Describe the normal function of CFTR protein

It acts as a chloride ion channel in the csm of mucus producing cells to allow Cl to move out of
these cells into the mucus lining. It has a binding site for ATP that is hydrolyzed to give energy
for opening the channel.

How does CF cause thicker and stickier mucus in the tubes?

The faulty CFTR protein causes chloride ions to accumulate in the mucus producing cells.
Sodium ion channels remain open and allow Na ions to diffuse from the mucus into the mucus
producing cells.
Na and Cl ions in the mucus producing cells create osmotic gradient causing water from the
mucus to move into the mucus producing cells by osmosis leaving behind thicker and stickier
mucus.

Gene mutation affects the structure and function of protein such as CFTR protein and enzymes. Gene
therapy can be used to treat a genetic disorder such as CF.

(a) Describe how a normal gene determines the structure and function of a
protein
The gene is transcribed into mRNA in the nucleus that is
translated on the ribosomes into amino acids in the primary
structure.
The R groups of the amino acids in the primary structure
determine the type of chemical bonds e.g. hydrogen which in
turn determine the structure/shape of the protein
The shape determines the function
(b)Describe how gene mutation affects the structure of a protein
Gene mutation is change in base sequence of the gene that
gives rise to a different gene
This gene is transcribed into different mRNA in the nucleus that
is in turn translated on ribosomes into different amino acids in
the primary structure
These amino acids have different R groups that determine
different types of chemical bonds e.g. hydrogen giving rise to
different shape of protein
(c) Describe how gene mutation affects the function of a protein
Gene mutation is change in base sequence of the gene that
gives rise to a different gene
This gene is transcribed into different mRNA in the nucleus that
is in turn translated on ribosomes into different amino acids in
the primary structure
These amino acids have different R groups that determine
different types of chemical bonds e.g. hydrogen bonds giving rise
to different shape of protein
This shape gives a different function or it has no function at all.
 (d)Describe how gene mutation affects the structure and property of
enzymes
Gene mutation is change in base sequence of the gene that
gives rise to a different gene
This gene is transcribed into different mRNA in the nucleus that
is in turn translated on ribosomes into different amino acids in
the primary structure
These amino acids have different R groups that determine
different types of chemical bonds e.g. hydrogen bonds giving rise
to different shape and property of the active site
The substrate can no longer bind to the active site to form
enzyme-substrate complexes
(e) Describe how gene therapy can be used to treat a genetic disorder, CF
Restriction enzyme cuts out the normal allele/gene from the DNA
The same restriction enzyme cuts open a plasmid/virus
The allele is introduced into the plasmid/virus using enzyme
ligase that catalyses the formation of phosphodiester bonds
between the allele and the plasmid/virus, forming recombinant
DNA (rDNA)
The rDNA molecules are inserted into the liposomes, forming
liposome-rDNA complexes
If the target cells are in the lungs, the complexes are takes as
aerosol/nasal spray using nebuliser to the target cells. If the
target cells are the digestive and reproductive cells, the
complexes are injected into the blood.
Inside the mucus producing cells, the allele is
expressed(Transcription and Translation) to form a normal CTFR
channel protein.
This protein allows chloride ions to leave the cells and move into
the mucus
Water leaves the cell by osmosis and enters mucus causing the
mucus to become less sticky and thick.

Cystic fibrosis is a genetic disease caused by mutations in the CFTR gene. This disease can be classified
according to the effect of the different gene mutations on the CFTR protein. The table below shows the
classification of cystic fibrosis.
 (a) For class I cystic fibrosis, suggest how a mutation in the CFTR gene could result in no CFTR protein
being synthesised. (2)
- Gene is transcribed into different mRNA that is translated into different amino acids of primary
structure.
- The amino acids have different R groups which determine different chemical bonds that in turn
give rise to different protein.

- Gene mutation occurs in start code (TAC) and so no protein formed.

(b) Class II cystic fibrosis results from the CFTR protein being located in the wrong place. Describe the
correct location for the CFTR protein.(2)

In the apical membrane (in the cell surface membrane) of mucus-producing cells / epithelial
cells lining respiratory, digestive and reproductive systems.

(c) The mutation causing class III cystic fibrosis results in a change in the primary structure of the CFTR
protein. Explain why this would result in the CFTR protein being mis-folded.(2)

- The primary has different amino acids that have different R groups that determine different
chemical bonds e.g. hydrogen bonds. They in turn give rise to a protein that is mis-folded/different
structure.

(d) For class IV cystic fibrosis, explain why a faulty opening of the CFTR protein would affect the
functioning of this protein. (2)
 CFTR is a channel protein so, more chloride ions will remain in the cell and less will be passed
through the CFTR protein to mucus.

(e) For a person with class V cystic fibrosis, describe the effect of having smaller quantities of CFTR
protein. (2)

The mucus will have less chloride ions and water

This will give rise to thicker and stickier mucus in the respiratory system (lungs), digestive
system (pancreatic duct in the pancreas), reproductive system (oviducts and cervix in females
and sperm ducts in males).

(f ) For class VI cystic fibrosis, suggest how the CFTR protein is broken down.(2)

Carried out by protease enzymes that break down peptide bonds through hydrolysis reaction.

List the tubes/area blocked by mucus in CF

Bronchioles
Pancreatic duct in the pancreas
Cervix
Oviducts
Vas deferens/sperm ducts
List the effects of CF in the human body

Breathing problems
Lung infection
Poor weight gain
Diabetes
Infertility in woman
Infertility in men

CF affects gas exchange. This is because thick and sticky mucus cannot be removed effectively by cilia
hence accumulate in the lungs. List the effects of this accumulation.

Reduction in airflow due to reduced lumen size of bronchioles

Reduction in gaseous exchange between the alveoli and lung capillaries
Difficulty in breathing/ shortness of breath as the body tries to take in more oxygen.
Bacterial infections in lungs
Fatigue due to lack of energy due to reduced aerobic respiration
Scarred lungs

CF affects digestion. This is because thick and sticky mucus blocks the pancreatic duct in the pancreas so
that digestive enzymes dont reach the duodenum. List the effect of this blockage:
This reduces digestion and absorption of food causing poor weight gain. Fatigue sets in due
to less energy.
 Enzymes trapped within the pancreas cause damage to insulin producing cells (-cells),
triggering diabetes.

Explain how CF affects reproduction

-In women, mucus block the cervix and oviducts are blocked hence infertility.
-In men, the vas deferens (sperm ducts) are blocked with mucus so sperm cannot leave the
epididymis.
Rhythmical tapping of the chest wall during physiotherapy can relieve the symptoms of cystic fibrosis in
the lungs. Suggest an explanation for this.

- This loosens mucus so that it is easily expelled from lungs

- This makes airways to be clearer for better breathing, lower risk of chest infections, larger surface area for gas exchange in
lungs.

Describe possible advantages and possible disadvantages of using gene therapy to treat CF.

Advantages
Treats the cause and not symptoms ;
no physiotherapy needed
No drugs such as antibiotics needed ;
less time consuming than other treatments ;

Disadvantages
The effect lasts for few days because when the cells with the alleles die, replacement cells
do not have the allele. In summary, only target lung cells at present have the allele hence
replacement cells do not have the allele.
Low uptake of the allele by target cells especially when liposomes are used as only 1 out of 1000
alleles will be delivered to the target cell.
Side effects due to use of viruses as vectors. Immune Reaction, Raised heart rate, Headache,
Fever and Fatigue.
The restored transport of chloride ions is only 25%.

Discuss the types of gene therapy

Somatic gene therapy

This is the introduction of a normal allele into the body cell/somatic cell using a vector.
It is permitted in many countries because:-
(a) It only affects target body cells
(b) It is not inheritable
(c) No consent is needed from future generations

Germ-line gene therapy

 This is the introduction of a normal allele into a sperm in the testis or egg in the ovary
using a vector such as a viral DNA.
This is illegal due to the following reasons:-
a) It affects all the body cells
b) It is inheritable
c) Consent from future generations is required yet they are unable to give
consent.
Describe the differences between somatic gene therapy and germ line gene therapy.
1. Somatic involves body (somatic) cells while germ line involves gametes (sperm in testes and eggs in the ovary)
2. Somatic cannot be inherited but germ line can be inherited
3. Somatic is legal but germ line is illegal
4. Somatic is temporary treatment but germ line could be a cure

Thalassaemia is the name of a group of inherited blood disorders that affect the bodys ability to produce
haemoglobin in red blood cells due to lack of all globin protein chains. Lack of enough alpha globin protein chains
causes alpha thalassemia and lack of enough beta globin protein chains causes beta thalassemia. Thalassaemias
are caused by recessive allele. Thalassaemias cause symptoms of anaemia that include: Shortness of breath as the
body tries to take in more oxygen and lack of energy due to insufficient oxygen in the blood hence fatigue. Using
the information given above and your knowledge of gene mutation, suggest why a person with beta
thalassaemia has symptoms of anaemia.

Gene mutation such as deletion and insertion cause different base sequence in a gene
The gene is transcribed into different mRNA that is translated into different primary
structure with different types of amino acids.
These amino acids have different types of R-groups that determine different types of
chemical bonds that then gives wrong shape of haemoglobin
Haemoglobin binds to less oxygen so that less oxygen is supplied to the cells reducing
respiration hence less energy.
Tiredness sets in due to lack of energy.
Breathlessness is due to the body trying to take in more oxygen

Gene therapy could potentially be used to treat beta thalassaemia. Suggest how gene therapy could be
carried out to treat this disorder.
Normal allele for hemoglobin is obtained from red bone marrow cells that synthesize
RBCs.
The allele is cut out from normal DNA using restriction enzyme.
The same restriction enzyme cuts open the vector, plasmid.
The allele is inserted into plasmid vector using ligase enzyme forming rDNA.
The rDNA is then inserted into liposome forming liposome-rDNA complexes.
The complexes are injected into red bone marrow to target stem cells that form RBCs.
There is repeated treatment to ensure success
Cystic fibrosis (CF) is an inherited disease. Explain briefly how two parents who do not have CF may
have a child with CF.
Parents are heterozygous/carriers and pass this recessive allele to the child. CF child must inherit
the two recessive alleles hence they are homozygous recessive for the condition.
Mutations occur in the gametes, after tests have been done.
Mutations occur in the zygote

Briefly describe the common gene mutation that causes CF.

It is called DF- 508, whereby the 508th triplet of bases (the CFTR gene consists of 1480 triplet of
bases) on the CFTR gene is deleted so that phenylalanine amino acid is not coded for in the
CFTR protein; and this faulty protein will not fold correctly, hence will not allow chloride ions
to diffuse from the epithelial cells into the mucus.

Another type of gene mutation affecting CFTR gene is loss of ATP binding site on the CFTR protein.
Explain the role of ATP and how lack of this site causes the closure of CFTR protein.

ATP binds to its site in the CFTR protein; It is hydrolysed to get energy; this energy causes the
chloride channel to open.
Lack of ATP means that there will be no energy to open the chloride channel protein (CFTR
protein), causing the accumulation of chloride ions in the epithelial cells.

Give reasons why most designed DNA tests for CF mutations give false negative results.

CF is caused by many different types of mutations, about 2000, and so any designed DNA tests
will detect few mutations and leave out others. So,the person maybe declared negative for CF,
but it is a false negative.

A couple is declared negative for CF. However, they gave birth to a child who is a genetic carrier for CF.
Suggest reasons for this.

False negative because the designed DNA tests did not detect other gene mutations, hence a
false negative result.
Mutation may have occurred in the egg or sperm cell, after the test was done.
Mutation occurs in the zygote.
Mutation occurs in formation of gametes.

CF analysis is done in the body cells (diploid cells) and not in the gametes (haploid cells). Suggest
reasons for this.

It is easier to collect body cells than gametes.

Body cells are genetically similar unlike gametes.
 Diploid cells have full no. of chromosomes and this allows mutations to be detected from all the
chromosomes; gametes, haploid cells, have half the no. of chromosomes.
Large number of cells can be collected.

Cystic fibrosis is a life-threatening condition that can affect many different parts of the body. It is a
recessive genetic trait. Genetic screening can be used to test for the presence of recessive alleles. A person
found to possess a recessive allele is called a carrier.
(a) Suggest why cells from mouth swabs or blood samples are used rather than gametes.
These cells are easy to collect
Large number of cells can be collected
Being diploid cells, they contain all the 46 chromosomes. Any recessive allele or mutated C.F
gene will be present in them. If gametes are used they may not contain all the mutated CF gene
because they are haploid( 23 chromosomes)
These cells are genetically identical, i.e., have the same alleles

(b) Explain why it is necessary to test for several different recessive alleles in the screening for cystic
fibrosis.
Cystic fibrosis is caused by different gene mutations.
So, testing for only one recessive allele will miss other recessive alleles.

(c) In the risk analysis, if neither partner is a carrier, then it is considered that the chance of having a child
with cystic fibrosis is low. Explain why the probability of having a child with cystic fibrosis is low and
not zero.
False negative because screening programme does not test for all possible mutations that can
cause CF.
Mutation may occur in the formation of gametes
Mutation may occur in both gametes
Mutation may occur after fertilization in the zygote.

(d) In the risk analysis, if one of the partners is found to be a carrier then screening for cystic fibrosis may
be offered to other family members. Explain why this screening is offered to other family members.
Any other family member could be a carrier.
informed choices can be made about having children (if they know that they
are carriers)

Describe 3 ways of reducing cases of CF.

 Pre-implantation Genetic diagnosis (screening) of embryos to select healthy embryos that
are then implanted in the uterus of a woman.
Pre-natal genetic screening, followed by abortions if the foetus has CF.
Adult genetic screening and if found to be carriers they are advised or forced not to
reproduce.