Cystic Fibrosis


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 the 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 two powerful techniques 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.
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

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 translation.4 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
carbohydrates.4 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
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 1x106 to 1x108 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


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