genetics Flashcards
(263 cards)
1
Q
State the 3 stages at which gene expression can be regulated.
A
1) transcription
2) splicing
3) translation
2
Q
Define transcription.
A
creates mRNA to allow the coding in DNA to be passed out of the nucleus
3
Q
In transcription, what are mRNA, tRNA, rRNA are all created by?
A
RNA polymerase
4
Q
State the role of each:
a) RNA Polymerase I
b) RNA Polymerase II
c) RNA Polymerase III
A
a) production of large ribosomal RNA (rRNA)
b) production of mRNA
c) production of tRNA & small ribosomal RNA molecules
5
Q
State the 7 stages of transcription.
A
- start of gene of DNA is marked at the promoter region
- RNA polymerase molecules randomly collide with DNA in the nucleus. They bind with specific DNA sequences called promoters. (e.g. TATA sequence/box)
- RNA polymerase breaks the H bonds betw/ the complementary BPs in the DNA helix exposing both sides of the DNA strand
- complementary base paring takes place on DNA template strand – creating a single RNA strand
- the nucleotides are joined together by RNA polymerase
- mRNA unwinds from DNA strand
- mRNA exits the nucleus via the nuclear pore
6
Q
What forms during transcription and only in which case does it occur?
A
- a transcription bubble It
- occurs when only a limited amount of DNA is unwound from the DNA helix
7
Q
As a part of RNA processing, during transcription, how are the ends of the mRNA is capped at the 5’ end?
A
- addition of methylated Guanine nucleotide, by the removal of a phosphate by phosphatase
- addition of GMP (Guanosine monophosphate) via guanylyltransferase
- addition of methyl group via a methyl transferase
- allows the mRNA to exit the nuclear membrane
8
Q
As a part of RNA processing, during transcription, how are the ends of the mRNA is capped at the 3’ end?
A
a tail of up to 200 nucleotides is added by poly-A polymerase.
9
Q
What is an intergenic region?
A
non-coding piece of DNA
10
Q
Explain how a gene and an intergenic regions (introns and exons) are edited before splicing.
A
- exon/gene region; codes for proteins; exits the nucleus
- introns are removed
- exons can also be removed if they are not the particular piece of DNA which is needed for that expression - protein
- pre-mRNA to mRNA
11
Q
What is splicing mediated by?
A
the spliceosome
12
Q
State the 7 stages of splicing.
A
- it consists of several protein-RNA complexes
- snRNPs and spliceosome bind on the RNA
- RNA is looped, more complexes bind
- the complex undergoes a conformational change
- intron is cleaved at 5’ end
- intron is cleaved at the 3’ end
- 2 exons are joined together
13
Q
What is alternative splicing?
A
Use of other proteins to aid the splicing process. Allows a single gene to produce multiple varieties of protein.
14
Q
What are the 5 possible ways alternative splicing can be done?
A
1) exon skipping – only including some exons and leaving some out.
2) mutually exclusive exons- taking either of the 2 consecutive exons in between
3) alternative 3’ acceptor sites – the splice junction at the 3’ end is used, changing the 5’ boundary of the downstream exon.
4) alternative 5’ donor sites – the splice junction at the 5’ is used, changing the 3’ boundary of the upstream exon.
5) intron retention – the intron is retained; stop codons
NB: (3) and (4) are opposites
15
Q
State the 4 main characteristics used to code gene structure.
A
- promoter
- transcriptional ‘start’ and ‘stop’ signal
- exons and introns
- upstream regulatory regions
16
Q
What does transcriptional control of genes usually involve?
A
- switching a gene on (promoter as genes are not always expressed)
- e.g. some genes may be expressed in response to stimuli or during a point in a cycle
17
Q
What drives gene expression/
A
RNA polymerase II
18
Q
What must bind in the promoter region in order for a gene to be expressed?
A
a number of DNA-binding proteins (transcription factors)
19
Q
How is gene expression fine-tuned?
A
- via the binding of other transcription factors
- to distal regions termed upstream enhancer sequences
20
Q
What do activators do when they bind, to enhancer sequences?
A
increase expression significantly than without them
21
Q
How can a gene be switched off?
A
- transcriptional repressors
- not expressing a gene
- bind to specific sites on DNA and prevent transcription of nearby genes
22
Q
How do the 2 types of repressors act?
A
1) interacts w/ a transcriptional activator (enzyme) - binds to the site next to the activator so that its function is blocked
2) overlapping binding sites - the repressor binds at the site where the activator would bind to, so the activator cannot bind
23
Q
Give an example of a transcriptional repressor and explain how it works.
A
- Wilm’s tumour protein (WTP)
- in a developing kidney, WTP binds to the promoter region of the EGR-1 gene (a transcriptional activator) – switching off its expression
- if the gene encoding WTP is mutated, it leads to uncontrolled expression of EGR-1
- this can lead to development of kidney tumours in early life
- its considered a tumour suppressor gene
24
Q
a) Write a piece of DNA which is 15 base pair long.
b) Make an RNA molecule out of the above DNA. – using 3’ to 5’ DNA strand
A
a) 5’-ACTGAGTACCTTTCG-3’ 3’-TGACTCATGGAAAGC-5’ b) 5’-ACUGAGUACCUUUCG-3'
25
State teh 3 main stages of translation (protein synthesis).
1. tRNA carrying a methionine (start codon) associates w/ a small ribosomal unit which is in association with eukaryotic initiation factor 2 (eIF2)
2. the ribosomal unit recognises the 5’ capped end of mRNA with 2 additional initiation factors (eIF4G and eIF4E)
3. the ribosome scans along the mRNA to find the start codon; this allows the large ribosomal subunit to bind
26
What is translation elongation driven by?
elongation factors EF1 and EF2
27
State the 4 stages of translation elongation.
1. tRNA (aminoacyl) binds to A site. tRNA molecule at E site is released.
2. The carboxyl end of the amino acid in the polypeptide chain is uncoupled from the P site on tRNA. it then is attached to the new amino acid (on the tRNA molecule at the A site) by a peptide bone - peptide transferase enzyme.
3. the large ribosomal unit steps one codon along the mRNA to carry on reading the code.
4. The small ribosomal unit steps 1 codon along so that there is a new tRNA at each site. generating a new A site.
28
What happens during translation termination?
- protein synthesis is stopped when the ribosome encounters a stop codon (UAA, UAG, AGA)
- cytoplasmic release factors bind to the stop codon. This frees the carboxyl end of the growing polypeptide chain.
29
How is translational control implemented/
- many ribosomes attach to each mRNA
- in time mRNA is degraded (due to its half-life)
- the half-life is a way in which the cell can regulate gene expression levels
30
What are isoforms?
- proteins which have similar functions and similar amino acid sequences, but not the same
- this can arise from alternative splicing of a single gene
31
What can errors in gene expression result in and what can understanding these errors allow?
- uncommon disorders (DMD, Spinal Muscular Atrophy, Cystic Fibrosis, Pulmonary Arterial Hypertension)
- influence predisposition of many common diseases (Breast cancer, lung cancer, prostate cancer & blood cancer)
- understanding of these errors can help to develop therapies
32
For each gene mutated state the genetic disease caused:
a) DMD
b) SMN1
c) CFTR
d) BMPR2
e) BRCA1 +BRCA2
a) duchene muscular dystrophy
b) spinal muscular atrophy
c) cystic fibrosis
d) pulmonary arterial hypertension
e) breast cancer
33
What can over-expression of transcription factor (TF) MYC cause?
- numerous cancers
| - if even 1 copy of the TF is mutated, it can still lead to disease
34
What is haploinsufficency ?
- where 1 copy of the gene is not enough (e.g. p53 gene)
- we have 2 alleles for each gene (mother & father)
- one of them can mutate, and one of them can stay the same. - one copy of the gene is not enough for protection
35
What is a dominant mutation?
- where a change in the gene sequence generates change in the protein that exerts dominance over the wild-type (phenotype)
- there is a change in AA and therefore a change in the protein produced
- i.e. TPCP2L3 gene & genetic deafness
36
Describe an example of mRNA processing errors that occurs during alternative splicing.
- cystic fibrosis
- mutations found in exon 7 of SMN1 gene
- exon 7 is skipped
- non-functional protein is produced.
- children only live for a few years
37
Describe example of mRNA translation errors that occurs during alternative splicing and state the type of TF and gene that are affected leading to each disease:
a) VWM – neurodegenerative
b) gastric cancer
c) rare types of anaemia & neurodegenerative diseases
- these mutations affect translational efficiency (mRNA into protein)
a) - translational initiation factors; eIF2
b) - release factors; eRF3
c) - N/A; affect tRNA transfer proteins/ ribosome itself
38
What are the techniques which detect DNA, RNA & proteins mutations used to do?
see the exact point where the mutation occurs
39
What is gel electrophoresis of DNA/RNA?
- lithium bromide binds to the DNA
- DNA is negatively-charged so will move from the anode (-) to the cathode (+)
- through the current passing through the (argarose) gel
40
What is western/southern blot used to detect and what are its 6 stages?
- detect proteins
1. sample preparation
2. gel electrophoresis
3. blotting (or transfer)- to a membrane
4. blocking
5. antibody Probing – attaches specifically
6. detection- labelled with dye or fluorescent markers
41
How can RNA be artificially created by PCR? State the method.
- RNA polymerase; DNA TO RNA
- for this to occur, a promoter region is needed on DNA to allow enzyme to attach and initiate the process
- free nucleotides are also needed
42
Describe how the splicing of pre-mRNA in a tube occurs.
- alternating splicing
- machines detect it
1. combine RNA with cytoplasmic extract (contains tRNA and ribosomes)
2. RNA to protein
43
How can PCR be used to detect gene expression?
- enables amplification of a specific region of DNA from a single molecule of starting material
- DNA polymerase is needed
- hydrogen bonds broken by heat
- from 5’ to 3’ the nucleotides are added to the parent strand by the aid of DNA polymerase
44
How can gene expression profiling be done by PCR?
- quantitative real-time PCR (qPCR)
- detects how many copies of a gene you have
- insight into regulation of gene expression at a given time
45
State 3 uses of PCR.
- diagnostics
- gene cloning
- legal disputes
46
What are nucleotides the building blocks of?
- DNA: deoxyribonucleic acid
- RNA: ribonucleic acid
- covalent bonds
47
State the 3 components of a nucleotide and state how they differ in DNA and RNA.
```
• Pentose sugar:
- deoxyribose (DNA)
- ribose (RNA)
• Phosphate group:
- acts as a bridge between adjacent ribose/deoxyribose groups
• Nitrogenous base:
- pyrimidine (C, T (DNA), U (RNA)
- purine (G, A)
```
48
Which type of bond links a ribose molecule to a nitrogenous base?
a glycosidic bond
49
What is the rule of base-pairing in DNA and RNA.
- a pyrimidine always pairs with a purine
- i.e. C-G and T-A
- A-U bonding occurs during gene transcription (RNA)
50
Which bonds are formed between nucleotides and is aG-C bond or an A-T bond stronger and why?
- hydrogen bonds a
| - G-C bond is stronger than A-T due to more H bonds (3-2)
51
State the structural features of the DNA double helix.
- hydrophobic bases are on the inside of the strand, away from the water stabilised by H-bonds
- hydrophilic-sugar-phosphate backbone is stabilised by electrostatic interactions & H-bonding with water
- bases which are stacked have weak transient (temporary) electrostatic interactions VDWF (pi-pi interactions)
52
What are the structural features of RNA and what can single-stranded RNA molecules form?
- stem: complementary base pairing
- loop: no base pairing
- can form secondary structures through base-pairing
53
How do DNA and RNA polymers differ in size?
- difference is the bases (monomer units) – U and T
- polymer is described by the sequence (of bases) and number of bases
- e.g. size is the number of monomeric units (X bases long)
54
How do DNA and RNA polymers differ in direction?
- nucleotides always add to the 3’ end
- the α-phosphate of new nucleotide attaches to the -OH group of polymer
- this forms a 3’-5’ phosphodiester bond
- chain grows in 5’ to 3’ direction (left to right)
- nucleic chain starts with 5’ phosphate and ends with 3’ OH
• e.g. 5’ACTGCT’
(see document for diagram)
55
Describe DNA organisation in a eukaryotic cell.
- to compact the DNA, it is held in the nucleus with the aid of proteins
- the DNA content in a human genome is much larger than the cell nucleus so its compacted to fit in the nucleus, whilst still being functional
- histone is a protein which is in chromatin
- linker DNA is double-stranded DNA in betw/ 2 nucleosomes
- H1 protein binds to the linker DNA, holding everything together
56
What is a:
a) chromatosome?
b) nucleosome?
c) chromatin?
a) nucleosome + histone H1
b) basic unit of compacted DNA
c) histone-bound DNA
57
Sort in order of complexity:
- chromsome territories
- nucleosome
- DNA duplex
- chromosomes
- chromatosome
DNA duplex → nucleosome → chromatosome → chromosomes → chromsome territories
58
What is core DNA?
(146 BP) wraps around the histone core
59
What is linker DNA?
(60-80 BP) leads to adjacent nucleosome
60
What does a histone core/octamer contain?
2 copies of 4 histone proteins:
- H2A
- H2B
- H3
- H4
61
Dseribe the histones and DNA backbone in an octamer.
- high Arg and Lys content (+ve charge)
| - which allows them to bind to the DNA backbone (-ve charge) easily
62
What does DNA replication involve, when doe sit occur and what is it vital for?
- involves replication enzymes and RNA
| - occurs in S phase of interphase during cell cycle
63
For which 3 things is DNA replication vital for?
1) cell growth
2) repair
3) reproduction
64
What does the semi-conservative model of DNA replication describe?
when each daughter strand contains ½ of parent strand
65
State and describe the 3 stages of the mechanism of DNA replication.
1. formation of replication fork
- base pairs are broken by DNA helicase
- unzips double strand
- the strands form a Y shaped replication fork – template for replication
- proteins bind to this replication fork to stabilise this area so the single stranded DNA does not join back together
- 5’ to 3’ replication
2. RNA primer binding (leading strand)
- a short piece of RNA (primer 3-4 BP) binds to the 3’ end of the parent strand
- this marks the starting point for replication
- primers are generated by DNA primase
3. Elongation (leading strand)
- DNA polymerase α binds to the parent strand at the site of the primer
- this enzyme adds new complementary BP to the parent strand
- after 20 BP, elongation is taken over by DNA polymerase ε
- DNA polymerase ε and 𝛿 = proofread 3’ to 5’ exonuclease activity. This prevents incorporation of incorrect nucleotides.
- produces 1 continuous DNA strand in the 5’ to 3’ direction
4. termination (both strands)
- RNA primers are degraded by RNAse H
- RNA primers are filled by DNA polymerase 𝛿
- DNA ligase joins any breaks in both strands; continuous strand
(see document for diagrams)
66
What is a bi-directional replication fork?
- one strand is orientated in the 3’ to 5’ direction on the parent strand (leading strand)
- the other is orientation 5’ to 3’ on the parent strand (lagging strand)
(see document for diagram)
67
Why does DNA replication occurs at multiple sites?
- replicate cannot originate from 1 site per chromosome – it would take too long
- 1 genome takes 8 hours to replicate
- there are multiple origins of replication with replication forks proceeding in different directions – replication bubbles
68
What is a replication bubbles?
- there are multiple origins of replication
- with replication forks proceeding in different directions
(see document)
69
How are replication bubbles joined?
by DNA ligase
70
Describe the inheritance of histones after replication.
- histones are removed after DNA replication (in front of replication bubble) – they are not needed as there only purpose is to compact the DNA
- H3/H4 tetramers (histone octamer) remain intact
- new H3/H4 tetramer cores bind
- new H2A /H2B bind
71
What is epigenetics?
- heritable changes in the phenotype/cell behaviour or gene expression in cells
- caused by changes other than changes in DNA base sequence that control the activity of genes
72
Give examples of epigenetics.
- histone modifications (acetylation of Lys, methylation of Lys and Arg)
- DNA modifications (methylation of cytosine)
73
What do epigenetic modifications alter and why?
- alter chromatin structure to control accessibility of transcription factors
- and co-activators necessary for gene transcription
74
What can epigenetic marks can be altered by and what does this provide a mechanism for?
- environmental stimuli (smoking, nutritional status)
| - provides a mechanism for environmental factors to be imprinted genetically
75
Explain the Dutch famine 1994 example of epigenetics.
- foetuses exposed to famine during 1994-45
- 50 years later less DNA methylation on the IGF2 gene compared with unexposed same sex siblings
- children developed obesity and schizophrenia later in life
- children born to these women 20-30 years later suffered from same problems – even though they were conceived and born in a normal dietary state
- the exposure took place after conception, therefore it suggests this period is important for establishing epigenetic marks that persist in generations
76
What can early life environmental conditions cause?
epigenetic changes that persist through life
77
State the mechanism for PCR.
- to amplify a single DNA strand into billions of identical copies
1. heat reaction mixture to 90 degrees. This denatures the DNA strands, breaking the H bonds between the complementary nucleotides.
2. temperature is reduced to 60 degrees. DNA primers anneal to their complementary base pairs.
3. temperature raised to 72 degrees. Taq polymerase attaches at the primer and starts to add the free nucleotides to the DNA strand.
4. 2 copies of DNA are now formed.
5. process is repeated many times, more identical strands are produced
78
Describe the stages of DNA 1st generational (Sanger Dideoxy) sequencing
1. 4 test tubes are labelled A, T, C, G. into each tube goes:
- the sample of DNA which is to be sequenced
- DNA primer
- 4 DNA nucleotides (normal)
- DNA polymerase
- for each test tube, the dideoxy nucleotide is added. This is a nucleotide which has no OH on the 3’ end, therefore DNA synthesis is prevented. (tube A which contain dideoxy A etc.)
2. after many cycles, DNA polymerase synthesises many copies of the DNA sample. The lengths are varied as once the dideoxy-nucleotide has been added, DNA synthesis is terminated e.g. in tube A, all DNA sequences will end in A etc.
3. contents of tubes are denatured and are run in an electrophoresis gel. This tells us the length of the DNA strands. Shorter DNA strands travel faster through the gel than the longer ones.
4. dye fluorescence is added – detected by computer programme to reveal the sequence
79
Describe improvements in genome sequencing and what they will allow.
- ongoing improvements in DNA sequencing technology & data mean that individual genome sequencing will eventually be affordable
- cost will be reduced the same as a sophisticated diagnostic test
80
What is precision/personalised medicine?
influence of genetic variation on drug response in patients by correlating gene expression or presence of single-nucleotide polymorphisms (SNPs) with a drug’s efficacy or toxicity
81
Describe the reasoning behind precision/personalised medicine?
- optimises drug therapy in accordance to the individual patient
- to ensure maximum efficacy with minimal side effects
82
Give an example of precision/personalised medicine in drugs.
e.g. drugs/drug combinations and doses are optimised for each individual’s unique genetic makeup
83
Give an example of precision medicine in clinical practice.
- cohort created: 1 million American volunteers that share genetic data, biological samples, diet and lifestyle info all linked
- sequence 100,00 genomes of patients with cancers, rare disorders & infectious diseases
- link data to extensible account of diagnosis, treatment and outcomes
- produce new capability in genomic medicine for transforming the NHS
84
What is a polypeptide?
amino acid monomers linked together by peptide bonds
85
What is a protein?
>40 AAs that can fold into a defined shape
86
State the 4 levels of protein structure.
- primary structure - AA sequence
- secondary structure - interactions betw/ adjacent AAs i.e. α helices, sheets, loops/random coils
- tertiary structure - 3D folding of single polypeptide chain
- quaternary structure - assembly of multiple proteins into complex
87
In what order is an AA sequence taken and what is a DNA sequence determined by?
- AA sequence from N-terminus to C terminus (left to right)
- determined by DNA sequence of gene for protein
- dictates final protein structure (sequential arrangement of R groups influences subsequent 2, 3 & 4 structures)
88
Give an example of a disease caused by a change in the primary structure of a protein.
- Example: Sickle cell disease
• caused by a single mutation in HbA haemoglobin gene
• single mutation in B-globin gene (T to A) changes 10 sequence (Glu to Val)
89
By which bonds is the overall 3-D shape of a protein held by?
1. hydrogen bonds betw/ R-groups
2. ionic bonds - electrostatic attraction betw/ CO2- and NH3+ of R Groups
3. disulphide bridges - betw/ cysteine –SH groups; strongest as they are covalent crosslinks
4. hydrophobic interactions - hydrophobic R-groups cluster
90
How long does it take for 3D confirmation of protein structure?
- attained within seconds
| - small regions of relatively stable secondary structure are formed first
91
Which 2 proteins does tertiary folding results in?
1) fibrous proteins
| 2) globular proteins
92
What are proproteins?
inactive peptides/proteins that need post-translational modifications to activate them
93
Describe the stages of production of insulin as an example of tertiary folding.
- ribosomes feed growing AA chain (preproinsulin) directly into the ER
- signal peptide is present to direct chain to its right location; once it has reached it, it is cleaved off by signal peptidase as this is no longer needed. – producing proinsulin
- oxidation of -SH groups to -S-S (Disulphide bridges) are formed allowing chain to fold – (covalent bonding)
- C-chain is then cleaved & removed to create active protein
94
What can post-translational modifications and what they can involve.
1. processing (proteolytic cleavage to an active form) AND/OR
2. covalent modification (occurs after translation & is a chemical modification)
95
What happens at the start of translation?
- during translation, 20 genetically-encoded AAs synthesised
- PT covalent modifications extend structure & function
- changes in chemical structure change in spatial structure & biological activity
96
Are all PT modifications reversible and how is this used?
- some PT modifications reversible (i.e. acetylation, phosphorylation, methylation) – allowing rapid dynamic regulation of protein activity
- controlling PTM allows control of their activity
- principle widely-used in nature to regulate numerous biological processes i.e. metabolism
- catalysed by enzymes, involved in regulation of target protein’s activity
97
PTMs are key mechanisms in increasing what and how?
- proteomic diversity
- our genomes comprised of 20,000-25,000 genes
- our bodies can produce many more proteins (proteome) than this by changes at transcriptional & mRNA levels
- increases size of transcriptomes (something which is transcribed into genes)
- this increases number of functional proteins in our bodies
98
State and classify all of the post-translational protein modifications (structural changes).
```
• proteolytic cleavage (irreversible):
- removed from N-terminus
- removed from internal part
• proline isomerisation
• addition of small functional groups:
- phosphorylation (reversible)
- methylation (reversible)
- acetylation (reversible)
- hydroxylation
- ubiquitination
• changes in chemical nature of AA
• addition of large functional groups & macromolecules:
- glycosylation
- addition of other peptides/proteins
- addition of fatty acids & lipid residues:
○ C-terminal glycosyl phosphatidyllinositol (GPI) anchor (irreversible)
○ N-terminal myristoylation (irreversible)
○ S-myristomylation (irreversible)
○ S-prenylation (irreversible)
```
99
What is PTM proteolytic cleavage?
occurs at a peptide bond; either at:
- N-terminus
- internal part of protein
100
What is PTM proline isomerisation?
the change in the AA proline residue spatial confirmation; produces cis and trans versions
101
What is PTM addition of small functional groups?
- phosphate is donated by ATP to an acceptor protein
- catalysed by protein kinase
- serine is most commonly phosphorylated AA, followed by threonine
- tyrosine phosphorylation (e.g. 2) leads to binding of specific proteins which is part of a signalling network
(see document for diagrams)
102
What is de-phosphorylation and how is it catalysed?
- removes phosphate group from protein and gives it back to ADP
- catalysed by protein phosphate
103
Explain the example of protein phosphorylation with pyruvate dehydrogenase
1. pyruvate dehydrogenase
- protein kinase is activated by high [NADH] : [NAD+] and [acetlyCoA] : [CoA]
- protein kinase is inhibited by pyruvate, so it can enter krebs cycle
104
How is an example of protein phosphorylation shown in the EGF (growth factor) pathway?
2. EGF (growth factor) pathway
a. EGF binds to receptor
b. receptor changes shape – dimerization as the 2 receptors join together
c. undergo autophosphorylation at C terminus
d. phosphorylation recruits proteins to the receptor
105
How is an example of protein phosphorylation shown in the cell cycle?
- cell cycle is controlled by cyclins and their cyclin dependent kinases (CDKs)
- G1 and S phase (checkpoints)
106
By which 2 methods can phosphorylated proteins bet detected?
- western blotting phosphor-specific antibodies
| - mass spectrometry (2D phosphopeptide)
107
What is protein acetylation and which enzyme is involved in each one?
- acetyl group is donated by acetyl Coenzyme A to an acceptor AA (lysine) in the protein.
- catalysed by Protein Acetyltransferase (PAT).
- deactylation is catalysed by Protein DeACetylase (PDAC).
108
Give examples of protein acetylation.
- Histones (packages of DNA- compress) are targets of protein acetylation
- PAT = histone acetyltransferases (HATs)
- PDAC = histone deacetylases (HDACs)
- non-histone substrates can also undergo acetylation
109
What is reversible histone acetylation important in?
control of gene transcription
110
Describe protein methylation and demethylation by:
a) SAM
b) how methylation is catalysed
c) how demethylation is catalysed
d) whether modifications is reversible
a) methyl group is donated by S-adenosylmethionine to an acceptor protein.
b) catalysed by protein methyltransferase
c) Demethylation is catalysed by protein demethylase
d) not all modifications are reversible
111
How can histones be modified and what does this allow?
by acetylation, phosphorylation, methylation to make them accessible to transcription factors
112
What is histone modification based on and what does it affect?
- the signal which is received from the outside of the cell
| - once these PMT changes are made to the histones, it can affect what protein is made.
113
State 'histone code hypothesis.'
- multiple histone modifications, acting in a combinatorial or sequential manner
- on one or multiple histone N-terminal tails specify unique downstream functions
114
What PTM is changing the chemical mature of an AA (citrullination or deamination)?
converts arginine into citrulline
115
What does the immune system do to citrullinated proteins and what can this cause?
- causes auto-immune response and arthritis disease
| - ligaments betw/ joints are attacked
116
What PTM is the addition of large functional groups & macromolecules?
- i.e. glycosylation – adding mono-/poly- saccharides to a protein (adding sugars)
- protein + sugar = glycoproteins
- significant effect on protein folding, confirmation, distribution, stability & activity
- biological functions – control of protein stability, trafficking, recognition (PM)
117
Give an example of a product of glycosylation.
carbohydrates on cell surface & secreted proteins (modifications are made in the ER and Golgi apparatus)
118
What is asparagine-linked (N-linked) glycosylation?
- 14-unit polysaccharide is added to asparagine AA of a newly synthesised polypeptide in the ER
- different sugars can be added to an asparagine AA
119
What is serine/threonine-linked (O-linked) glycosylation?
- a sugar is added one at a time in the Golgi (secreted proteins) or cytoplasm (cellular proteins).
- usually consist of a few AA residues
- sugar added to hydroxyl-group of serine/threonine
- in some proteins, the polysaccharide can be added to hydroxy-lysine or hydroxyproline
120
How can types of glycosylation be classified into groups?
depending on the nature of the sugar-peptide bond
121
How can proteins be modified by addition of other peptides/proteins?
- mono-/poly- ubiquitination
- attachment of mono-ubiquitin changes protein structure
- attachment of poly-ubiquitin = marks the protein for degradation by a proteasome or lysosome
122
What is ubiquitin?
- a small protein containing 76AA
| - the last glycine in ubiquitin is attached to lysine in proteins
123
Which enzymes are required for ubiquitination?
1. E1 – ubiquitin-activating enzymes (uses ATP to attach ubiquitin onto the substrate)
2. E2- ubiquitin conjugating enzymes (catalyses function of ubiquitin)
3. E3- ubiquitin ligase enzymes
124
State the biological functions of polyubiquitination & proteasome degradation.
- removal of damaged and mis-folded proteins
- control of lifespan of different proteins
- control cellular processes by regulating the availability of regulatory proteins (cell cycle, mitosis, response to DNA damage)
- controls neuronal excitability and synaptic transmission
(see document for diagram0
125
Wahat is addition of fatty acids and lipid residues (lipidation)?
target proteins to membranes in organelles (ER, Golgi apparatus, mitochondria), vesicles (endosomes, lysosomes) and the plasma membranes
126
What are the 4 types of lipidation?
1. C-terminal glycosyl phosphatidylinositol (GPI) anchor
2. N-terminal myristylation
3. S-myristylation
4. S-prenylation
127
What does each type of lipidation modification give?
- proteins w/ distinct membrane affinities by increasing the hydrophobicity of a protein
- allowing it to travel through the PM
128
How many types of lipidation can one protein undergo and what does this allow?
- more than 1 type of lipidation
| - allowing it to travel through multiple membranes.
129
How can defects in PTMs be used in medicine?
- defects in PMT & cell signalling crucial in pathobiology of diseases
- enzymes controlling PMTs often used as therapeutic targets
130
Explain GPCR (G-protein-coupled receptor) regulation as an example of how PTMs can be used in medicine.
1. ligand binds to the GPCR
2. activates enzymes – phosphorylation
3. phosphorylation induces endocytosis – internalisation
4. recycled to bind to a new drug
5. if ubiquitin is added, it follows the lysosomal pathway where it is degraded
(see document for diagram)
131
What does a mutation represent?
represent changes in the base sequence
132
Compare a mutation and a genetic variation as:
a) the % of the population
b) what they arose from
c) how often they occur
d) if the mutation varies among individuals
e) what it changes
f) how often it causes disease
- mutation:
a) <1% of the population
b) arose alone
c) tend to come in one form for that single mutation
d) mutation can also vary among individuals (spectrum of effects)
e) change DNA & protein sequences
f) may occasionally cause disease
- genetic variation:
a) >1% of the population
b) arose from mutation positively-selected during evolution
c) described as DNA polymorphism (many forms)
d) rare variants can occur at <1%, but are distinct from mutations
e) change DNA & protein sequences
f) may occasionally cause disease
133
How do mutations arise?
1. strand breakage; DNA helix strands separate and cell mechanism to fix this does not function correctly so nucleotides are lost before end-joining
2. base loss
3. base change;
4. DNA crosslinking
5. DNA replication error
134
Which health consequences arise when DNA isn’t repaired or is repaired inaccurately?
- cancer susceptibility
- progeria (accelerated ageing)
- neurological defects
- immunodeficiency
135
How do mutations arise?
1. strand breakage; DNA helix strands separate and cell mechanism to fix this does not function correctly so nucleotides are lost before end-joining
2. base loss; a base is lost as glycosidic bond betw/ sugar & base is broken or enzymatically cleaved
3. base change; G oxidised to 8-oxoguanine so base pairs w/ A, C loses amine to become U and base pairs w/ A, thymidine glycol blocks replication
4. DNA crosslinking
by agents which cause DNA to cross-link i.e. UV light into cyclobutane dimers or cis-platin causes adjacent guanine to cross link
5. DNA replication error (not corrected)
136
Which health consequences arise when DNA isn’t repaired/ is repaired inaccurately?
1. cancer susceptibility
2. progeria (accelerated ageing)
3. neurological defects
4. immunodeficiency
137
What is a mutation?
hereditable change in DNA sequence, chromosomes number, form or structure
138
What do changes in DNA sequence arise from?
errors in DNA replication
139
Why must mutation rate be maintained in the middle?
- too low; organisms cannot adapt
| - too high; info cannot be retained
140
Which 2 things can cause genetic variation?
1. mutation
| 2. recombination (crossing over to swap DNA segments during meiosis) – hereditary
141
Classify all types of mutations into 3 groups.
1. point mutation (only affects a single nucleotide):
- substitution
- missense
- nonsense
- insertion
- deletion
2. indels (only affecting a couple nucleotides)
- insertion
- deletion
3. chromosomal mutations
- polyploidy
- aneuploidy
- chromosome rearrangements
142
What is a point mutation?
changes to a single nucleotide (substitution)
143
What is an indel?
an insertion and deletions of a few nucleotides or several kb
144
What are the 3 types of chromosomal mutations and what does each one mean?
- polyploidy - multiple sets of chromosomes
- aneuploidy (abnormal number) - extra or missing chromosome
- chromosome rearrangements - parts moved to other chromosome
145
What are missense mutations and what do they include? Give examples.
- change in nucleotide sequence which results in a change to the AA sequence
- includes point mutations (single nucleotide) and frameshifts (addition & deletion)
- may cause:
• loss of function; e.g. pulmonary hypertension is caused by many mutations
• gain of function; e.g. achondroplasia (restricted growth-dwarfism)
146
What are nonsense mutations and what do they include? Give examples.
- change in nucleotide sequence that results in a premature stop codon
- caused by point mutations & frameshifts
- usually results in non-functional protein
- i.e:
• PAH (pulmonary hypertension)
• duchenne muscular dystrophy; introduce premature stop codon in dystrophin
147
What are indel mutations and what do they include? Give examples.
(insertions & deletions)
- removal of 1-several mill. nucleotides
- 5-10% of all mutants
- 50% of Duchenne muscular
- small indels cause frameshifts which then result in missense/nonsense
148
What is a haplosufficient disease? Give examples of diseases caused by this type of mutation.
- situation in which total level of a gene product (protein) produced by cell is about 1/2 of normal level which is not sufficient to permit normal cell function
- 1 of 2 copies of gene may be missing due to a deletion
- i.e.:
• α-Thalassemias (haplosufficient)
• associated w/ melanoma (haplosufficient)
149
What is an electropherogram and what does it use?
– a plot of DNA sequencing results generated by Sanger sequencing
- uses results obtained by electrophoresis
- used to determine DNA sequence
- uses fluorescent dye; different nucleotides have different colours
- can be used to identify mutations
(i. e. see figure)
150
Identify the mutations shown in the diagram.
see document
151
What is pulmonary arterial hypertension and its histopathological features?
- lumen is open
- mutations cause arteries to become blocked as cell apoptosis is prevented
- cells not dying as apoptosis is not occurring
- instead there is cell proliferation (division/increase lie in tumours)
- cells migrate from other places and block lumen
- therefore, heart cannot pump enough blood around and oxygenate itself
152
What are the effects of mutations in BMPR-II functional domains?
failure of anti-proliferative effects on vascular cells
153
What are simple tandem repeats?
the same part of sequence repeated lots of times one after the other
154
State some expanding trinucleotide repeats found throughout the human genome and the effects they can have.
- CGG, CAG, CTG
- however, during replication these can increase in copy number
- lead to e.g. Huntington’s Disease, Fragile X syndrome, Kennedy disease, myotonic dystrophy
155
What is Huntington’s disease and what is it caused by?
- causes involuntary muscle movements & dementia
- in IT15 gene encoding huntingtin protein, region consists of 6-35 CAG repeats
- 36+ repeats cause neurodegenerative huntington’s disease which encode poly-glutamine region in number of proteins
156
Which issues are raised by Huntington's disease?
- a number of ethical issues around genetic screening
- “What ethical justification can be found for informing a person that he/she will later develop a lethal disease for which no therapy is available?”
157
What are transposons?
sequences of DNA which can move around the gene that are regularly repeated throughout the genome & act as recombination hotspots
158
What are the 3 types of transposon?
1. retrotransposon
- ‘copy & paste’
- occur at an intermediate RNA stage, prior to insertion
2. DNA transposons
- cut and paste the transposable element (TE)
3. Alu repeats
- short interspersed elements
- most abundant mobile element in human genome
- LDL receptor has a large no. of Alu repeats; may be responsible for the large number of pathogenic deletions in 45kb gene
- LDL receptors remove ‘bad cholesterol’ from body; atherosclerosis
159
Which organisms also possess transposons and what does this suggest?
- viruses
| - a distant evolutionary ancestor
160
What is a selective pressure? Give an example.
diseases which have a protective effect
- i.e. Malaria: selective pressure for erythrocytes with sickle cell haemoglobin (Hb S) which causes sickle cell anaemia but provide protection against malaria
161
What do cancers acquire during their evolution and how does this happen?
- distinct characteristics
• tumours are derived from a single ancestral cell
• this requires more mutations to evolve from benign proliferation to malignant
• these mutations are selected in favour as they proliferate more and lead to genome instability (accelerated mutation rate)
162
What is haplionsuffiency?
- when one gene is deleted or inactivated by a mutation
| - so you only have 1 functioning gene which is not enough.
163
Why are loss of function haploinsufficient mutants usually recessive?
- feedback loops that upregulate production of normal gene in heterozygous
- 50% of gene product is sufficient
164
In some haploinsufficient situations why may dosage effect be seen?
- gene product is part of quantitative signalling system
- gene products compete to determine a metabolic/developmental switch
- gene products combine in a fixed stoichiometry (composition)
o e.g. in anaemia, both α and β globin in haemoglobin need to combine. If the α globin chain is affected by mutation = α thalasseamia (same with β)
165
How much of genetic variation is single nucleotides and how many represent structural changes?
- 75%
| - 25%; copy number variation
166
How often do differences between parental genomes occur and where?
- every 1000bp
| - most of them are in non-coding regions (introns)
167
In which 3 ways can genetic changes increase risk/susceptibility to a disease?
1. rare high risk variant and high penetrance
2. rare moderate risk- low penetrance mutation/variation
3. common low risk and low penetrance variant
168
What is penetrance/
how frequently a disease is manifested
169
How much of our DNA encodes for proteins and what do most mutations and some mutations do?
- only 1.2%
- most mutations have a little effect or are silent (don’t change AA sequence) or are in introns
- some mutations are harmful and if the carriers do not reproduce, will gradually be eliminated
- some mutations are beneficial & become prevalent by positive selection
170
Give a brief timeline of the progress in drug-receptor interactions.
- 1900- concept that compounds exhibited biological activity; by binding to “receptive substances”
- 1926- receptor occupancy theory; model to explain “drug action on receptors”
- 1970- development of radioligands; visualisation & quantification of hormone & neurotransmitter bindings sites for drugs in tissues & cells
- 1980 – receptor-cloning; application of molecular biology to isolate receptor genes; expression of mutant receptors to identify drug binding sites
- 1990s onwards – deorphinisation and pharmacogenomics
171
What is deorphinisation?
receptors which functions are unknown
172
What do patients expect from drugs and what problems do they sometimes get from drugs?
- expect drugs which work, are safe and are right for them
| - problems w/ drugs individual variability in efficacy and susceptibility to adverse drug reactions
173
What are Adverse Drug Reactions (ADRs)?
unintended events that occur at drug doses in humans for prophylaxis (treatment given to prevent disease), diagnosis, therapy or modification of physiological functions
174
What is pharmacogenomics (precision medicine)?
influence of genetic variation on drug response in patients by correlating gene expression or presence of SNPs w/ a drug’s efficacy or toxicity
175
In pharmacogenomics, drugs and drug combination doses would be optimised for each individual's unique genetic makeup. What would be the rationale behind this?
- optimisation of drug therapy with respect to an individual patient
- to ensure maximum efficacy & minimal ADRs
176
Is precision medicine hard to achieve?
- no
- DNA microarray device using drop of blood (8hrs turn-around time) – used to identify polymorphisms in cytochrome P450 drug metabolism genes
- CYP450 genes are highly variable betw/ individuals
- variations in genes can affect pharmacokinetics & pharmacodynamics of specific drugs
177
Isn’t pharmacogenomics a bit far-fetched?
- not Really
- ongoing improvements in DNA sequencing technology & data analysis mean that individualised genome sequencing will eventually be affordable
- i.e.:
- human genome project (completed 2003) = $2.7 billion and in 15 years
- genome sequencing in 2017 = $1,000 and in 26 hrs; the cost equivalent of a sophisticated diagnostic test
178
Which 3 things and known and which 3 things are unknown about the human genome project?
Facts
- 2% of human genome contains genes (instructions for making proteins)
- 20,000 genes – half of the functions are unknown
- approx. 99.9% of human genome is the same in everyone
Unknown
- correlation of individual DNA variations with health and disease
- disease-susceptibility prediction based on gene sequence variation
- genes involved in complex traits and multi-gene diseases
179
Explain each predicted benefit and how it may reduce the overall cost of healthcare:
a) advanced screening
b) better drugs
c) design/administration of customised drugs
d) improved dosing regimens
e) improved drug discovery & approval process
a) - early detection of genetic predispositions for disease
- lifestyle changes to minimise risk
b) - molecular info
c) - based on an individuals genetic profile to reduce ADRs & maximise efficacy
d) - drug trials; target specific genetic populations
- reviving failed drugs – why? how can it be fixed
180
Describe variation in the human genome.
- 3mil variable locations in the 0.1% human genome
- 100 new mutations
- variations passed down- hereditary
- it is the variation in the remaining tiny fraction of the genome, 0.1 percent (roughly several million bases) that makes a person unique
- this small amount of variation determines attributes such as how a person looks or the diseases he/she develops
181
Where do most mutations occur and what are silent variations?
- in non-coding regions
| - silent variations – changes that occur in coding & regulatory regions with no known effect
182
Which variations cause harmless changes and what can they influence?
- variations in coding & regulatory regions w/ no harmful effect
- can influence benign (non-harmful) individual characteristics;
• eye colour
• stature (height)
• shape
183
Which variations cause harmful changes? Give examples.
- specific variations in coding & regulatory regions
- alter function/expression of important proteins needed for health
- e.g.
- haemophilia = ‘simple’ disease = variation in 1 gene
- diabetes/heart disease; ‘complex’ = symptoms only seen after many variations in different genes in the same cell
184
What are variations that cause latent changes and what could this explain?
- variations in coding & regulatory regions which aren’t harmful on their own
- changes apparent/higher risk if there is exposure to environmental factors
- this could explain differing drug responses between individuals
185
What are SNPs?
- DNA sequence variations that occur when a single nucleotide (A,T,C,or G) in the genome sequence is altered at same genetic location betw/ different chromosomes (either w/in an individual or betw/ individuals)
- must occur at >=1% of population to be an SNP.
186
What are SNPs responsible for and what effect do they have?
- responsible for 90% of all human genetic variation
- most SNPs don’t have effects on proteins
- can act as biological markers; help identify genes associated w/ disease
187
How can SNPs help with precision medicine?
- diagnose; heart disease, cancers, diabetes
- track inheritance of disease
- latent effects; risk/susceptibility
188
how can SNPs be used to inform drug treatment in precision oncology?
disease treated and medicine
- HER2 & breast cancer; trastuzmab (herceptin)
- oncogenic V6ooE B-Raf melanoma; vemurafenib
- chronic myelogenous leukaemia; imatinib (gleevee)
189
For each SNP location state the drug/treatment for it:
a) CYP2C9 & VKORC1
b) CYP2C19
c) VKORC1
a) warfarin = anti-coagulant
b) clopidogrel = anti-platelet
c) Vitamin K epoxide Reductase complex subunit 1
190
How can the disease enhanced coagulation cause venous thrombosis and how can this be treated?
- e.g. Deep Vein Thrombosis
- intravascular clot formation in deep veins (particularly legs) when blood flow is sluggish
- fragments may bud off (venous thromboembolism) which can block blood vessels such as the pulmonary artery, which can be fatal
- treatment = anti-coagulants
191
How can the disease enhanced coagulation cause arterial thrombosis and how can this be treated?
- e.g. myocardial infarction, ischaemic stroke
- platelets aggregate and then are encapsulated by blood clot formation
- this blocks flow to target tissues
- blockage of the coronary artery causes MI
- blockage of the cerebral artery causes IS
Treatment:
- immediate = fibrinolytic (clot busters)
- long term/prophylaxis = anti-coagulants, anti-platelets
192
What is warfarin and how does it act?
- vitamin K antagonist which acts in several parts of the coagulation pathway
- vita K is a co-factor for the coagulation process
- for it be to work as a co-factor, it must be in its reduced form
- therefore, vit K reductase (VKORC1) does this
193
Who is warfarin prescribed to, what is it prescribed for and what is the main adverse effect?
- most widely prescribed oral anti-coagulant
- administered to patients with ↑ thrombotic tendency
- prescribed to those with:
• atrial fibrillation
• pulmonary embolisms
• heart valverecipients
• (+ DVT)
- administered as a prophylactic (protection/ prevention of disease)
• slow onset of action (days)
• narrow therapeutic range - difficult to maintain patients w/in a defined anti-coagulation range
• main adverse effect = bleeding/haemorrhage
194
What are the dosage issues with warfarin?
- dose too low = ineffective
- dose too high = risk of bleeding (esp. in brain)
- dosing algorithms must consider many complicating factors:
• weight/diet/disease state/other medications/genetic factors
195
What is the variable pharmacokinetics/efficacy due to?
the genetic variability of:
- CYP2C9 = liver enzyme responsible for inactivation
- VitK reductase, VKORC1 (site of warfarin action)
196
Describe the structure of warfarin and its metabolism.
- has a chiral centre at C9
- a racemate; the 2 different isomers act differently
- S isomer is >5 -fold more potent anti-coagulant
- cytochrome P450 (CYP) 2C9:
• converts S-isomer to inactive 6- and 7-hydroxy derivatives
197
The CYP2C9 gene influences polymorphisms. For each allele, state the:
a) AA change
b) mutation
c) effect on enzyme activity
d) effect on warfarin dosing
- CYP2C92:
a) Arg144Cys
b) non-synonymous variation in protein sequence
c) 30% less active
d) 8-16% lower
- CYP2C93:
a) Ile359Leu
b) non-synonymous variation
c) >90% less active
d) 20-36% lower
198
What does the net effect of CYP2C9 gene polymorphisms depend on?
whether variant is of heterozygous (1 copy) or homozygous (2 copy) nature
199
State the timeline of discovery of the link between VKORC1 (enzyme) and warfarin resistance.
- 1974; VKORC1 enzyme identified
- 2004; VKORC1 gene isolated in from genetic mapping of rare heritable recessive bleeding disorder (combined deficiency of Vit K-dependent clotting factors Type 2)
200
How many commonly-occurring VKORC1 SNPs contribute to the variability of the warfarin response/
- >10
| - in coding & noncoding regions
201
What are the functional consequences of warfarin?
- functional consequences unclear despite clear association w/ differing dosing requirements
- Example: 1173C>T polymorphism in gene intron 1
(numbering = nucleotide position from translation start site)
202
In which patients is warfarin dose lower in?
- in patients with TT genotype
| - vs CT/CC genotypes
203
Describe what is shown in the diagram in terms of VKORC1.
- the diagram shows the SNPs which occur in the noncoding regions which is shown by the green section
- they can be given a wide range of dosages
204
CYP2C9 and warfarin dosing
- CYP2C9*3 homozygotes have the highest sensitivity to the drug, therefore need a lower dosage
- those with VKORC1-coding SNP homozygotes have the highest resistance to warfarin, therefore they need a higher dose
205
Organisms with which gene has the:
a) highest sensitivity
b) highest resistance
a) CYP2C9*3 homozygotes
| b) VKORC1-coding region SNP homozygotes
206
What is the commonly-occurring noncoding region is the main genetic contributor to variability of warfarin dosing across the “normal” range?
- VKORC1 SNPs
207
What is the link between ethnicity & warfarin dosing?
- the frequency of SNPs are subject to ethnic variation also
- caucasians have SNPs in cytochrome enzymes >> Asian & African populations
- Asians have SNPs in non-coding regions > Caucasian> Africans
208
What is the future of precision medicine?
- genomics-first approach: drug administration decisions are predicted by genomic analysis of individuals
- genomics → phenotype → lifestyle/environment
209
What was the study undertaken for 'moving precision medicine into clinical practice' and what were the outcomes?
- cohort of >1million American volunteers to share genetic data, biological samples, diet, lifestyle – link to electronic health records; goals:
• short-term goals: expanding precision medicine in cancer research
• sequence 100,000 genomes of patients with cancers, rare diseases, infectious diseases
• link sequence data to standardised account of diagnosis, treatment & outcomes
• produce new capability & capacity in genomic medicine for transforming NHS
210
For each type of point mutation; substitution (missense), nonsense, insertion and deletions state the:
a) disease
b) mutated gene
c) effect of mutation
d) case/examples
```
• substitution (missense);
a) sickle cell anaemia
b) β-globin gene
c) E6V
(glutamic acid (E) replaced by valine (V) at position 6 in protein)
d) N/A
• nonsense
a) duchenne muscular dystrophy
b) dystrophin gene
c) K1524X
(Lysine (K) is replaced by unknown/unspecified codon e.g. stop codon (X))
d) N/A
• insertion
a) Familial Hypercholesterolemia (FH) – elevated levels of blood lipids
b) LDLR
c) c.2416_2417InsG
c. = for cDNA sequence
no._no. = position
Ins = insertion
G = nucleotide
d) from genetic study carried out on large consanguineous (w/ 1 common ancestor) Pakistani family with a history of FH
• deletions
a) cystic fibrosis
b) CTFR
c) ΔF508
(Δ= deletion, F = phenylalanine (F), number = position in protein)
d) info applies to approx. 80% of patients w/ CF in Western Europe
(AUUU codon on chr. 7)
```
*when counting the sequence only exon sequence is counted
211
Give more examples of point mutation disease and the genes involved.
• p. = amino acid sequence
• c. = genetic code (DNA base sequence)
- e.g. C.2292InsA = Insertion of Adenine at 2292 position
• e.g. C.1276-6T>C = At DNA sequence position (exon) 1276, -6 in intron from the 3’ splice site, Thymine is substituted by Cytosine (>); this means mutation has occurred betw/ nucleotides 1275 and 1276.
• *If it was +6 = it would mean substitution would take place -6 in intron from the 5’ splice site. Therefore, betw/ nucleotides 1276 & 1277.
• + means sites positions from the 5’ splice site & - means positions from the 3’ splice site
212
How can a deletion mutation in the intron of the DMD gene cause Duchene Muscular Dystrophy?
- DMD patient found to have large deletion (12,000 BPs) in intronic (non-coding) region
- deletion did not affect classical 5’ splice site, 3’ splice site & branch point; theoretically should not have affected splicing but patient did not produce enough dystrophin protein
- reason unknown; decided to investigate how deletion mutation in intron affects gene expression
- cloned exon 11, some portion of intron 11 & exon 12 → then looked at splicing
- in event of constitutive splicing, exon 11 should splice w/ exon 12 & intron 11 should be removed
- in event of alternative splicing (intron retention) intron 11 is incorporated betw/ exon 11 and exon 12
- found intron 11 was incorporated → led to intro. of pre-mature termination codon dystrophin was not sufficiently produced
213
Identify the symbols of a genetic family pedigree.
```
A - male
B - female
C - sex unstated
D - unaffected
E - affected
F - obligate carrier (won't manifest disease)
G - carrier who may go on to manifest disease
H - deceased
I - mating
J - consanguineous mating; 2 people related as second cousins/closer
K - siblings
L - twins
M - identical twins
N - 4 children; sex unstated
O - spontaneous abortion
P - termination of affected pregnancy
```
214
What is autosomal dominant inheritance according to single gene inheritance? Give an example of this.
- both sexes affected and equally-likely to pass on affected characteristic e.g. PAH
- characteristic is manifested in heterozygote of both sexes
- not linked to a sex chromosome – both sexes equally affected & likely to pass on affected characteristic
- affected individuals ½ chance of passing to offspring
- homozygotes (both parents having disease) are v. rare & have more severe phenotype or show characteristic much earlier (individuals tend to die at young age & do not produce offspring)
- e.g. Achondroplasia – babies die shortly after birth and homozygous for BMPR2 embryonically lethal
215
Analyse the family pedigree of autosomal dominant. inheritance
- blue shows affected individuals- carry the mutation & therefore disease
- roman numerals show generation & normal numbers show amount of people in that generation (used to identify individual) i.e. 2-5; generation 2, person 5
216
What is autosomal recessive inheritance according to single gene inheritance? Give an example of this.
– both sexes affected & parents of affected children in IV are both carriers
- characteristic is NOT manifested in heterozygote of both sexes
- not linked to sex chromosome
- double defect – mutations in both mother & father
- affected individuals are born to unaffected parents (parents are carriers)
- chance of 2x affected parents passing disease onto children
- each parent has one Wild Type (WT = normal) & one mutated allele (Disease allele)
o = ½ x ½ = ¼
217
What is an compound heterozygote? Give an example.
- compound heterozygote; an affected individual w/ 2 different mutant alleles from the same gene
- i.e. like Mm and M’m
218
What is a true heterozygote? Give an example.
- where genes in the affected individual arise from a consanguineous (related) relationship and so the 2 different mutant alleles will be the same
- i.e. like 2x Mm
219
Analyse the family pedigree of autosomal recessive inheritance.
A) shows production of 2 affected individuals via a non-consanguineous relationship. This shows compound heterozygotes as affected individuals have 2 different mutant alleles
B) shows production of 2 affected individuals via a consanguineous relationship. This shows true homozygotes as there has been previously 2 different mutant alleles, but when affected individuals were produced, only 1 mutated allele was inherited
220
What do mutations responsible for recessive traits usually lead to?
- lack of gene expression (promoter regions affected)
- lack of protein production e.g. frameshift mutations which lead to premature termination of translation
- production of protein with reduced/absent function e.g. missense AA substitution
221
What is sex-linked (x-linked) dominant inheritance according to single gene inheritance?
(Hint – most is logic, farther is XY and mother is XX)
- characteristic manifested in heterozygote (one allele) of both sexes
- 1 parent is affected to cause disease
- more affected females than males
- affected females typically show milder characteristics & more variable expression than males
- each child of affected mother has ½ chance of being affected
- each male child of affected father is unaffected (he only receives Y chromosome from father)
- every daughter of an affected father is affected
222
What is sex-linked (x-linked) recessive inheritance according to single gene inheritance? Give examples.
(Hint – HDR)
- characteristic is not manifested in heterozygote of both sexes
- mostly male
- affected males are born to unaffected parents (carriers)
- no male-male inheritance
- sons have ½ chance of being affected
- daughters are not affected, have ½ chance of being carriers
- i.e. red-green colour blindness, duchenne muscular dystrophy, haemophilia A & B
223
What are manifesting heterozygotes?
females through non-random mosaicism can show affects
224
What is non-random mosaicism?
the X-chromosome is inactivated
225
Distinguish the type of sex-linked inheritance in each pedigree.
A - sex-linked recessive
| B - sex-linked dominant
226
Are there more diseases or genes mutated? What does this mean?
- more diseases attributed to mutations in single genes than no. of genes mutated
- we have genetic heterogeneity (= different mutation in the same gene can lead to different diseases)
- i.e. FGFR-3 gene: both are autosomal dominant inheritance:
1. muenke’s syndrome (premature closure of skull bones) in nucleotide P250R
2. achondroplasia (dwarfism) in nucleotide G308R
( 3/5 affected P = Proline, A = Arginine and G = Glycine )
227
What is Achondroplasia?
- mutation in fibroblast growth factor type 3 receptor (FGFR3)
- leads to constitutive activity (= activity of a receptor w/o presence of a ligand) un-regulated by receptor – causes Achondroplasia
- FGFR3 promotes differentiation of cartilage bone, but unregulated so cannot occur
- gain of function mutation constitutively activates FGFR3 receptor causing premature conversion of the growth plate into bone – can no longer grow any further (hence shortened bones)
228
How can multiple genes be affected by one mutation and lead to one disease? Include an example.
- xeroderma pigmentosum (XP)
- hypersensitivity to sunlight & often develop carcinomas (skin cancer)
- mutations in 8 different genes that all function in DNA-damage repair lead to XP (8 variants)/DNA replication
229
For gene types A-G and V state the gene affected and the locus affected.
```
A - XPA - 9q22.3
B - XPB - 2q21
C - XPC - 3p25
D - XPD ERCC6 - 19q13.2-q13.3, 10q11
E - DDB2 - 11p12-p11
F - ERCC4 - 16p13.3-p13.13
G - RAD2 ERCC5 - 13q33
V - POLH - 6p21.1-p12
```
230
State 5 other factors influencing the phenotype of single gene disorders.
1) penetrance – frequency w/ which person manifests (express) gene they possess
- low/medium/high
- not all dominant mutations display 100% penetrance
- penetrance is determined via genetic & environmental factors
2) expressivity – variation in severity of symptoms caused by that mutation (spectrum of genes)
- e.g. sickle cell anaemia (always caused my same mutation) – symptoms range from v. severe to extremely mild
3) phenocopy – an environmental modification that mimics a genetic disease
- e.g. confusing genetic deafness w/ deafness caused by rubella during pregnancy
4) environmental effects – expression of genetic conditions can be influenced by environmental conditions
- e.g. Phenylketonuria; treated via provision of low phenylalanine (in most protein-rich foods) diet
- phenylalanine AA cannot be broken down, which builds up in blood & brain; high levels can damage brain: learning disabilities, behavioural difficulties, epilepsy
- e.g. hypoxia can induce pulmonary arterial hypertension (PAH) & chemical agents i.e. monocrotaline can also induce this
5) other factors
- mutations in BMPR2 gene PAH
- a pathogenic nonsense mutation has been introduced to mice, which develop PAH
- mice exposed to hypoxia develop PAH
- findings of level of BMPR2 expression:
- reduced in mutant mice compared w/ the wild-type mice
- reduced in hypoxic mice compared w/ normoxic mice
- taken together, environmental conditions can develop symptoms similar to those caused by genetic mutations
231
Which type of tests are carried out to identify single gene disorders?
• new-born screening
- uses dried blood spot collected on filter paper from baby of 5 days
- screening for phenylketonuria, CF, congenital hypothyroidism
• diagnostic testing
- used to identify/rule out a specific genetic condition
- in some cases, used to confirm a diagnosis when condition suspected based on physical symptoms
• carrier testing
- major objective of this is to identify careers of mutant allele for severe autosomal recessive disorder
- if it happens both parents are career, can elect to have prenatal diagnosis & termination of pregnancy
• prenatal testing
- carried out during pregnancy/expectant mothers to determine if baby is likely to have specific birth defects (such as Down syndrome)
- needle inserted to extract amniotic fluid containing cells of baby – extract DNA trisomy, Downs & others
• preimplantation genetic diagnosis (profiling of embryos before being implanted)
- test includes removal of 1+ cells from in vitro fertilised embryo to prevent transmission of harmful genetic defects
• predictive & pre-symptomatic testing
- pre-symptomatic – before/without symptoms & may appear later in life e.g. when patient carrying mutant allele will develop symptoms (Huntington disease)
- predictive – if at high risk of developing symptoms, e.g. careers of BRCA1/BRCA2 alleles for developing breast cancer
232
For each environmental cause of disease state the incidence and a description of the disease:
a) single gene mutation in disease
b) multifactorial/complex
c) chromosomal changes
d) environmental factors
a) rare;
- mutations in a gene
- dominant/recessive pedigree patterns; this indicates probability of disease
- structural proteins, enzymes, receptors, transcription factors
b) common; >1 gene affected (gene variants)
c) common; 100’s of genes affected e.g. loss/gain of chromosome (large pieces of DNA)
d) very common; includes viral/bacterial infections, affect disease penetrance – set of changes which will result in change in phenotype (observed characteristics)
233
State the 3 key features of multifactorial inheritance.
- each gene contributes small amount to a final phenotype that is also significantly influenced by environmental factors
- multiple genes not viewed as being dominant/recessive to each other
- phenotype doesn’t segregate, but multiple genes do
234
State the key differences between monogenic and polygenic (complex) diseases.
monogenic:
- single, strong & highly-penetrant phenotype
- very rare
- other genes (modifier genes) affect phenotype severity (e.g. regulate expression of disease allele), but primary gene dominates
polygenic (complex)
- very common
- lower penetrant (genetic changes may not always result in disease)
- not dominated by one gene a genetic locus could have a predominant effect – predisposition increases risk of disease (e.g. BRCA1/2 genes in breast/ovarian cancers)
- environmental contribution to penetrance (risk)
- pedigrees much more complex as multiple genes involved
- some complex diseases have related phenotypes
235
What effect does a condition have one genes it is:
a) fully penetrant
b) low penetrance
c) multifactorial
a) no effect of other genes and environmental factors
b) genes play small part along w/ other genetic & environmental factors in determining a person’s susceptibility to disease
c) genetic factors play a major part in determining susceptibility; each individual factor has a very low penetrance
236
What does the causation of genetic disorders depend on? Include examples.
1) genetics
2) environmental
- the interaction betw/ the 2 is multifactorial:
- eye colour is completely genetic
- car accident chance is completely environmental
- heart disease (like most diseases) is caused by both genetics and environment; genetically-proven as hereditary but susceptibility can increase due to environmental factors such as; smoking, diet, stress
237
State some examples of polygenic complex diseases.
- hypertension
- coronary artery disease
- obesity
- crohn’s disease
- schizophrenia
- autism
- alzheimer’s
- cancer
- asthma
- migraine
- diabetes mellitus
- arthritis
- multiple sclerosis
238
Why study genetics of multifactorial disease?
- identify individuals w/ increased risk of disease
- understanding to help identify targets for better therapy
- untangle complexity of gene-environment interactions
- complex inheritance is the underlying factor for most common human diseases (makes up 60% of the population) due to:
- multifaceted interactions of genotypes at multiple loci
- environmental factors that trigger, accelerate or worsen disease development
239
What is polygenic inheritance?
- traits are quantified by measurement
- >= 2 genes contribute to phenotype
- phenotype variation varies across a wide range
- better analysed in populations than individuals
- interaction of genes w/ environment produces range of phenotypes
240
Give an example of polygenic inheritance.
- e.g. Adult Male Height
- adult male height is governed by quantitative traits at 180 genetic loci
- bell curve shows most individuals are clustered around a median (70 inches)
- few individuals are at extremes of the phenotypes (v. tall, v. small)
241
State 2 methods that can be used study multifactorial traits and label on a bell curve.
- threshold model: frequency of disorder among relatives is compared w/ the frequency of the disorder in the general population
- recurrence risk: estimates the risk that the disease will recur
- see document
242
What is a liability threshold?
measure of the disease state of an individual
243
When does an allele become a 'risk alleles’/susceptibility allele'?
- genes which are associated with increasing the risk of a disease
- push distribution towards the liability threshold they are termed as ‘risk alleles’/susceptibility alleles
244
Not all alleles are additive. What does this mean for some alleles?
they may not lead to disease
245
Which 3 alleles may not lead to disease?
1. dominant alleles
2. epistatic alleles (modifier genes)
3. protective alleles
246
How do we know a complex disease has a genetic component?
- by heritability, defined as number 0-1
- number reflects the extent to which phenotype is influenced by genetics
- 1= high genetic influence and 0= no genetic influence
- high heritability doesn’t mean that phenotype is solely determined by genetics → environment may also have effect
247
Why are twin studies used to study complex disease heritability?
allows us to identify the effects of genetics, shared environment & non-shared environment contributions
248
Compare monozygotic and dizygotic twins.
```
Monozygotic twins:
- single fertilisation (1 sperm)
- share all the same genes
- genetically identical
Dizygotic twins:
- independent fertilisation events (2 sperms fertilising 2 eggs)
- share approx. ½ genes
- non-identical
```
249
Why do we study concordance?
- probability twins will both have a disease given one has the disease
- the difference between % concordance in dizygotic twins vs monozygotic twins estimation of heritability
- by looking at the concordance and discordance between mono- and dizygotic twins possible to estimate the genetic, shared environment + non-shared environment contributions
250
Explain concordance studies of in terms of the example of familial breast cancer.
- familial BC accounts for 5-10% of the populations BC
- known genes (e.g. BRCA1 and 2) involved in familial BC and account for <30% of familial risk
- most contributing genetic factors unknown – may be interactions w/ environmental agents
251
What is a SNP?
- DNA sequence variations that occur when a single nucleotide (ATCG)
- in the genome sequence is altered at same location betw/ different chromosomes (can be w/in 1 individual or betw/ many individuals)
252
Describe an SNP.
- variation must occur in >=1% of population, otherwise invalid
- responsible for 90% of all human genetic variation
- most have no effect on protein function (silent)
253
State some biological uses of SNPs.
- act as biological markers – helps to identify genes associated with disease
- biomedical research
- developing pharmaceutical products
- medical diagnosis (allows us to understand differing susceptibilities to complex but common diseases) – e.g. heart disease, diabetes, cancers
- track inheritance of diseases in families
- can act as markers to map halotypes
254
How can we identifying variants associated with disease using Genome-wide association study (GWAS)?
- Uses sample of 1000+ people
1. take DNA from both patients and non-patients
2. compare SNPs in both DNA to identify particular SNPs associated w/ that disease
3. create Manhattan plot to do the comparisons (see document)
- Manhattan Plot – P (y-axis) is the probability of the association w/ the disease phenotype
- arrow denotes a specific SNP which is strongly associated with the disease (each dot = specific SNP)
255
Describe chromosomal disorders.
- 0.6% of the people w/ this disease are born affected (live born)
- usually de novo (during embryogenesis) – but can be inherited
- thousands of genes may be involved
- multiple organ systems affected at multiple stages in gestation
- involves trisomies, deletions, duplications, chromosome translocations
256
For each sex chromosome aneuploidy (humans normally have 46 chromosomes) state the number of chromosomes, chnage in chromsomes and probability of it occuring.
a) Triple-X
b) Klinefelter
c) XYY
d) Turner
a) 47; XXX; 1/1000
b) 47; XXY; 1/1000
c) 47; XYY; 1/1000
d) 45; XO; 1/2,000-5,000
257
What is the quality of aneuploidy due to genetic imbalance and what does this mean?
- nearly always deleterious (causes harm/damage)
| - ratio of genes are different from normal; difference interferes w/ genome function
258
For each autosomal chromosome syndrome (humans normally have 46 chromosomes) state the triosmy affected and probability of it occurring.
a) down syndrome
b) edward syndrome
c) patau syndrome
a) 21 x 3
b) 18 x 3
c) 13 x 3
259
What property do all other trisomies and monosomies have?
usually fatal
260
Explain the chromosomal deletion DiGeorge Syndrome
- disease occurs during embryogenesis
- deletion of band 11.2 on long arm of chr. 22
- 3 MBs are deleted containing 45 genes
- prevalence 1:4000
- 0.025% risk in the population
261
What are the symptoms of DiGeorge Syndrome?
- congenital cardiac defects
- facial dysmorphia
- thymic aplasia (underdeveloped thymus; affects immune system)
- cleft palate
- hypocalcaemia (low blood [Ca2+])
- learning disabilities
- T-Cell immunodeficiency
262
What is the cause and probability of Down syndrome?
- cause: meiotic non-disjunction (failure of chromatids to separate into 2 chromosomes)
- probability of non-disjunction increases with maternal age
263
Compare mendelian and complex diseases.
see document
