Chapter 4: Molecular biology in medicine.

Background

The beginning…

• The first link between genetic

inheritance and a human

condition was made in 1902 when

alkaptonuria was identified as an

inherited disorder. (51 years prior

to Watson & Crick discovering DNA)

• Alkaptonuria is a condition in which

urine turns black on exposure to air.

• Sir Archibald E. Garrod (1857–1936)

related this and similar diseases to

a lack of particular enzymes in

the body.

CausesGenetic defects can be due to problems with an entire chromosome, multiple genes, or even a single gene (section of a chromosome).

The human –Globin gene is 626 base pairs long. If a single base substitution mutation occurs in a particular place, the person will develop Sickle Cell Anaemia.Eg: small section of gene

ACT CCT GAG GAG AAG (unaffected person)

ACT CCT GTG GAG AAG (sickle-cell individual)

Inherited diseases

1. Phenylketonuria (PKU) is a disorder characterized by an inability to produce the enzyme phenylalanine hydroxylase, resulting in a potentially fatal or damaging build up of the amino acid phenalalanine, and occurs in one in every 10000 babies.

1. Hypothyroidism is a disorder caused by a small or improperly functioning thyroid gland, or even its complete absence, and occurs in one in every 3500 babies.

2. Galactosaemia is an inherited disorder and occurs in one in every 40,000 babies. Lactose is digested into galactose and glucose. A baby with galactosaemia lacks the enzyme that metabolises galactose and will die if untreated because of the build-up of galactose in the blood.

3. Cystic fibrosis (CF) is an inherited disorder and occurs in one in every 2500 babies. A person with CF produces abnormal secretions that have a serious adverse effect on the function of lungs and digestion. Recent advances in treatment have greatly improved the prognosis for these babies so early diagnosis and treatment are important

The following conditions can be tested by conductiong a simple heel-prick test on a baby 2-3 days after birth,

Early detection

PKU – a case study• The aforementioned conditions are tested for at birth as

early detection is essential in successful treatment.

• Individuals born with PKU can’t produce phenylalanine

hydroxylase (an enzyme).

• Many products contain the amino acid phenylalanine, which

can’t be broken down, the build-up starts to destroy brain

tissue, thereby having a detrimental effect on development.

• Initial detection technique was the “wet nappy” test using

ferric chloride but this often occurred too late

PKU – a case study• An alternative technique was the

Guthrie plate. Blood was collected via

the heel prick test.

• Foetal blood was tested for high levels

of phenalalanine. Using bacteria that

would only grow under certain

concentrations

• Blood is still collected via a heel prick

test but can be tested far more

efficiently via mass spectrometry (a

biochemical analysis of the make-up of

the babie’s blood)

Gene Therapy

Gene therapy• Many genetic diseases are caused by a single gene.

• Eg: PKU

• this single gene defect, could be ‘treated’ by consuming

a special diet.

• This approach is not possible with many conditions.

• Why not try to insert a normal functional gene

into cells that contain a defect?

• Such a procedure is called gene therapy and is a medical

procedure that modifies the genetic material of living cells of

an individual so that a genetic defect is corrected.

Gene therapy

Transferring a piece of DNA into a cell

• The functional piece of DNA inserted into a cell

is specially prepared and is called a cloned

gene.

• How does the cloned gene, get into a patient’s

cells?

• This action is most successful when a vector

is used to carry the gene into a cell.

• The most commonly used vectors for gene

therapy are modified viruses. (Retroviruses

and adenoviruses are the main types used.)

Gene therapy using a retrovirus vector:• Some viruses have DNA, some have

RNA, Retroviruses have RNA that codes

for DNA once it enters a cell

• 1 – remove harmful RNA but leave bits

that code for infection and

transformation to DNA

• 2 – insert RNA version of normal gene in

to virus

• 3 – introduce virus to cells ex-vivo and

allow cells to replicate

• 4 – inject infected cells in to patient

ISSUES

• If inserted in the wrong place could disrupt a

healthy gene

• May cause a severe immune response

Gene therapy using an adenovirus• Adenoviruses contain DNA, in to which a normal copy

of a gene can be integrated

• When the adenovirus enters the host cell, its DNA

enters the nucleus and starts coding for the required

proteins.

• Adenovirus DNA does not integrate with host DNA, so

when mitosis occurs, it does not replicate.ISSUE

• If life span of host cell is

short, positive effects of

this type of gene therapy will be very short-li

ved.

Prenatal testing

Success of gene therapy• Many successful experiments have been conducted with

animals such as mice…

• Still very experimental in humans and can pose risks to

patients’ safety.

• Non-viral gene therapy would be very difficult as this is one of

the few ways of bypassing our immune system

Ultrasound is commonly used to view the uterus and fetus during pregnancy.

Scans are often performed at 18 to 20 weeks into a pregnancy.

Ultrasound maybe performed earlier if there are signs or indications that the fetus is not growing normally.

Ultrasound is also usedto diagnose:

multiple pregnancies(twins, triplets)

gross fetal abnormalitiessuch as trisomy(e.g. Down, Klinefelter,and Turner syndromes)

Screening during Pregnancy

ISSUE

• Not a very useful technique for detecting foetal

abnormalities that have no outward signs

Other types of pre-natal testingCSV (Chorionic Villus Sampling)

Performed at 6-8 weeks

Material gathered can be used for both metabolic and genetic testing

AmniocentesisAmniotic fluid contains both skin cells and urine

Material gathered can be used for both metabolic and genetic testing

Miscarriages are often not random occurrences but actually the result of the foetus dying doue to a genetic abnormality

Thin tube extracts fetal tissue

Amniotic fluid

Genetic Testing

Electrophoresis• DNA collected during screening procedures can be cut in

specific locations using REs (restriction enzymes).

• The DNA can be run through an agarose gel. A current is put

through the gel.

• DNA is negatively charged and will be drawn towards the

positive terminal.

• Smaller fragments will move faster while larger fragment will

take more time to move through the gel

• Every person has 2 copies of every gene and depending on the

abnormality, these can be observed on the gel

• In this case the disease form of the gene is smaller than the

normal version

• The gel shows that both parents were carriers for the condition

and that their first child received each parent’s diseased gene

and therefor developed the condition.

• Fortunately their unborn child received both normal versions of

the gene

OTHER CONDITIONS

The age of onset of many genetic conditions can

vary:

Genetic disease vs predispositionDISEASE

• If you have two affected copies the Huntingtons gene you are

guaranteed to get the disease.

• Onset is usually around the age of 40 and manifests in fairly

rapid brain degeneration. There is no currently available

treatment

PREDISPOSITION

• If you have affected copies of the BRCA1 or BRCA 2 genes for

breast cancer, there is no guarantee that you will get the

disease.

• You have a much higher chance of it developing it, but the

right environmental conditions are required to trigger its

onset

Genetics and DiseaseDown syndrome is often characterized by impairment of cognitive ability and physical growth, as well as some very recognizable facial features

It is caused by an individual being born with a 3rd copy of chromosome 21, due to a separation error during meiosis.Occurrence is estimated at approximately 1:750 births.

Ph

oto

: A

rt T

od

ay

21

Who should be screened

• Anyone with a history of genetic disease

• Two unaffected individuals who have had an affected

child are both carriers of a recessive disease.

• Their chances of having another affected child are 1 in

4

• An affected individual with a dominant disease has one

or both of their alleles carrying the disease

• They have a 1 in 1 or at best a 1 in 2 chance of having

an affected child

Rational Drug Design

Rational drug design• When our immune system fights off the flu it develops a

memory of it, the next time we encounter the same

strain we may not even develop symptoms before it is

fought off.

• The problem is that new strains are constantly

appearing, so the process must be repeated time and

time again.

• Two surface proteins on influenza virus:

• Haemagglutinin is active in gaining entry to a cell.

• Neuraminidase allows the exit of new virus

particles from a cell, freeing them to infect other

cells.

Rational drug design

• Neuraminidase is an enzyme that varies in

structure from strain to strain.

• Examination of strains of the virus from past years

demonstrated that although most of the

molecule changed dramatically, one small

part remained constant.

• Fortunately, this non-variable part is the active

site of the enzyme.

Using the active site

• If a drug was to be designed to

inhibit the active site of

neuraminidase,

• the molecular structure of the

site had to be worked out.

• This was done by computer

modelling so that the active site’s

exact shape and the spatial

arrangements of the atoms

surrounding it became known.

Computer representation of the anti-flu drug in the

active site of neuraminidase.

Designing the drug• Once the detailed structure of the active

site was known

• a molecule could be designed to fit and

bind to the active site,

• Then an anti-flu drug was created!

• This technique, in which the active site

of a molecule is determined and a

second molecule (the drug) is

constructed to fit into that active site to

inhibit the activity of the first molecule,

is called rational drug design.Ph

oto

: C

DC

How does it work?

Developing Vaccines

Types of VaccineThere are two basic types of vaccine:subunit vaccines and whole-agent vaccines.

Recombinant vaccines

Toxoids

Conjugated vaccinesAcellular vaccines

Attenuated(weakened)

Inactivated(killed) ie. just antigen

Subunit Vaccine

Contains some part or product of micro-organisms that can produce

an immune response

Whole-Agent

VaccineContains whole,

nonvirulent microorganisms

Case study: Malaria

MalariaThere are a number of strains, all carried by the Anopheles mosquito1/3 of the world’s population is at risk. Every year there are 4 million new cases and 2 million deaths

Involves a very complex life cycle

Illness mainly due to:Capillaries becoming blocked by “sticky protein” on red blood cells

Release of the toxin GPL

Healthy blood cells being overtaken by the parasite

Only Plasmodium Faliciparum curable as in other strains parasites lay dormant in gut

ResistanceMosquitoes becoming resistant to DDT

Plasmodium becoming resistant to Chloroquine

Developing VaccinesMALARIA

Developing a vaccine is difficult due to Malarial DNA constantly changing and therefore its recognizable protein coat aswell.

A potential vaccine could pre-expose people to a microscopic quantity of the GPL toxin

Another option is to create an inhibitor to block the receptor site on the “sticky protein” so that it is unable to bind to tissue and block capillaries

MEASELSStill a major cause of death in developing countries due to unavailability of vaccine and difficulty in storing it

Current trials have spliced Measles antigen into a strain of lettuce

Lettuce is freeze-dried in to powder and then packaged in to tablets

Cheap and does not require refrigeration

Manufacturing Biological Molecules

Manufacturing biological molecules

Insulin:

• produced by the pancreas, a hormone that

controls the level of glucose in the blood by

controlling its uptake from the blood by

cells…

• A deficiency of this hormone results in an

abnormally high level of glucose in the blood

- diabetes.

• Because the amino acid sequence of the

active molecule of insulin is known, a piece

of DNA carrying the instructions (code) for

insulin can be built and inserted into a

plasmid vector.

Manufacturing biological molecules

Manufacturing biological moleculesApplication for manufactured molecules

Insulin (for diabetics)

Growth hormone

Factor VIII (blood clotting agent missing in hemophiliacs)

AdvantagesCan be produced in very large quantities

If viral antigen can be copied, can be produced using no materials of human biological origin – minimises risk of associated disease

Eg. in the past haemophiliacs receiving transfusions of Factor VIII from blood risked HIV

Eg. patients receiving growth hormone from cadavers risked the brain disease CJD

Nanoparticles

Manufacturing biological moleculesVery small human-made particle (0.1-100 nM in

diameter)

Being used to deliver drugs directly to the cells requiring them

In cancer trials with mice, survival chances increased by 30%

Made up of 3 parts

Fluorescent stain – to follow progress

Methotrexate – a drug that destroys cancerous cells

Folic Acid – a vitamin required by rapidly reproducing cells