7. Future of genomic medicine

Question 1

What is Monogenic disease caused by?

Answer 1

Single gene mutation
or Many mutations in 1 gene
Penetration not affected by environment

Question 2

What is Polygenic disease caused by?

Answer 2

Many mutations in many genes

Environment has strong effect

Question 3

Principles of DNA test

Answer 3

DNA isolation from sample
PCR (amplification of selected DNA region + digestion, if needed)
Visualisation on an agarose gel

Question 4

Cystic fibrosis

Answer 4

Monogenic autosomal recessive disorder

Caused by CFTR mutations

Question 5

What is the consequence of a CFTR mutation?

Answer 5

Affect chloride ion channels, leading to excessive mucous production in airways and pancreas
Effects breathing and secretion of pancreatic enzymes

Question 6

What is the most common mutation in cystic fibrosis?

Answer 6

Delta F508

Deletion of 3 nucleotides at position 507-508 on CFTR gene

Question 7

How can we test for delta F508 mutations?

Answer 7

Design primers either side of the delta F508 site for PCR
Normal length of DNA produced will be 63 bp
Because of the deletion, any chromosomes with delta F508 will be 3 bp shorter (60bp)

Question 8

How can several single nucleotide changes can causing CF be identified?

Answer 8

With PCR
Restriction Fragment Length Polymorphism(RFLP) digestion
Restriction endonucleases recognize specific short DNA sequences

Question 9

What can we use if the mutation is unknown to us?

Answer 9

Sanger sequencing

Question 10

Whole genome next generation sequencing

Answer 10

Using NGS to sequence the entire genome

Very expensive

Question 11

Targeted capture next generation sequencing

Answer 11

Using NGS on a single region/gene
Cheaper
Sensible for mutations in a known region
Exome is just 1% of whole genome but contains 85% of disease causing mutations

Question 12

Genome panels

Answer 12

Contain gene regions associated with a disease/ phenotype
Focus on genes most likely to be involved
Minimises data analysis

Question 13

Epigenome

Answer 13

Identify methylation of DNA which can explain the basis of monogenic disease

Question 14

Transcriptome

Answer 14

convert mRNA to cDNA to sequence only transcribed proteins

Question 15

Precision medicine

Answer 15

Use of genomics to tailor medical care to individuals based on their genetic makeup

Question 16

What is the 100,000 genome project?

Answer 16

sequencing 50K cancer + 50K rare disease patients to identify causal genetic mutations
To improve diagnostics and therapeutics

Question 17

Biomarkers

Answer 17

Identify presence/ severity of disease/ risk

e.g. miscarriage

Question 18

How can NGS be used for rapid diagnostics in infectious diseases?

Answer 18

Sequence bacterial 16S rRNA - to identify bacterial species

Can sequence whole viral sequence

Question 19

Nanopore technology

Answer 19

e.g. MinION
Very small sequencing machine
Used for small genomes e.g. viruses: Ebola

Question 20

What are the 2 methods of gene therapeutics?

Answer 20

Direct delivery

Cell-based delivery

Question 21

Describe direct delivery of gene therapeutics

Answer 21

Correct/ missing gene added to a viral vector

Directly injected into patient to incorportate into genome

Question 22

Describe cell-based delivery of gene therapeutics

Answer 22

patients stem cells removed, cultured and gene introduced using retrovirus
Stem cells that incorporate gene are returned to patient

Question 23

CAR T cells

Answer 23

Chimeric antigen receptors are added to patients T cells, modifies them so they recognise and bind to cancer cells
Leads to destruction of tumour cells
CAReceptors are specific to tumour antigens
Very effective in blood cancers
A living drug, can stay in the body for life

Question 24

What dangerous side effect of CAR T cells could arise and how is this combatted?

Answer 24

Cytokine release syndrome (sCRS)

Combat with modification of T cells by CRISPR/Cas9

Question 25

CRISPR/Cas9 enzyme

Answer 25

Can be guided by sgRNA to complementary DNA sequences

Here can insert genes, cause epigenetic modification or introduce single/ double strand DNA breaks

Question 26

List 6 challenges of gene therapy

Answer 26

Delivering the gene to correct place and switching it on
Avoiding immune response
Making sure the new gene doesn’t disrupt the function of other genes, off target effects
Size of delivered constructs
Expensive
Ethics

Question 27

Read through oglionucleotides

Answer 27

Treat disease caused by premature stop codons

Question 28

Nonsense suppression therapy (NMD)

Answer 28

If premature stop codons are introduced into mRNA transcripts they are degraded in NMD
If NMD inhibitors are administered protein production is increased

Question 29

mRNA therapeutics

Answer 29

possible to insert mRNA into cytoplasm for immediate translation but rapidly degraded and causes immune response

Question 30

miRNA therapeutics

Answer 30

injection of miRNA to interact with DICER and target cancer mRNA can be used for silencing cancer mRNA

Question 31

What does personalised medicine allow?

Answer 31

Precision diagnostics
Tailored therapeutics
Genetic risk assessment