Metabolic diseases biochem Semester 2
Why do genetic mutations form
This is due to a change in the nucleotide sequence of the DNA via a substitution, insertion or deletion of 1 more base pairs
How are muations caused
This can be due to spontaeneous reasons like- Replication errors, tautomerisation, deamination or depurination
Induced reasons like intercalting agents, base analogues, deaminating agents, alkylating agents, oxidising agents and UV
What are replication errors
Normal replication introduces the wrong base pair every 10^10 base pairs so there is a goof change it may be repaired
However in some repeating regions of the DNA it will cause base pair slippage and insertion of more repeats that cant be repaired
There can be deletion of a repeating region or the expansion of a repeating region
What are tautomeric shifts
The bases are subjective to spontaeneous structural alterations called tautomerization
There is the ability of being 2 exisitng forms between DNA that can shift between the 2 forms
Amino group and imino group can shift between the 2 for adenine and cytosine as well as Keto groups can shift to enol groups for guanine and thymine Both are bad due to change the hydrogen bonding of the nitrogenous base effected and this then may effect the complimentary binding patterns due to gain or lose a hydrogen bonding site so bind with bases its not supposed to
What is deaminiation
This is where there is the loss of an amino group of a nucleotide and change the nucleotide structure to a different nucleotide so then bind to different base
C --> Uracil
A--> Hypoxanthine
G --> Xanthine and these are all reversible
5 methly C --> T whihc is unreversible
What is depurination
This is the cleavage of a base sugar bond and this will then lead to change in the overall DNA sequence that then will lead to change in the overall DNA sequence and this may cause underlying mutation
What are intercalating agents
They are a chemical reason for induced genetic changes
These are able to insert themselves between the bases of the DNA
It can cause both insertion and deletion mutations that are then further consilidated in further replication rounds
What are base anologues
These are a chemical reason for induced genetic change
These are molecules that have a similar shape to certain bases and so may be added to DNA instead of the base
This will then destabilise the DNA and make tautomeric shifts more likely to happen all leading to changng hydrogen bonds
What are alkylating agents
These are a chemical reasons for indiced genetic change
They will add an alkyl group to the nucleobases that will then cause a speed up of depurination but it can be repaired
Can also damage hydrogen bonds and the base pairing this allow
Whar are deaminating agents
These are chemical reasons for induced genetic change
They will remove the amino group from a base and it will take much less time than spontaeneous deaminiation
What are oxidising agents
These are chemical reasons for induced genetic change
They can oxidise a base and then leads to the base being able to bind to more than 1 base pair
This will cause the most mutations
What is the physical reason for the induced genetic change
This is due to UV and raditation, as they will cause the formation of free radicals that then lead to base pairs being chemically altered, linked or detached
What are in born errors of muations - IBEM
This is a class of genetic diseases involving the altering of metabolic function
There are 3 types:
Anabolic - Where unable to build up smaller units to then lartger units due to Metabolite B defficiency
Catabolic - Where unable to break larger unit down to smaller units due to excess metabolite G
Storage - Cant store large units for later usage as a smaller unit in stroage organs due to monomer defficiency and excess larger unit
What is Phenylketonuria
This is a type of IBEM and is a catabolic mutations causes by autosomal recessive disorder in PAH gene and effect phenylalanine hydroxylase
This then cause inabiolty to metabolise phenylalanine and so get excess phenly ketones in the urine that causes mental retatdation, organ damage and bad posture
There are 3 mutation site on the PAH gene:
40% at the intron/.exons splicing site
20% at the coding region
20% at promotor region and these all cause type 1-3
What is type 4 phenylketonuria
This is where have fully functioning PAH enzyme so can produce the tyrosine from the phenylaline but then there is no Dihydropteridine reductase to remake the co-factor BioH4
IT will manifest post birth
It can cause death in early adulthood if not detected and the patient must have a low phenylalanine diet
What is glycogen storage disease
There are 14 types and it cause the inability to convert glycogen to glucose and can be detected by an enlarged liver and also prolonged low blood glucose levels
There will be excess glycogen in liver and muscles
How to break down glycogen
This can be done glycogen phosphatase: This is glycogejn + Pi --> Glucose + Glucose 1 Phosphate
Then there glycoegn debranching enzyme
Then phosphoglucomutase will be used where glucose 1 phosphate goes to glucose 6 phosphate
Glucose 6 phosphatase will convert glucose 6 phosphate to glucose
What is Von Girke disease
Phosphorylated glucose produced by glycogen breakdown is not readily transported out of the cell
The liver has enzyme glucose 6 phosphatase to cleave glucose 6 phosphate to release glucose
In this disease there will be a deficient of glucose 6 phosphates so less glucose released by the liver
But there will be more glycolysis producing more pyruvate and so more lactate will be produced and blood glucose will stay low to cause hypoglycaemia
What is Macardle Disease
The muscle will be absent on glycogen phosphorylase
This is due to myophoshorylase gene that is prone to 33 mutations
This will then limit glycogen + Pi àGlycoegn + Glucose 6 phosphate
It will cause:
Muscle cramps and inability to perform strenuous exercise
Why is Cholesterol important
Modulate the cell membrane fluidity
Precursor of steroid hormones
It is made in the liver and is transported in the blood as lipoproteins particles
This is made of core of hydrophobic lipids surrounded by core of polar lipid and proteins
What will the defect in the LDL-R gene cause
Heterozygous so there is 50% reduction in LDL-R
There is then less cholesterol uptake in the liver so then higher cholesterol level in the blood and may cause coronary vascular events
If the gene mutated is homozygous then there is full deficiency of LDL-R receptor so then even more cholesterol stay in the blood due to no uptake in the liver
What is the progress in combatting LDL-R defects
Statins: will lower production of lDL cholesterol and produce more receptors
This decrease amount of cholesterol in the liver cels so then they take up more LDL cholesterol from the blood due to less normal cholesterol production so more is taken from the blood
What are IEMs diseases examples
Phenylketonuria can be detected by phenylalanine level in the blood
Glycogen storage disease - can be seen by enlarged liver, no glycolysis or prolonger low blood sugar
Tar sachs disease - measured lipid levels
Familial hypercholesterolaemia - measure the cholesterol levels
How can genetic cause of IEM be found
S1 -Tissue samples
S2 - Chromosomal DNA being exctracted
S3 - DNA analysis for known or unknown mutations of the extracted chromosomal DNA
How are tissue samples obtained
From the fetus: can gather tissue from as easly in fetus development as 12 weeks via aminocentisis so extract the amniotic fluid and then centrifufe the sample to get skin cells and then cutlure and grow the skin cells for analysis
Chronic Villius sampling by using catheter to gather sample of mother chorion that is made of embyronic cells
In new borns can use skin prick to test for PKU,CF and sickle cell
In adults can use blood sample or skin biopsy
How to test for PKU
Guthrie test is based on the measurement of Phe concentration In the blood and then can sequence the PAH gene as well to test for PKE
How to test for GSD
Can be confirmed by full sequencing of G6PC gene
How to test for Tay sachs disease
Blood test that detect the absent or low level of beta hexosaminidase A enzyme activity
Molecular genetic testing for HEXA gene can be used to find specific mutation present
How to test for Familial hypercholesertolaemia
Genetic testing for inherited genetic changes in 3 different genes – LDLR, APOB and PCSK9
How is PCR used to detect unknown mutations
This is for mainly insertion or deletion mutations
The cells are pelleted and lysed ready for PCR
There are 2 primer designed to anneal at either end of the gene, and then the PCR is carried out, and if lighter sequence then is a deletion and if heavier then is an insertion muatations
How to test for base subsititions
Can carry out an ARMS test that will use a specific oligonucleotides as primers to test for the presence of mutations, this also uses PCR test to compare to known mutations
How to scan genes for unknown mutations
Many genes can be effected by wide range of different mutations and its important to scan the gene and detect differwences from the wild type of that gene
Can use very specific wild types of genes where they have no mutatations and so can compare to patient genes to show where the mutations is there and what gene it is in
What are the treatements for IEMs
Can use biochemical methods: The control of metabolites for PKU, Supply of metabolites for GSD, Can use statins to lower cholesterol in the blood form FH but have to be a heterozygous patient
There is no treatment for Tays sachs
The biochemical may not work due to potential genetic treatment will need detail info of muations so have to carry out DNA sequencing first
What is gene therapy
This is used when biochemical methods dont work by either removing the falty gene or by altering the mutations and fixing them backl to the normal gene so the gene works again
If the mutations is recessive add the normal gene and if mutations is dominant then switch off the gene all together
S1= Prepare the corrective nucleic acids
S2 = Gene transfer
How to prepare the corrective nucleic acid for gene therapy
This is done by PCR to produce the normal version of the gene and then a carrier is needed for the gene + the specific promotor sequence so the gene can be inserted to the patient genome and is expressed
This is done by inserting them in a plasmid vector
What is direct delivery
This is where the vector is in injected direct to the patient with the new gene and promotor sequence, and it then go straight to the target organ - the transgene is delivered direct by delivery vehicle like a virus
What is cell based delivery
The therapeutic transgene is inserted to virus again, before then being introduced to a delivery cells like a stem cells
These cells are then genetically modified with the transgene in them before being delivered to the patient and going to the target organ
What is ex vivo gene therapy
This involved the manipulation of a target cell population outside of the body in which the patient own cell are genetically modified and then engrafted back to patient
There is no immune response, no off target effects, and tgere is the intergration of the transgenic DNA into the genome
However there may be surgery needed, and some cell types are hard to culture in ex vivo methods and there is a poor engraftment rate
What is in vivo gene therapy
The injection of a vector encoding the corrective gene or gene editing tools directly into a tissue or into the systemic circulation
There is no surgey needed and easy to redo many times but there is non specific targeting and there will be immune response to the vector
What are the different types of vectors
Non viral ones such as liposomes that are membrane bound and have a phospholipid bilayer capsule with an aqueous media section
Viral ones such as adenovirus, AAV, Retrovirus and lentivirus as virus have evolved to be effcient at inserting their DNA into a host cells DNA genome and getting it expressed
What are the pros and cons of lipossome vectors
Pros = They are non toxic
Cons = Ineffcient DNA transfer, non specific uptake, cannot target certain cell type, lead to poor expression
What are viral vectors
They cannot replicate on their own and all virus contain nucleic acid genome
The genetic material is surrounded by a capsid
How to make the viral vectors
To make the viral vector use a replication deficient virus so it can divide and harm the patient:
The transgene will be inserted to the viral genome in the capsid
The different viral vectors vary the amount of genetic material
can carry and also have different methods of entry to the cell
What are the pros and cons of viral vectors
Pros= They easily get into cells and also can get their materila into the nucleus and get it expressed easily
Cons = There may be immune response to them, there is limited duration, there may have to be small inserts and can get insertational mutagenesis from the retrovirus