
 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.