122 A Case Study: Cystic Fibrosis

Key Idea: Cystic fibrosis results from a triplet deletion from the CFTR gene, causing a non-functioning protein that is unable to regulate chloride ion transport.

` Cystic fibrosis (CF) is an inherited disorder caused by a mutation of the CFTR gene. It is one of the most common lethal autosomal recessive conditions affecting people of European descent (4% of people are carriers). ` The CFTR gene's protein product, the cystic fibrosis transmembrane conductance regulator, is a membrane-based protein that regulates chloride transport in cells. Over 1900 mutations of the CFTR gene have been reported, causing disease symptoms of varying severity.

The D(delta)F508 mutation is particularly common and accounts for more than 70% of all defective CFTR genes. This mutation leads to an abnormal CFTR, which cannot take its proper position in the membrane (below) nor perform its transport function.

CF patient receiving physiotherapy treatment

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The CFTR gene on chromosome 7

The DF508 mutation of the CFTR gene on chromosome 7 is a deletion of three bases spanning the 507/508th triplets. The net effect is the loss of a single amino acid from the gene’s CFTR protein which normally regulates chloride transport in cell membranes. The mutant protein fails to do this. The template strand of the DNA containing the mutation is shown below:

CFTR gene

DNA template strand Base 1630

This triplet codes for the 500th amino acid These three bases are deleted from the 507/508th triplets in the ΔF508 mutation

CFTR protein

The DF508 mutant form of CFTR fails to take up its position in the membrane. Its absence results in defective chloride transport and leads to the cell absorbing more water. This causes mucus-secreting glands, particularly in the lungs and pancreas, to become fibrous and produce abnormally thick mucus. CFTR is widespread throughout the body explaining why CF is a multisystem condition affecting many organs.

Normal CFTR (1480 amino acids)

Correctly controls chloride ion balance in the cell Cell exterior

Abnormal CFTR (1479 amino acids)

No or little control of chloride ion balance in the cell Mucus build up

CFTR protein Chloride ions effectively removed from the cell.

Cell interior

Cl-

Water

The DF508 mutation causes the CFTR protein to degrade rapidly, stopping it from inserting into the plasma membrane. Water

Cl-

Chloride ions build up inside the cell.

(b) Use the mRNA table on page 154 to identify the amino acids encoded by the mRNA for the normal DNA strand:

2. (a) Rewrite the mRNA sequence for the mutated DNA strand:

(b) Use the mRNA table on page 154 to identify the amino acids encoded by the mRNA for the mutant DNA strand:

(c) What amino acid did the 507th triplet encode in the normal strand and in the mutant strand:

(d) What was the net effect of the three base deletion?

3. (a) Explain why the abnormal CFTR fails to transport Cl- correctly:

(b) What effect does this have on water movement in and out of the cell?

4. There are over 1900 different CFTR mutations described so far. One mutation is the W1282X mutation. This occurs at base pair 3846. The sequences below show the correct DNA sequence for the coding strand (upper line) and the mutated DNA sequence (lower line).

Normal DNA: ACT TTG CAA CAG TGG AGG AAA GCC TTT GGA

Mutated DNA: ACT TTG CAA CAG TAG AGG AAA GCC TTT GGA

(a) Circle the mutation in the mutated DNA sequence:

(b) What general type of mutation is this?

(c) Write out the mRNA sequence for the normal DNA:

(d) Write out the mRNA sequence for the mutated DNA:

(e) Use page 154 to work out the amino acid sequence of the normal DNA:

(f) Use page 154 to work out the amino acid sequence of the mutated DNA:

(g) Explain why the mutated DNA leads to a non-functional CFTR protein:

(h) Recall your answer to (b). You can now give a more specific answer to the question: What type of mutation is this?

Give reasons why this kind of mutation can produce non-functional proteins: