Cystic Fibrosis Transmembrane Conductance Regulator Protein

Mutation R117H

 

GROUP NAME: FLAWLESS FIJIANS.

Figure 1: representation of the Cystic Fibrosis transmembrane conductance regulator protein spanning through the phospholipid bilayer membrane of a cell.

As a part of a study in the Lyman Briggs College at Michigan State University, we have developed a diagnostic test to test for the R117H CFTR mutation. In collaboration with multiple other Lyman Briggs researchers and under the supervision of Dr. Douglas Luckie, our laboratory has created diagnostic tests for several of the known Cystic Fibrosis mutations. Various methods were utilized during the development process, including polymerase chain reaction (PCR), gel electrophoresis, ultraviolet imaging, and purification of genomic DNA from Cystic Fibrosis Cells. Once we found the loci of the R117H mutation in CFTR, two primers were  designed (mutant & wild-type) so that they would anneal to the mutation site at the 3’  end on the sense strand of DNA. A third primer was also designed to anneal 780 base pairs away on the anti-sense. From this we ran various PCRs and gels, continuously increasing the stringency as much as possible to obtain optimal results.

Abstract

 

             Cystic Fibrosis is a life threatening recessive genetic disease that leads to mucus buildup and infections (Welsh and Smith, 1995). R117H is a mutation that occurs in various phenotypes: ranging from the absence of Vas Deferens, the fatal disease of Cystic Fibrosis, to no symptoms at all (Gervais et al, 1993). We hypothesized that a single base mutation in the CFTR gene can be tested for using primer design and a PCR assay. This was based on a previous study (Wittwer et al, 1993). Using Polymerase Chain Reaction (PCR), we amplified a target region of DNA containing the CFTR gene using Taq polymerase and primers to direct the replication of the DNA.  Three primers were made, P₁ (primer #1) is a forward primer and would attach only to the mutant sequence, P₂ (primer #2), the reverse primer, attached 772 base pairs away at the site of mutation, and P₃ (primer #3), the third primer, also forward, annealed to the wild-type sequence. Once our DNA was amplified, our PCR product was viewed using agarose gel electrophoresis (Wright et al., 2009). PCR with carefully designed primers can detect a single base-pair mutation in DNA, which can be used to test for the CF mutation. With epithelial tissue from the lung of a patient with Cystic Fibrosis, we obtained a positive result with the wild-type primers; indicating the absence of the R117H mutation. The product sequence was confirmed using restriction enzymes. We conclude that testing for Cystic Fibrosis and the R117H mutation is important to develop new assays for the fatal disease that has yet to be cured. 

Cystic Fibrosis is a disease that creates thick mucus in the body, which can lead to lung infections. The parts of the body that cystic fibrosis generally affects are the airways, liver, pancreas, small intestine, reproductive tract, and the sweat glands in the skin (Welsh and Smith, 1995). Children who receive two recessive alleles of the Cystic Fibrosis gene will likely become diagnosed with the disease. The consequence of this illness is an abnormally high concentration of extracellular chloride, which causes the mucus to become more thick and sticky than normal (Campbell et al., 2008). People diagnosed with Cystic Fibrosis have an average lifespan of 40 years (Elborn et al., 1991). Every year, new research and findings are helping to extend the average lifespan of a person diagnosed with Cystic Fibrosis (Campbell et al., 2008). 

The CFTR gene has about 170,000 base pairs; it is reduced to 4,440 bases once the mRNA leaves the nucleus because the introns are spliced out by the spliceosomes (Campbell et al., 2008). The Cystic Fibrosis Transmembrane Conductance Regulator (CTFR) codes for 1,480 amino acids (Dean, 2009). The CFTR protein is a chloride channel that is found on the cell membrane (Welsh and Smith, 1995). Normally found in the epithelial cells, the absence or malfunction of the CFTR channel prohibits chloride movement. This causes cells to give off extra sodium because the epithelial sodium channels remain open, while the CFTR channels do not function properly.  This causes mucus to become thicker and more resistant to removal, which allows the bacteria to grow in the lungs (Welsh and Smith, 1995).

CYSTIC FIBROSIS & CFTR

Figure 2: This is a depiction of the CFTR protein, showing the Amino Acid Sequence throughout. The squared region at the top is a representation of where the protein would traverse through the membrane. The two “loops” at the bottom represent the Nucleotide binding domains.

Group Members: Stephen Humes (humesst1@msu.edu), David Maison (maisonda@msu.edu), Lauren Priniski (prinisk2@msu.edu), Andrew Rygiel (rygielan@msu.edu)

Discussion

The inherited disorder of Cystic Fibrosis is a genetic disease that causes the inhibition of physiologically significant chloride channels. It is an ailment that creates excess mucus; mucus that possesses the capability of disturbing the function of several organs and ducts within the body. Children born with a pair of recessive alleles of the gene will likely have Cystic Fibrosis. When treated early, these individuals have the potential to live beyond the age of 40 (Elborn et al., 1991). The CFTR mutation of interest in this experiment is identified as R117H. It is present in approximately 7% of Cystic Fibrosis occurrences in the Caucasian population (Watson et al., 2004). This mutation occurs at amino acid 117 in CFTR and involves the mutation of base pair 483 from guanine to adenine; which alters the three base pair to code for Histidine, rather than Arginine (Dean et al., 2009). 

             The techniques utilized in this experiment include polymerase chain reactions, sequence-specific primer design, annealing conditions testing, and optimization of conditions pertaining to band visualization in gel electrophoresis. Thermo-cycle Polymerase Chain Reaction (PCR) is a process with improved product specificity and completed cycles consisting of various time fragments for denaturing, annealing, and extension. PCR amplifies DNA; it essentially and effectively creates carbon copies of the targeted gene. This process requires a template and primers in order to be conducted (Wright et al., 2009). Primer design is the most important factor when conducting allele-specific amplification. Combinations of primers are intended to amplify a specific region in order to help differentiate between mutant and wild type. When a single-base alteration is present, the most effective forward primers will correspond, at their 3’ end, to the anticodon for the mutation locus (Wittwer et al., 1993). The level of stringency is also important to eliminate the possibility of false-positives. A high stringency is ideal, whereas a low stringency allows for a greater possibility of error. The inclusion of magnesium chloride and raising the annealing temperatures are the primary, and most effective ways to raise the stringency in PCR.

After consulting with Dr. Douglas Luckie and our colleagues from the Cystic Fibrosis laboratory at Michigan State University, our original hypothesis was that the primer design would give definitive results as to whether the R117H mutation was present or not. This hypothesis was founded on the ideals that annealing and extension would be interrupted and inhibited if the 3’ site of the mutation locus primers does not anneal.

Before beginning our investigation, we had to first purify genomic DNA to use in PCR. It took several attempts to do this. During these attempts we made numerous changes to the protocol, including changing centrifuge speed and duration of incubation. We concluded that the fault was in our experimental protocol. The original protocol (Quigen, 2001) called for incubation in a sand bath; however, in our initial unsuccessful trials the incubation was done in a water bath. When this was corrected, the purification occured as expected. We believe this was due to the consistency of heat provided by the sand bath rather than water. Using the original PCR products, obtained from the purified bronchial epithelial cells of cystic fibrosis genomic DNA, we carried out our first set of tests to see if the R117H mutation was present (DNA is mutant). The results we obtained showed that this CFTR gene did not possess the R117H mutation. As seen in the agarose gel picture, there was no band in the P₁/P₂ lane. Conversely, a band of 772 base pairs in length was observed in the P₃/P₂ lane. Our hypothesis was verified by this particular gel due to the presence/lack of presence of bands in specific lanes. Once we achieve the task of distinguishing the type (wild) of the DNA, we proceeded with further testing to provide additional evidence to support these findings. Our primary objective, after achieving a working PCR, was to maximize the clarity results. In order to accomplish this, we needed to maximize the stringency for each PCR. The addition of MgCl and the alteration of annealing temperatures proved to increase the stringency enough to be able to distinguish which PCR was most effective (Figure 5). Thus, we found that our proposed annealing temperature of 57°C was negated for the reason that the band corresponding to that well was dull and barely visible. The optimal annealing temperature was 4°C higher than anticipated. This refuted our predictions since we anticipated the annealing temperature to be 3-5°C lower than the annealing temperature (Wright et al., 2009).

Restriction endonucleases are enzymes used to cut single or double stranded DNA. These are sequence specific and each enzyme cuts according to its corresponding codon. The two restriction endonucleases that were used in this experiment were EcoR1 and Dra1. These were chosen because they cut our 772 base pair length into distinguishable lengths. EcoR1, the first enzyme that was used, cut lengths of 90 and 682. This experiment was unsuccessful in several trials. We were unable to determine the cause of this so we decided to create a thick agarose gel to run the enzyme product in. It was believed that adding a higher percent of agarose would prevent the short band from running of the edge; but that was unsuccessful. After these trials, we concluded that the cause of this was the product lengths. The longer cut band is close in length to our 772 base pairs length product and therefore hard to differentiate the two; the shorter cut band also had a high potential to run off the gel due to its size. EcoR1 also has the potential to bind to various sites in the presence of magnesium (Woodhead et al., 1980). This could also be a contributing factor to the reason no band was observed. The restriction enzyme Dra1 was then used due to the position that it would cut at in our sequence; band lengths of 592and 180. The trial with this enzyme was successful and the desired band lengths were observed (Figure 6).

The investigation comparing the R117H and ΔF508 mutations yielded unexpected results.  The original hypothesis for the relationship between these mutations was that their frequencies would move and change together. However, the results showed that this was not the case; but rather they were completely independent of each other. The plotted data points for these mutations gave a Least Squares Line with a correlation coefficient value ~0.3. This result indicates that this line is not a proper representation of the changing frequencies for each R117H and ΔF508, respectively. It was concluded that the reason our hypothesis was refuted was due to the data points collected. If the investigation were to continue we would like to use many more data points to create more accurate Least Squared Lines, to represent the mutations.

Some research that we would like to conduct in the future would include several investigations testing our primers and conjoining assays. We would like to test the DNA we are expecting from Paul Quinton which contains the R117H mutation in order to obtain positive results for our mutant primers. Also, as previously stated, the expansion of our statistical examination of mutations would be ideal as it would give more definitive results. Eventually, future research could also include combining this R117H assay with other mutation assays from our colleagues; creating a single assay for many mutations.

Statistical Analysis

After researching the CFTR mutation R117H, our original investigation was concerning the rate of recurrence of the R117H mutation throughout the preceding years. Using statistical analysis, it was planned to determine if there was a trend between the frequencies throughout years, and to be able to predict, depending on the strength of the relationship, future frequencies. However, as the R117H mutation was researched, in numerous scientific journals and articles, a trend was noticed by the expression of the R117H in conjunction with ΔF508 mutation; primarily noted in studies related to pancreatic dysfunctions (Massie et al., 2000). We found this relationship intriguing since the pancreas is a major organ affected by Cystic Fibrosis. In Cystic Fibrosis the blocking of ducts inhibits the pancreas from excreting significant digestive enzymes; this can result in diabetes (Welsh and Smith, 1995). As a result of these findings, we decided to broaden our statistical investigation to include the trend of the ΔF508 mutation throughout the same years. We hypothesized that as the frequency of ΔF508 mutation increases, the rate of occurrence of the R117H mutation will also increase due to the common expression of the two mutations. 

The investigation comparing the R117H and ΔF508 mutation s yielded unexpected results.  The original hypothesis for the relationship between these mutations was that their frequencies would move and change together. However, the results showed that this was not the case; but rather they were completely independent of each other (Figure 7). The plotted data points for these mutations gave a Least Squares Line with a correlation coefficient value ~0.3. This result indicates that this line is not a proper representation of the changing frequencies for each R117H and ΔF508, respectively. It was concluded that the reason our hypothesis was refuted was due to the data points collected. If the investigation were to continue we would like to use many more data points to create more accurate Least Squared Lines, to represent the mutations.

Figure 7: The graph above is a representation upon completion of collecting data on the frequencies for !F508 and R117H mutations. The data was plotted to show the relationship between the frequencies for !F508 and R117H over time. This chart is showing the unaltered data for both mutations as well as a linear trend line to show the general change over time for the mutations. An inverse trend appears between the linear trend lines for each mutation. After acquiring and plotting the data we obtained, we concluded that due to our low correlation coefficient value, that R117H and !F508 are not related.

Additional Figures

Figure 4: The top image (Dean, 2009) is a depiction of CFTR Genomic DNA Sequence which is the site in DNA where mutations occur that cause the disease Cystic Fibrosis. Exon 4, squared in red, is where the R117H mutation occurs. The image below (Dean, 2009) is a magnified view of exon 4. At codon 117, when the R117H mutation is present, the guanine will mutate into adenine; causing an amino-acid change of Arginine to Histidine.

Figure 5: This gel picture incorporates 1KB ladder in lane 1, PCR with primer 1 (forward primer that will attach to the mutant sequence) and primer 2 (reverse primer attaching 800 bp away from mutation) in lanes 2,3, and 4 with varying annealing temperatures at 57ºC,59ºC and 61ºC respectively. In lane 5 is another 1KB ladder and in lanes 6, 7, and 8 primer 2 (reverse primer attaching 800 bp away from mutation) and primer 3 (control primer annealing to wild-type sequence) were used with varying annealing temperatures at 57ºC, 59ºC and 61ºC respectively. This confirmed the presence of wild type DNA. Lane 8 clearly depicts that 61ºC is the most effective annealing temperature and that the DNA given in lab tested as wild-type because a band was present at 772 bp.

Figure 6: This gel figure shows the restriction digest from our DNA sample after PCR was completed with enzyme EcoR1 in lanes 2 and 3 and Dra1 in lanes 6 and 7.  Faint bands can be detected at 600 bases in the Dra1 lanes. This enzyme cuts the 800 base pair band into a 600 and 200 DNA fragments. In lane 8, this bright band was the product of the previous PCR which has more bases than the two products in lanes 6 and 7. This signifies that the restriction enzyme Dra1 cut at the predicted location confirming this sequence.

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