Other Genomes In Our Body

Question 1

What is a meta genome

Answer 1

It is the genetic material in the environment

Question 2

What is a microbiota

Answer 2

It’s the community of organisms

Question 3

What is a micro biome

Answer 3

It is the collective genomes of microorganisms in an environment as well as their proteins and metabolites

Question 4

What are the two metagenomic approaches

Answer 4

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

Question 5

Why 16S rRNA PCR

Answer 5

16S = component of the 30S prokaryotic ribosome

Largely conserved with variable regions different between genus and species

The conserved regions allow primer design

Question 6

Describe the use of the 16S variable regions

Answer 6

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

Question 7

What are the drawbacks of 16S targeted PCR amplification

Answer 7

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

Question 8

How can you mitigate contamination in 16S Targeted PCR amplification

Answer 8

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

Question 9

What is whole genome SHOTGUN sequencing

Answer 9

Sample > extract > sequence WITHOUT PCR > analyse

You can either reassemble genomes or simply bin reads into specifies to find out what the species are

Question 10

What are the pros and cons of whole genome shotgun sequencing

Answer 10

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)

Question 11

How can you overcome whole genome shotgun limitations

Answer 11

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

Question 12

Compare 16S targeted PCR amplification and whole genome shotgun sequencing

Answer 12

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

Question 13

What is dysbiosis

Answer 13

This is when the microbiota changes which could be due to new eating habits or antibiotics

Question 14

Why is the microbiome important

Answer 14

It is integral to host digestion

Question 15

What is autochthonous and allochthonous bacteria

Answer 15

Autochthonous = resident
Allochthonous = passenger

Question 16

What are the benefits of a diverse microbiome

Answer 16

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

Question 17

What can the microbiome tell you

Answer 17

It can identify if someone is lean or obese with 90% accuracy

Associated with IBD, depression, cancer etc. and allergic reactions e.g. asthma

Question 18

Describe the role of the microbiome in IBS/IBD

Answer 18

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

Question 19

How is the microbiome changed in colorectal cancer

Answer 19

Higher proportion of pseudomonas, heliobacter, acinetobacter

Less beneficial bacteria such as butyrate producing bacteria and bifidobacterium

Question 20

What are some interventions to correct the gut microbiota

Answer 20

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

Question 21

How does clostridium difficile infection affect the miccrobiome

Answer 21

The microbiome is very different and has a greater effect than host genetics

Individuals are most susceptible if the microbiota diversity is low

Question 22

What bacteria does FMT increase/decrease after clostridium difficile infection

Answer 22

Increases firmicutes and bacteroides

Decreases proteobacteria

Question 23

Describe the role of dysbiosis in psychiatric disorders

Answer 23

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.

Question 24

What is bacterial vaginosis

Answer 24

BV = increased discharge

Often asymptotic with risk of STI and can cause preterm birth

Question 25

How is the microbiome changed in Bacterial Vaginosis

Answer 25

BV = overgrowth of anaerobic bacteria, gardnerella vaginalis Healthy = lactobacillus

Question 26

What kind of environment is the skin and how does this relate to its microbiome

Answer 26

It is a nutrient poor acidic environment where the microbiota remain stable overtime Sebaceous sites - propionibacterium Moist - staph and cornyebacterium

Question 27

What diseases are associated with skin microbiome dysbiosis

Answer 27

Cutaneous disease such as acne, a topic dermatitis, psoriasis

Question 28

What is the importance of the oral microbiome

Answer 28

It’s contained oral homoeostasis and protects the oral cavity and prevents disease development It can be affected by smoking, alcohol, spicy food, antibiotics

Question 29

What diseases are associated with oral microbiome dysbiosis

Answer 29

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

Question 30

What is a rare disease

Answer 30

With disease that affects less than 1 in 2000 individuals

Question 31

List systems where mitochondrial gene mutations may have a large impact

Answer 31

Brain, heart, muscle, guts It can be multisystem or isolated It can start at any age and have a wide disease spectrum

Question 32

List how drugs can influence mitochondrial disease - in nuclear and mitochondrial DNA

Answer 32

Epilepsy treatment valproate can cause fatal hepatopathy in POLG disease (nuclear gene) Mitochondrial DNA mutations predisposing to aminoglycosides induced deafness (mitochondrial DNA)

Question 33

Describe sodium valproate (VPA) and POLG VPA toxicity

Answer 33

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

Question 34

What is aminoglycoside induced ototoxicity

Answer 34

A1555G predisposed to aminoglycoside induced ototoxicity It is the most common genetic predisposition to aminoglycosides induced deafness (33-59%)

Question 35

What are aminoglycosides and give some examples of them

Answer 35

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

Question 36

What are some examples of side-effects of aminoglycosides

Answer 36

Side-effects include ototoxicity and nephrotoxicity These occur due to similarity of bacterial and mitochondria ribosomes as these drugs can bind each other

Question 37

Describe the mechanism of action of chloramphenicol/macrolides and lincosamides

Answer 37

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

Question 38

Describe the antibiotic mechanism of action of tetracyclines

Answer 38

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

Question 39

Describe the mechanism of action of aminoglycosides

Answer 39

They bind to the 30S ribosomal subunit They impair proofreading resulting in production of faulty protein which misfold and aggregate which is toxic

Question 40

Are there any cure for mitochondrial diseases

Answer 40

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

Question 41

What symptoms or secondary consequence of generic therapies for mitochondrial disorders target

Answer 41

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)

Question 42

Why are some mitochondrial disease clinical trials not successful

Answer 42

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

Question 43

What targeted therapies for genetic diseases could be considered for mitochondrial disease

Answer 43

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

Question 44

List some mitochondrial mutation specific therapies

Answer 44

Treating premature stop codons (10% inherited) Therapy for splicing mutations - antisense oligonucleotide therapy Therapies for missense mutations causing protein misfolding - pharmacological chaperone

Question 45

What are TALENS and Zinc finger nucleases

Answer 45

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

Question 46

Describe why premature stop codons are bad, and the therapies for premature stop codon treatment

Answer 46

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

Question 47

Describe therapies for splicing mutations

Answer 47

Antisense oligonucleotide therapy targets splicing It has been approved in Duchenne’s muscular dystrophy and spinal muscular atrophy

Question 48

Describe therapies for missense mutations causing protein misfolding

Answer 48

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

Question 49

What does the mutation in MRPL44 cause

Answer 49

It causes protein misfolding and infantile onset cardiomyopathy