metabolism

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Genetic Mutations

DNA- is the moelcule that contains genetic information

Genetic mutations changes in the nucleotide sequence of the DNA (Substitution, insertion or deletion of the one or more base pairs)

Gene- nucleotide sequence required to direct proteins synthesis.

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Genetic disease

- 30% of individual will suffer from a genetic based disease during their lifetime.

- cystic fibrosis, thalessemia, huntingtons, cancer, heart disease and inborn errors of metabolism.

In born errors- a class of disease involving latered metabolic functions (archibald Garrod 1909)

Mutation in single gene- encoding a metabolic enzyme

Unable to: break down substances- toxic/ synthesise vital substances- deficiency

Chromosomes

Eukaryotic DNA is containined withina chromosomes

- in the nucleus of the cell

Number and appearance of chromosomes

- Variation may lead to developmental abnormalities

22 pairs of autosomes (1 pair of sex chrhomosomes)

XX females, XY- male

Single gene defects (classified)

Classified according to whether:

1) gene is ont he X chromosome- X linked

or on one of the 22 autosomes.

2) Whether a gene is dominant or recessive

- 2 alleles can then classify if dominant or recessive (2 copies, carriers have 1 copy whic is the domininant one)

Cystic fibrosis

- is an autosomal recessive diesease

- cauused by a defection CFTR gene (cystic fibrosis transmemebrane conductane regulator)

- CFTR encondes a Cl- ion transporter

In born errors of metabolism

Mayple syrup urine disease- autosomal recessive

- also called the branced chain ketoacidura- type of organic academia.

Smelly wee after eating asparagus

- most people produce the odurus compounds after eating asparagus

- 22% of the population have the autosomal genes required to smell them.

genetic mutation

- changes in the nucleotide sequence of DNA (adenine, guanosine, thymine and cytosine)

Types of mutation

- mutaions may be spontaneous or induced by an outside factor.

- they come in two types: 1) point mutations (substitutions) 2) insertion/ deletion mutations( length changing, disurpt during replication)

Point mutation (substitutions)

Transitions (purine to purine, same for pyrimidine)

TRansversions (purine to pyrimidine, vice versa)

eg. .....GATTACA... ----> .....GATTAGCA....

----> ...GATTCA....

Causes of mutations:

spontaneous- replication errors, tautomerisation, deamination, depurination

Induced- intercalating agents, base analogues, deaminiating agents, alkylating gets, oxidation agents, Radiation, U/V

Spontaneous mutations

1) Replication errors- normal repication introduces the wrong base once every 10^10 bp, good chance it will get repaired.

- some repetitive regions cause slippage and insertion of more repeats- cannot be repaired.

2) Tautomeric shifts- the bases of DNA are subject to spontaneous structural alterations called tautomerization

- capable of existing in two forms between which thry interconvert.

e.g. Amino group of A or C (NH2) <---------------------> Imino group = NH

Keto group of G or T (C=O) <-------------------------------> Enol group (c-OH)

- tautomers can form non standard base pairs (shifterd (enol) form binds G instead of A

- certain chemicals can induce the rate

- chaning B.p will carry on with that mutation

mutation consolidted on next replication (slow rate spontantoeus)

3) deamination

- loss of amino group U pairs with A C pairs with G

C, A and G deaminations can be repaired

5-Methyl- C- T which cannot be repaired ( effects transcription ruins replication

4) Dupurination ( A or G)

- cleavage of base sugar bond.

Causes of induced mutation- mutages (external agents that can cause mutation, chemical or physical

Mutagens

Chemical #1- intercalating agents- insert themselves between bases and can cause deletion shortening the DNA. (e.g. eithidium bromide)

Chemicall #2- base analogues- incorporated into DNA, more prone to tautomeric shifts. (e.g bromouracil)- more unstable so changing will occur at greater rate changing the H bond causing it to slip htorugh the next step.

- tricks the cell, looks like one of the bases eg T

Chemical #3- alkylating agents- add alkyl groups to nucelobases e.g.. nitrosamines

- alkylation can also speed up deprivation and it can be repaired.

Chemical #4 - deaminating agents- remove amino groups e.g. nitrous acid, nitrosamines, nitrite ( far quicker then spontaneous deamination)

Chemical #5- oxidising agents (e.g superoxide ions (o2^-), H202

- cause of most mutations

many possibe nucleotide alterations (OH+G----> 8-oxo G -----> base pairs A and C

Physical- Radiation, U/V, x rays

- break bonds and creat free radicals

- bases chemically altered, linked or detached

- leading source of mutation

- Number of mutations proptional to a radiation dose (radiation exposure leads to a larger number of mutations)

Consequence of mutagens

1) Mutation in regulatory regions of gene- alters the rate of transcritopns (protein deficiency excess)

- muation in regulatory areas- can cause increase. decrease in transcription if protein deficiency/ excess

2) mutation in into/ exon boundary mutation in coding region of gene- alters protein function (protein dysfunction)- 1 AA change can have major effects (DNA mutation to Altered Codon to Altered amino acid, Sickle cell (Glu to Val)

- alter length of messenger RNA within the intron, cause cell make wrong mRNA- rubish protein

Mutation in coding areas of gene

base substitution in the coding region

silent mutation ATG TGG CAC -----> ATG TGG CAC no affect

misense mutation TGG ----> GGG ( new Amino acids)

nonsense mutation ATG TGG ----> ATG TAG (stop codon)

Base deletion- ATG TGG CAT C... ----> ATG GGC ATC (frameshit mutation)

Base insertion- ATG TGG CAT -----> ATG CTG GCA T... (Frameshit mutation)

In born errors of metabolism (IEMs)

- Mutation in single gene encoding a metablic enzyme

- Unable to: break down substances- toxic/ synthesise vital substances- deficiency.

- Archibald Garrod 1909- errors in amino acid metabolism e.g. alcaptonuria (pee came out brown contained homogentisate)

- alcaptonuria- mutation in single gene autosomal recessive and ha sno homogentisate oxidase to break it down. Mutation was int he enzyme so it is inborn.

- change in pathways, eg Metabolite conversions A to B (has deficiency- anabolic) and can no longer be converted because the enzyme has the issue.

Types of metabolic disorders

Anabolic

Catabolic- F (excess) to G

Storage macromolecules X to monomers- deficiency of monomer and cellular deposits of macromolecules X - no break down

Examples of IEM- phenylketonuria (PKU)- Amino Acid (catabolic) buildup of intial product

Glycogen storage disease (GSD)- (CHO)- storage/ anabolic

Tay- Sachs disease- lipids

Familial hypercholesteroleamia - cholesterol

phenylketonuria (PKU)-Autosomal (12) recessive disorders

- enzyme affected Phenylalanine HYdroxylase

Inability to metabolise the essential amino acid phenylalanine.

excess phenyl- ketones in urine

Builds up in blood > 20mg/dL (normal < 2mg/L) - consequences metal retardation organ damage unusual posture

Mutant- Classic PKU (types I, II, III) due to point mutations

- 40% of mutations in intron/ exon splice site

- 20% of mutations in coding region

- 40% in promoter

- other PKU (types IV) not due to PAH

Normal: relys on the reductase enzyme

Progress of PKU: PKU manifests after birth

- during pregnancy mother breaks down Phe

- Neonate develops HyperPhenyalaninaemia a few days after birth (1 in 20,000 new borns)- consequences 5 IQ/month, death by 20/30y

- sufferer must be put on a low Phe diet.

Glycogen storage disease

- 14 diferent types: autosomal recessive

Inability to convert glycogen- glucose

Excessive levels of glycogen in liver and muscles

glycogen occurs occurs as intracellular granules that contain up to 120,000 glucose units

glycogen is a glucose store for later ,metabolic use

Glucose from glycogen stores in liver- provides a constant supply of glucose

glycosidic linkages in between glucose polymer

Muscle- Glycolysis

Liver- Bloood glucose

Four enzymes involved in glycogen breakdown

1. Glycogen phosphorylase

2. Glycogen debranching enzyme

3. Phospogucomutase

4. Glucose ^ phosphatase ( becmomes hypoglycaemic)

Glycogen storage disease 1- von Gierke disease

- phosphorylated glucose produced by glycogen breakdown is not readily transported out of cells

- the liver contains glucose 6 phosphate which cleaves glucose 6 phosphate into free glucose

- Glucose 6 phospate ------> Glucose + Pi

Glucose 6 phosphate if deficient in von Gierke disease

- leads to server hypoglycaemia within 2-4hrs after last meal.

- patients kept alive by regular carbohydrate feeding day and night

- shows that without the synthesis of glucose by the liver, humans would die of hypoglycaemia within hours of last meal.

- The muscle glycogn phosphorylase is absent

Myophosphorylase (PYGM) on chromosome 11

33 different mutations throughout the gene

1 in 100,000 20/30 yrs old

Muslce- glycolysis

muscle weakness and cramps

ability to performs strenuous exercise is limited

- shows that utilisation of muscle glycogen is not essential for life.

Tay- Sachs disease

- lipid storage disorders (sphingolipids)

- Autosomal recessive chrmosome 15

Deficiency of Hexo saminidase A

Failure of lipid degradation- Accumulation of ganglioside GM2 (gangliosidosis)

Consequences- mental retardation weakness blindness.

Normal: Gm2 (breaks up)

Mutant: you have an buildup of insoluble non- excretable spingolipid in lysosomes and neurons becomes swollen with lipid filled lysosomes

Progress: normal at birth

Year 1

- Neurological disorders

- blind, deaf, unable to swallow

----> progress to death by 3yrs

Family hypercholesterolaemia

Cholesterol- modulates the fluidity of membranes

- it is the precursor of steroid hormones

- cholesterol is produced in the liver ( as well as diet)

- transported in body fluids as lipoprotein particles- consist of a core of hydrophobic lipids surrounded by a shell of more polar lipids and proteins.

- cholesterol can be broken down in the liver

Defect in LDL- R gene (effect charge) - Heterozygous 50% reduction in LDL- R, double normal plasma LDL (250- 500mg/L) ----> Coronary atherosclerosis; 1 in 2 chance of myocardial infarction by age of 50.

Homozygous- No LDL-R at all/ Plasma LDL > 600mg/L----> Coronary atherosclerosis: high chance of death before age of 20.

Progress- heterozygous

statins- decreased production of LDL cholesterol/ produces more receptors

- this decreases the amount of cholesterol in the liver cells, causes them to take up LDL cholesterol from the blood.

decreased cholesterol production/ increased uptake from the blood

Treatment of IEMs

Biochemical

treatments

§Control of metabolite consumption (PKU)

§Supply of missing metabolite (GSD)

§Drugs (FH)

§Biochemical treatment may not be applicable

§Potential genetic treatment requires detailed knowledge of mutation

§Need DNA sequence

Determining the genetic tissue:

1) Tissue

samples taken from sufferer

2)

Chromosomal extracted

3)

DNA analysed for known/unknown mutation

Obtaining a DNA sample

Fetus

Amniocentisis

(12th week+)

§Fetal skin cells in amniotic fluid- epthelial cells in liquid

§Centrifuge and culture

- Risky process

-during pregnancy can take a sample

- look for genetic mutation in cultured samples.

Chorionic

villus sampling (8-12th week)

§Chorion is embryonic

§Risk of contamination by maternal tissue damage

getting close to the wall so internal damage

- Newborn

§Skin prick - Guthrie test (blood good for metabolism)

§PKU, CF, sickle cell anaemia

- Adult

§Blood sample (WBC)

§Skin biopsy- small number of cells culture.

Cells are washed and the nuclear DNA is extracted and purified by centrifugation

and ethanol precipitation.

1. blood on filter paper

2. place on culture of bacteria

3. agar inhibits bacteria to grow , if a lot phenylalanine.

Testing for known mutations- Polymerase chain reaction (PCR)

- allows you to amplify a sequence of DNA from a single cell.

- Invented by Kary Mulllis 1985 (nobel prize 1993)

- Cell- free DNA replication system.

Template DNA

DNA polymerase

Oligonucleotide primers

dNTPs

Buffer

- replicates chosen region of DNA (up to ~3000 b.p)

Quick way of producing lots of DNA

Requires: ds DNA template, DNA polymerase nucleotides, pair of oligonucleotide primers.

want primer to stick to DNA that has your gene in it.

PCR Powerful

- PCR can amplify up a single molecule of DNA into a visible amount of DNA in approx 2hrs

- Template DNA need not be highly purified

- PCR product can be digested and sequenced

PCR Method

Step 1- denature template DNA to ssDNA with heat (Heat to 95 C)

Step 2- Annealing (55C- 60C depends on number of A and C's compared to G's and T's)- lower temp. to allow primers to anneal

Step 3- Extension- separation, DNA polymerase extends primer

- lots of cycles, amplifying DNA x2

DNA polymerase

- Originally bacteria DNA polymerase was used.

- Subsequently (1988) DNA polymerase from the thermophillic bacteria Thermus aquatics was used.

- Taq DNA polymerase has a temp optimum of 72 C and is relatively stable at 94 C.

Polymerase Chain Reaction

- Denature

- Annealing

- Extension

Material required for PCR

- Sample preparation minimal

- only ngs of DNA need

- Cells are pelleted, lysed and the lysate used for PCR

Design 2 primers to anneal to either end of the gene- for your gene of interest run on gel 1 band successful PCR for gene of interest.

- may get 1 or more products

- always test to get 1 mutation from PCR

Testing for known mutations

a) deletions/ insertions

Run on polyacrylamide gets 1 b.p change can be detected

smaller means run through further, gel will run according to size

How is PCR used to detect known mutations

- detecting the F508 mutation is the cystic firbosis transmembrane conductance regulator gene (CFTR) gene

- good to detect deletion in this disease

Trinucleotide repeat expansion within the huntington gene

- any repeat more prone to slippage when translated will cause nonsense cause extra letters from repeat.

- you know its larger region cause of expansion / smaller region when less expansion

b) Base substitutions- uses allele (gene) specific oligonucleotides as primers to test for the presence of a mutation.

- ARMS (Amplification refractory mutation system) - use specific primers that contest for presence of mutation

ARMS: 2 PCR Reactions:

A) Primer A~ specific to wt dna

(A + C amplify wt)- binds only to wt

B) Primer B ~ specific to mutant dna (B+C amplify mutant)- will only bind if mutation at region.

Workout what samples are homozygous and heterozygous to the wild type/ will detect heterozygous and homozygouse version of protein

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Scanning genes for unknowing mutation mutations

- many genes are affect by a wide range of different mutations

- therefore to scan the gene and detect differences from the wild type gene

a) Single-strand conformational polymorphisms (SSCP)

- single stranded DNA molecules form extensive 3D structure in solution because of intramolecular pairing

- even a single base change can radically alter the structure. (shape b.p)

- this 3D has large effect on the rate of migration during electrophoresis

- PCR up strand stop at 95C

- good way to detect if there is a mutation in gene.

- don't want anything in gel that will disrupt it

mutation cause of single change in b.p

b) Denaturing gradient gel electrophoresis (DGGE)

- Tells you if you have mutant

- splits open at lower concentration more A and T

- at end of gene will get tightly bond strip of DNA in agarose gel will denature DNA splits open DNA and forms Y

c) Protein trunctuation test

- many mutations result in the formation of a stop codon- and a truncated protein product

- this type of mutation can be detected by using the product pf a PCR reaction as a template in a transcription/ translation reaction.

Advantage: will only detect change that are biologically significant, will not detect silent mutations

- used in screening for mutation in BRCA1 and BRCA2, where over 90-95% of mutations are chain terminating

d) DNA sequencing

- expensive technique

- time consuming

- used in cancer related tests

- take sample through DNA template machine

- colour associate with nucleotide

- sequence that DNA

- Run patient sample if you have a particular change that is associated with particular order.

1) Known mutations

a) PCR amplification of gene

b) ARMS

2) Unknown mutations

a)

single strand conformational polymorphism

b)

denaturing gradient gel electrophoresis

c) protein truncation test

d) sequencing

Known mutation

- PCR & run on gel – size change

(insertion/deletion)

- ARMS (Amplification refractory mutation system)

with allele specific primers (substitution)

Compare to known image for that mutation

(e.g. parent)

Unknown mutation

- PCR & SSCP (Single-strand conformational polymorphisms)

- PCR & denaturing gradient gel

- PCR and protein truncation test

-DNA sequencing

-Compare to known image for that mutation

(e.g. parent)

Designing

a therapy

Strategy

§Dominant - switch off mutant gene

§Recessive - add normal gene

ØPreparing the corrective nucleic

acid

Blocking

mutant gene

Antisense

- DNA complementary to

mutant mRNA

- Hybridises and

prevents translation

RNAi

RNA interference

Endonuclease that cleaves dsRNA to

short interfering (si) RNA (20-25bp)

- RNA-Induced Silencing ComplexBlocking mutant gene: Familial

Hypercholesterolemia

Gene Therapy (2014) 21,

60–70. Therapeutic expression of hairpins targeting apolipoprotein B100

induces phenotypic and transcriptome

changes in murine liver

Addingnormal gene

§PCR up normal version of gene

§Need carrier for gene + promoter

§Inserted into plasmid

vector

Gene transfer- In vivo vs in vitro

In vivo

- May only require an injection

- easy re-treat

-needs specific targeting

- may have immune complications

In vitro

- Rquires surgery

- little wastage

- no need for targeting

- no immune system

Vectors: Non-viral- microinjection, liposomes

Viral- adenovirus, AAV, retrovirus, lentivirus

Microinjection

Injection of 'naked' plasmid

- inefficient

Liposomes

In vitro

Drawbacks:

- Inefficient transfer of DNA to target

cells

- Non-specific uptake - mostly endothelial

-Cannot target particular cell types

- Poor expression

Advantages of liposomes

-Non-toxic

Introduction to viruses

§Intracellular particles that cannot

replicate on their own

§All viruses contain a nucleic acid genome

Viral

genomes: ssRNA ssDNA 7kb-----> 200kb

§The genetic material is surrounded by a

protein coat known as a capsid

Retroviral vectors

§Most studied viral vector system

- ssRNA viruses 7-9 kb

Viral vectors

Replication- deficient

Transgene inserted into viral genome

Pros: get into cells, get into nucleus, get expressed

Cons: Toxicity, immune response, limited duration and small inserts

Viral vectors

1) Adenovirus

§DNA virus

§Efficient transfection

§Limited cell types - epithelium

§Toxic

§Not integrated into genome - temporary

2) Adeno-associated virus (AAV)

§Wide variety of cells

- Integrate DNA into chromosome 19

§Non-toxic, no immune response

§Only small inserts

3) Retrovirus - Based on MMLV (mouse leukaemia)

§Integrate into genome

§7-7.5kb inserts

§Only infect dividing cells

§Insertional

mutagenesis

§Toxic?

insertional mutgenesis

4) Lentivirus

§Subclass of retrovirus (inc.

HIV)

§Infect non-dividing cells

§Variety of cell types, inc.

liver

§Toxic??

Improvements:

- Gutless’ forms lack nearly all viral

DNA/RNA

- Alter coat protein Þ Infect other cells

Combined hiv/ebola

virus used to treat CF in rats

→quarter of lung cells took up gene