Cystic fibrosis is the most common recessive hereditary disease amongst Caucasians that causes disruption of chloride movement and due to this commonality it is important to design assays to test for it (Welsh and Smith, 1995).  The 3272-26AàG mutation is a CFTR splicing mutation that causes cystic fibrosis; to test for this mutation a diagnostic assay was created using three primers, PCR, and gel electrophoresis (Beck et al, 1999).  If PCR and agarose gel electrophoresis were conducted correctly, the electrophoresis will produce a gel with illuminated bands that represent the size of DNA segments complementary to three specifically designed primers. Primers 1 and 2 will produce a DNA segment approximately 800 base pairs in length if the mutation is present with the mutant DNA.  If wild type DNA is used with these primers, no bands should show up. Primers 2 and 3 will produce an 800 base pair long DNA segment with wild type DNA if the mutation is not present.  Also it is predicted that there is a link between cystic fibrosis and health related quality of life in the areas of depression, pain, family and cost.

Several PCR trials were run to test for the 16S rDNA genes in E-Coli and for the Rz Gene in the lambda virus (Wright et al, 2006). The trials were conducted until the optimum cycle times and temperatures (not including annealing) were determined and visible bands were achieved at the 500 bp marker, indicating the presence of the targeted genes (Abilock and Stephenson, 2008).  Running PCR with E.Coli and Lambda helped create a template for future PCR experiments.  When PCR was run, both e-coli and lambda cocktails were prepared. Following electrophoresis, bands were seen at approximately the 500bp marker for all four columns containing lambda, which indicated the presence of the Rz gene. There were, however, no bands seen for the column containing e-coli. The e-coli columns had no smears or glowing bands, indicating that not enough DNA was used in the cocktails.  This positive result gave a good template for running PCR for CFTR mutations.

The PCR cocktails run with wild type DNA and primers 2 and 3 yielded bands at 786 base pairs indicating that those primers annealed correctly and amplified the correct section of DNA.  An additional band was seen at 509 base pairs, this indicated a smaller unspecified product that could be gotten rid of by increasing extension time and temperature.  Since bands appeared for these primers with the wild type DNA this indicates that the 3272-26AàG mutation was not present.  This can be surmised since the same DNA ran with primers 1 and 2, which were supposed to anneal if the mutation was present, gave no bands indicating a lack of presence of the mutation in the wild type DNA.  These results indicate that the assay created for the wild type DNA was a success since it showed a negative result for the presence of the mutation.  This reaction was also completed a second time with the same results, indicating a lack of presence of the mutation in the wild type DNA.  These same cocktails were also run with mutant DNA.  When PCR was run with mutant DNA, primers 1 and 2 attached to the DNA template and after gel electrophoresis a band was observed at 903 base pairs.  When primers 2 and 3 were run with the same mutant DNA, no bands appeared.  These results show that the assay to test for the presence of the 3272-26A->G mutation was successful since it showed bands at the correct number of base pairs showing with wild type DNA that the mutation was not present and with mutant DNA that the mutation was present.  The results support the hypothesis that an 800 base pair band will illuminate on the gel when the primers anneal to the mutant and wild type DNA.

It has been shown that the quality of life in patients with cystic fibrosis is markedly decreased compared to that of people without the disease (Abbott, 2003).  It has been shown that people with the disease have a higher risk of depression and often exhibit depression and anxiety symptoms.  These psychological issues are also often present, and worse, in the parents or caregivers of the CF patients, this can be due to watching a child grow up sick and knowing that they will not grow old and also seeing the child in pain (Quittner et al, 2008).  The increase in depression in parents and caregivers leads to interfamily and household problems and may lead to increased problems in the child with CF.  With depression in patients with CF, the worse these symptoms the more the decrease in health related quality of life since patients with CF and who are depressed are less likely to comply with therapy and to go to their various doctors appointments (Quittner et al, 2008).  This amounts to a decrease in physical and health wellness which would lead to an increase in pain and decrease in functional abilities in the patient.  Due to the high occurrence of depression in patients and families with cystic fibrosis, it is recommended that regular screening be done and, if a patient is diagnosed, that appropriate therapy and counseling be made readily available.  Cost is also a big factor in quality of life.  Cost for medical care for patients with CF varies by the severity of the disease, but the cost is still substantial in patients with a mild disease.  With mean costs ranging from $6,300 for mild to $43,300 for severe, the ability to be cost effective with new and needed treatments is important (Lieu et al, 1999).  It is very important to have available effective and high quality treatment for all people with this condition.

Since the 3272-26AàG CFTR mutation causes such a mild case of cystic fibrosis, 40% of CFTR channels from this mutation will function normally, the health related quality of life problems studied do not have as much of an effect on overall quality of life than other more severe mutations (Beck et al, 1999).  Patients with this mutation are healthier than other CF patients so such things as depression, which can be influenced by the severity of a disease, will be less pronounced and less of a problem.  Due to the mildness of this mutation, costs will also be reduced and the treatment itself will be more cost effective because of it (Lieu et al, 1999).  Also, since the disease is less severe, the parent care-givers will be less likely to have depression problems and overall family harmony will be increased because of this and the less cost associated with a healthier child.  Though regardless of the severity of the disease, knowing the risk factors associated with cystic fibrosis treatment can be tailored to patients based on the their psychological or pain complications.  Screening for psychological problems and encouraging genetic counseling will help to facilitate adaptation to the condition and also it will increase awareness for of health related quality of life risk factors (Quittner et al, 2008).  Doing this will prepare both the parents and child for future costs and complications associated with the disease and, hopefully, will help reduce or eliminate some of the problems by prior knowledge of them.  Along with this, genetic counseling can help to advise and counsel parents on the risks of having another child with the same condition.

Continuing this research would allow more definitive answers to be found as to the presence and effects of the 3272-26AàG mutation.  Reverse transcriptase PCR could be run with different primers, this would allow for the 25 extra nucleotides in the RNA to be observed when the alternative splicing does happen (Beck et al, 1999).  It would be expected that two bands would appear: one when alterative splicing occurred and the other when alternative splicing did not occur.  Within this, sequencing can also be done to know the specific nucleotides present in the bands to see if the extra ones are present and to see if they match up with the nucleotides from intron 17a that didn’t get spliced out.  Additionally, more research and interviews can be conducted regarding the health related quality of life in patients and families with CF to both further current knowledge and to add additional factors that could influence it.