Genetic variation Flashcards
1
Q
What are alleles?
A
- different forms of the same gene
- usually dominant or recessive
- some are phenotypic
- can predispose people to disease
2
Q
What are autosomal dominant alleles?
A
will express itself in the phenotype of a heterozygous individual, eg huntingtons
3
Q
What are autosomal recessive alleles?
A
both parents must be at least heterozygous for condition, is most common if parents are related
eg sicle cell, CF
4
Q
What are X-linked recessive alleles?
A
most often expressed in males, females must be homozygous
eg haemophilia, colour blindness
5
Q
What are single nucleotide polymorphisms?
A
DNA sequence variation of a single nucleotide at same position in genome between members of the same species
6
Q
What 3 things can SNPs cause?
A
- changes in amino acid codon > altered codon function
- affect levels of expression of protein
- affect where/when protein is expressed
7
Q
What is the one requirement to properly classify an SNP?
A
- ## sequence variation must be found into at least 1% of the population
8
Q
What are SSRs?
A
Simple sequence repeat - tandem repeat of 2-8 base sequences
9
Q
What is pharmacogenetics?
A
influence of an individual’s genetic variation on response to medicines
- may be due to difference in expression levels of proteins
- different gene sequences
10
Q
What is pharmacogenomics?
A
- studying drug interactions with multiple genes in large patient groups
11
Q
What is personalised medicine?
A
- individuals screened for commonly occuring SNPs
- SNP profile used to develop patient specific profile for response to specific drugs
- informed drug choices
12
Q
What are the 6 advantages of personalised medicine?
A
- more powerful medicines - targeted, maximising affects
- moving away from one-size-fits-all approach
- better screening for disease
- improvement in drug discovery and approval
- better vaccines
- overall decrease in healthcare costs
13
Q
What are the 4 normal control mechanisms to prevent mutation?
A
- Heterozygosity: two copies to minimise loss of funtion due to mutation
- Apoptosis: damaged cells killed to prevent transmission of mutated genes
- Cell cycle control: check points during cell division to ensure cells are healthy
- regulation of gene transcription: requirement for appropriate activation signals to induce gene expression
14
Q
What are the three risk factors for mutation?
A
- environmental
- chemical (smoke)
- radiation (UV)
- inherited
- mutations in germline
- viral
- rous sarcome virus
- HPV
15
Q
What are 2 the types of sequence mutations?
A
- deletions or insertions
- single base mutations
16
Q
What are inherited mutations?
A
monogenic diseases
- cystic fibrosis
- sickle cell anaemia
- X-SCID
17
Q
What are oncogenes?
A
- mutated forms of normal cellular genes that control cell growth
- usually dominant - only need mutation in one allele to increase function
- oncogenes usually encode growth factor, receptor, signal transducers or nuclear transcription factors
18
Q
What are epidermal growth factors? (EGF)
A
- important proto-oncogenes involved in many cancers
- EGF is important, drives cell proliferation
- intrinsic kinase domain leading to activation of downstream signalling pathways
19
Q
What 4 things can mutations of EGF receptors cause?
A
- ligand dependence
- consititutive dimerisation
- overexpression
- gene amplification
20
Q
What are tumor suppression genes?
A
- exert negative effects on cell growth
21
Q
What is the result of mutations on tumor suppressor genes?
A
- mutations inactivate them and promote aberrant cell growth
- usually autosomal recessive - require loss of both alleles
22
Q
What are translocations?
A
- aberrant cross-overs between chromosome during cell division
- can lead to cells becoming separated from this and instead controlled by another
- can dysregulate gene expression may lead to alterations in cell growth
23
Q
How does lung cancer develop?
A
- mutations commonly caused by environmental carcinogens, particularly those in cigarette smoke
- 60% of lung cancer cases involved p53 mutations
- benzo(a)pyrine in cigarette smoke in metabolised in liver, generating a potent mutagen
24
Q
What is eukaryotic gene regulation?
A
- controlled expression of a gene in a temporal and spatial manner
- important for specialised function
- most common mechanism for gene regulation
25
What are promoters?
- DNA sequence which determines the site of transcription initiation for an RNA polymerase
- composed of set of conserved DNA sequences
- present in DNA upstream of transcriptional start
- recognised by transcription factors and polymerases
26
What is the gene transcriptional process?
- driven by availability/activation of transcription factors
- accessibility of DNA
- chromatin structure - DNA + histone proteins
- requires co-activation proteins to unwind
27
What is histone acetylation?
- histone proteins contains many basic amino acids with +ve change
- enables interaction between with -ve phosphate backbone of DNA
- acetylation blocks ability to bind DNA by masking +ve change
- relaxes interaction of DNA with nucleosome
- uses histone acetyle transferases (HAT) and histone deacetylase (HDAC)
28
What is chromatin?
- heterochromatin
- densely packed nucleosomes
- not actively transcribed, deacetylated histones
- euchromatin
- extended 'beads on a string' appearance
- being actively transcribed, acetylated histone
29
How common is CF?
1 in 25 are carriers
1 in 25,000 live births are affected
30
What is the most common cause of death in CF sufferers?
Lung dysfunction and infection
31
What is affected in CF sufferers?
Impaired function of organs with secretory function
32
How is the pancreas affected in CF?
- lack of pancreatic enzyme secretion
- reduced insulin
33
How is the digestive system effected by CF?
- meconium ileus + obstruction
- failure to thrive in newborns
34
What are the 3 ways you screen for CF?
- immunoreactive trypsinogen (IRT) - guthrie test
- all newborns in uk
- trypsinogen made by pancreas and secreted into gut
- elevated in blood/
- genetic screening
- IRT positive + siblings are then screened for mutations
- sweat test
- elevated skin Cl- levels
- below 30mM CF not likely
- above 60mM CF likely
35
How is nutrition mananged in CF?
- formerly the most common cause of death in CF
- nearly all patients have pancreatic insufficiency
- lack of pancreatic protease, amylase and + lipase
- unable to digest food properly
- managed by diet + enzyme supplement
- pancreatin
- lipase, protease, amylase
- usually porcine
- inactivayed by capsules
- enteric coating
36
How are the lungs affected by CF?
- chronic infection
- mucus plugging
- structural changes
- massive neutrophil infiltration in airways
- failure to clear bacteria and dying neutrophils
- inflammatory to airway tissue
- decline in lung function
- epithelial damage
37
What causes sticky mucus in CF?
- low water volume
- difficult to clear by cilia
- provides growth environment for bacteria
- partial correction with inhaled hypertonic saline
38
How are antibiotics used in CF?
- prophylactic floxacillin in infants
- reguilar swab or sputum culture
- oral oral/iv abx intervention
- inhaled abx useful
39
What are the 5 established treatments of CF?
- abx against pathogens
- inhaled bramycin
- b-adrenoceptor agonists
- activation of residual CFTR activity
- anti-inflammatories
- osmotic agents
- hypertonic saline, mannitol
- mucolytics
- recombinant DNAase
- breaks down very viscous mucus
- CF mucus contains large amounts of DNA from inflammatory cells
- N-acetyl cysteine
40
What are the 4 CFTR modulators?
- supressors - force through stop codon
- correctors - overcome folding defects
- potentiators - modulate gating characteristics
- amplifiers - enhance amount of function protein
41
What are the 4 anti-inflammatory therapies for CF?
- Glucocorticoids
- inhaled no proof of reduction
- IV prednisolone some benefit but significant adverse
- Macrolides
- non-antibiotic effect of azithromycin
- High dose ibuprofen
- Brensocatib
- inhibits neutrophil elastase
- phase ii clinical trials
42
What are the 2 ways gene therapy works for CF?
- mRNA therapy
- adding extra protein making templates
- gene editing
- repairing and correcting defective sections
43
What is NFkB?
Nuclear Factor of Kappa (light chain) in B cells (5 family members)
Ubiquitous Transcription Factor
Generally associated with the activation of inflammatory genes
All contain conserved sequence known as Rel domain which regulates dimerization and nuclear localisation
Inhibited by IkB which binds across the Rel domain
44
What is AP-1?
Activator Protein -1
Regulates immediate early response genes (~20min from signal to transcription)
Regulates a number of cellular functions including cell growth
Comprises a dimer of the Fos and Jun families
Requires transcription of fos family member and phosphorylation of jun family member
45
What are the functions of AP-1?
- Regulates many genes involved in cell growth and differentiation
- Role in bone development
- Implicated in inflammation
- May have a role in long term memory
46
What is cellular proliferation?
Cells are signalled to proliferate through the binding of growth factors to receptors
Activation of intracellular signalling pathways leads to gene transcription
Gene transcription results in the expression of proteins required to drive the cell
through the cell cycle
Process is tightly controlled to prevent aberrant proliferation
47
What are the two families of proteins involved in cell cycle control?
- cyclins
- cyclin-dependent kinases
48
What are the 5 ways that the cell cycle is inhibited?
- CDK inhibitors
- INK4 inhibits CDK4/6
- CIP/Kip - inhibit all CDKs
- transcriptionally regulated
- bind cyclin/cdk complexes
49
What is the MoA of glucocorticoids?
Glucocorticoids enter cells and bind to cytoplasmic receptors.
Complex translocates to the nucleus to act as transcription factor
Can bind to response elements and activate anti inflammatory gene transcription
Can bind and repress pro-inflammatory gene activation
Can interact and inhibit binding of other transcription factors
50
What are the anti-inflammatory affects of steroids?
Inhibit both early and late stages of inflammatory response
Decrease vasodilation and extravasation
Inhibit cell activation- TH (inhibit IL-2 and clonal expansion)
Decrease production of inflammatory mediators (ROS, prostanoids and leukotrienes, complement and histamine)
Endogenously prevent ‘overshoot’ of immune response
51
How can steroids be toxic?
Generally associated with high doses, systemic long-term admin
Immunosuppression can increase risk of infections
Impaired leucocyte traffic can delay wound healing
Suppress HPA axis through feedback inhibition
52
What is chronic inflammation?
- a manifestation of the body's response to tissue damage and infection
- main purpose is to bring cells from blood to site of infection
- level of response must match injury in severity + duration
53
What is the role of the immune system in chronic inflammation?
- largely mediated by cytokines
- cells are activated to produce cytokines on exposure to antigen
- the efficacy of the immune response affects the ability of the body to eliminate antigen
- prolonged activation of the immune system due to failure to eradicate can lead to chronic inflammation
54
What happens when the skin barrier is destroyed?
Allergens- largely bypass innate causing direct activation of B cells- IgE
Bacteria- activation of innate system then adaptive
55
What is eczema?
Inflammation and intense irritation
In the UK, one in five children and one in twelve adults have eczema while eczema and contact dermatitis account for 84-90% of occupational skin disease.
Symptoms tend to be caused by dry skin which fails to retain moisture
In eczema- reduction of fats and oils in skin leaves gaps- more water loss, cells become flaccid, more gaps for entry of bacteria and allergens
Irritants contribute to the breakdown of skin (surfactants in washing up liquid)
56
What is psoriasis?
An autoimmune condition resulting in increased production of skin cells.
Skin cells are normally made and replaced every 3-4 weeks, but in psoriasis this process only lasts about 3-7 days. The resulting build-up of skin cells creates the patches associated with psoriasis.
Symptoms start or become worse because of a "trigger". Possible triggers of psoriasis include an injury to your skin, throat infections and using certain medicines.
57
How is chronic inflammation linked to autoimmunity?
Endogenous antimicrobial peptides such as LL37 are produced as the body’s own antibiotics, protecting injured skin, providing an important defence against harmful bacteria
LL37 is overexpressed in psoriatic skin, and is involved in the activation of dendritic cells, acting as an autoantigen.
LL37 triggers an immune cell response in some psoriasis sufferers, and the immune cells triggered by LL37 produce key cytokines, such as IL-17
58
How is genetic mutation linked to psoriasis?
Mutations on a gene, called IL36RN, have been linked to pustular psoriasis.
This gene helps regulate inflammation by suppressing inflammatory cytokines such as interleukin-1
59
What are the first line treatments for chronic inflammatory conditions?
Moisturisers and emollients to restore skin barrier function and hydration
60
What are the second line treatments for chronic inflammatory conditions?
Topical corticosteroids/ hydrocortisone(glucocorticoids) – induce anti-inflammatory gene transcription
Steroids act as transcription factors to alter gene transcription
Vitamin D is absorbed from diet or made in skin from sunlight - metabolised to active form (calcitriol) by liver and kidney, vitamin D deficiency can lead to chronic inflammatory skin conditions and cancer, vitamin D and its analogues are effective treatment options for psoriasis and other skin diseases.
61
What are the third line treatments for chronic inflammatory conditions?
Biologicals – adalimumab (anti TNF: reduce circulating pro-inflammatory TNF)
62
What are anti-TNF agents and how can they work in psoriasis?
Anti-TNF monoclonal antibody treatments or recombinant receptors can be used to treat moderate to severe psoriasis as TNF play a role in inflammation in immune system diseases
However, anti-TNF therapy can increase development of psoriasis
Mechanism not fully established but may be due to imbalance of cytokines
(TNF inhibits IFNa, high levels of which can induce psoriasis)
63
What is gene silencing?
To determine the role of a gene you can knockout or knockdown expression of a gene
64
What are the 3 approaches to gene silencing?
antisense (knockdown)
short interference RNAs (knockdown)
CRISPR (knockout)
65
What are the 3 rules for antisense?
- Single stranded DNA of 15 – 30 nucleotides in length
- Strand that is complementary (antisense) to the target mRNA
- Use predictive RNA structure programmes to target areas of mRNA that are single stranded and accessible
66
How is antisense applied?
- Difficult to identify effective antisense sequences and therefore siRNAs is approach of choice when targeting mRNAs in cell models
- Commonly employed for non-coding RNAs such as miRNAs (short sequences) and long non-coding RNAs (located in nucleus) that are less amenable to siRNA mediated RNA interference
- Often employed in animal models and as therapeutics due to advantageous pharmacokinetic properties (stability and size)
67
What is CRISPR?
- A bacterial defence mechanism against virus infection
- Following infection, the viral is cleaved by Cas complex to produce short spacer regions that are inserted into the CRISPR region of the bacterial genome (between repeats).
- CRISPR region is transcribed (primary transcript) and tracrRNA (via repeats).
- tracrRNA binds to the primary transcript and these are cleaved to produce crRNA. Each crRNA recognises a specific virus.
- crRNA guides the Cas9 protein to complementary virus DNA. The Cas9 then cleaves viral DNA and destroys the virus.
68
What are the disadvantages of using antisense, siRNAs and gRNAs for mRNA knockdown?
- Delivery
- May induce immune response
- Maybe no correlation between mRNA and protein levels
- How much protein knockdown is required to see phenotypic effect
- How stable is the protein i.e. how long does it take mRNA knockdown to result in a reduction in protein
69
What are the advantages of using antisense, siRNAs and CRISPR
- Only require knowledge of the mRNA or DNA sequence
- C an get potent knockdown of mRNA (although this will not always results in changes at protein level)
- Cheap and easy to identify effective antisense, siRNA and gRNA sequences
70
What are stem cells?
Unspecialised cells that can self-renew and that can be turned into many other cell types
71
What are the 3 major types of stem cells?
- Adult (somatic) stem cells
- Embryonic stem cells (ESC)
- Induced pluripotent cells (iPC)
72
What are totipotent cells?
Can form all tissues required for an organism including extra-embryonic tissue such as placenta e.g. fertilized egg
73
What are pluripotent cells?
Can form all cells comprising an adult organism e.g. embryonic stem cells
74
What are multipotent cells?
can form a limited number of cell types e.g. most adult stem cells
75
What are unipotent cells?
can only form one type of cell e.g. committed progenitor cells
76
What is Self-renewal?
When a stem cell divides, at least one of its daughter cells is also a stem cell
77
What are the 5 characteristics of Adult stem cells?
1. Can proliferate but this will not continue indefinitely
2. Can only differentiate into a limited number of cells
3. Located in organs and tissues in very small numbers in areas called ‘stem cell niches’
4. Important in organ and tissue homeostasis
Vast majority of body cells are terminally differentiated
Replace worn out or lost cells
Repair damaged tissue
5. Difficult to identify and culture
Demonstrate ability to produce specific cell types
Demonstrate ability to re-populate organ or tissue following re-injection
78
How can stem cells be used for bone marrow transplants?
First experiments conducted to treat patients exposed to radiation from nuclear accidents/explosions – abnormal production of blood cells since bone marrow has been damaged
Pioneered by E. Donnall Thomas at the Fred Hutchinson Cancer Research Center between 1950 – 1970’s
Now used extensively to treat patients whose bone marrow has been destroyed during the treatment for myelomas and lymphomas (obtained from bone marrow or peripheral blood)
Major problem is graft-versus-host rejection (HLA) which is reason that autologous (self) transplants are preferred against allogenic (non-self)
79
How can stem cells be used to treat MS?
Multiple Sclerosis (MS) is a disease that affects the brain and spinal cord.
Autoimmune disease that causes damage to the myelin sheaf that surrounds and protects nerve cells
Begins with relapsing-remitting MS that can eventually proceed to primary/secondary progressive
Effect 2.3 million people worldwide
Treatment with autologous haematopoietic stem cell transplant (AHSCT)
MIST trial (110 people) for highly active relapsing treated with autologous haematopietic stem cell transplantation (AHSCT) and compared with disease modifying drugs (DMDs) (March 2018)
After 12 months – 1 relapse with AHSCT group compared 39 with DMD group - AHSCT had 1.1 EDSS improvement whilst DMD group had -0.6 EDSS
At 3 years treatment failure was 6% (3 of 52) for AHSCT compared with 60% (30 of 50) for DMD Autoimmune disease that causes damage to the myelin sheaf that surrounds and protects nerve cells
80
How can stem cells be used to treat age-related macular degeneration?
Age-related macular degeneration (AMD) leads to loss of cell on the retina that are involved in central vision
Common is older people (> 65 years) leading to partial blindness. ~ 700,000 people have some form of AMD in the UK
Transplanted stem cells into 2 patients who recovered eye sight (Nat Biotechnology 36, 328-337 (2018) (Pete Coffrey – UCL)
81
What are the characteristics of embryonic stem cells?
Continue to proliferate (self-renewal) almost indefinitely (years)
Can be differentiated into all > 200 cell types (pluripotent)
Form tumours when injected into immuno-compromised mice (immune system has been destroyed)
82
What is somatic nuclear transfer?
First successful cloning approach (Dolly the Sheep)
Remove nucleus from egg, inject in nucleus from somatic cell and expose to electrical shock
Need to produce cocktail of chemicals that drive the egg to divide past 16 cell stage (includes caffeine)
Research showed good success rate (> 10%) but still need large numbers of good quality human eggs (paid donors!!!)
83
What is one of the ways that somatic cell nuclear transfer can be used?
Three person IVF for mother with mitochondrial defects
Small number of women have changes in mitochondrial DNA that cause severe muscle degenerative disease
Fertilise defective egg (mother) with father’s sperm (IVF)
Remove nucleus from donor egg (that contains normal mitochondrial DNA)
Inject in fertilised nucleus derived from egg of mother
84
What are the characteristics of induced pluripotent cells?
1. No ethical issue since derived from adult cells
2. Can proliferate (self-renew) almost indefinitely
3. Can be differentiated into > 200 cell types (pluripotent)
4. Chances of graft-versus-host rejection is reduced since these are derived from individuals to be treated
5. May develop into cancers
85
What is an animal model?
Animals that can be used to model either physiological or pathological (disease) response in order to understand what occurs in humans
Animal models allow researchers to investigate disease states that are inaccessible in human patients
Animal models allow researchers to development new drugs and therapeutic approaches
86
What are transgenic organisms?
Organisms harbouring exogenous (extra) genetic material (the transgene) in their genomes
Transgene transmitted in a stable mendelian fashion to offspring
Can create transgenic plants, mice, cattle, flies, frogs, fish, swine….
Mice are species of choice
- lower cost
- small size
- rapid reproduction rates
- genetically well characterised
87
Why are trangenic organisms generated?
To test gene function - typically gain of function
e.g. action of oncogenes > tumour formation
To develop animal models of human disease
e.g. role of cytokines in inflammatory conditions, IL-13
To tag (often fluorescent) and track genes of interest
For the large scale production of therapeutic proteins
e.g. antibodies in bovine milk
Agricultural purposes
e.g. pesticide resistance (tomatoes, soya beans...)
88
What are the advantages of embryonic stem cells?
can be grown for many generations
Transgenes can be inserted into a specific site (i.e. using CRISPR and/or homologous recombination)
Presence of transgene can be detected using qRT-PCR
Transgenic ES cells can be rapidly produced and stored
Can obtain pre-made transgenic ES cell from Sanger Institute (UK) and Jackson Labs (USA
89
What are tissue specific promoters?
Include promoter that is driven by a transcription factors that is ONLY expressed in specific cell/tissue
90
What are inducible promoters?
Cis-acting elements that allow timing and sometimes location and level of expression of a transgene to be controlled
Most common is Tetracycline-Controlled Transcriptional Activation (TET)
