Using PCR primers as diagnostic assay for mutation in human MAOA gene and in mapping a piece of fainting goat genome

Connor McCormick, Katharine Ramp, and Sally Wicks

Abstract

           A deficiency of the enzyme monoamine oxidase A (MAOA), responsible for catalyzing the deamination of the neurotransmitters serotonin, dopamine, and norepinephrine is linked to the presence of antisocial and aggressive behavior in both mice and humans (Alia-Klein et al., 2008 and Cases et al., 1995). A point mutation at nucleotide position 936 of the MAOA gene on the X chromosome results in drastically lowered levels of MAOA, leading to exhibition of aggressive behavior and decreased mental capabilities (Brunner et al., 1993). Polymerase chain reaction (PCR) was conducted utilizing published primers to determine the presence of the 8th exon of the MAOA gene in our human sample (Tadic et al., 2001). Additionally, we designed primers that anneal to this mutation in human DNA. We hypothesized that discriminating primers could detect the presence of a point mutation causing Brunner syndrome in humans as well as to predict the MAOA nucleotide sequence of myotonia congenita, commonly known as the fainting goat. This allowed us to predict the genome of a previously unmapped species as well as to develop a diagnostic assay for a psychological illness using the same technique. After conducting PCR and running gel electrophoresis with wells containing a positive and negative control, published human primers, diagnostic primers for the MAOA gene in humans, and primers to detect the MAOA gene in fainting goats, we were unable to conclude the band lengths of our products because we were unsuccessful in producing a well separated ladder to compare our bands to.

Results

Figure 4: Final gel following gel electrophoresis containing homolog, diagnostic, and published PCR products. All PCR products were placed in same 0.8% agarose gel in order to avoid any potential discrepancies created by minor differences that could occur between gels. Well 1 contains 6 µL of 1 Kb+ molecular weight ladder. Well 2 contains 7 µL of lambda bacteriophage PCR product, as seen in Figure 3A, functioning as a positive control. Well 3 contains 7 µL of the PCR product created using the published for the 8th exon of the MAOA gene (Tadic et al., 2001). Well 4 contains 7 µL of the product of the PCR primers designed as a diagnostic assay for the point mutation resulting in Brunner’s syndrome and well 5 contains 7 µL of the PCR product of the primers designed to bind to the homologous sequence in goats. Well 6 contains 7 µL of water, functioning as a negative control. Each of the aforementioned samples had the addition of 3 µL of 6x gel loading dye prior to being inserted into the well. Both controls functioned correctly, however a clear ladder is not present, and the relative position on the gel of the four bands is not correct; HP, HD, and G have base pair lengths of 130, 210, and 125, respectively, while the lambda control has an experimental base pair length of 377 and an actual base pair length of 395. HP, HD, and G therefore did not function correctly, as they are closer to their respective wells than the lambda control, meaning they have a longer base pair length.

Discussion

           While variance in MAOA protein levels are not uncommon when comparing individuals and does not always result in the presence of antisocial behavior, an association has been noted between antisocial behavior and low MAOA protein activity when combined with environmental factors, such as childhood maltreatment (McDermott et al., 2009). The individuals that have this MAOA deficiency are more likely to develop disruptive behavior following childhood maltreatment than individuals with high MAOA activity (Caspi et al., 2002). In addition, the presence of a rare point mutation at the 936th nucleotide position in the MAOA gene results in a consistent phenotype of aggressive behavior related to drastically lowered MAOA production, commonly known as Brunner syndrome (Scott et al., 2008). This mutation results in a glutamine codon changing to a termination codon due to a cytosine to thymine change, causing premature termination of the protein (Brunner et al., 1993). Our purpose when designing this study was to create an effective diagnostic test for this point mutation that would allow for the identification of individuals with Brunner syndrome which has the potential to be a target for gene therapy (Palmer et al., 2015). We hypothesized that a PCR primer that anneals to nucleotide 936, which is the site of mutation in the mutated strand of DNA, containing the corresponding nucleotide sequence for the wild type genotype, will create bands following gel electrophoresis that correspond to the 210 base pair length of DNA amplified by these primers in a non-mutated sample. 210 is the anticipated base pair band length because from the 5’ end of the forward primer to the 5’ end of the reverse primer is 210 base pairs in length. Two other sets of primers were utilized, one being a published set of primers used on a Dutch family that were the first studied cases of this mutation that replicate the exon 8 of the MAOA gene (Tadic et al., 2001). These primers served to confirm that this segment of the MAOA gene was present in our sample. The second set of primers are designed primers that were also intended to amplify a segment of DNA that can contain a point mutation on the MAOA gene in a fainting goat homolog. Primers were designed using conserved DNA sequences across several species of mammals.

Predicted Results

           The central focus of our study was surrounding the PCR primer that we designed to detect the point mutation that results in Brunner syndrome and decreased MAOA levels. This primer, which was predicted to be displayed via a 210 base pair band during gel electrophoresis, was designed to anneal during PCR only to the wild type sequence of this segment of DNA. This was controlled by having a guanine in the primer that is complementary to position 936 in the wild type DNA sequence rather than an adenine that would be complementary to the mutant thymine at nucleotide position 936 in the MAOA gene (Brunner et al., 1993). This band showed up in gel electrophoresis representing human DNA with the wild type genetic sequence.

           To ensure the presence of the specific genetic sequence of the MAOA gene in study, we utilized a set of published primers that have replicated exon 8 of the MAOA gene in previous studies. A 130 base pair band was predicted to be represented in the gel which was a result from this set of primers in our gel electrophoresis. MAOA production occurs primarily in mitochondria; this means genetic material from human cell cultures is likely to contain MAOA genetic material, as mitochondria are in most human cells (Binda et al., 2011). Additionally, this primer was previously applied and successfully amplified genetic material.

            The third set of primers are derived from published DNA sequences of the MAOA gene that are conserved in humans, boars, wolves, and gorillas. These were found using the NCBI Nucleotide BLAST program, and suggest that there is likely a similar conservation of DNA sequence in fainting goat, another mammal (Boratyn et al., 2013). Therefore, we predicted that the 125 base pair band seen in the previously mentioned study will be observed when PCR with those primers is replicated in the fainting goat gene, allowing us to conclude that the conserved regions present in the other mammal species are also conserved in the genome of myotonic goat.

Ultimate results and final thoughts

           The diagnostic test reiterated our expectations by showing a band. The human sample came from two people who have never been diagnosed with anger issues or impulsive behavior. It was predicted that if there was the mutation in the DNA, then the primers would not bind and the band would not be present. Both the designed primer for the goat and the published primer for the human also successfully bound to the DNA. With this in mind, the lengths of each band are not what we predicted. Since we used KB+ ladder which can go all the way down to 100 bp the bands for all our PCR products should be far lower in the gel. With MAOA deficiency having a known genetic link, ethical dilemmas have recently been raised regarding the idea that some individuals are genetically predetermined to be criminals or in some other way an antisocial individual (Schmidt et al., 2000).

Future Directions

           Although this experimental design has been done multiple times, the specific PCR-based MAOA diagnostic-based experiment for humans is a novel experiment alongside the designed primers for the myotonic goat. Just like with all new experiments there were some setbacks. As shown in Figure 3, one can note the particularly curved bands. This was a common occurrence. We tried changing the amount of loading dye, the voltage that we ran the gel electrophoresis at, running the gel for a shorter period of time, the ratio of loading dye to PCR cocktail in each well, we had another group make a gel for us. All of this with no avail. We did get straight bands one time. For this to happen, we ran the gel a little bit shorter than we usually did: 12 minutes. The acceptable thing about having bent hands is that they are still bands and the ladder was separated enough to make a semi-log plot.

Nonetheless, with a statistically significant semi-log plot, we moved forward. Once we made the PCR products with the human diagnostic primer, published human primer, and designed primer for the fainting goat DNA, we began to run gels. This was immediately problematic. In the first gel, none of the PCR products showed up except the ladder. This is most likely due to the fact that the annealing temperatures for this ‘batch’ of products were too high; leading us to believe that the primers may not have bound to the DNA set to this temperature.

On the other hand, referring to figure 4, it can be observed that there are bands across the gel in all wells. Although this is the case, the band sizes are the wrong length. This can indicate that the primers were not specific enough and bound to the wrong section of DNA (Sommer and Tautz, 1989). When the DNA went through PCR, a much larger portion was amplified than intended (PCR troubleshooting, 2017). Everything else in this gel was kept constant with the gel that failed to get bands except the annealing temperatures indicating that the annealing temperatures were too high.

If more time was permitted, more gels would be run with fresh PCR products at annealing temperatures close to the successful gel shown in figure 4 which had a published human primer annealing at 63.4°C, a diagnostic human primer annealing 52.6°C, and the designed myotonic goat primers annealing at 45°C.