Identification of ΔG529 Mutation in the ALD Protein of S9

Epithelial Cells Using PCR Allele Specific Amplification

 

 

 

 

Team Helix: Alex McNeill, Brooke Pallas,

Eric Kontowicz, Sophia Herrera

 

 

 

 

 

 

 

Abtract

The ΔG529 mutation responsible for Cerebral Adrenoleukodystrophy (CALD) is found on the X chromosome within exon six of the ABCD1 gene. This mutation is a deletion of the three nucleotides GGT, coding for Glycine, the 529th amino acid within the ATP-binding domain of the ALD protein (Braun et al, 1995).  A PCR assay was designed to consistently show the absence of the GGT deletion through the use of specific wild-type and mutant primers.  We hypothesized that by using these primers, the absence of the GGT deletion can be detected due to the wild-type primer’s ability to only anneal when the GGT codon at the 3’ end is present. It was predicted that by controlling the PCR environment, the absence of the ΔG529 mutation would be represented by a gel electrophoresis band at 222 base pairs.  Our assay design, modeled after the successful PCR techniques performed by Mosser et al (1993), holds purpose in its ability to ascertain that a patient’s DNA does not possess the GGT deletion, signaling the absence of this CALD causing mutation.  Although bands were created, our results were inconclusive due to failure to produce bands at the correct hypothesized lengths.  Additional sociological experiments were performed to gain a deeper understanding of possible patient symptoms.  Statistical analysis of pre, during, and post difficulty quizzes revealed a trend that participants undergoing paralysis and selective mutism recorded a significant increase in all difficulty questions, due directly to the experience of operating under the given disability.

 

 

Discussion

Experimental Summary

Cerebral adrenoleukodystrophy is the most common X-linked peroxisomal disorder with symptoms clinically recorded in approximately 1:20,000 males (Braun et al, 1995).  CALD has devastating effects on the central nervous system with the onset of neurological symptoms that usually begin between the ages of five and twelve.  Death often occurs around ten years after the onset of symptoms (Mosser et al, 1993).  A deletion of the codon GGT, coding for the amino acid Glycine, in exon six of the ABCD1 gene located on the X chromosome causes a loss of the 529th amino acid in the sequence of the ALD protein.  This deletion causes a malfunction in the ATP-binding domain of the ALD protein (Braun et al, 1995).  This malfunctioning peroxisomal membrane transporter protein can no longer actively transport VLCFAs into peroxisomes, where they are broken down into individual organic components (Kemp et al, 2001).   These organic products of the broken down VLCFAs are normally used in the synthesis of the neuron insulating material, myelin. Myelin acts as a medium surrounding long stretches of neurons and allows for the efficient conduction of electrical impulses (Braiterman et al, 1999).  It is the inability of CALD patients to digest VLCFAs that result in their inability to produce needed myelin, further resulting in the degradation of neurological body control.  Although PCR has been proven to help diagnose CALD, the question under study is if PCR is useful in the detection of this specific mutation of the disease. It is hypothesized that through the use of our allele specific primers the absence of the GGT (Glycine) deletion in the 529th amino acid can be detected because the wild-type  primer will only anneal with the presence of the 529th GGT nucleotides at the three prime end. With the use of these three allele specific primers, a strong band at 222 base pairs was predicted to represent a positive identification of the absence of the GGT deletion, through agarose gel electrophoresis (Braun et al, 1995). While PCR is projected to be able to help detect the absence of the mutation, there are potential weaknesses in the assay. Due to the fact that the entire human genome is being used, versus just the X-chromosome, it is possible that the primers could anneal to a similar DNA sequence that is present elsewhere (Gu et al, 2000).  In addition, the natural repetitive nature of the ABCD 1 gene makes it possible to produce a band at a length other than the predicted 222 base pairs (Braun et al, 1995).  As seen in Figure 1, there are no bands within any well.  This was likely due to the use of too high of an annealing temperature and the resulting lack of primer annealing.  In Figure 2, streaking and non specific bands were observed in all utilized lanes.  These results were likely to be due to the lower, more successful annealing temperatures of 53ºC for lanes 2-4 and 55ºC for lanes 6-8, as well as to the addition of mineral oil and an extended initial denaturation period of five minutes to the PCR cocktail.  The bands in all lanes of Figure 2 are too small in length to be the desired region, and appear to be primer dimers or the result of primers annealing to themselves.  The apparent streaking within the lanes is representative of poor primer annealing and non specific binding.  Figure 3, represents the most successful gel produced, and is attributed to the increased agarose gel and DNA concentrations as well as the extended gel electrophoresis time.  The faint bands produced in lanes two and five appears to be 100 base pairs in length, and although expected, are not at the hypothesized lengths.  The presence of the control band at 511 base pairs is also absent within lanes two, three, five, and six.  The incorrect heights of produced bands and the presence of undesired bands can be attributed to the repetitive nature of the ABCD1 gene, as well as non specific binding of primers.  The removal of the primer dimers in the “All Primers” lane represents the greatest achieved improvement in gel results.  This leads to the conclusion that the optimal annealing temperature of the designed assay lies with the range of 53ºC-55ºC.

To complement the laboratory investigation of this disease, a sociological experiment was developed in order to help further the understanding of what patients with CALD must endure everyday of their lives after the onset of symptoms.  Due to the fact that CALD leads to the deterioration of the myelin within the nervous system, a vast number of phenotypic symptoms are seen in patients who test positive for CALD.  With possible symptoms ranging from the loss of vision and hearing, to the inability to speak, swallow, and control both gross and fine motor movements, patients lead an incredibly debilitated life (Carmant et al, 1998).  With these disabilities in mind, it was decided that two group members will spend a week without the ability to use their legs and the other two members will spend a week unable to use their voice to speak.  All members took a pre-quiz before the experiment began, regarding the perceived difficulties of their specific disability.  This same quiz was then also taken after their loss of that ability, and their responses were charted and statistically compared in order to understand the actual difference that the experience had upon them.  It was predicted that post experience quiz results would show drastic differences when compared to that of the pre quiz results.  Jeff Chu’s article in Time magazine discusses his thirty day experiment and how, by undergoing this sociological experiment, the group gained further understanding, and an interpersonal intelligence of how patients with CALD live their lives and how people around them are affected by the disease as well.

            The sociological experiments in which two participants underwent paraplegia, and two participants underwent selective mutism for five consecutive days, was accomplished with the use of a wheel chair and no verbal communication respectively. In addition, members also took, on average, two quizzes per day during their periods of disability, and members kept a journal of times inhibited by disability per hour (see Appendix for quiz questions). The “during” disability quizzes were also taken during a normal week of the participants’ life to allow for a baseline, control reading. The pre and post quiz results were analyzed through an interaction plot in which the influential factor for the change in the quiz results was determined by examining the produced slope of the numerical change in each question for before and after the experiment.  The significance of the change was then analyzed through an ANOVA, and produced a p-value, allowing for the support or rejection of the null hypothesis. The p-value of 0.00014 allows for the rejection of the null hypothesis and shows that the effect of the disability plays a crucial role in the change of the quiz results (Figure 5).  The quizzes taken during the experiment were charted against those taken not during the experiment, and the mean quiz results were analyzed through a statistical paired T-test, which also produced a p-value of 0.0206 and allowed for the rejection of the null hypothesis (Figure 4).  Finally, the journal of inhibitions was analyzed through the production of histograms for each disability, charting amounts of inhibitions against the hour of the day in which they occurred (Figure 6). This allowed for the visual representation of the times of day that became most difficult for each disability.  These spikes in inhibition rates tend to be centered around meal times, and evening hours.

Original Predictions

It was originally predicted that through the use of the designed allele specific primers, and the control of the PCR environment, that the absence of the GGT deletion within exon six would be represented by a gel electrophoresis band at 222 base pairs.  Through actual laboratory research, the proposed predictions were never achieved, due to the production of bands at undesired lengths, as well as in unexpected lanes.  Although the bands were produced at undesired lengths, they do indicate that the optimal annealing temperature of the PCR products to be within the range of 53ºC-55ºC. 

The original prediction for the sociological experiment was that the change in the pre and post quiz results would reflect an increase in the participant’s difficulty to perform daily life activities.  The actual found data supports these predictions and showed that the disability was the direct cause of the increase in participant’s daily life activities.

Future Studies

          There are many things that could be altered if this assay was ever to be followed in the future.  Overall, conclusive data was not obtained, but some things that may have attributed to this could potentially be corrected.  For one, mutant DNA could be obtained, and the mutant primers could be tested for success.  Another pitfall that could be avoided, was that the primers were unintentionally over-diluted by pipetting errors, so it would be very beneficial to reorder primers. The DNA concentration yield was also low (0.005mg/mL) so rerunning DNA extraction would potentially result in a higher concentration and more DNA in the PCR cocktail for the primer to anneal to.  A positive control that would be effective would be to run the gel electrophoresis with lambda virus primers.  This would be effective, due to having already proven their success. It also would confirm that the fundamentals, such as the gels and the PCR machine of the assay, are working properly.  An additional idea for future studies would be to run PCR using digestive enzymes to verify that the band present is the band desired (Braun et al, 1995).  Furthermore, the produced bands could be genetically sequenced, in order to determine if they are made up of the actual hypothesized region (Kemp et al, 2001).  These ideas identify problematic areas of our research, and offer alternative routes for future researchers to potentially obtain successful definitive results.

 

 

 

 

 

 

 

 

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