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 withan entire chromosome, multiple genes, oreven a single gene (section of achromosome).The human –Globin gene is 626 base pairslong. If a single base substitution mutationoccurs in a particular place, the person willdevelop Sickle Cell Anaemia.Eg: small section of gene ACT CCT GAG GAG AAG (unaffected person) ACT CCT GTG GAG AAG (sickle-cell individual)
The following conditions can be tested by conductiong a simple heel-prick test on a baby 2-3 days after birth,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
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• 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.
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 aretrovirus 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
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.
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
Screening during PregnancyUltrasound is commonly used to view theuterus 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 used to diagnose: multiple pregnancies (twins, triplets) gross fetal abnormalities such as trisomy (e.g. Down, Klinefelter, and Turner syndromes)
Other types of pre-natal testing CSV (Chorionic Villus Sampling) Performed at 6-8 weeks Material gathered can be used for both metabolic and genetic testing Amniocentesis Amniotic fluid contains both skin cells and urine Thin tube extracts fetal tissue 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 Amniotic fluid
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 CONDITIONSThe age of onset of many geneticconditions 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 treatmentPREDISPOSITION• 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 oftencharacterized byimpairment of cognitiveability and physicalgrowth, as well as somevery recognizable facial 21featuresIt is caused by an individualbeing born with a 3rd copyof chromosome 21, due to aseparation error duringmeiosis. Photo: Art TodayOccurrence is estimated atapproximately 1:750 births.
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• 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 Computer representation of the spatial arrangements of the atoms anti-flu drug in the active surrounding it became known. 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, Photo: CDC• 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.
Types of Vaccine There are two basic types of vaccine: subunit vaccines and whole-agent vaccines. Recombinant vaccinesSubunit VaccineContains some part or Toxoids product of micro- organisms that can Conjugated produce an immune response vaccines Acellular vaccines Attenuated Whole-Agent (weakened) Vaccine Contains whole, nonvirulent microorganisms Inactivated (killed) ie. just antigen
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 casesand 2 million deathsInvolves a very complex life cycleIllness 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 parasiteOnly Plasmodium Faliciparum curable as in other strains parasites laydormant in gutResistance Mosquitoes 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 capillariesMEASELS Still 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 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 Application for manufactured molecules Insulin (for diabetics) Growth hormone Factor VIII (blood clotting agent missing in hemophiliacs) Advantages Can 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
Manufacturing biological molecules Very 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