Huntington’s disease detected in Homo sapien Calu-3 lung epithelial cells based on CAG microsatellite in HTT via PCR

By: Olivia Allen, Amber Faasen, Mitch Kelley, and Anthony Lai

Abstract

Huntington’s disease is a neurodegenerative disorder characterized by many physical as well as psychological symptoms. Huntington’s is caused by the trinucleotide repeat CAG that is 36 to 120 repeats long at chromosome 4p16.3 on exon 1 (Ravina et al., 2008). PCR was utilized to detect the presence of a mutation in the Huntington gene, HTT that causes the long sequence of CAG repeats. It was hypothesized that the designed primer would be able to determine if someone has Huntington’s disease. This hypothesis was tested by using PCR on purified DNA of mutant and wild-type patients. Once the PCR products were analyzed by gel electrophoresis, the bands allowed the determination of wildtype or mutant DNA. The presence of bands at a base pair length above 203 that include the CAG repeats and the length of the primer will indicate that the patient has Huntington’s disease (Kremer 1994). The length of DNA is predicted to be below 200 bp for wildtype DNA and anything over that would be considered mutant. Gel electrophoresis provided no definite determination of a band length of below 200 bp, so further troubleshooting was planned out. A social experiment was carried out in order to become familiarized with the symptoms of a Huntington’s patient. This experiment consisted of walking on crutches and not speaking for four consecutive days, and the Quality of Life was rated. With a linear regression and an R² value of 0.9058 it was concluded that as time progressed with symptoms of a HD patient, the quality of life decreased steadily and consistently. This PCR assay is significant because many people are affected by this dominant disease and the detection of this mutation is crucial for diagnosis of HD. Additionally, the sociocultural experiment gives insight on the plights of HD patients.

Quality of life all group members after completing four days of moderate impairment similar to a Huntington’s patient. This graph is a good indicator that the quality of a persons’ life greatly decreases due to symptoms of Huntington’s disease. Series 1-4 represent the quality of life rating for each of the group members throughout a span of 4 days. Series 5 represents the average Quality of Life rating for all of the participating members on their respective days. The activities for a normal day were carried out while incorporating symptoms of a Huntington’s patient (the use of crutches and inhibited talking). A quantity that symbolizes a certain quality of life rating was chosen at the end of each day by the participants to signify the effects of being impaired. The Quality Of Life Scale was used from the American Chronic Pain Association and ranges from 0 (non-functioning) to 10 (normal quality of

Gel electrophoresis of purified wildtype Calu 3 epithelial cells and DNA segment containing HTT mutation. Thermocycling conditions consisted of denaturation at 95°C for 5 minutes. 30 cycles were run for each of the samples at 94°C for 45 s, 62°C for 30 s and 72°C for 60 s, with an extension of 72°C for 7 minutes. Lanes 1 and 8 contain a 1 kb plus ladder. Lanes 2 through 4 consisted of the wildtype DNA while lanes 5 through 7 consisted of the HTT mutation DNA. The ladder once again did not seem to run properly through the gel, creating not very distinct bands. Lanes 5, 6, and 7 all show the HTT mutated products. It is difficult to distinguish the actual band length due to the faulty ladder products. However, there are distinct bands in all three lanes at about 1,000bp and 850bp. Lanes 5 and 7 have fainter bands at 400bp as well as 300bp and just above 200bp. Those of which are similar to the bands seen in lane 6 of figure 3.

Discussion

Experiment Summary

            Huntington’s disease affects 3 to 7 per 100,000 people of European descent. It has a detrimental effect on the various neurological functions. Huntington’s disease is an autosomal dominant trait, so if one of the parents of the patient has it, then the patient has a 50 percent chance of having it (Bates et al., 2003). This attack on the neurological system causes various symptoms such as uncontrollable muscle movements and degenerative cognitive function (Bourne et al., 2006). The median survival time is about 15 to 18 years after diagnosis, and the majority of people die around age 55 (Harper 2005). This mutation on chromosome 4p16.3 on exon 1 affects the HTT gene (Harper 2005). The HTT gene of a healthy individual usually repeats the trinucleotide sequence (CAG) about 10 to 35 times. The CAG trinucleotides for mHTT or mutant HTT repeats 36 to 120 times. This microsatellite leads to Huntington’s disease. (Margolis et al., 2003). This repeat causes the normal protein huntingtin, created by the HTT gene, to elongate. This elongated protein eventually breaks into small, toxic pieces that bind together and accumulate in the nucleus and cytoplasm of neurons. This accumulation disrupts the neuronal function, leading to neuron cell death and the subsequent symptoms of Huntington’s disease (Palidwor et al., 2009). Certain PCR methods have been shown to diagnose various genetic diseases (Levin et al., 2006), but the question in this specific assay is to determine if an unknown individual has Huntington’s disease. It was hypothesized that both allele specific primers, which were designed to be 53 base pairs upstream and 45 base pairs downstream from the CAG repeat, respectively, would be able to target the correct sequence on mutant HTT DNA to amplify. From this, the correct diagnosis of a proband should be able to be determined.            

To further the experimenter’s understanding of Huntington’s disease, experimenters lived the life of a person with Huntington’s disease for four days each. They acted out a major symptom of Huntington’s patients -immobility. This was done by each experimenter being confined to crutches. Data on various thoughts and feelings was recorded in a journal daily (see appendix for entries). Also, feelings were measured using the quality of life scale. The results of this sociological portion of this experiment show the correlation between time spent on crutches and staying silent with a lower quality of life scale rating.

Original Predictions

Calu-3 epithelial cells from a patient who was known to contain the wild-type HTT gene were amplified by PCR. The DNA was tested for Huntington’s disease by analyzing the bands made by gel electrophoresis. A separate Calu-3 epithelial cell from a diseased person with Huntington’s disease was also tested. In order to target the HTT part of the human genome two different primers were designed. The two different primers start at the 5’ end and are 16 base pairs long each.  Because many parts of the genome contain the repeated trinucleotide sequence, it is important to ensure that the right part of the genome is targeted and amplified. The gel electrophoresis of the known person with Huntington’s disease was predicted to have an increased trinucloetide sequence for the HTT gene (36 to 120 repeats), thus indicating that this person was positive for Huntington’s disease. The homozygous wild type DNA was predicted to have a normal amount of the trinucloetide sequence of the HTT gene (10 to 35 repeats). The two different primers were designed to ensure that the specific sequence within the DNA was targeted and to see if the primer was able to detect if a person has the mutated HTT gene.

Ultimate Findings

Various temperatures were tested in multiple experiments to find the optimal annealing temperature, which can affect the annealing of certain primers. The concentration of the buffer solution can also affect polymerase activity in PCR. The ideal annealing temperature was determined to be 60-62ᵒC based upon the PCR products that were made visible by gel electrophoresis. The designed primers were able to decisively tell if a person had the elongated segment of the CAG repeat; thus they were able to tell if a person has Huntington’s disease. PCR and gel electrophoresis were shown to be an effective method in determining whether or not a person has Huntington’s disease (Potter et al., 2004). However, PCR has been shown to not detect Huntington’s disease if the person has more than 115 repeats (Levin et al., 2006). While greater than 115 CAG repeats is rare, misdiagnosis of patients that may have the worst symptoms can be harmful. However, it is possible to accurately profile a lengthy microsatellite by end-labeling restriction fragments that contain the expanded repeat (Sathisivam et al., 1997). In this specific diagnostic assay, the DNA with the mutant HTT gene that was ordered was a typical Huntington’s disease sample, meaning that it contained less than 115 CAG repeats. Also, because the sequence of CAG repeat could be in multiple places of person’s genome, primer 1 was created that stretched out 53 base pairs before the repeat.

The sociological investigation experiment revealed that the disease had a direct correlation to lesser degree of happiness. The more severe the conditions, the less happy the person was.  Everyday activities were also found to be much more difficult and time consuming which is discussed in detail in the experimenter’s journals (see Appendix). This negative impact of just one of the symptoms of Huntington’s disease is unmistakably evident in the results from this independent experiment. Each experimenter’s quality of life rankings decreased as the days went on, correlation between the time spent being immobile and the quality of life ranking –the longer spent on crutches, the lower the quality of life ranking.

A preliminary PCR test was run using lambda virus DNA in order to determine if a successful PCR product could be obtained. Successful bands were produced at 25, 30, and 35 cycles. These PCR products were run in a gel and the bands showed up in the correct spot (data not shown).

Following the preliminary PCR test, an extraction and purification test using Calu-3 human lung epithelial cells was conducted. The DNA was purified using the QIAGEN DNA extraction kit and method and was then run through gel electrophoresis to determine the concentration of the DNA. A “purity ratio” of 1.8 is considered to be an ideal ratio of DNA to protein (CRAIC 2011). The PCR product of this was unsuccessful after running it through a gel. This may be because of the absence of dye used when running a gel. The dye can be important to a successful gel because it holds the DNA within the wells. The unsuccessful product may also be due to inaccurate loading of the gel. Another way to determine the concentration of DNA is to use spectrophotometry. Three trials were run and the purity of the DNA in each trial was found to be an average of 1.2 (Table 1). This is not the most ideal ratio of DNA to protein. In order to ensure a good DNA concentration is obtained next time, the time the DNA spends incubated in a warmer temperature can be extended. The range for incubation was anywhere from 1 minute to an hour (Qiagen 2010). However, the longer the time spent incubation, the better the results will be.

Both wild type DNA and mutant DNA were run at 54°C, 57°C, 60°C, and 62°C annealing temperatures. 60°C and 62°C were determined to be the best temperatures and yielded the best results. Successful PCR products were created at this temperature, however non-specific binding was evident. This could be that the annealing temperature was still too low or that primer dimers occurred.

Future Directions

There are many factors of this assay that could be altered that could lead to more conclusive results. The optimum DNA used in order to easily obtain a visible band is typically 20 ng (Lewis 2001). The DNA that was run in the gels was 15ng so maybe for the DNA used in this assay, it was necessary to use the optimum amount of DNA because too little DNA may cause the smallest bands in gel to be too faint. Too small of bands for this particular assay poses a problem because the expected band sizes is above and/or below 200 base pairs. Another way to get better resolution of small band lengths is to use a thicker gel (Lewis 2001). 2% agarose gels, rather than 1% gels should be used in subsequent tests. Other alterations that may be necessary for use in further experiments would be additives in the PCR cocktails such as betaine and dimethyl sulfoxide (DMSO) to increase specificity of primers and optimize yields of the amplification products (Frackman 1998). DMSO facilitates DNA separation by disrupting the improper base pairing that can occur in GC rich regions on DNA, like those seen in this particular assay. Betaine equalizes the ratio of AT-GC base pairing to stabilize the DNA (Frackman 1998). The results of the Frackman paper revealed that without one of these additives, no specific products were visible.  Another protocol that would be beneficial for future experiments would be the use of hot starts in the PCR assay. This allows for greater specificity and amplification as well. There are multiple hot start approaches but basically pre-amplification heating would be done. This would be done by heating the PCR cocktail up prior to adding taq polymerase to ensure that it does not begin extension at too low of temperatures before the DNA has been completely denatured (Paul 2010).  Also, further tests could be executed that would alter PCR and gel electrophoresis to account for more than 115 repeats by end-labeling restriction fragments. These experiments would ensure that even the small percentage of people with more than 115 could be tested with PCR and gel electrophoresis to see if they had Huntington’s Disease. This is because the length of CAG repeats correlates to age of onset and severity of symptoms (Andresen et al., 2007).