CYSTIC FIBROSIS

What happens when development goes wrong?

APRIL 20, 2014
By Maksim Richards
Figure 1 the Stages of development (Macfarlan, et al., 2012)
Figure 2 an example of mitosis (Anon., 2006)
Figure 3 an example of meiosis
(Anon., 2014)
Cystic Fibrosis (CF) is a congenital abnormality. Congenital means present at birth. Cystic Fibrosis is inherited
disease, this means that you have received a mutation from your parents. Congenital abnormalities can also occur through
problems during development caused by environmental or genetic factors. In this essay I aim to explain to you, the reader,
Cystic Fibrosis as though you have little understanding of genetics, development and Cystic Fibrosis. However In order to
explain to you the complexity of cystic fibrosis I must first explain early development so that you can understand what goes
wrong where. I will explain Cystic Fibrosis and its symptoms; the cause and what treatments are available; the treatments
of the future and is there a cure?
Let us begin with early development. During a pregnancy a women will have a person growing inside of her.
Adult Humans have an estimated 3.72x10
13
cells in their entire body (Bianconi, et al., 2013). Thats 37,200,000,000,000 or
37 trillion 200 billion. When a baby is conceived there are only two cells. The sperm and the egg. These are called gametes.
How do two cells turn into 37 trillion? This takes place in the womb of the mother.
The first step is fertilization; the sperm reaches the egg and they fuse to form a single cell. This cell then divides
into more cells of different kinds. Forming muscle, nerves, skin, bone and everything in your body. This happens over a
period of 9 months until the baby is developed enough to emerge into the real world. During early development there are
several stages each of which have their own name: We have the embryo, which is throughout early development. We then
have the Zygote, the single celled organism. This then becomes the morula which is the result of the cleavage (division) of
the zygote and contains 16 cells, this moves on to 32 cells then to 64. 5-6
days after fertilization we get something called the blastocyst. This is formed
of a trophoblast, which is the wall of the blastocyst. This supplies the embryo
with nourishment and will become one of the major parts of the placenta.
We also have the blastocoel the fluid region inside the trophoblast. Inside the
Blastocoe is the inner cell mass. This is what will become the tissues of the
embryo. After the blastocyst the embryo is called the fetus. During this entire
process the cells of the inner cell mass have a special name and property,
they are known as stem cells.
Embryonic stem cells (ES) can divide into another stem cell or any
other kind of cell that is in the human body. We call this differentiation. There
is another type of stem cell which can only change into a certain group of
cells; these are called Adult stem cells. We call embryonic stem cells
totipotent, this means they can differentiate into anything whereas adult
stem cells are multipotent meaning they can differentiate into all sorts of cell
but not every cell. (Hall, et al., 2005, pp. 120-124) Stem cells divide in a
process called mitosis one of the two types of cell division.
In mitosis the cell replicates all its 23 pairs of chromosomes the cell then splits leaving one of these 23 pairs in
each daughter cell. Mitosis is the type of cell division that is used during the cleavage stages of development and
throughout are lives to repair tissues. Eventually mitosis creates billions of cells.
Once one cell has split into two, each cell can split again making 2 divisions
which will add up to 4 cells after 2 divisions. Once there are 1 million cells
one division of each cell will create 2 million cells. Meiosis is the other type
of division. There are two stages to meiosis, or two divisions. The beginning
is the same as mitosis.
The chromosomes duplicate themselves so there are 46 pairs, 92
chromosomes in total then the two pairs of chromosome 1 pair up and then
separate into two new cells. The next bit is different. The new cells, with two chromosomes of each
different chromosome, split again this time leaving only 1 chromosome of each type (figure 2). At the
first division, the chromosomes of the same kind pair up and then separate. This makes a total of 2 cells.
During the next division these two cells split again this time forming 4 cells with only 23 chromosomes.
Each pair splits apart and then they create their own cells called haploids. (Hall, et al., 2005, pp. 108-
109)
Gametes are formed using meiosis as each gamete has 23 chromosomes inside of it. When
the sperm fertilizes the egg the 23 chromosomes from the sperm pair up with the 23 from the egg;
chromosome 1 from the sperm with chromosome 1 from the egg etc... This makes a total of 46
chromosomes in the zygote. Gametes are called haploid cells as they have only 23 chromosomes. Skin
and bone cells are called 46 chromosomes and are called diploid cells.
Now what are chromosomes and what do they do? DNA (Deoxyribonucleic acid), is split into
small sections we call genes. Genes are stored within chromosomes. DNA is made of a unique order of
nucleotides. Nucleotides are made up of; a base, a deoxyribose sugar, and a phosphate. There are four bases: adenine,
guanine, thymine and cytosine. Each base has a complementary base. In other words they can only pair
with a certain other base. For example adenine can only pair with thymine and guanine can only pair
with cytosine. This is very important for the replication of DNA during cell division. The other
components of nucleotides are unimportant for the subject of this essay.


Figure 4 Cilia Cell (Anon.,
n.d.)
Figure 5 tree of inheritance, the red
lowercase c indicates the CFTR
mutation. (Kaneshiro, n.d.)

DNA does not come in one long strand as we are lead to think. It comes in little snippets held within genes. All
genes contain a little packet of DNA. Within each one a blueprint if you like of how to build a certain enzyme or protein.
Genes are laid out on chromosomes. Each gene lies along one of the 23 chromosomes and within each gene the plan for an
enzyme or a protein. We have given a number to each chromosome. These range from 1-23 if you hadnt of guessed.
Chromosome 1 can only pair up with another chromosome 1 etc...
Genetic Mutations will come up a lot in this as Cystic Fibrosis is a genetic mutation, however the mutation is one
of a planned nature that is to say that the DNA from both parents hold instructions to create a protein with a fault.
Sometimes there will be no protein at all. There are four types of genetic mutation: substitution, deletion, insertion and
inversion. In brief, substitution is where a base is swapped for another. Deletion is where are base is completely
eradicated. Insertion is where a gene is put between two others and inversion is where two adjacent genes swap places.
These mutations all happen when DNA is replicated; sometimes the replication goes wrong and you get these changes to
the base order.

Cystic Fibrosis

Symptoms and Incidence
Cystic Fibrosis kills about 114 people each year in the United Kingdom; there are over 10,078 cases in the UK
alone (Burton & Owen, 2013). Cystic Fibrosis is characterized by patients having trouble breathing, coughing etc Cystic
Fibrosis results in a shorter life expectancy on average the person will live to be 37 (Flume,
et al., n.d.). The lungs and trachea have cells lining their walls called cilia cells (figure 4). The
Cilium are covered in a mucus used to catch bacteria and other microorganisms. This mucus is
then moved up and out of the lungs to avoid infection.
With patients with cystic fibrosis something else is the case. The mucus becomes
very sticky and very thick, this means it cannot be moved by the cilium. The cells still produce
mucus at a normal rate which leads up to layers of the thick mucus. The mucus has low levels
of Oxygen partly because oxygen goes slowly through it; and partly because the cells use up
more oxygen in CF patients. Harmful bacteria can thrive in these anaerobic (low or no
oxygen) conditions. White blood cells fight the infections in the mucus but as they die they
are broken down, releasing DNA which makes the mucus even stickier and thicker. Repeated
infection weaken the bodys ability to fight the pathogens, and can lead to damage in the gas
exchange system. This can lead to trouble breathing.



The CFTR Gene
At a genetic level scientists would say Cystic fibrosis (CF) is an autosomal recessive disease
caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance
regulator (CFTR) anion channel. The most common CF-associated mutation is F508, which deletes
a phenylalanine in position 508. (Ostedgaard, et al., 2011) What does this mean?
Autosomal recessive means that two copies of the gene must be present in the zygote
(Kaneshiro, n.d.). This tells us that both of the parents must have had the mutation of the
gene. If both parents are carriers you have a 1 in 4 chance of inheriting this disease. If only
one parent is a carrier there is a slight chance of inheriting CF (Anon., n.d.).

Encoding, in this context it refers to the plans for the creation of the Cystic
Fibrosis Transmembrane Conductance Regulator (CFTR) protein. The anion channel is a
channel in which ions can travel through. Ions are atoms with either a negative or a
positive charge not with a neutral charge.

F508, or its full name CFTRF508, is a mutation
within a gene on chromosome 7. This mutation is of the
type deletion. It deletes 3 nucleotides (including 3 bases).
This means that at position 508 on the chromosome, 3
bases are deleted. These make up the codon for
phenylalanine. This mutation is the most common mutation
associated with CF. Codon is the word used for an order of
three bases which encodes the pieces of proteins (amino
acids). This means that if you were to change the amino
acid within the CFTR protein you could change the structure
or function of the protein.

Figure 6 the regulation of water in a system with excess water (Hall, et al.,
2005)
Figure 7 the regulation of water in a system with too little water (Hall,
et al., 2005)
Figure 8 a person with CF and their water regulation (Hall, et al., 2005)
This is all well and good but what effects does this
mutation cause. Firstly the thickness of the mucus along the
trachea is defined by how much water is in it. How does the
regulation of water differ in lungs with CF to those without? Let us first talk about how water is regulated in normal lungs.

If there is too much water (an excess) then it is detected by the epithelial cells membranes (the outmost part of
the cells along the airways).



Carrier proteins in the basal membrane (figure 6) actively pump Na
+
(sodium with a positive charge) out of the epithelial
cells into the mucus. A gradient means in two places that are joined, there is a different amount of something. The forms
a hill, if you like from the peak being in the area with a high level of the substance and the bottom where there is a low
amount. In this case there are more Na
+
molecules in the mucus then in the cell. Between the tissue fluid basal membrane
(figure 6) and the apical membrane (figure 6) there is this gradient. There is more Na
+
in the mucus on the apical
membrane than on the tissue fluid. The sodium ions roll down the gradient into the fluid. The higher concentration of
sodium ions in the fluid within the basal membrane causes a potential difference (a difference in electrical charge) There
are more positively charged ions in the fluid than on the apical membrane. This gradient allows negatively charged Cl
-
atoms (chlorine with a positive charge) to diffuse (move from high to low concentration until equilibrium reached) out of
the mucus and into the tissue fluid by moving through the gaps in between the epithelial cells.

The increase of concentration of Na
+
and Cl
-
draw water out of the cell by osmosis (form of diffusion involving
free water molecules) across the basal membrane and into the tissue fluid. The lack of water in the cell increases the
overall concentration of Na
+
and Cl
-
in the cell this means that there is a higher concentration in the cell than in the mucus
and now water is drawn out of the mucus by osmosis again. This means that there is a higher concentration of Na
+
at one
place and lower one in another. This gradient is over the apical membrane. This is membrane on the side facing the
trachea.

The higher concentration of sodium ions in the tissue fluid on the basal membrane (figure 5) the cell creates a
potential difference (this means there is a difference in electrical charge). The basal membrane contains more positively
charged ions then the mucus does. This creates an electrical gradient between these two. This gradient causes negatively
charged chloride ions to diffuse (move due to different concentration until equilibrium is reached) out of the mucus and
into the tissue fluid (Hall, et al., 2005, pp. 72-73).

If there is a need for water in the mucus chloride ions are moved over the basal membrane into the epithelial
cell. This would then again create a gradient of concentration across the apical membrane however now it is chloride that
is of a higher concentration in the cell than out. At the same
time as this transportation the CFTR channels open. Chloride
ions go through these channels down the concentration
gradient and into the mucus. When CFTR channels open they
block the sodium channels stopping sodium from entering the
mucus. We do not know what causes this to happen. These
negatively charged chloride ions create a concentration
gradient between the mucus and the tissue fluid on the basal
membrane. Sodium ions diffuse out of this fluid and move
down this electrical gradient passing between the epithelial
cells and entering the mucus. This movement of sodium and
chloride ions takes water from the cells by osmosis until the
concentration of water is the same on both sides of the
membrane of the cell (Hall, et al., 2005).

In a person with CF, the CFTR protein is either
missing or does not function correctly (figure 7). When there
is too little water in the mucus. Cl
-
cannot be moved across
the apical membrane, and there is no blockage of the
epithelial sodium ion channels. Since Na
+
channels are always
open the epithelial cells constantly absorb sodium. Sodium
draws water out of the mucus into the cells. This makes the
mucus more sticky and thick. This means that this person will
get bacteria stuck in the mucus and will cause airway
infections and damage (Hall, et al., 2005).


Treatment
How can Cystic Fibrosis be treated? A person with CF will be given medication which lessen the struggle but are
not a cure; these come in the form of:
Bronchodilators This medication is inhaled using a nebuliser (it blows air through the drug making a
fine mist) this medicine relaxes the muscles in the airways, this opens them up stopping tightness of
the chest.
Antibiotics The early diagnosis and treatment of lung infections is very important for the treatment of
CF. Several antibiotics are required to kill bacteria in the airways or to stop it growing all together.
DNAase enzymes if the luings are infected often the amount of white blood cells builds, as I have said
before if the white blood cell dies it releases DNA increasing the stickiness of the mucus. These drugs
can be inhaled using a nebulizer. They break down the DNA, so that the cilium can clear the airways
more easily.
Steroids Used to decrease inflammation of the lungs making it easier to breath.
It is recommended for adults with CF to high energy foods along with double the amount of protein
that a person without CF. Some CF patients may also need salt supplements.
Digestive Enzyme Substitutes: These are if the pancreatic duct is blocked, as sticky mucus can easily
block it. They help dissolve food so it can be absorbed more easily.
Physiotherapy: Tapping the chest in a rhythmical fashion can loosen the mucus.
Heart and lung transplant: If the lungs become badly damaged and very inefficient they may need to be
replaced with a heart and lung transplant. (Hall, et al., 2005)

The Cure
What research is being done toward new treatments and cures? Well at the moment scientists are trying to
develop the most commonly heard of treatment; gene therapy. They use pigs and ferrets to further their research. Now
why would you use pigs and ferrets? Scientists often use model organisms (organisms used to represent some other
creature). But they are nothing like us! Looks wise maybe, but genetically we share this same CFTR gene. Also research has
shown that pigs and ferrets can show signs of Cystic Fibrosis and other lung diseases similar to humans. (Ostedgaard, et al.,
2011) In gene therapy they attempt to alter the very gene that causes CF, this is done by inserting normal alleles (variations
of the same gene) into the target cells in our case the epithelial cells, this is mostly done by using a genetically modified
virus to infect these cells. Then the normal form of the gene is replicated. Finally a functioning CTFR protein is produced in
the target cells. In terms of CF the functioning protein is produced and incorporated into the cell membrane of the
epithelial cells this restores the ion channel. In turn the patient ceases to have the symptoms of CF (Hall, et al., 2005).

How do they insert the genes using the viruses? This is actually quite simple, the sequence which allows the virus
to replicate itself they replace this with the allele of the gene they want to incorporate. With this they add a promoter
sequence which is responsible for duplicating the gene.

When we become ill with a virus, the virus has either mixed its DNA with ours or it can remain independent of it.
This depends of the virus. When treating CF scientists use the second of these. During trials of this gene therapy started in
1993 they only saw a correction of chloride secretion and no signs of sodium secretion. In 1999 this chloride transportation
was restored to 25% of the normal. The longest this fix lasted 15 days (Hall, et al., 2005).

Cystic Fibrosis is a terribly debilitating disease and even if the current gene treatment was to be put in place this
treatment would have to be repeated throughout the patients life. Hopefully another form of gene delivery will offer a
more permanent solution or even a cure. The patients life is constantly having to take medication and endless hospital
appointments. The end result is a short, painful and unpleasant disease. Even if both have the CF gene even if they
personally dont have CF the child may still have it. Is the answer to mandate who mates with who? Parental genetic
testing is also carried out (testing if the baby will have the disease). There are several ethical issues associated with this
however. Should you abort a baby just because it will have Cystic Fibrosis? Let us hope that in the future they come up
with a solution for a cure. Not only would this offer a cure for Cystic Fibrosis but maybe it would offer a cure for all genetic
diseases. Thank you very much for reading.

Maksim Richards




















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