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 lethal genetic disorder in Caucasians, affecting one out of 2,500. On the bioethical front, CF was the first human genetic disease to be cloned by geneticists. The intent of this paper is to describe how the cystic fibrosis gene was identified, how the gene is defective, its physical manifestations, and to discuss possible treatments of the disease.
The classical approach to finding the gene that is responsible for causing a genetic disease is to first characterize the bio-chemical defect within the gene, then to identify the mutated protein in the gene, and finally to locate the actual gene. However, this classical approach proves to be impractical when searching for the CF gene. To find the gene responsible for CF, the principle of “reverse genetics” is applied. Scientists accomplish this by linking the disease to a specific chromosome. After this linkage, they isolate the gene of interest on the chromosome, and then test its product. Before the disease can be linked to a specific chromosome, however, a marker needs to be found that will 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. The RFLP for cystic fibrosis was first discovered through somatic cell hybridization (cell fusing) and southern blot electrophoresis (gel separation of DNA within an electric field). By using these techniques, three RFLP’s were discovered for CF: Doc RI, J3.11, and Met. Utilizing hybridization (rapid heating and cooling of RNA with denatured DNA so that the RNA permanently associates with the DNA,) scientists discovered the CF gene to be located on the long arm of chromosome 7q. Soon after identifying these markers, another marker was discovered that frequented more often with CF than the did the other markers. This meant the new RFLP was closer to the CF gene. At that time, two scientists named Lap-Chu Tsui and Francis Collins were able to isolate probes (radioactively marked DNA/RNA sequences used to detect the presence of a complimentary sequence by hybridization) from the gene interval. In order to determine the exact location of the CF gene, probes were taken from the nucleotide sequence obtained from chromosome jumping. To find these probes, horse, cow, chicken, and mouse DNA was separated using Southern Blot electrophoresis. Two probes were found to bind to all of the vertebrates DNA. This meant that the base pairs within the probes contained related information, possibly even the gene.
The Northern Blot electrophoresis technique was then used to distinguish between the two probes still remaining in order tofind out which one actually contained the CF gene. This was successful because Northern Blot electrophoresis tests RNA, not 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 (all CF-affected cells,) 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 number two did. From this evidence, these scientists determined that probe number two contained the CF gene.
While isolating the CF gene and screening the genetic library made from mRNA (cDNA library – the gene sequence cloned by mRNA and reverse transcriptase), it was discovered that probe number two did not hybridize. The chances for hybridization were suspected to have been decreased either because of low levels of the CF gene present within the probe, or 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 (CF patients commonly have elevated levels of sodium and chloride ions in their sweat.) By using this new cDNA library, probe number two was found to hybridize excessively. They 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 same sequences base pair in a non-affected cell. The entire CF cDNA sequence is approximately 6,000 nucleotides long. In those 6,000, three base pairs were found to be missing in affected cells, all three were in exon (coding sequence) #10. This absence effects the loss of a phenylalanine residue and accounts for seventy percent of the CF mutations. In addition to this three base pair deletion pattern, up to 200 different mutations have since been discovered in the gene accounting for CF, all to varying degrees.
Treatment for CF varies greatly from case to case, but is usually substantial enough to double a patients lifespan (most untreated cases die by age thirteen.) One method is to provide missing chemicals to the epithelial cells (inner nose, throat, and lung surface), such as adenosine 3′,5′-monophosphate (cAMP), or the nucleotide triphosphates ATP or UTP. This has been proven to raise the rate of Cl- secretion (in CFTRs) by removing a layer of fluid common to CF patients from the respiratory tract. Chloride conductance channels have dramatic transfer potentials.
Specifically, one channel can conduct from 1,000,000 to 100,000,000 ions per second. This is particularly impressive when considering the fact that there are not many channels present on cells to perform these 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. Accordingly, even the mildest of treatments altering the cystic fibrosis Transmembrane Conductance Regulator in CF afflicted people can lead to significant improvements in an individuals health. The most fascinating treatment that has been attempted is to administer solutions of genetically engineered cold viruses, in aerosol form, into the nasal passages and lungs of those 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. This known as gene therapy.
A drastically different approach taken in an attempt to cure cystic fibrosis, performed as a method of preventive medicine, involves correcting the disease while the affected “person” is still an embryo. Test tube fertilization from affected parents and gene diagnosis during embryonic development can be accomplished through a biopsy of a cleavage-stage embryo and amplification of DNA from single embryonic cells. After this treatment, only unaffected embryos would be selected for implantation into the uterus. Unfortunately however, affected embryos would be discarded.
These treatments are all remarkable breakthroughs in medical science and provide geneticists with a highly positive outlook, but they are nowhere near the miracles yet to come with more research. Since cystic fibrosis is the most common lethal genetic disorder among Caucasians, intense research efforts towards better treatments and its cure are invaluable. The discoveries made from researching this disease may very well help the treatment or cure of other, unrelated diseases.