Biology - Genetics

The Cystic Fibrosis Gene

 Introduction:

         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.

         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

 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

 inactive. 

        

 An Account of the Absorption and Secretion of Cl-, Na+, and

 Proteins:

         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 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.

 Conclusion:

         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 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.