Download Cystic fibrosis: molecular genetics and pathophysiology - PBL-J-2015

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An Interesting Proposal
Cystic fibrosis: molecular genetics and pathophysiology
Classify the common mutations which are found in CF. Explain how different mutations result in
differing severity of clinical disease, according to whether cAMP stimulated chloride secretion is
reduced or absent.
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Abnormality of CFTR (cystic fibrosis transmembrane conductance regulator), gene dysfunctional or absent.
CFTR functions as a chloride channel and is ATP activated.
CFTR gene located on the long arm of chromosome 7.
1 in 25 Caucasians carriers, incidence is 1 in 2500 CF babies.
There are ≈1000 different mutations of the CFTR gene, the phenotypes vary from normal to severe.
Most common mutation is ΔF508- indicating missing amino acid (3 nucleotides) to be phenylalanine in
position 508 (NB: delta= Δ=deletion).
 Mutations most commonly occur at the nuclear binding domain.
 A ΔF508 deletion produces a mutant CFTR protein which cannot be folded into its proper shape and is
therefore dysfunctional and is degraded.
 Autosomal recessive inheritance.
The clinical manifestation of CF is a result of either absent or defective Cl- channels at the apical epithelial
surface of the cells in the lungs, sweat glands, pancreas and reproductive tract. There are over 1000
mutations of the CFTR and depending on the mutation (ie channel function/expression) will determine the
severity, varying from asymptomatic to a severe form which may be fatal. The CFTR Cl- channel is unusual
and requires both ATP and cAMP dependent phosphorylation in order to open. In normal individuals,
increases in concentration of the intracellular second messenger cAMP causes  Cl- transport, but not in CF
patients.
CLASSES OF CF GENE MUTATIONS
CFTR production
CFTR functionality
Examples
Severity
Complete
lack
of
CFTR.
Nil.
No
Cl
transporters
G542X
?
most
severe?
CLASS
Truncated mRNA due to stop
at the apical cell
(X represents
mutations.
membrane
stop codon)
Nil. No Cl transporters
ΔF508
Clinically most
CLASS CFTR fails to mature, thus
degraded by proteases in ER
at the apical cell
(70% of CF
severe form of CF
membrane
individuals)
Correctly
processed
CFTR
but
Cl
transporters
are
G551D (3% of CF) Less severe than
CLASS
defective regulation due to
present but poor
ΔF508
abnormal protein substitution. opening/closing
regulation.
Cl- movement
R117H (2% of CF) Mild phenotype
CLASS Mature CFTR, protein
normally activated.
diminished.
I
II
III
IV
CLASS Mature CFTR at apical
V
epithelial surface, normal,
decreased abundance.
Defect in intron splicing
or promoter->
decreased amount of
full length mRNAs.
(Very rare these
mutations make
up <1% of CF)
Mild phenotype.
May go
unnoticed.
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An Interesting Proposal
Outline the current hypothesis for the prevalence of the CF gene.
The Genetic Advantage Hypothesis: It is suggested that CF heterozygotes (ie carrier) withstand
secretory diarrhoea better than normal individuals and so are genetically advantaged. The hypothesis is
logical since the CF gene codes for the epithelial chloride channel, the CFTR which is necessary for
intestinal fluid secretion. It is postulated that CF heterozygote individuals have an improved survival
following infections such as typhoid and cholera, with less chloride loss with diarrhoea.
NB: This was not mentioned in the lecture, this is information from an old paper (1995) plus Gordos
notes!
Describe the cellular and molecular basis of strategies for gene therapy.
Gene therapy has been explored as a potential cure for CF. Ideally, gene therapy attempts to place a normal
copy of the CFTR gene into affected cells. Gene therapy uses a virus to insert the missing genes for CFTR into
affected lungs. A virus is found which replicates by inserting its genes into the host cell’s genome. The virus is
modified in order to transfer the functioning gene into the host cell, without causing disease.
There are two forms of gene therapy:
 Germ-line gene therapy involves gene manipulation of all the individuals cells, or a subset of the
germ cells. Therefore the manipulated genes with the functioning CFTR will pass on to future
generations.
 Somatic-cell gene therapy involves treating the cells in the individual. (Except the gametes, which
are corrected either due to an absent or malfunctioning gene at the cellular level).
 Ex-vivo: involves removing cells from the individual, altering the DNA and re-inserting
into the patient.
 In-situ: requires the vector to be placed directly into the affected cells.
 In-vivo: involves injecting the vector (the altered virus for example) into the bloodstream,
the vector must find its’ way to the affected region.
There are a number of ways genes can be introduced into human cells. For the CFTR, research has targeted
liposomes (non-viral) and viral vectors as their transport vehicles.
The non-viral liposomes have contained an inner genetic material coated with an outer lipid layer. Liposomes
are not at risk of any infection to the host, however have shown to be of low efficiency, targeting limited
tissues, with transient expression of the CFTR.
Viral vectors transfer the altered DNA via a non-pathogenic virus to the affected epithelial cells. The first CF
gene therapy research focused on adenoviruses, where the modified virus injected the gene into tissues->
protein produced. There have also been adeno-associated viruses, which focus on injecting the DNAcontaining parovirus together with the adenovirus (co-infected). These have shown to elicit a mild immune
response, but not cause disease. The adeno-associated viruses have shown a preference to inserting into
chromosome 19. Research has found that the adenoviruses cannot easily enter into the epithelial cells of the
lungs. Research has also explored the use of retroviruses, however has found the results to be non-specific
and have inserted elsewhere other than the ‘target’ tissue. Due to its’ lack of specificity, the retrovirus has
been associated with the adverse effect of leukaemia.
The current problems with gene therapy have been associated with persistent expression of the ‘good
genes’ and a mode of administration. Research of viral vectors is continuing and current studies are now
focusing on aerosol delivery, in the hope that a greater epithelial cell uptake will be observed. Studies have
shown that even 15% restoration of CFTR function could prevent CF and thus there is great hope for future
CF sufferers.