Genome variation 2

Question 1

Give an example oof a genetic variant that can be advantageous

Answer 1

The CCR5 protein is a co-receptor used by the HIV virus to infect its target cells
* The mutation in CCR5 prevents the HIV virus from entering its target cells, thus providing resistance to HIV infection.
* The CCR5 Δ32 mutation is common in the human population: ~10%
* The CCR5 Δ32 may have conferred protection against the pathogen
causing the bubonic plague (Yersinia pestis).

Question 2

How many human genes go through alternative splicing?

Answer 2

Genome wide studies estimated that 90–95% of human genes undergo some level of alternative splicing

Question 3

Do all alternative transcripts result in a protein?

Answer 3

Not all alternative transcripts result in the production of a protein and if a protein is produced it can be non-functional !

Question 4

Alternative pre mRNA Splicing

Answer 4

Ø Alternative splicing is an ubiquitous regulatory mechanism of gene expression that allows generation of more than one unique mRNA species from a single gene
Ø Alternative splicing can generate mRNAs that differ in their coding sequence
Ø Mechanisms leading to alternative splicing include: exon skipping, intron retention or the use of alternative splice sites
Ø The different transcripts may differs in terms of mRNA stability, localization or translation (different protein sequences)

Question 5

What does alternative splicing contribute to?

Answer 5

§ species diversity
§ cell differentiation
§ tissue identity and organ development § disease

Question 6

Different mRNA products (isoforms) from the same gene are often selectively expressed and translated in:

Answer 6

§ different cell and/or tissues
§ during different stages of embryogenesis § in different metabolic conditions

Question 7

What is splicing regulated by?

Answer 7

Ø The acceptor and donor splice site “strenght”
Ø Regulatory splicing elements on the pre mRNA, e.g. enhancers and silencers (exonic and intronic)
Ø Additional splicing elements, e.g.:
§ serine and arginine-rich (SR) proteins, which promote the recognition of alternative exons and their inclusion in the mRNA
§ heterogeneous nuclear ribonucleoproteins (hnRNPs), which promote the exclusion of the exon from the mRNA

Question 8

How does alternative splicing shape the life of neuronal cells?

Answer 8

Alternative splicing helps to shape the life of neuronal cells
Alternative splicing is a fundamental process in neuronal cell life and guides:
* The initial cell differentiation into a neuronal cell
* Neuronal cell migration in post natal life
* Neuronal cell maturation into a mature neurone, which has an axon and dendrites and is able to make synaptic connections and signal

Question 9

Do mutations affect alternative splicing?

Answer 9

Ø 15–50% of human mutations causing monogenic diseases, are estimated to affect pre mRNA splicing
Ø Splicing mutations can occur within exons and introns

Question 10

Where can the mutations that alter alternative splicing be located?

Answer 10

§ The donor and/or acceptor splice sites (5ʹ and 3ʹ)
§ The branch point
§ The polypyrimidine tract
§ The regulatory elements that modulate spliceosome recruitment, e.g. exonic splicing enhancer (ESE), exonic splicing silencer (ESS), intronic splicing enhancer (ISE) and intronic
splicing silencer (ISS) elements
Gene
§ Additional proteins that coordinate splicing

Question 11

Pre mRNA splicing and disease

Answer 11

• Deep intronic mutations can modify pre-mRNA splicing by causing the activation of a pseudoexon
• The activation of a pseudoexon can result from the creation of de-novo splice sites, or the strengthening of existing “weak” splice sites
• The resulting transcripts can be:
  – subjected to premature degradation, e.g. the new nucleotide sequence introduced in the mRNA results in an amino acid frameshift and a premature stop codon. These aberrant transcripts are likely degraded through a process known as nonsense- mediated decay
  – produce a protein with a modified function, e.g. the mutant mRNA results in the in- frame inclusion of a new amino acid sequence in the protein. This mutant protein may be non-functional or may undergo misfolding

Question 12

Splicing defects and neurological disorders

Answer 12

• Neurodegenerative diseases are often caused by loss of function of neurons
• One of the main mechanisms leading to neurodegenerative diseases is the
  accumulation of misfolded proteins that aggregate
• Neurodegenerative disorders result from the interplay of genetic and enviromental factors
• Splicing mutations are often the cause of neurodegenerative disorders and have been identified in patients with Parkinson’s disease, Alzheimer’s disease and spinal muscular atrophy (reviewed in Dunhui et al. Translational Neurodegeneration 2021; 10,16)

Question 13

In silico predictions of splice variants

Answer 13

• There are several established algorithms to predict the effect of genetic variants on pre mRNA splicing
• The VEP (Variant Effect predictor) website at EBI has the option to predict splice variants using established tools
• The GeneSplicer and SpliceRegion algorithms are available from VEP but only from the locally installed version of VEP

Question 14

Splicing defects and disease – case 1

Answer 14

Case presentation:
* Two unrelated children with Familial Glucocorticoid Deficiency (FGD, OMIM identifier MIM: 202200)
* FGD results in low blood levels of a hormone called cortisol (also known as stress hormone), which has a fundamental role in metabolism and immune response
* Affected individuals lack the hormone cortisol and, if they are not identified and treated, are likely to die because of hypoglycemia (low blood sugar) or infections in early childhood

The DNA was extracted from from the blood cells of the children
The exons and flanking introns of the MRAP gene were amplified and sequenced
Several genetic variants at the splice site of MRAP exon 3 wereidentified

Question 15

What is pharmacogenetics?

Answer 15

• Pharmacogenomics is the study of how genetic factors affect the interindividual variability to drug response
• The goal of pharmacogenomics is to use information on a patient’s genetic background to predict the response to certain drugs
• This can lead to optimization of drug treatment and minimization of side effect

Question 16

Pharmacogenomics in clinical practice

Answer 16

• Azathioprine inhibits DNA synthesis (immunosuppressor)
• It is used for the treatment of blood cancers, such as leukaemia and of
  inflammatory bowel disease
• Azathioprine is a prodrug and therefore needs to be converted into its active form 6-MP
• 6-MP is then inactivated by an enzyme called TPMT
• Genetic variants in TPMT that result in decreased or absent TPMT
  protein, are present in 5%–10% of the general population
• Patients with a non-functioning TPMT protein are, therefore, at increased risk of 6-MP toxicity.
• TPMT testing is recommended to identify patients at risk of azathioprine toxicity

Question 17

Cystic fibrosis and genetic variants in the CFTR gene

Answer 17

• An example of genetic variants causing disease are those occurring in the CFTR gene (Cystic Fibrosis Transmembrane Conductance Regulator)
• The CFTR protein is an ion channel and mediates the transport of chloride ions and water across the cell membrane
• Mutations in the CFTR gene that disrupt the function of the CFTR protein cause cystic fibrosis, an inherited conditions, which causes progressive damage to the respiratory and digestive system

Question 18

Classes of CFTR mutations

Answer 18

Mutations that result in a CFTR protein that does not reach the cell surface due to
* impaired protein translation in the cell nucleus (Class I) or
* misfolded protein in the Golgi apparatus (Class II)
Mutations that result in a CFTR protein that does reach the cell surface but its function is impaired due to
* a gating defect (Class III) or
* decrease conductivity (Class IV)
* overall reduced production of normal CFTR (Class V)

Question 19

Cystic fibrosis and Ivacaftor

Answer 19

• Ivacaftor is a drug that acts as a “potentiator of the CFTR protein”
• It increases the probability the CFTR chloride channel will open, thus restoring ion flux
  across the membrane
• It is only effective if the CFTR protein is produced and can reach the cell surface
• It can be prescribed to patients with genetic defects in the CFTR gene that lead to a CFTR protein normally expressed and able to reach the cell surface (for example class III and IV defects: G551D, R117H)

Question 20

What does LDL do to you?

Answer 20

High levels of LDL-cholesterol in the blood markedly increase cardiovascular risk
Drugs that can reduce the production or increase the degradation of LDL-cholesterol are used to reduce cardiovascular risk

Question 21

What is PCSK9 protein and what does it do?

Answer 21

• PCSK9 is a glycoprotein that is mainly expressed in the liver
• Overexpression of PCSK9 in mice results in a marked
  reduction in LDL receptors
• These mice have increased LDL cholesterol levels in the bloods
• Mice lacking Pcsk9 have increased levels of hepatic LDL receptors, they can remove LDL-cholesterol at a fast rate and have low LDL cholesterol levels

The authors compared the incidence of heart attacks over a 15-year interval according to the presence or absence of genetic variants in the PCSK9 gene that are associated with reduced plasma levels of LDL cholesterol
They studied two populations and showed that individuals with nonsense mutations in PCSK9 had a significant reduction in mean LDL cholesterol as well as a significant reduction in the risk of heart attacks (p=0.003)
The authors concluded that: “The results of the current study, make PCSK9 an attractive new target for LDL-lowering therapy.”