibility prior to development of the disease.
Pharmacogenomics is the study of how an individual’s entire genetic makeup determines the body’s response to drugs. The term pharmacogenomics is used interchangeably with pharmacogenetics, which refers to the study of how sequence variation within specific candidate genes affects an individual’s drug responses.
In pharmacogenomics, scientists take into account many aspects of drug metabolism and how genetic traits affect these aspects.
Aspect: (a part of something) the direction that something faces or points towards

3. Variations in patient response to drugs
When a drug enters the body, it interacts with various proteins including carriers, cell-surface receptors, transpor-ters, and metabolizing enzymes. These proteins affect a drug’s target site of action, absorption, pharmacological response, breakdown, and excretion. Because there are so many interactions that occur between a drug and proteins within the patient, many genes and many different genetic polymorphisms can affect a person’s response to a drug.
Figure: A general summary of the percentages of
patients for which a particular class of drugs is effective.

4. Optimizing drug therapies
On average, a drug will be effective in only about 50% of patients who take it (see Fig. in previous slide), which means that physicians often must switch their patients from one drug to another until they find one that is effective.
Not only does this waste time and resources, but also it may be dangerous to the patient who is exposed to a variety of different pharmaceuticals and who may not receive appropriate treatment in time to combat a progressive illness.
One of the most common current applications of personalized pharmacogenomics is in the diagnosis and treatment of cancers.
Large-scale sequencing studies show that each tumor is genetically unique, even though it may fall into a broad category based on cytological analysis or knowledge of its tissue origin.
Cytology is that branch of life science that deals with the study of cells in terms of structure, function and chemistry. Robert Hooke is sometimes seen as the father of cytology.

5. One of the first success stories in personalized medicine was that of the human epidermal growth factor receptor 2 (HER-2) gene and the use of the drug Herceptin® in breast cancer.
Because Herceptin will only act on breast cancer cells that have amplified HER-2 genes, it is important to know the HER-2 phenotype of each cancer. In addition, Herceptin has potentially serious side-effects. Hence, its use must be limited to those who could benefit from the treatment.
EGFRs (eidermal growth factor receptors). The K-RAS gene encodes a small signaling protein that is part of the EGFR pathway.
Chronic myelogenous leukemia (CML), also known as chronic granulocytic leukemia (CGL), is a cancer of the white blood cells. It is a form of leukemia characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow and the accumulation of these cells in the blood. CML is a clonal bone marrow stem cell disorder in which a proliferation of mature granulocytes (neutrophils, eosinophils and basophils) and their precursors is found.
B-lymphocyte antigen CD20 or CD20 is an activated-glycosylated phosphoprotein expressed on the surface of all B-cells beginning at the pro-B phase (CD45R+, CD117+) and progressively increasing in concentration until maturity.
The non-Hodgkin lymphomas (NHLs) are diverse group of blood cancers that include any kind of lymphoma except Hodgkin's lymphomas (also known as Hodgkin's disease, is a type of lymphoma, in which cancer originates from white blood cells called lymphocytes).
Types of NHL vary significantly in their severity, from slow growing to very aggressive types. Lymphomas are treated by combinations of chemotherapy, monoclonal antibodies (CD20), immunotherapy, radiation, and hematopoietic stem cell transplantation.

6. Determining the gene and protein status of breast cancer cells
A number of molecular assays have been developed to determine the gene and protein status of breast cancer cells. Two of the most commonly used tests are based on immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).
In IHC assays, an antibody that binds to HER-2 protein molecules is added to fixed tissue on a slide. The antibody is bound to another molecule that reacts to produce a visual stain.
After washing and staining, the tissues are observed under a microscope. The level of HER-2 staining is assessed from “0” (fewer than 20,000 HER-2 molecules per cell) to “+3” (approx. 2 million molecules per cell).
Histology is the study of the microscopic anatomy of cells and tissues of plants and animals.
Immunohistochemistry or IHC refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.

7. Immunohistochemistry (IHC)
IHC is a method to stain the tissue sections/cells and is perhaps the most commonly applied immunostaining technique. While the first cases of IHC staining used fluorescent dyes, other non-fluorescent methods using enzymes such as peroxidase and alkaline phosphatase are now used.
These enzymes are capable of catalysing reactions that give a coloured product that is easily detectable by light microscopy. Alternatively, radioactive elements can be used as labels, and the immuno-reaction can be visualized by autoradiography.

8. Enzyme Linked ImmunoSorbent Assay (ELISA)
ELISA is a diagnostic method for quantitatively or semi-quantitatively determining protein concentrations from blood plasma, serum or cell/tissue extracts in a multi-well plate format (usually 96-wells per plate). Broadly, proteins in solution are adsorbed to ELISA plates. Antibodies specific for the protein of interest are used to probe the plate.
Background is minimized by optimizing blocking and washing methods (as for IHC), and specificity is ensured via the presence of positive and negative controls. Detection methods are usually colorimetric or chemilumi-nescence based.
Chemiluminescence (sometimes "chemoluminescence") is the emission of light (luminescence), as the result of a chemical reaction.

9. In FISH, DNA or RNA molecules with sequence complemen-tarity to the HER-2 gene sequence are added to the fixed tissue on the slide.
These DNA or RNA probes are labeled with a fluorescent tag molecule. After hybridizing the probes to the tissue and washing off excess probe, the location and intensity of the probe are determined by observing the tissue under a fluorescence microscope.
The number of HER-2 genes is assessed by comparing the fluorescence signal of the HER-2 probe with a control signal from another gene that is not amplified in the cells.
Herceptin has had a major effect on the treatment of HER-2 positive breast cancers. When Herceptin is used in combination with chemotherapy, there is a 25 to 50 percent increase in survival, compared with the use of chemotherapy alone. Herceptin is now one of the biggest selling biotechno-logy products in the world, generating more than $5 billion in annual sales.

10. HER-2 gene and protein assays
HER-2 gene and protein assays. (a) Normal and breast cancer cells within a biopsy sample, stained by HER-2 immunohistoche-mistry. Cell nuclei are stained blue. Cancer cells that overexpress HER-2 protein stain brown. (b) Cancer cells from the same tumor assayed for HER-2 gene copy number by FISH. Cancer cell nuclei appear green under the fluorescence microscope and the HER-2 gene DNA appears bright yellow. Large clumps of yellow stain indicate HER-2 gene amplification (>20 copies per nucleus).

11. Reducing adverse drug reactions
Every year, about 2 million people in the United States suffer serious side-effects from pharmaceutical drugs, and appro-ximately 100,000 people die from that adverse side-effects.
The costs associated with these adverse drug reactions (ADRs) are estimated to be $136 billion annually.
Although some ADRs result from drug misuse, others result from a patient’s inherent physiological reactions to a drug.
Sequence variations in a large number of genes can affect drug responsiveness (see Table in the next slide).
Of particular significance are the genes that encode the cytochrome P450 families of enzymes. These family members are encoded by 57 different genes.
The products of the CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 genes are responsible for metabolizing most clinically important pharmaceutical drugs.
Cytochromes P450 (CYPs) belong to the superfamily of proteins containing a heme cofactor and, therefore, are hemoproteins.
Cytochrome P450 is a family of isozymes responsible for the biotransformation of several drugs. Drug metabolism via the cytochrome P450 system has emerged as an important determinant in the occurrence of several drug interactions that can result in drug toxicities, reduced pharmacological effect, and adverse drug reactions.

12. Examples of variant gene products that
affect drug responses
Lupus erythematosus is a name given to a collection of autoimmune diseases in which the human immune system becomes hyperactive and attacks normal, healthy tissues. Symptoms of these diseases can affect many different body systems, including joints, skin, kidneys, blood cells, heart, and lungs. Systemic lupus erythematosus (SLE) is an autoimmune disease in which the body's immune system mistakenly attacks healthy tissue. It can affect the skin, joints, kidneys, brain, and other organs.
Serotonergic or serotoninergic means "working on the neurotransmitter serotonin". A synapse is serotonergic if it uses serotonin as its neurotransmitter. A serotonergic neuron produces serotonin. A substance is serotonergic if it produces its effects via interactions with the serotonin system.
Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter. Biochemically derived from tryptophan, serotonin is primarily found in the gastrointestinal tract (GI tract), blood platelets, and the central nervous system (CNS) of animals, including humans. It is popularly thought to be a contributor to feelings of well-being and happiness.

13. Personalized medicine and disease diagnosis
Growth of gene tests and testing laboratories from 1993 to (from the GeneTests Web site at
As of 2009, there were genetic tests for approximately 2000 different diseases.

14. Table: Some single-gene defects for which genetic
tests are available.
Predisposition: the fact or condition of being predisposed (to dispose in advance).
Achondroplasia is a form of short-limbed dwarfism. The word achondroplasia literally means "without cartilage formation.“
Fibroblast growth factor receptor (FGFR) 3 is a protein that in humans is encoded by the FGFR3 gene. FGFR3 has also been designated as CD333 (cluster of differentiation 333).
Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy, affecting around 1 in 3,600 males, which results in muscle degeneration and premature death.
Muscular dystrophy (MD) is a group of muscle diseases that weaken the musculoskeletal system and hamper locomotion. X-linked: In mammals, the female is the homogametic sex, with two X chromosomes (XX), while the male is heterogametic, with one X and one Y chromosome (XY). Genes on the X or Y chromosome are called sex-linked.
Fragile X syndrome (FXS) is a genetic condition that causes a range of developmental problems including learning disabilities and cognitive (is the set of all mental abilities and processes related to knowledge: attention, memory and working memory, judgment and evaluation, reasoning and "computation", problem solving and decision making, comprehension and production of language, etc) impairment. Also known as Martin–Bell syndrome, or Escalante's syndrome.
Friedreich's ataxia is an autosomal recessive inherited disease that causes progressive damage to the nervous system. Ataxia (lack of order) is a neurological sign consisting of lack of voluntary coordination of muscle movements that includes gait abnormality. Autosome (any chromosome other than a sex chromosome). An allele is one of a number of alternative forms of the same gene or same genetic locus.
Spasticity (meaning "drawing, pulling") is a feature of altered skeletal muscle performance with a combination of paralysis, increased tendon reflex activity and hypertonia (damage to the central nervous system). It is also colloquially referred to as an unusual "tightness", stiffness, or "pull" of muscles.
Hemophilia is a rare disorder in which your blood doesn't clot normally because it lacks sufficient blood-clotting proteins (clotting factors).
Huntington's disease is an inherited disease that causes the progressive breakdown (degeneration) of nerve cells in the brain.
Lesch-Nyhan syndrome (LNS) is a condition that occurs almost exclusively in males. It is characterized by neurological and behavioral abnormalities and the overproduction of uric acid. LNS is the most severe form of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency.

15. Although these genetic tests are extremely useful for detecting some future diseases and guiding treatment, it is clear that most disorders are multifactorial and complex.
It is likely that diseases such as diabetes, Alzheimer’s, and heart disease are caused by interactions between many genes, as well as by factors contributed by epigenetic effects, lifestyle, and environment.
These diseases tend to be chronic and have a significant burden on health-care systems.
Genome sequencing, SNP identification, and genome-wide association studies (GWAS) are beginning to reveal some of the DNA variants that may contribute to the risk of developing multifactorial diseases such as cancer, heart disease, and diabetes.
In genetics, epigenetics (prefix epi - over, outside of, around)) is the study of cellular and physiological trait variations that are not caused by changes in the DNA sequence; epigenetics describes the study of dynamic alterations in the transcriptional potential of a cell. Examples of mechanisms that produce such changes are DNA methylation and histone modification, each of which alters how genes are expressed without altering the underlying DNA sequence.

16. The Pharmacogenomics Knowledge Base (PharmGKB): Genes, Drugs, and Diseases on the Web
PharmGKB is a publicly available Internet database and information source developed by Stanford University. It is funded by the National Institutes of Health (NIH)
On the PharmGKB Web site ( you may search for genes and more than 650 variants that affect drug reactions, information on a large number of drugs, diseases and their genetic links, pharmaco-genomic pathways, gene tests, and relevant publications.

17. Pharmacogenomics and Rational Drug Design
Homozygous is a genetic condition where an individual inherits the same alleles for a particular gene from both parents. TPMT: thiopurine S-methyltransferase.
A diploid organism is heterozygous at a gene locus when its cells contain two different alleles of a gene. The cell or organism is called a heterozygote specifically for the allele in question, therefore, heterozygosity refers to a specific genotype.
An allele is one of a number of alternative forms of the same gene or same genetic locus. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation. However, most genetic variations result in little or no observable variation.
Fig. Different individuals with the same disease, in this case childhood leukemia, often respond differently to a drug treatment because of subtle differences in gene expression. The dose of an anticancer drug (6-MP) that works for one person may be toxic for another person.

18. Several methods are being developed for expanding the uses of pharmacogenomics. One promising method involves the detection of SNPs.
Perhaps researchers will be able to identify a shared SNP sequence in the DNA of people who also share a heritable reaction to a drug.
If the SNP segregates with a part of the genome containing the gene responsible for the drug reaction, it may be possible to devise gene tests based on the SNP, without even knowing the identity of the gene responsible for the drug reaction.
In the future, DNA microarrays may be used to screen a patient’s genome for multiple drug reactions.
Knowledge from genetics and molecular biology is also contributing to the development of new drugs targeted at specific disease-associated molecules.
Most drug development is currently based on trial-and-error testing of chemicals in lab animals, in the hope of finding a chemical that has a useful effect.

19. Fig. Microarray analysis for analyzing gene-expression patterns in a tissue.

20. Rational Drug Design (RDD)
RDD involves the synthesis of specific chemical substances that affect specific gene products.
An example of a rational drug design product is the new drug imatinib, trade name Gleevec, used to treat chronic myelogenous leukemia (CML).
Geneticists had discovered that CML cells contain the Philadelphia chromosome, which results from a reciprocal translocation between chromosomes 9 and 22.
Gene cloning revealed that the t(9;22) translocation creates a fusion of the C-ABL proto-oncogene with the BCR gene. This BCR-ABL fusion gene encodes a powerful fusion protein that causes cells to escape cell-cycle control.
The fusion protein, which acts as a tyrosine kinase, is not present in non-cancer cells from CML patients.

21. Gleevec is now used to treat CML and several other cancers.
To develop Gleevec, chemists used high-throughput screens of chemical libraries to find a molecule that bound to the BCR-ABL enzyme.
After chemical modifications to make the inhibitory molecule bind more tightly, tests showed that it specifically inhibited BCR-ABL activity.
Clinical trials revealed that Gleevec was effective against CML, with minimal side effects and a higher remission rate than that seen with conventional therapies.
Gleevec is now used to treat CML and several other cancers.
With scientists discovering more genes and gene products associated with diseases, rational drug design promises to become a powerful technology within the next decade.
Remission: a period of time during a serious illness when the patient's health improves.

22. Gene Therapy
Although drug treatments are often effective in controlling symptoms of genetic disorders, the ideal outcome of medical treatment is to cure these diseases.
In an effort to cure genetic diseases, scientists are actively investigating gene therapy - a therapeutic technique that aims to transfer normal genes into a patient’s cells.
In theory, the normal genes will be transcribed and translated into functional gene products, which, in turn, will bring about a normal phenotype.
Human gene therapy began in 1990 with the treatment of a young girl named Ashanti DeSilva who has a heritable disorder called severe combined immunodeficiency (SCID). Individuals with SCID have no functional immune system and usually die from what would normally be minor infections.
Ashanti has an autosomal form of SCID caused by a mutation in the gene encoding the enzyme adenosine deaminase (ADA). Her gene therapy began when clinicians isolated some of her white blood cells, called T cells. These cells, which are key components of the immune system, were mixed with a retroviral vector carrying an inserted copy of the normal ADA gene.
Autosomal: refers to any of the chromosomes other than the sex-determining chromosomes (i.e., the X and Y) or the genes on these chromosomes.

23. (a) Ashanti DeSilva, the first person to be treated by gene therapy
(a) Ashanti DeSilva, the first person to be treated by gene therapy. (b) To treat SCID using gene therapy, a cloned human ADA gene is transferred into a viral vector, which is then used to infect white blood cells removed from the patient.

24. To date, gene therapy has successfully restored the health of about 20 children affected by SCID. Although gene therapy was originally developed as a treatment for single-gene (monogenic) inherited diseases, the technique was quickly adapted for the treatment of acquired diseases such as cancer, neurodegenerative diseases, cardiovascular disease, and infectious diseases, such as HIV.
In the case of HIV, scientists are exploring ways to deliver immune system-stimulating genes that could make individuals resistant to HIV infection or cripple the virus in HIV positive persons.
There are nearly 1000 gene therapy trials actively underway in the United States alone.
Over a 10-year period, from 1990 to 1999, more than 4000 people underwent gene therapy for a variety of genetic disorders. These trials often failed and thus led to a loss of confidence in gene therapy.
Neurodegenerative: Neurodegenerative disease is an umbrella term for a range of conditions which primarily affect the neurons in the human brain. Neurons are the building blocks of the nervous system which includes the brain and spinal cord. Neurons normally don’t reproduce or replace themselves, so when they become damaged or die they cannot be replaced by the body. Examples of neurodegenerative diseases include Parkinson’s, Alzheimer’s, and Huntington’s disease.
Neurodegenerative diseases are incurable and debilitating conditions that result in progressive degeneration and/or death of nerve cells. This causes problems with movement (called ataxias), or mental functioning (called dementias).
Cripple: one that is disabled or deficient in a specified manner.

25. Gene replacement approaches
Scientists are also working on gene replacement approaches that involve removing a defective gene from the genome.
Recent work with enzymes called zinc-finger nucleases have shown promise in animal models and cultured cells.
These enzymes can create site-specific cleavage in the genome and when coupled with certain integrases may lead to gene editing by cutting out defective sequences and introducing normal homologous sequences into the genome.
Encouraging breakthroughs have taken place in this area using model organisms such as mice; however, this technology has not advanced sufficiently for use in humans.
Attempts have been made to use antisense oligonucleo-tides in order to inhibit translation of mRNAs from defective genes, but this approach to gene therapy has generally not yet proven to be reliable.
Antisense RNA (asRNA) is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell. Some authors have used the term micRNA (mRNA-interfering complementary RNA) to refer to these RNAs but it is not widely used.

26. Gene silencing approaches
The recent emergence of RNA interference as a powerful gene-silencing tool has reinvigorated gene therapy approaches by gene silencing. RNA interference (RNAi) is a form of gene-expression regulation.
In animals, short and double stranded RNA molecules are delivered into cells where the enzyme Dicer chops them into 21-nt long pieces called small interfering RNAs (siRNAs).
siRNAs then join with an enyzme complex called the RNA inducing silencing complex (RISC), which shuttles the siRNAs to their target mRNA, where they bind by complementary base pairing.
The RISC complex can block siRNA-bound mRNAs from being translated into protein or can lead to degradation of siRNA-bound mRNAs so they cannot be translated into protein.
A main challenge to RNAi-based therapeutics so far has been in vivo delivery of double-stranded RNA or siRNA. However, several RNAi clinical trials to treat blindness are underway in USA.
Reinvigorated: made or become fresh in spirits or vigor. Synonyms: energized, freshened, invigorated, newborn, reanimated, reborn, recreated, reenergized, refreshed, regenerated, reinvigorated, renewed, resuscitated, revived.

27. Fig. Mechanisms of gene regulation by RNA-induced gene silencing.
RNA-induced silencing complex (RISC).
RNA-induced initiation of transcription silencing complex (RITS).
In the cytoplasm, double stranded RNA molecules are recognized by an enzyme complex known as Dicer and are cleaved by Dicer into siRNAs.
A microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses, which functions in RNA silencing and post-transcriptional regulation of gene expression. The miRNAs are derived from single-stranded RNAs that are transcribed within the nucleus from the cell’s own genome and that contain a double-stranded stem-loop structure. Nuclease enzymes within the nucleus recognize these stem-loop structures and cleave them from the longer single-stranded RNA.
Stem-loop (intramolecular base pairing) is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a hairpin or hairpin loop.

28. What is Bioinformatics?
In today’s world, computers are as likely to be used by biologists as by any other professionals - bankers or flight controllers, for example.
Many of the tasks performed by such professionals are common to most of us: we all tend to write lots of memos and send lots of s; many of us use spreadsheets, and we all store immense amounts of never-to-be-seen-again data in complicated file systems.
However, besides these general tasks, biologists also use computers to address problems that are very specific to biologists, which are of no interest to bankers or flight controllers.
These specialized tasks, taken together, make up the field of bioinformatics. More specifically, we can define bioinformatics as the computational branch of molecular biology.

30. What bioinformatics can do for us?
Analyzing DNAs
Analyzing RNAs
Analyzing proteins
Analyzing others such as complex pathways, in silico simulation, bioimaging, etc.

31. How most people use bioinformatics?