The Cystic Fibrosis Gene

Biology – Genetics
The Cystic Fibrosis Gene
Cystic fibrosis is an inherited autosomal recessive disease
that exerts its main effects on the digestive system and the
lungs. This disease is the most common genetic disorder
amongst Caucasians. Cystic fibrosis affects about one in
2,500 people, with one in twenty five being a heterozygote.
With the use of antibiotics, the life span of a person
afflicted with CF can be extended up to thirty years
however, most die before the age of thirteen.1 Since so
many people are affected by this disease, it’s no wonder
that CF was the first human genetic disease to be cloned by
geneticists. In this paper, I will be focusing on how the
cystic fibrosis gene was discovered while at the same time,
discussing the protein defect in the CF gene, the
bio-chemical defect associated with CF, and possible
treatments of the disease.
Finding the Cystic Fibrosis Gene:
The classical genetic approach to finding the gene that is
responsible for causing a genetic disease has been to first
characterize the bio-chemical defect within the gene, then
to identify the mutated protein in the gene of interest, and
finally to locate the actual gene. However, this classical
approach proved to be impractical when searching for the CF
gene. To find the gene responsible for CF, the principle of
“reverse genetics” was applied. Scientists accomplished
this by linking the disease to a specific chromosome. After
this linkage, they isolated the gene of interest on the
chromosome and then tested its product.2
Before the disease could be linked to a specific
chromosome, a marker needed to be found that would always
travel with the disease. This marker is known as a
Restriction Fragment Length Polymorphism or RFLP for short.
RFLP’s are varying base sequences of DNA in different
individuals which are known to travel with genetic
disorders.3 The RFLP for cystic fibrosis was discovered
through the techniques of Somatic Cell Hybridization and
through Southern Blot Electrophoresis (gel separation of
DNA). By using these techniques, three RFLP’s were
discovered for CF; Doc RI, J3.11, and Met. Utilizing in
situ hybridization, scientists discovered the CF gene to be
located on the long arm of chromosome number seven. Soon
after identifying these markers, another marker was
discovered that segregated more frequently with CF than the
other markers. This meant the new marker was closer to the
CF gene. At this time, two scientists named Lap-Chu Tsui
and Francis Collins were able to isolate probes from the CF
interval. They were now able to utilize to powerful
technique of chromosome jumping to speed up the time
required to isolate the CF gene much faster than if they
were to use conventional genetic techniques.3
In order to determine the exact location of the CF gene,
probes were taken from the nucleotide sequence obtained from
chromosome jumping. To get these probes, DNA from a horse,
a cow, a chicken, and a mouse were separated using Southern
Blot electrophoresis. Four probes were found to bind to all
of the vertebrate’s DNA. This meant that the base pairs
within the probes discovered contained important
information, possibly even the gene. Two of the four probes
were ruled out as possibilities because they did not contain
open reading frames which are segments of DNA that produce
the mRNA responsible for genes.

The Northern Blot electrophoresis technique was then used
to distinguish between the two probes still remaining in
order to find out which one actually contained the CF gene.
This could be accomplished because Northern Blot
electrophoresis utilizes RNA instead of DNA. The RNA of
cell types affected with CF, along with the RNA of
unaffected cell types were placed on a gel. Probe number
two bound to the RNA of affected cell types in the pancreas,
colon, and nose, but did not bind to the RNA from
non-affected cell types like those of the brain and heart.
Probe number one did not bind exclusively to cell types from
CF affected areas like probe number two did. From this
evidence, it was determined that probe number two contained
the CF gene.

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While isolating the CF gene and screening the genetic
library made from mRNA (cDNA library), it was discovered
that probe number two did not hybridize. The chances for
hybridization may have been decreased because of the low
levels of the CF gene present within the probe.
Hybridization chances could also have been decreased because
the cDNA used was not made from the correct cell type
affected with CF. The solution to this lack of
hybridization was to produce a cDNA library made exclusively
from CF affected cells. This new library was isolated from
cells in sweat glands. By using this new cDNA library,
probe number two was found to hybridize excessively. It was
theorized that this success was due to the large amount of
the CF gene present in the sweat glands, or the gene itself
could have been involved in a large protein family.
Nevertheless, the binding of the probe proved the CF gene
was present in the specific sequence of nucleotide bases
being analyzed.
The isolated gene was proven to be responsible for causing
CF by comparing its base pair sequence to the base pair
sequence of the same sequence in a non-affected cell. The
entire CF cDNA sequence is approximately 6,000 nucleotides
long. In those 6,000 n.t.’s, three base pairs were found to
be missing in affected cells, all three were in exon #10.
This deletion results in the loss of a phenylalanine residue
and it accounts for seventy percent of the CF mutations. In
addition to this three base pair deletion pattern, up to 200
different mutations have been discovered in the gene
accounting for CF, all to varying degrees.

The Protein Defect:
The Cystic Fibrosis gene is located at 7q31-32 on
chromosome number seven and spans about 280 kilo base pairs
of genomic DNA. It contains twenty four exons.4 This gene
codes for a protein involved in trans-membrane ion transport
called the Cystic Fibrosis Transmembrane Conductance
Regulator or CFTR. The 1,480 amino acid protein structure
of CFTR closely resembles the protein structure of the
ABC-transporter super family. It is made up of similar
halves, each containing a nucleotide-binding fold (NBF), or
an ATP-binding complex, and a membrane spanning domain
(MSD). The MSD makes up the transmembrane Cl- channels.
There is also a Regulatory Domain (R-Domain) that is located
mid-protein which separates both halves of the channels.
The R-Domain is unique to CFTR and is not found in any other
ABC-transporter. It contains multiple predicted binding
sites for protein kinase A and protein Kinase C.4

Mutations in the first MDS are mainly found in exon #4 and
exon #7. These types of mutations have been predicted to
alter the selectivity of the chloride ion channels.4
Mutations that are in the first NBF are predominant in
CFTR. As previously mentioned, 70 percent of the mutations
arising in CF cases are deletions of three base pairs in
exon #10. These three base pairs give rise to phenylalanine
and a mutation at this site is referred to as DF508.5 Such
a mutation appears not to interfere with R-Domain
phosphorylation and has even been reported to transport
chloride ions.6;7
There are five other frequent mutations that occur in the
first NBF. The first is a deletion of an isoleucine
residue, DF507. The second is a substitution of glycine or
amino acid #551 by aspartic acid/F551D. The third involves
stop mutations at arginine #553 and glycine #542. The
fourth is substitutions of serine #549 by various other
residues. The fifth is a predicted splicing mutation at the
start of exon #11.7
Mutations within the R-Domain are extremely rare. The only
reason they do occur is because of frameshifts. Frameshifts
are mutations occurring due to the starting of the reading
frame one or two nucleotides later than in the normal gene
Mutations in the second membrane spanning domain of the
CFTR are also very rare and have only been detected in exon
#17b. These have no relevance to mutations occurring in the
first membrane spanning domain. They apparently do not have
a significant impact on the Cystic Fibrosis Transmembrane
Conductance Regulator either.4
Mutations in the second nucleotide-binding fold occur
frequently in exon #19 and exon #20 by the deletion of a
stop signal at amino acid number 1282. Exon #21 is
sometimes mutated by the substitution of asparagine #1303
with lysine #N1303K.4
The Bio-Chemical Defect:
Studies of the chloride channels on epithelial cells lining
the lungs, sweat glands, and pancreas have shown a consensus
in that the activation of chloride secretion in response to
cAMP (adenosine 3′, 5′-monophosphate) is impaired in cystic
fibrosis cases. Another affected, independently regulated
chloride channel that has been discovered is activated by
calcium-dependent protein kinases. Sodium ions have also
been noted to be increasingly absorbed by apical sodium
channels.8 Therefore, the lack of regulated chloride ion
transport across the apical membranes and apical absorption
of sodium ions, impedes the extracellular presence of water.
Water will diffuse osmotically into cells and will thus
cause the dehydration of the sol (5- mm fluid layer of the
cell membrane) and the gel (blanket of mucus) produced by
epithelial cells.9 As a result of this diffusion of water,
airways become blocked and pancreatic proteins turn
An Account of the Absorption and Secretion of Cl-, Na+, and
An inward, electrochemical Na+ gradient is generated by the
Na+, K+-ATPase pump located in the basolateral membrane (the
cell side facing the organ it is lining). A basolateral
co-transporter then uses the Na+ gradient to transport Cl-
into the cell against its own gradient. This is done in
such a way that when the apical Cl- channels within the
membrane spanning domain open, Cl- diffuse passively with
their gradient through the cell membrane.4
In pancreatic duct cells, a Na+, H+-ATPase pump is used and
a bicarbonate secretion is exchanged for Cl- uptake in the
apical membrane. Chloride ions then diffuse passively when
the Cl- channels are opened. Such secretions also allow for
the exocytosis of proteins in the pancreas which will later
be taken into the small intestines for the breaking down of
In addition to the pump-driven gradients and secretions,
there exists autonomic neurotransmitter secretions from
epithelial cells and exocrine glands. Fluid secretion,
including Cl-, is stimulated predominately by cholinergic,
a-adrenergic mechanisms, and the b-adrenergic actions.4
Such chemical messengers cannot enter the cell, they can
only bind to specific receptors on the cell surface and
transmit messages to and through an intracellular messenger
such as Ca2+ and cAMP by increasing their concentration.
The intracellular message is transmitted across the cell by
either diffusion or by a direct cascade. One example of a
directed cascade is the following:
Possible Treatments For Cystic Fibrosis:
One suggested treatment for CF has been to provide the
missing chemicals to the epithelial cells. This can be
accomplished by the addition of adenosine
3′,5′-monophosphate (cAMP) or the addition of the nucleotide
triphosphates ATP or UTP to cultures of nasal and tracheal
epithelia. This has been proven to alter the rate of Cl-
secretion by removing the 5-mmeter sol layer of fluid in the
respiratory tract.9 Moreover, luminal application of the
compound amiloride, which inhibits active Na+ absorption by
blocking Na+ conductance in the apical membrane, reduced
cell secretion and absorption to a steady state value.

Another treatment that has been suggested is to squirt
solutions of genetically engineered cold viruses in an
aerosol form into the nasal passages and into the lungs of
people infected with CF. This is done in hopes that the
virus will transport corrected copies of the mutated gene
into the affected person’s airways so it can replace the
mutated nucleotides.10 This form of treatment is known as
gene therapy.

A different approach taken in an attempt to cure cystic
fibrosis involves correcting the disease while the affected
“person” is still an embryo. Test tube fertilization (in
vitro fertilization) and diagnosis of F508 during embryonic
development can be accomplished through a biopsy of a
cleavage-stage embryo, and amplification of DNA from single
embryonic cells.5 After this treatment, only unaffected
embryos would be selected for implantation into the uterus.
Affected embryo’s would be discarded.

Chloride conductance channels have dramatic potentials.
One channel can conduct from 1×106 to 1×108 ions per
second.8 This is particularly impressive when you consider
the fact that there are not many channels present on cells
to perform the required tasks. As a result of this, a
mutation of one channel or even a partial mutation of a
channel, that causes a decrease in the percentage of channel
openings, can exert a major effect.

Even the mildest of cures altering the Cystic Fibrosis
Conductance Regulator in CF afflicted people would lead to
significant improvements in that individuals health. Since
cystic fibrosis is the most common genetic disorder,
particularly amongst Caucasians, in today’s society, intense
research efforts towards its cure would be invaluable. When
will cystic fibrosis be completely cured? No one can say
for sure but, strong steps have already been taken towards
reaching this goal.


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