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BIOM30002 - M2M > Lecture 8 - CF: Molecular Basis > Flashcards

Lecture 8 - CF: Molecular Basis Flashcards

1
Q

In general, what does the mutation in CFTR lead to?

A

Abnormal cAMP-mediated regulation of chloride transport

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2
Q

Which gene is mutated?

A

CFTR gene

CF transmembrane conductance regulator

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3
Q

What is the CFTR protein?

A

Chloride channel protein

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4
Q

To which family does CFTR belong?

A

ABC superfamily of membrane transporters

ATP-binding cassette

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5
Q

Describe the energy requirements of the chloride channel

A

ATP required to open and close the channel

Ion movement through the channel is downstream (no ATP)

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6
Q

Describe and draw the structure of the CFTR protein

A

5 domains:
• 2 x MSD’s (MSD 1, MSD 2) which form the channel
• Regulatory domain; R domain
• Nucleotide binding domains: NBD 1, NBD 2

Order:
 • N terminus
 • MSD 1
 • NBD1
 • MSD2
 • NBD 2
 • R domain
 • C terminus
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7
Q

What is the important feature of NBDs?

A

ATP binding → channel opens

NB when ATP is hydrolysed, the channel closes again

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8
Q

What is the function of the R domain?

A

(Regulatory domain)
Phosphorylated by cAMP
Phosphorylation leads to fine tuning of the channel function

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9
Q

Describe the opening and closing of CFTR

A

ATP binds → opening of channel

ATP hydrolysis → closing of channel

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10
Q

What are some secondary roles of the CFTR channel?

A 
Carboxy terminal region:
 • regulation of ENaC (a sodium channel)
 • conductance
 • signal transduction
 • cytoskeleton
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11
Q

Describe the role of CFTR in airways

How is this disrupted in CF?

A

Normally:
• Cl- ions moving out of the cell down a concentration gradient
(Na+ and water moving into cells)

In CF:
• Cl- ions maintained within the cell
• Increased Na+ and water movement into the cell
• Dehydrated mucous

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12
Q

Describe the role of CFTR in sweat ducts

How is this disrupted in CF?

A

Normally:
• Cl- ions moving into the cell down a concentration gradient

In CF:
• blockage of Cl- movement into the cells
• Na+ and water maintained outside the cell

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13
Q

What is the CF Mutation Database?

A

Collection of mutations that cause Cystic Fibrosis of varying and unknown significance

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14
Q

What are mutations of ‘varying clinical consequence’?

A

Mutations that have decreased penetrance

Difficult for clinicians, because the severity of the mutation is often not known

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15
Q

Which type of mutation is most common in CF?

A

Missense (40%)

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16
Q

Which exons are most commonly mutated in CF?

A

Membrane spanning domains: 4, 8

Regulatory domain: 14

20

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17
Q

What are the classes of mutation?

A 
6 classes
 • Class I: no protein production
 • Class II: Defective processing
 • Class III: Defective regulation
 • Class IV: Reduced ion conductance
 • Class V: reduced protein production
 • Class VI: accelerated turnover from the cell surface
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18
Q

What happens in Class I mutation?

A

Premature nonsense mutation
• STOP codon before the end of the protein
• truncated protein produced
• defective protein is unstable and tagged for degradation

There are also mutations whereby the mRNA is not produced properly

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19
Q

What happens in Class II mutation?

A

Gene is transcribed and translated into the ER
However, the protein never makes it out of the ER due to e.g. mutation in folding, targeting to cell surface

→ Degradation of the protein in the ER

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20
Q

What happens in Class III mutation?

A

CLOSED CHANNEL

  • Protein makes it to the cell surface
  • ATP doesn’t bind properly to NBD
  • Channel never opens
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21
Q

What happens in Class IV mutation?

A

REDUCED CONDUCTANCE
• Protein makes it to the cell surface
• Dysfunction of phosphorylation of the R domain
• Channel only opens somewhat, may be partially open / closed

22
Q

What happens in Class V mutation?

A

Protein is produced, but not in the amounts required

23
Q

What happens in Class VI mutation?

A

Protein is functional and makes it to the cell surface, but is removed too soon

24
Q

What is the most common mutation in CF?

Describe it

A 
F508del
 • Class II mutation
 • In NBD 1
 • (Defective processing)
 • Protein missfolds
 • doesn't get out of ER and is targeted for degradation
25
Q

What happens in S1255P?

A
  • Class III mutation
  • In NBD 2
  • Defective regulation

NB don’t need to memorise this

26
Q

What happens in Q1412X?

A
  • Class VI mutation
  • Instability at the cell surface

NB don’t need to memorise this

27
Q

What happens in Splice mutation intron 4 donor site?

A
  • Class I mutation
  • In MSD 1
  • Absent protein

NB don’t need to memorise this

28
Q

Why is important to know the mutations?

A

Therapies can be targeted to the particular mutation for better results

29
Q

Which therapies are used for Class I mutation?

A

Aminoglycoside antibiotics:

• allow ‘read through’ of mRNA despite nonsense mutations

30
Q

Which therapies are used for Class II mutation?

A

‘Correctors’

• improve processing

31
Q

Which therapies are used for Class III mutation?

A

‘Potentiators’

active proteins, keep the channel open

32
Q

Which therapies are used for Class IV mutation?

A

‘Flavanoid’

Augment channel function

33
Q

Which therapies are used for Class V mutation?

A

Increase levels of correctly spliced RNA

34
Q

What is happening in F508del?

A

Phenylalanine n° 508 deletion
• three bases are deleted
• NB only two bases from the F508 codon deleted, and one for Isoleucine

35
Q

How is CFTR mutation tested for?

A 
Earlier:
1. RFLP analysis
S23
2. Multiplex PCR; SNP analysis
3. MALDI-TOF Mass Spectrometry
36
Q

How many mutations are screened for in newborns?

A

12 mutations

More being added all the time

37
Q

Describe the different frequencies of F508del in people of different ethnicities

A

Different ethnicities are affected to greater and lesser degrees by certain mutations.
For example Northern Europeans (esp. Danes) are particularly affected by the F508del mutation

38
Q

How is Mass Spectrometry used for screening?

A

(MALDI-TOF MS)
• Large number of samples can be tested simultaneously
• Looking for a medium n° of SNPs

39
Q

What is an RFLP?

A

Restriction fragment length polymorphism

• When DNA from different individuals is digested with restriction enzymes, one gets fragments with different lengths.

40
Q

Describe RFLP analysis

A
  • PCR + Restriction enzymes
  • Run on an acrylamide gel
  • Analysis of RFLPs
41
Q

Describe Multiplex PCR

A

• Fluorscent tagging to see SNPs

42
Q

Where is CFTR expressed?

A

Apical membrane of epithelium

In a wide range of tissues

43
Q

What is the function of the MSD’s?

A

Form the ion channel through the membrane

44
Q

Draw a rough diagram of the CFTR gene

A 
S 18
 • 27 exons
 • 2 x MSD coding regions
 • 2 x NBD coding regions
 • R domain coding region
45
Q

What is the R117H mutation?

A
  • Class IV mutation
  • In MSD 1
  • Defective conduction through the channel
46
Q 
Describe the difference between class III and IV mutation.
Which mutation is more severe?
A

Class III: ATP doesn’t bind, hence channel doesn’t open

Class IV: dysfunctional phosphorylation of R domain, channel has reduced conductance

Class III mutation is more severe

47
Q

What is the importance of the external projections of the CFTR?

A

These are loops of the MBD that are located on the outside of the cell
These can interact with other, external molecules, altering the function of the CFTR channel

48
Q

Which class of mutation is F508del?

A

Class II

49
Q

How and when was the CFTR gene identified?

A

1989

Identified by positional cloning

50
Q

How does the CFTR regulate ENaC?

A

The TRL (Threonine-Arg-Leu) sequence on the cytoplasmic C-terminus interacts with it

(Inhibition of ENaC)

BIOM30002 - M2M (36 decks)

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