Other Genomes In Our Body Flashcards
What is a meta genome
It is the genetic material in the environment
What is a microbiota
It’s the community of organisms
What is a micro biome
It is the collective genomes of microorganisms in an environment as well as their proteins and metabolites
What are the two metagenomic approaches
Targeted PCR amplification
This targets a single gene marker to identify variations between species e.g. 16s rRNA or 18s rRNA
Whole genome SHOTGUN sequencing
Why 16S rRNA PCR
16S = component of the 30S prokaryotic ribosome
Largely conserved with variable regions different between genus and species
The conserved regions allow primer design
Describe the use of the 16S variable regions
9 variable regions, combinations include:
V1-2, V1-3, V3-4, V4-5, V1-9
You choose the combination according to the regions that are different enough to differentiate species common in the tissue/environment of investigation
Smaller regions = better as NGS short reads are more likely to overlap (2*300bp)
Long read can be used but these have high error rates
What are the drawbacks of 16S targeted PCR amplification
It is sensitive to contamination as it can pick up bacteria on the environment, operator, reagent. This effect is larger when biomass is smaller
Biased to bacteria
It is not reliable below genus, and requires suitable choice of variable region
How can you mitigate contamination in 16S Targeted PCR amplification
Randomise samples - test controls and cases at same time
Note batch number of reagents - use same batch
Sequence negative controls - this effectively sequences any contamination on the reagents or tools
What is whole genome SHOTGUN sequencing
Sample > extract > sequence WITHOUT PCR > analyse
You can either reassemble genomes or simply bin reads into specifies to find out what the species are
What are the pros and cons of whole genome shotgun sequencing
There is no bias to a single organism
However host cells are often in excess and can overpower (depends on tissue e.g. faecal = <10%, while skin = >90% human cells)
How can you overcome whole genome shotgun limitations
Pre extraction = differential lysis of mammalian cells
This is biased to gram +ve bacteria as they have thick cell walls that can resist lysis
Post extraction = enzymatic degredation of methylated DNA (mammalian methylation pattern is different)
This is biased to AT rich bacterial genomes, with GC rich being weakest
Compare 16S targeted PCR amplification and whole genome shotgun sequencing
16S = taxonomic diversity BUT only bacteria and PCR introduces bias
WGSS = taxonomic diversity and gene function determined, and unbiased to species UNLESS enriched. Has high throughput for low cost
What is dysbiosis
This is when the microbiota changes which could be due to new eating habits or antibiotics
Why is the microbiome important
It is integral to host digestion
What is autochthonous and allochthonous bacteria
Autochthonous = resident Allochthonous = passenger
What are the benefits of a diverse microbiome
Increased energy and nutrient extraction via provision of unique enzymes e.g. to digest fibre
Alteration of appetitive signals
The competition of good V bad pathogen is protective to host
What can the microbiome tell you
It can identify if someone is lean or obese with 90% accuracy
Associated with IBD, depression, cancer etc. and allergic reactions e.g. asthma
Describe the role of the microbiome in IBS/IBD
Altering the microbiota through dietary changes, probiotics or antibiotics can be beneficial
The gut brain access can alter symptom perception in the brain
Whether IBS does not have a single cause so some patients benefit but it can be used as a diagnostic tool
IBD is inflammation in the gut - with environmental and genetic risk factors
How is the microbiome changed in colorectal cancer
Higher proportion of pseudomonas, heliobacter, acinetobacter
Less beneficial bacteria such as butyrate producing bacteria and bifidobacterium
What are some interventions to correct the gut microbiota
Prebiotic - consumption of undigestable food to increase beneficial bacterial growth
Probiotic - increased beneficial bacteria, FMT is a probiotic
Symbiotic - mix of the above
HOWEVER THERE IS NOT MUCH EVIDENCE
Lactobacillus and bifidobacteria is the most common beneficial bacteria
How does clostridium difficile infection affect the miccrobiome
The microbiome is very different and has a greater effect than host genetics
Individuals are most susceptible if the microbiota diversity is low
What bacteria does FMT increase/decrease after clostridium difficile infection
Increases firmicutes and bacteroides
Decreases proteobacteria
Describe the role of dysbiosis in psychiatric disorders
It increases translocation of gut bacteria into lymphoid tissue, provoking immune response, and activation of the vagus nerve and spinal afferent neurons
It is associated with autism, depression etc.
What is bacterial vaginosis
BV = increased discharge
Often asymptotic with risk of STI and can cause preterm birth
How is the microbiome changed in Bacterial Vaginosis
BV = overgrowth of anaerobic bacteria, gardnerella vaginalis
Healthy = lactobacillus
What kind of environment is the skin and how does this relate to its microbiome
It is a nutrient poor acidic environment where the microbiota remain stable overtime
Sebaceous sites - propionibacterium
Moist - staph and cornyebacterium
What diseases are associated with skin microbiome dysbiosis
Cutaneous disease such as acne, a topic dermatitis, psoriasis
What is the importance of the oral microbiome
It’s contained oral homoeostasis and protects the oral cavity and prevents disease development
It can be affected by smoking, alcohol, spicy food, antibiotics
What diseases are associated with oral microbiome dysbiosis
Because associated with dental caries, periodontal diseases and systemic diseases as well as cancer
Systemic diseases include cardiovascular disease, pneumonia, rheumatoid arthritis
Cancers include pancreatic, colorectal, oesophageal cancer
What is a rare disease
With disease that affects less than 1 in 2000 individuals
List systems where mitochondrial gene mutations may have a large impact
Brain, heart, muscle, guts
It can be multisystem or isolated
It can start at any age and have a wide disease spectrum
List how drugs can influence mitochondrial disease - in nuclear and mitochondrial DNA
Epilepsy treatment valproate can cause fatal hepatopathy in POLG disease (nuclear gene)
Mitochondrial DNA mutations predisposing to aminoglycosides induced deafness (mitochondrial DNA)
Describe sodium valproate (VPA) and POLG VPA toxicity
VPA is used to treat epilepsy as a first-line therapy but is also used for migraines, headaches, and bipolar disorder
POLG mutations predispose to VPA toxicity
Rare mutations in POLG, which codes for the mitochondrial DNA polymerase γ (polγ), cause the Alpers-Huttenlocher syndrome (AHS)
Polymerase gamma is the mitochondrial DNA polymerase that is most commonly mutated in mitochondrial disease
Backlight toxicity can induce fatal liver failure
What is aminoglycoside induced ototoxicity
A1555G predisposed to aminoglycoside induced ototoxicity
It is the most common genetic predisposition to aminoglycosides induced deafness (33-59%)
What are aminoglycosides and give some examples of them
These are antibiotics including streptomycin, kanamycin, neomycin
These are used in the treatment of gram-negative sepsis
They are bactericidal, targeting the bacterial ribosome, where they bind to the A-site and disrupt protein synthesis
What are some examples of side-effects of aminoglycosides
Side-effects include ototoxicity and nephrotoxicity
These occur due to similarity of bacterial and mitochondria ribosomes as these drugs can bind each other
Describe the mechanism of action of chloramphenicol/macrolides and lincosamides
These bind the 50S ribosome subunit
They prevent peptide bond formation and stop protein synthesis
It is important to note that resistance can rise when there are mutations in the rRNA
Describe the antibiotic mechanism of action of tetracyclines
These bind to 30S ribosomal subunit
They blocked the binding of tRNA thus inhibit protein synthesis
It is important to note that mutations in the 30S ribosomal subunit may cause resistance
Describe the mechanism of action of aminoglycosides
They bind to the 30S ribosomal subunit
They impair proofreading resulting in production of faulty protein which misfold and aggregate which is toxic
Are there any cure for mitochondrial diseases
No treatments are largely supportive and only treat the symptoms
As they can cause toxicity in patients with specific gene mutation there are no approved therapies yet
What symptoms or secondary consequence of generic therapies for mitochondrial disorders target
They can target oxidative stress and eliminate reactive oxygen species
There may be drugs that increase the number of mitochondria who overcome problems (mitochondrial biogenesis)
Why are some mitochondrial disease clinical trials not successful
This is because there are few patients with the same mutations in the same gene
Heterogeneous phenotypes that fluctuate over time
They may be poor trial design and inappropriate endpoints
What targeted therapies for genetic diseases could be considered for mitochondrial disease
Enzyme replacement therapy
Dietary therapy for inborn errors of metabolism
Stem cell therapy
Gene therapy
You cannot just insert new gene
CRISPR guide RNA cannot enter mitochondria
Therefore TALENS or zinc finger nucleases are used which target disease mtDNA so healthy mtDNA replicate
Mitochondrial replacement therapy a.k.a. three parent babies
Mutation specific therapy
List some mitochondrial mutation specific therapies
Treating premature stop codons (10% inherited)
Therapy for splicing mutations - antisense oligonucleotide therapy
Therapies for missense mutations causing protein misfolding - pharmacological chaperone
What are TALENS and Zinc finger nucleases
Zinc finger and TALENs are
Proteins with a DNA cutting region and a DNA grabbing region
It can be programmed to recognise different genes
TALEN is easier to design than zinc finger
Describe why premature stop codons are bad, and the therapies for premature stop codon treatment
Normally the ribosome proceeds to the stop codon before dissociating and releasing the protein
Premature stop codon = early release = smaller protein and might result in nonsense mediated decay
The therapy allows the ribosome to bypass premature termination cover and continue to synthesise a full length protein
These drugs are not specific for any particular protein so it can be effective for many stop codon mutations
Describe therapies for splicing mutations
Antisense oligonucleotide therapy targets splicing
It has been approved in Duchenne’s muscular dystrophy and spinal muscular atrophy
Describe therapies for missense mutations causing protein misfolding
Pharmacological chaperone find specific protein helping it fold and retains its activity
It is currently used in lysosomal storage disease and cystic fibrosis
Mutation is MRPL44 courses protein this folding an infantile onset of cardiomyopathy
What does the mutation in MRPL44 cause
It causes protein misfolding and infantile onset cardiomyopathy
