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GENES and MEDICINE:

APPLICATIONS of MOLECULAR GENETICS

APPLICATIONS of RECOMBINANT DNA TECHNOLOGY (GENETIC ENGINEERING)

A. Commercial Gene Products
- use genetically modified organisms to produce recombinant proteins
- replacement therapy (e.g., insulin in diabetes)
- disease prevention (e.g., vaccines)
- diagnostic tests (e.g., monoclonal antibodies)
- medical or industrial applications

B. New Phenotypes
- use gene technology
- alter the characteristics of organisms (farm animals or crops)
C. Gene Therapy
- use gene technology on humans to treat a disease

COMMERCIAL GENE PRODUCTS

- most successful form of genetic engineering (products of medical, agricultural or commercial value)
a. Strategy
- transfer a gene (often human) to a host organism (usually a microbe) products made quickly,
cheaply and ethically
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A. Human Insulin
1. Synthetic cDNA Gene
- inserted into
a. E. coli
- make pro-insulin
- post-translational conversion to insulin carried out chemically
b. Saccharomyces cerevisiae
- yeast
- capable of post-translational modification  simplified human insulin
production
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B. Bovine Somatotrophin (BST)

- growth hormone produced by cattle
- gene cloned in bacteria to produce large quantities of BST
- injection with BST  10% increase in mass in beef cattle and 25% increase in milk production in dairy
cows
C. Rennin
- enzyme used in the production of cheese
- produced in the stomach of juvenile mammals (including humans)
- aids the digestion of the milk protein casein
1. Artificial cDNA Gene for Rennin
- made from mRNA extracted from calf stomach cells
- inserted into a variety of microbes
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Synthesis and Use of a Cosmid

- DNA is obtained from the bacteriophage (phage)lambda
- phage DNA is opened with a restriction enzyme  cDNA fragment is joined to it
 cosmid formation
Cosmid - contains base sequences for proteins that encourage entry into a bacterium
- penetrates a bacterium
- circularizes to form a plasmid-like body that carries the cDNA
D. AAT (-1-Antitrypsin)
- human protein (glycoprotein) made in the liver and found in the blood
- inhibitor of protease enzymes (trypsin, elastase)
1. AAT Gene Mutation
- rare
- single base substitution
a. Result
- inactive AAT  uninhibited protease enzymes
b. Consequence
- digestion of tissue by elastase  pulmonary emphysema
c. Treatment
- aerosol spray inhalation containing AAT
2. AAT Production
- produced in genetically modified sheep
- AAT gene coupled to promoter for milk protein β-lactoglobulin (only activated in mammary
gland cells  AAT harvested from sheep’s milk)
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GENE REPLACEMENT THERAPY
- most significant and most controversial kind of genetic engineering
- evolving technique for correcting defective genes
- least well developed
A. Goal - to genetically alter humans in order to treat a disease (altering the genotype of a tissue or even a whole
individual)
- insert the normal, cloned DNA for a gene into the somatic cells of a patient who is defective in that
gene as a result of disease-causing mutation
- DNA must become permanently integrated into the patient’s chromosomes  proper
expression  production of correct protein
B. Two Major Factors Involved in Gene Therapy
1. Introduction of the Functional Gene Sequence into Target Cells
a. Several Approaches
- a normal gene may be inserted into a nonspecific location within the genome to
replace a non-functional gene (most common)
- an abnormal gene can be swapped for a normal gene through homologous
recombination
- the abnormal gene can be repaired through selective reverse mutation returning the
gene to its normal function
- the regulation (the degree to which a gene is turned on or off) of a particular gene may
be altered

2. Expression and Permanent Integration of the Transfected Gene into the Host Cell Genome
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C. Vectors
- carrier molecule
- deliver the therapeutic gene to the patient’s target cells
1. Viruses
- most common vector used
- evolved a way of encapsulating and delivering their genes to human cells in a pathogenic
manner
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- genetically altered (portion of viral genome replaced with cloned gene  virus can infect but
not complete its replication cycle) to carry normal human DNA

- early vectors used in gene delivery

- adenoviruses
- retroviruses
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- newer strategy to take advantage of vectors with better properties  use of adeno-associated
viruses (AAV)
- no disease association in humans
- limited innate immunity
- AAV restricts expression to specific tissues
- tissue-specific promoter in AAV is genetically engineered to control transcription of the
inserted transgene

2. Two Strategies to Deliver a Therapeutic Gene (Transgene) into an Individual

a. In Vivo Gene Replacement Therapy
- direct delivery of therapeutic gene into patient’s body  target cells  inserted
transgene expressed into therapeutic protein
b. Ex Vivo Gene Replacement Therapy
- genetic manipulation of patient’s target cells outside the body  target cells
infected with recombinant virus harboring therapeutic transgene  genetically
modified target cells (harbor and express the therapeutic protein) reintroduced
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D. Other Non-Viral Options for Gene Delivery

- use of liposomes to carry the therapeutic DNA through the target cell’s membrane
- delivery of therapeutic DNA by chemically linking the DNA to a molecule that will bind to target cell
receptors
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E. Facts
1. Leber Congenital Amaurosis
- inherited blindness
- reported success in using gene therapy
2. Severe Combined Immunodeficiency (SCID)/Adenosine Deaminase (ADA) Deficiency
- rare immunodeficiency disease
- gene therapy entails introducing a normal human ADA gene into the patient’s lymphocytes 
reconstitute the function of the cellular and humoral immune system in severe
combined immunodeficiency (SCID)
- follow-up study of children concluded that 8 of 10 treated seemed to have been cured
a. Mutations
- in either
- adenosine deaminase gene (SCID)
- gene coding for interleukin receptor subunit (X-linked severe combined
immunodeficiency, SCID-X1)
b. Retrovirus-Mediated Gene Transfer
- successful in treating SCID-X1
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3. Cystic Fibrosis
- good candidate for gene therapy
a. Cystic Fibrosis Transmembrane Regulator (CFTR)
- chloride channel found on the apical surface of epithelial cells
- malfunction  cystic fibrosis
- regulate the fluidity of the extra-epithelial mucous layer
- channel opens  Cl- flow out of the cell down their electrochemical gradient 
induces Na+ to flow between the cells of the epithelium (via a paracellular
pathway) to balance the electrical charge  water follows the efflux of sodium
chloride by osmosis  maintain mucus fluidity

b. Cystic Fibrosis Transmembrane Regulator (CFTR) Gene

- gene defect is a recessive disorder
- gene responsible for cystic fibrosis
- localized to chromosome 7 by linkage analysis
- spans about 250 kbp
- contains 27 exons
- DNA sequence analysis  polypeptide sequence of 1480 amino acids
- also encodes a simple chloride ion channel within the cystic fibrosis transmembrane
regulator
- commonest defect
- single mutation with 3 bp deletion in exon 10  removal of a codon
specifying phenylalanine (F508del)  no normal gene product
- other defects
- over 1000 different minor mutations of the CFTR gene with most mapping
to the ATP-binding domains
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b. Gene Therapy Experiments to Restore CFTR Function

i. Transfection of Cells with Wild Type Receptor
ia. Two Different Routes
- placing the CFTR gene in an adenovirus vector
- placing the CFTR gene into a liposome
- can be conveyed to the lung using an aerosol spray  fatty
surface of the liposome fuses with the epithelial cell
membrane  deliver CFTR DNA into the cell 
make functional CFTR chloride channels
- if about 10% of the cells can be corrected  will ‘cure’ the
disease
ii. Alternative Methods
ia. Suppress Premature Termination Codons
- translation continues
ib. Topical Nasal Gentamicin
- aminoglycoside antibiotic
- shown to result in the expression of functional CFTR channels
4. Familial Hypercholesterolemia
- defective low-density lipoprotein (LDL) receptor gene
- gene therapy entails insertion of receptor gene into hepatocytes removed by liver biopsy from
the patient  gene-corrected hepatocytes are then reinjected into the portal circulation
 migrate back to the liver  reincorporated  should start to produce LDL
receptor protein  would dramatically lower the patient’s cholesterol level
F. Muscle-Cell-Mediated Gene Therapy
1. Problem Associated with Gene Therapy of Chronic Genetic Disease
- how long the transfected cells will survive
a. Myoblasts Transfected with a Retrovirus
- shown to function and live for the lifetime of mouse models (2 years)
- myoblasts fuse with the animal’s muscle fibres  express the transfected
protein
- long life of the muscle fibres and rich blood supply  ideal site for
treatment of diseases in which functional serum-born factors are
missing (ex: human growth hormone, coagulation factors,
erythropoietin)
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b. Duchenne Muscular Dystrophy
i. Myoblast Transfection
- appear to be the best option for the treatment
ii. Duchenne Gene
- far too large to fit into any current viral vector
iii. Reimplanted Myoblasts
- do not colonize muscles fibres distant from the site of injection
G. Present Experiment
- introduce a 47th (artificial human) chromosome into target cells  large vector capable of carrying
substantial amounts of genetic code
H. Current Status of Gene Therapy
- experimental
- no approved human gene therapy products
1. Problems to Overcome
a. Short-Lived Nature of Gene Therapy
- therapeutic DNA introduced into target cells must remain functional
- rapidly dividing nature of cells prevents gene therapy from achieving any long-term
benefits
- patients need to undergo multiple rounds of gene therapy
b. Low-Level or Transient Expression of the Therapeutic Gene
c. Immune Response
- the “immune memory” makes repeated gene therapy more difficult
d. Viral Vectors
- carrier of choice in most gene therapy studies
- present a variety of potential problems to the patient
- toxicity
- immune responses
- inflammatory responses
- gene control
- targeting issues
d. Problems Caused by Random Insertion of the Therapeutic Gene into the Host DNA
e. Multigene Disorders
- account for many of the most commonly occurring disorders
- heart disease
- high blood pressure
- Alzheimer’s disease
- arthritis and diabetes
- difficult to treat effectively using gene therapy
I. Ethical Questions
1. What is normal and what is a disability or disorder, and who decides?
2. Are disabilities diseases? Do they need to be cured or prevented?
3. Does searching for a cure demean the lives of individuals presently affected by disabilities?
4. Is somatic gene therapy (which is carried out in the adult cells of persons known to have a disease) more
or less ethical than germline gene therapy (which is carried out in egg and sperm cells and
prevents the trait from being passed on to further generations)? In cases of somatic gene therapy,
the procedure may have to be repeated in future generations.
5. Preliminary attempts at gene therapy are expensive. Who will have access to these therapies? Who will
pay for their use?
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TREATMENT of SOMATIC DISEASES
- gene therapy which requires only a transient expression of the transfected genetic material
circumvents the problems currently plaguing gene therapy of inherited disorders  front-line of
gene therapy
A. Vascular Disease
1. Ideal Therapy
- regeneration/neovascularization  increase blood flow
- repair of cardiac tissue after a myocardial infarction
2. Temporary Expression of Angiogenic Factors
- at the site of a blockage  induce new blood vessels
3. Local Temporary Expression of Clot-Disintegrating Enzymes
- ex: streptokinase
lipases
- may repair damaged and diseased arteries
4. Possible Routes
- liposomes loaded with DNA
- directly inject DNA plasmids into the tissue
 protein will be expressed by the cells which take it up
5. Result
- only 1-3% take-up
- sufficient for the local effect required
- transient expression
 controllable gene therapy
B. Neuronal Disease
1. Neurotrophic Factors
- can be transiently expressed
- local expression of neurotrophins is essential for nerve cell regeneration and maintenance
2. Transfected Myocyte Injection
- extend the expression period of the neurotrophin into the damaged area
- will fuse with any adjacent muscle tissue  prolonged expression of the factor gene
C. Cancer
- genetic disease
- many genes are deregulated
1. p53 - tumour suppressor gene
- reintroduction and overexpression of a functional p53 in tumours is being investigated with
some success
- induce apoptosis in cells with damaged genetic material  transient expression of high
levels in a tumour cell with p53 pathway defects  induce own apoptosis
- only likely to occur in rapidly dividing cells  perfect target for cancer gene therapy
2. Inhibitors of Angiogenesis
- tumour growth depends on the development of new blood vessels
- used in trials
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STEM CELL THERAPY
A. Human Embryonic Stem Cells (HESCs)
- can differentiate into all of the specialized embryonic tissues
- can be induced to differentiate into the three neural lineages
- astrocytes
- oligodendrocytes
- mature neurons
- when transplanted into new born (mouse) brains  migrate along established neuronal tracts 
differentiate to the appropriate cell type in a region-specific manner (totipotentiality)
1. Human Embryonic Stem Cell Research
- controversial as the establishment of a stem cell line requires the destruction of a human
embryo and/or therapeutic cloning
- recently  embryonic stem cell lines can be generated without destroying embryos using
single-cell biopsy  may circumvent the ethical blockade on using HESCs in stem cell
therapy
B. Adult Stem Cells and Progenitor Cells
- act as repair system for the body  replenish specialized cells
1. Bone Marrow Transplant
- currently existing adult stem cell therapy
C. Umbilical Cord Blood
- readily available
- rich source of hemopoietic stem cells (ex: CD34-positive, CD38-negative)
- invaluable source of stem cells for bone marrow transplants
1. Cord Blood Stem Cells
- readily colonize the bone marrow  rapidly populate the marrow with all the various cells
(erythrocytes and leukocytes) required of a fully functional blood system -
pluripotentiality
2. Cord-Blood-Derived Embryonic-Like Stem Cells (CBEs)
- able to differentiate into more types of tissue than simply hemopoietic cells (super
pluripotentiality)
D. Peripheral Blood Monocytes
- believed to be the unipotent precursors of phagocytes
1. Primitive Monocyte-Derived Multipotential Cell (MOMC)
- claimed that it can be isolated from adult peripheral circulating monocytes
- can be induced (given the correct paracrine, environmental and adhesion signals) into endothelia,
neurons, cardiomyocytes and mesenchymal lineages
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CREATING and USING ANIMAL MODELS
- test gene therapies prior to use in patients
A. Targeted Gene Disruption
- experimental inactivation of a gene in order to investigate its function
1. “Knockout” Animal
- usually in mice
- the gene under study is inactivated in the germ line by disrupting it (gene knockout)
- the effects can be studied at different embryonic stages and after birth  understand the effects
of mutations in homologous human genes as seen in human genetic diseases
i. Gene Knock-In
- variant of knockout
- the targeting construct contains a normal gene that is introduced either in addition to
or instead of the gene to be studied
2. Preparation of Embryonic Stem Cells with a Knockout Mutation
- the target gene is disrupted (knocked out) in embryonic stem cells (ES) by homologous
recombination with an artificially produced non-functional allele
- isolation of ES cells with disrupted gene requires positive and negative selection
- a bacterial gene conferring resistance to neomycin (neoR)  introduced into DNA of
the artificial allele partially cloned from the normal target gene
- DNA containing the thymidine kinase gene (tk+) from herpes simplex virus is added to
the gene replacement construct outside the region of homology
- the selective medium contains the positive and the negative selectable markers
- neomycin
- ganciclovir
- nonrecombinant cells and cells with nonhomologous recombination at random sites
cannot grow in this medium
- nonrecombinant cells remain sensitive to neomycin
- recombinant cells are resistant (positive selection)
- thymidine kinase (tk+) gene confers sensitivity to ganciclovir (nucleotide
analog)
- enzyme derived from herpes simplex virus is able to convert
ganciclovir  monophosphate form  triphosphate form 
inhibits cellular DNA replication
- nonhomologously recombinant ES cells contain the tk+ gene at
random sites  sensitive to ganciclovir  cannot grow in its
presence (negative selection)
- only cells that have undergone homologous recombination can
survive because
- they contain the gene for neomycin resistance (neoR)
- do not contain the tk+ gene
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3. Gene “Knock-Out” Experiments in Mice
- provide new animal models of disease
- the normal gene of interest in a mouse  targeted using recombinant DNA techniques  gene
function is impaired
- embryonic stem cells (ES) from a mouse blastocyst are isolated after 3.5 days of gestation (19.5
days)  transferred to a cell culture grown on a feeder layer of irradiated cells that are
unable to divide
- ES cells heterozygous for the knockout mutation are added
- derived from a mouse that is homozygous for a different coat color (ex: black) from that
of the mouse that will develop from the blastocyst (ex: white)
- the recombinant ES cells are integrated into the recipient blastocyst
- early embryos  transplanted into a pseudopregnant mouse
- the offspring that have taken up ES-derived cells are chimeric
- consist of two types of cells  some with and some without the disrupted gene
- the transgenic mice can be recognized by black coat color spots on a white (or brown)
background
- chimeric mice  backcrossed to homozygous white mice
- black offspring from this mating are heterozygous for the disrupted (mutant)
gene
- further breeding of the heterozygous mice  some of their offspring (knockout
mice)  homozygous for the disrupted gene
B. Transgenic Animals
- contain foreign DNA which has been injected during early embryonic stages
- produced by injecting a cloned gene into the fertilized egg  present in the germline of the resulting
animal  can be passed from generation to generation
- can also use mutant gene to replace normal gene  enzyme deficiencies produced  models for further
study of corresponding human disease
- mutant dystrophin gene  study of muscular dystrophy
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The HUMAN GENOME PROJECT
- international cooperative effort
A. Goal - sequence the euchromatic portion of the human genome
B. Architecture of the Human Genome
- outstanding structural features are different types of repetitive, noncoding sequences
- each chromosome contains blocks of duplicated sequences (segmental duplications)
- genomic DNA is associated with nuclear proteins called histones
- coiling around histones and structural regions such as the centromeres and telomeres requires
regions of DNA devoted specifically to the purpose of packaging
1. Types of Sequence
- 3.2 billion base pairs per haploid set of chromosomes
- coding DNA in genes (exons) accounts for 1.2% (34 Mb)
- about 3/4 of all known human genes have counterparts in other species
- about 1/4 found only in other vertebrates
- 1/4 are common to all prokaryotes and eukaryotes
- untranslated regions of transcripts account for 21 Mb (0.7%) of the total amount of DNA
a. Expressed Sequences
- there are around 23,000 protein-encoding genes in mammalian genomes, of which
almost 42% are of unknown function (orphan genes)
- approximately 60% of the human genome is transcribed into RNA
- over 4000 genes encoding
- rRNA
- tRNA
- snRNA
- other small RNAs
- at least another 10,000 loci resulting in the transcription of noncoding RNAs
(ncRNAs)
- most have no known function
- many are involved in RNA interference (form of
posttranscriptional gene silencing)
b. Unexpressed Sequences
- experience little selective pressure  accumulate mutations at a great rate  can be
used to trace evolutionary relationships
i. Prokaryotes
- predominantly promoter and operator sequences adjacent to genes
- insertion sequences
- remnants of integrated viruses
ii. Eukaryotes
- promoters
- other regulatory regions
- the bulk are repetitive DNA of unknown function
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2. Tandem Repeats
- short (~10 bp) sequences
- present in millions of copies
- often tandemly repeated thousands of times
- comprise about 3% of the human genome

3. Moderately Repetitive Sequences

- < 106 copies per haploid genome
- occur in 100 to several thousand bp segments
- most are retrotransposons
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a. Retrotransposons
- about 42% of the human genome
i. Long Interspersed Repeat Sequences (LINEs)
- mammalian retrotransposons
- lack long terminal repeats (LTRs)
- account for 21% of the genome
- consist of repetitive sequences up to 6500 bp long that are adenine-rich at
their 3’ ends
- encode two open reading frames (ORF1 and 2) which are translated
- in addition to a 5’ promoter (P) they have an internal promoter
ia. LINEs 1
- approximately 600,000 L1 elements are dispersed throughout the
human genome
- can result in genetic disease if one is inserted into a gene (ex:
hemophilia A)
ib. LINEs 2 and 3
- inactive because reverse transcription from the 3’ end often fails to
proceed to the 5’ end
ii. Short Interspersed Repeat Sequences (SINEs)
- repetitive segments
- about 100-400 bp with tandem duplication of CG-rich segments separated by
A-rich segments
- do not encode a protein
- not capable of autonomous insertion
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iia. Alu Family (Alu Sequences)

- most abundant type of SINE sequences in humans
- about 1200000 copies (about 6% of the genome)
- one Alu repeat occurs about once every 3 kb in the human genome
- a full length Alu (Arthrobacter luteus) repeat is a dimer of about 280
bp (120 bp for each monomer) followed by a short sequence
rich in A residues
- specific for primate genomes
iii. Long Terminal Repeats (LTRs)

b. Autonomous Retrotransposons
- contain gag and pol genes (encode proteins required for retrotransposition)
c. LTR Retroposons
- flanked by long terminal direct repeats (LTRs) containing transcriptional regulatory
elements
d. Mobile DNA Elements/Transposons
- move in an essentially random manner from one site to another on the same or a
different chromosome
- resemble bacterial transposons by having terminal inverted repeats and encoding a
transposase
- at least seven major classes exist
- can be subdivided into families of independent origins
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i. Transposase
- enzyme responsible for the transposition of transposable elements or
transposons
- involved in site-specific DNA recombination required for transposition in
bacteria and other organisms
ii. DNA Transposition
iia. Direct
- transposase cuts out and then inserts the transposon at a new site
iib. Replicative
- transposon is copied
- the copy inserted elsewhere while the original remains in place
- frequently involves an RNA intermediate
- in this case the transposon is called a retrotransposon
4. Segmental Duplications
- blocks of 1-200 kb genomic sequence
- also present at another site in a chromosome (intrachromosomal) or another chromosome
(interchromosomal) usually in subtelomeric regions
- the human genome contains 1077 duplicated blocks containing 10310 pairs of genes
- when sequence identity exceeds 95% and size of 10 kb or more  unequal crossing-
over may occur  predisposition to duplication and deletion  genomic
disorders
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C. Concerns
1. Functional Genomics
- functions of genes and genomes
2. Transcriptome
- entire process of transcription
3. Proteome
- analysis of all human proteins
4. Comparative Genomics
- human genome compared with that of other organisms
5. Bioinformatics
- development of new techniques for handling the vast amount of data
6. Epigenome
- epigenetic functions
D. Ethical, Legal, and Social Implications
- covers a wide range of issues
- confidentiality and fairness in the use of individual genetic information
- prevention of genetic discrimination
- use of genetic methods in clinical diagnostics
- conditions for genetic testing
- public and professional education
E. Medical Relevance
- important implications for the theory and practice of medicine
- complete knowledge of human genes 
- more precise diagnoses
- better assessment of genetic risk
- development of treatments
- certain allelic haplotype combinations that predispose to common complex disease are likely to be
identified
F. Some Interesting Facts and Figures About the Human Genome
1. Human Genome
- contains 3164.7 million chemical nucleotide bases (A, T, C and G)
2. Average Gene
- consists of 3000 bases
- sizes vary greatly
a. Dystrophin Gene
- largest known human gene
- 2.4 million bases
3. Chromosome 1
- has the most genes (2968)
4. Y Chromosome
- has the fewest genes (231)
5. Total Number of Genes
- estimated at 30,000 (much lower than previous estimates)
6. Nucleotide Bases
- almost all (99.9%) nucleotide bases are exactly the same in all people
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7. Functions
- unknown for over 50% of discovered genes
8. Proteins Coded
- < 2% of the genome codes for proteins
9. “Junk DNA”
- repeated sequences that do not code for proteins make up at least 50% of the human genome
10. Gene Concentration
- genes appear to be concentrated in random areas along the genome
- large expanses of non-coding DNA in between
11. CG Islands
- stretches of up to 30,000 C and G bases repeating over and over
- often occur adjacent to gene-rich areas
- form barrier between the genes and the junk DNA
- believed to help regulate gene activity
G. Human vs. Other Organisms
1. Gene Distribution
a. Humans
- seemingly random distribution of gene-rich areas
b. Other Organisms
- more uniform genomes
- genes evenly spaced
2. Gene Products
a. Humans
- greater because of mRNA transcript “alternative splicing” and chemical modifications
to the proteins yielding different protein products from the same gene
3. Protein Families
- the number of gene family members has expanded in humans particularly for those proteins
involved in development and immunity
4. Human Genome
- greater portion of repeat sequences
5. Single-Base DNA Differences
- some 1.4 million locations at which single-base DNA differences (SNPs) occur have been
identified in humans  promises to revolutionise the processes of finding chromosomal
locations for disease-associated sequences and tracing human history
6. Ratio of Germline (Sperm or Egg Cell) Mutations
- 2 : 1 in males vs. females
H. The International HapMap (Haplotype Map) Project
- similar projects for other organisms
- initiated to investigate common DNA variants
- SNPs close together are inherited in blocks (haplotypes)
- each haplotype has a few SNPs that can be identified (tag SNPs) which reduces the number of SNPs in
the genome that have to be identified and typed and correlated with a phenotype
- will enable studies on the role of genetic factors in different diseases
- used to decide which therapy is likely to have a beneficial effect
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I. The Wellcome Trust Case Control Consortium
- was set up to analyse thousands of DNA samples from patients with different diseases in which there is
thought to be a genetic component
- genetic markers have been found in many diseases
- diabetes
- hypertension
- Crohn’s disease
- many polymorphic markers being studied in
- breast cancer
- multiple sclerosis
- autoimmune thyroid disease
J. In Silico Cloning
- information arising from the human genome and similar animal programmes  used for bioinformatic
(semi-automated) identification of genes
- largely based on the abundance of databases of expressed sequence tags (ESTs)
1. ESTs - represent portions of expressed genes
- produced by one-shot sequencing of cDNA clones taken from a cDNA library
- rapidly generated
- represent markers of active genes present in the specific tissue from which the cDNA library
was generated
2. Bioinformatics
- allows the searching and matching of ESTs to complementary sequences (spliced or not) in the
huge databases of the genome project
- position of the gene can be defined
- other ESTs in libraries throughout the world can be matched to the same location, mapping out
the expressed gene
- can be achieved by searching and manipulating computer-based databases are
- predicting the protein product
- shape and function
- tissue specific expression

FUNCTIONAL GENOMICS
- the use of genome data to explore how DNA and proteins work with each other and the environment to
create complex, dynamic living systems
A. Exploratory Studies
1. Transcriptomics
- involves the analysis of messenger RNAs, following when, where and under what conditions
genes are expressed
- provide a clearer understanding of what is actually happening in the cell
2. Structural Genomics Initiatives
- to generate the three dimensional structures of one or more proteins from each protein family
thus offering clues to function and biological targets for drug design
3. Knockout Studies
- used to inactivate genes in living organisms and monitor any changes that could reveal their
functions
4. Comparative Genomics
- analysis of DNA sequence patterns of humans and other model organisms
- one of the most powerful strategies for identifying human genes and interpreting their
function
B. Genome
- organism’s complete set of DNA
C. Genomics
- study of organisms’ genomes
- endeavors to elucidate the functions of all genes
- also explores evolutionary relationships between genes, organelles, and organisms
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D. Proteins
- products of a cell’s genome
- the genome’s proteome
- the dynamic proteome changes from minute to minute in response to tens of thousands of intra- and
extracellular environmental signals
E. Proteomics

The HUMAN PROTEOME PROJECT

A. Proteome
- protein expression characteristics of normal and diseased cells
- more direct route to understanding genetic and somatic disease is by studying the proteome
- relies on the separation of proteins expressed by a given tissue by molecular size  charge on a simple
two-dimensional display (using two-dimensional gel electrophoresis)
- the pattern of dots corresponds to the different proteins expressed
- non-, over- and underexpression of a given protein
- detected by a corresponding change on the proteome two-dimensional
electrophoresis image
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1. Rheumatoid Arthritis
- protein profile of synovial fluid from a diseased joint shows a new protein arising with disease
(inflammatory cytokines)
- post-translational modifications of the protein show up as a change in either size or charge on
the proteome picture
2. Discovery of New Protein Markers
- looking for changes (appearance of new proteins) as above  discovery of new protein markers
for the diagnosis of
- Creutzfeldt-Jakob disease
- multiple sclerosis
- schizophrenia
- Parkinson’s disease (spinal fluid protein)
- Alzheimer’s disease (blood and brain proteins)

METABOLOMICS
- the study of the repertoire of non-proteinaceous, endogenously synthesized small molecules present in
an organism
A. Small Molecules
- include
- glucose
- cholesterol
- ATP
- lipid signalling molecules
- the ultimate product of cellular metabolism
B. Metabolome
- refers to the catalogue of those molecules in a specific organism
- the analysis of the pattern of change of such molecules in urine samples of individuals with and without a
particular disease and those treated with specific drugs represents a change in the metabolome
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PHARMACOGENOMICS
- study of how an individual’s genetic makeup will affect their response to drugs (holds the promise that
drugs may one day be tailor-made for an individual)
A. Facts
- there is no simple way to determine whether an individual will respond well, badly or not at all to a
medication
- heterogeneity in patient response to chemotherapy is consistently observed across patient populations
- pharmacogenomics may be important for oncology
- severe systemic toxicity and unpredictable efficacy are hallmarks of cancer therapies
- genetic polymorphisms in drug-metabolising enzymes are responsible for much of the inter-individual
differences in the efficacy and toxicity of many chemotherapeutic agents
B. Anticipated Benefits of Pharmacogenomics
- facilitation of new drugs with specific targeting, based on the proteins, enzymes and RNA molecules
associated with genes and diseases
- safer drugs and avoidance of adverse drug reactions, and more accurate drug dosage
- advanced screening for disease, to allow for lifestyle and environmental changes at an early age, and to
ascertain the most appropriate timeline for therapy
- better vaccines, made of genetic material, which will avoid the current risks, be inexpensive, stable and
easy to store, and capable of being engineered to carry several strains of a pathogen at once
- facilitation of drug discovery and approval, since trials will be targeted to specific genetic population
groups  cost and risk should also be reduced
- decreasing overall costs of health care, in line with a reduction in adverse drug effects, number of failed
drug trials, time for approval, length of time patients are on medication and time to find an
effective therapy
C. Cytochrome P450 and Pharmacogenomics
1. Cytochrome P450 Isoenzymes (CYPs)
- superfamily of hemeprotein enzymes
a. Function
- catalyze the metabolism of a large number of endogenous and exogenous compounds
b. Location
- predominantly in the liver
- also - intestine
- lungs
- kidneys
- brain
c. DNA Variations in Genes That Code for CYPs
- can influence their ability to metabolise certain drugs
d. Nomenclature
i. Cytochrome P450
- have a heme group
- maximum absorption wavelength of 450 nm in the reduced state in the
presence of carbon monoxide
ii. Family
- denoted by arabic numeral
- ex: CYP1
CYP2
iii. Subfamily
- denoted by letters A, B, C and so on
- ex: CYP3A
CYP3C
iv. Gene/Isoenzyme
- denoted by another arabic numeral
- ex: CYP3A4
CYP3A5
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2. Polymorphism
- genetic variation in a population
a. Polymorphic Forms
- all genes coding CYP enzymes in families 1-3 are polymorphic
- responsible for the development of a significant number of adverse drug reactions
(ADRs)
- 56% of drugs reported in ADR studies are metabolised by polymorphic phase
1 enzymes
- 86% are CYPs
- CYP2C9, CYP2C19 and CYP2D6
- main polymorphic forms responsible for almost 40% of
P450 mediated drug metabolism
For Your Eyes Only
3. Clinically Important Major Allelic Variants of P450 Genes
- four phenotypes
a. Poor Metabolism
- lacking functional enzymes
- poor metabolism will increase the likelihood of adverse events at normal doses because
of resulting increased levels of the drug
- prodrugs activated by CYP enzymes  poor metabolism may result in little response
b. Intermediate Metabolism
- heterozygous for one deficient allele
c. Extensive Metabolism
- with two normal alleles
d. Ultrarapid Metabolism
- with multiple gene copies
- ultrarapid metabolism of an active drug  therapeutic levels are not reached
- prodrugs activated by CYP enzymes  ultrarapid metabolism may increase adverse
effects