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Introduction to Biochemistry > Metabolic Diseases > Flashcards

Metabolic Diseases Flashcards

Lectures 37-38

1
Q

Describe replication errors.

A
  • When repetitive regions cause slippage and insertion of more repeats (this cannot be repaired)
    Normal replication can be repaired and occurs every 10 base pairs.
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2
Q

What is a tautomeric shift?

A

Tautomerisation: When something is capable of existing in two forms between which they interconvert.
- so it changes the base pairs it can bind to by making it an enol group instead of keto, imino instead of amino, etc.

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3
Q

What is deamination?

A

Loss of amino group that changes the bonds the base can make. This changes base pairing, which is carried on when replication occurs.

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4
Q

What is depurination?

A
  • Cleavage of base-sugar bond
  • Wrong bases are paired together
  • This is common and repaired a lot
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5
Q

What are intercalating agents?

A

Chemical mutagens that insert themselves between bases.

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6
Q

What are base analogues?

A

Chemical mutagens that incorporate into DNA.
- These are more prone to tautomeric shifts

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7
Q

What is an alkylating agent?

A

Chemical mutagen that adds alkyl groups to nucleobases and can speed up depurination. These can be repaired.

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8
Q

What are deaminating agents?

A

Chemical mutagens that remove amino groups. This is a lot faster than spontaneous deamination.

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9
Q

What are oxidising agents?

A

Chemical mutagen that oxidises.
- Cause of most mutations
- Can cause many possible nucleotide alterations

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10
Q

Describe what physical mutagens can do.

A
  • Leading source of mutations, with mutation amount being proportional to the dose of radiation.
  • Bonds broken, free radials created
  • Bases chemically altered, linked or detached
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11
Q

What are anabolic genetic diseases?

A

Metabolite A cannot → into (BIGGER) metabolite B
- Causes deficiency of metabolite B

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12
Q

What is a catabolic genetic disease?

A

Metabolite F cannot → into (SMALLER) metabolite G
- Causes excess of metabolite F

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13
Q

What is a storage related genetic disease?

A

Macromolecule X cannot transform into monomers, so there are cellular deposits of macromolecule X and deficiency of the monomer.

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14
Q

Describe the disease Phenylketonuria.

A
  • Autosomal (12) recessive disorder in PAH gene
  • Causing inability to metabolise phenylalanine which builds up in the blood.
  • Symptoms include: Mental retardation, organ damage and unusual posture. They start at birth as during pregnancy the mother breaks down Phe.
  • If undiagnosed, 5iq is lost each month and life expectancy is 20-30 years.
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15
Q

What is a Glycogen storage disease?

A
  • Disease that causes inability to convert glycogen into glucose meaning excess glycogen in liver/muscles.
  • 14 types
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16
Q

What are the 4 enzymes involved in glycogen breakdown?

A
  1. Glycogen phosphorylase
  2. Glycogen debranching enzyme
  3. Phosphoglucomutase
  4. Glucose 6 phosphatase
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17
Q

Describe Von Gierke disease (Glycogen Storage disease 1).

A
  • Phosphorylated glucose produced by glycogen breakdown is not transported out of cells properly.
  • Deficiency of glucose 6 phosphatase.
  • Patients kept alive via constant carbohydrates.
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18
Q

Describe McArdle’s disease (Glycogen Storage disease 2).

A
  • Absence of glycogen phosphorylase.
  • Caused by my phosphorylase on chromosome 11. 33 mutations occur on this gene (0.0001%)
    Symptoms include: Muscle weakness/cramps, inability to exercise, muscle breakdown
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19
Q

Describe Familial Hypercholesterolaemia (Glycogen Storage disease 3).

A
  • Defect in LDL-R gene
    Heterozygous: 50% reduction in LDL-R, double normal plasma (250-500mg/L). 50% chance in myocardial infarction by 50 yrs.
    Homozygous: No LDL-R. Plasma over 600. High chance of death before 20yrs.
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20
Q

What are the 3 ways of determining the genetic cause of IEMs?

A
  • Tissue sample from sufferer
  • Chromosomal DNA extracted
  • DNA analysed for known/unknown mutations
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21
Q

How are tissue/DNA samples taken from foetuses?

A

8-12 weeks: Chorionic villus sampling. Chorion is embryonic. High risk of contamination by maternal tissue damage.
12+ weeks: Amniocentesis. Foetal skin cells in amniotic fluid collected, centrifuged and cultured.

22
Q

How are tissue/DNA samples taken from babies and adults?

A

Newborns: Skin prick (Guthrie test) for PKU, CF, sickle cell anaemia. Always done.
Adults: Blood sample and skin biopsy.

23
Q

How is Phenylketonuria tested for?

A
  • Guthrie test based on measurement of Phe concentrations in blood spots
24
Q

How are Glycogen storage diseases tested for?

A

Full gene sequencing of:
- G6PC gene (Von Gierke disease)
- PYRL gene (McArdle disease)

25
Q

How is Tay-Sachs disease (lipid) tested for?

A
  • Blood test that detects absent or very low levels of beta-hexosaminidase-A enzyme activity
  • Molecular genetic testing of HEXA gene may identify specific mutation present
26
Q

How is Familial Hypercholesterolaemia tested for?

A
  • Genetic testing looking for inherited genetic changes in genes LDLR, APOB and PCSK9)
27
Q

What are the first two steps of screening for IEMs (Genetic metabolic diseases)?

A
  • Suspect the disease
  • Lyse cells and extract DNA
28
Q

How are IEMs screened for if the mutation is known?

A
  • PCR and run on gel, see a size change
  • ARMS (Amplification refractory mutation system) with allele specific primers
  • DNA sequencing
  • Compare to known image of mutation
29
Q

How are IEMs screened for if the mutation is unknown?

A
  • PCR and SSCP (Single strand conformational polymorphisms)
  • PCR and denaturing gradient gel
  • Protein truncation test
  • DNA sequencing
  • Compare to known image for that mutation
30
Q

How is PCR used to detect known insertions/deletions?

A
  1. Cells are pelleted, lysed and the lysate used for PCR.
  2. Design 2 primers to anneal either end of the gene.
  3. Generates enough DNA for sequencing and to detect mutations.
31
Q

How is PCR used to detect known base subsitutions?

A

ARMS: Uses allele specific oligonucleotides as primer to test for the presence of a mutation
- Primer A will be specific to normal DNA, B to mutant DNA. Both are amplified and then compared in gel electrophoresis.

32
Q

What are the biological treatments of IEMs?

A
  • Control of metabolic consumption (for PKU)
  • Supply of missing metabolite (for GSDs)
  • Drugs (Statins etc)
  • No treatment available (Tay-Sachs)
  • Biochemical treatment not always applicable, detailed knowledge of mutation needed.
33
Q

How is gene therapy carried out?

A

Recessive = Add normal gene
Dominant = Switch off mutant gene
- Prepare corrective nucleic acid and gene transfer.

34
Q

What is Ex Vivo gene therapy?

A

Gene therapy involving manipulation of a target cell population outside of the body in which a patients own cells are genetically modified and then engrafted back into the patient.

35
Q

What is In Vivo gene therapy?

A

Injection of a vector encoding the corrective gene or gene editing tools directly into tissue or systemic circulation.

36
Q

What are the pros and cons of Ex Vivo gene therapy?

A

Pros:
- No immune response
- No off target response
- Integration of transgenic DNA into genome
Cons:
- May require surgery
- Some cell types hard to culture Ex Vivo
- Poor engraftment rate

37
Q

What are the pros and cons of In Vivo gene therapy?

A

Pros:
- May only require an injection
- Easy to re-treat
Cons:
- Non specific targeting
- Immune responses to the vector

38
Q

What are the two types of Vectors?

A

Non-viral: Liposomes
Viral: Adenovirus, AAV, Retrovirus, Lentivirus

39
Q

How are liposomes used as vectors?

A
  1. Lipid bilayer and wanted gene are mixed
  2. Transferred into target cell
    3.Fusion with nucleus of target cell
    - Cationic lipids react with anionic lipids in cell membrane, aiding in delivery.
40
Q

What are the pros and cons of Liposomes as vectors?

A

Pros:
- Non toxic
Cons:
- Inefficient transfer of DNA to target cells
- Non specific uptake, mostly endothelial
- Cannot target particular cell types
- Poor expression

41
Q

What are the characteristics of viral vectors?

A
  • Viruses cannot replicate on their own
  • All viruses contain a nucleic acid genome
  • Genetic material is surrounded by protein coat called a capsid.
42
Q

What is the Therapeutic Nucleotide Sequence (TNS) of a DNA called?

A

Adeno-associated virus TNS.

43
Q

What are the sections of Adeno-associated virus TNS?

A
  • Enhancer + promoter + intron = drives transgene expression
  • Transgene = corrects genetic defect
  • Polyadenylation signal = Proper translation
  • ITR = (Inverted terminal repeats) for packaging of the TNS into the virus
43
Q

What is the Therapeutic Nucleotide Sequence (TNS) of a RNA called?

A

Retroviral TNS.

44
Q

What are the sections of Retroviral virus TNS?

A

LTR = (Retroviral Long Terminal Repeats) Mediates integration of retroviral DNA and has promoter activity.
Ψ = Retroviral packaging signal.
RRE = (Rev-responsive element) For post transcription transport of viral RNA during retroviral packaging.
+ Enhancer, Promoter, Intron, Gene

45
Q

How are viral vectors inserted into a cell?

A
  1. Replication deficient viruses are used.
  2. Transgenes inserted into the viral genome, alongside a viral packaging signal.
  3. Viral coat proteins (no nasty stuff) + enzymes inserted into the viral genome.
  4. Translated and inserted into the capsid, followed by the transgene and viral packaging signal also entering the capsid.
  5. Tells capsid to package viral particle, so it is then secreted and we can collect it.
46
Q

What are the pros of viral vectors?

A
  • Gets into cells
  • Gets into nucleus
  • Gets expressed
47
Q

What are the cons of viral vectors?

A
  • Toxicity
  • Immune response
  • Short term response
  • Small inserts only
  • Insertional mutagenesis (Retrovirus)
48
Q

How does CRISPR work?

A
  • Finds and replaces/disables defective gene.
  • System produces DNA cleavage at target gene
  • Insertion at donor template can lead to gene correction via the Homology Directed Repair pathway (HDR)
  • Used for both In Vivo and Ex Vivo.
49
Q

What is the disadvantage of CRISPR?

A

Risk of introducing off target mutations in genome regions with similar sequences to the target gene site.

Introduction to Biochemistry (5 decks)

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