Spinal Muscular Atrophy

The Bleu Team

Jordyn Winship, Brandon Heins, Christopher Salas, and Sami Tatrow

            Spinal Muscular Atrophy (SMA) is a degenerative motor neuron disease caused by mutations in the SMN1 gene (Lunn and Wang, 2008). SMA Type I is the most severe case and is caused when the 6^th nucleotide of exon 7 in the SMN1 gene changes from C to T, resulting in a faulty SMN1 protein (Lorson et al, 1999). To identify the presence of this mutation, polymerase chain reaction (PCR) was used to amplify a target sequence of human DNA and the products were analyzed using gel electrophoresis. Wild-type DNA was purified from S9 cells of a cystic fibrosis patient and no mutant DNA could be obtained. We hypothesized that primers designed using the Yaku method (Mprimer/Wprimer, Rprimer) would increase binding specificity. The intentional mismatches on the 1^st and 3^rd bases in from the 3’ end of the forward primers reduce the chance of nonspecific binding by not allowing any of the three bases on the 3’end to anneal to the DNA, resulting in an extremely low chance that Taq polymerase can extend the primer (Yaku et al, 2008). Neither Wprimer nor Mprimer amplified a 757 base pair sequence of wild-type DNA; Wprimer was expected to while Mprimer was not. A sociological experiment was also conducted to experience the challenges of SMA as well as to visualize how students with different academic backgrounds, science and non-science, react to a person displaying symptoms of SMA. To measure this, students’ reactions were observed and recorded as either normal or adverse and the results of the two different groups were compared using a chi-square test. Although statistically insignificant (p=0.08), we correctly predicted that the students with science backgrounds would react less adversely than the students without science backgrounds because they are exposed to and have more knowledge about disabilities (Corrigan et al, 2001).

              Spinal Muscular Atrophy (SMA) is an autosomal recessive genetic disorder, causing motor neurons to degrade (Akbari et al, 2011). The severity of SMA varies from Type I to Type IV, with Type I being the most severe; our study focuses on SMA Type I (Lunn and Wang, 2008). It is caused by a cytosine to thymine change on the 6^th base pair in exon 7 of the SMN1 gene, which is located on chromosome 5 (Monani et al, 1999). This C to T change activates an exon splicing enhancer and when the pre-mRNA is spliced, exon 7 is cut out (Nlend et al, 2010).                                           
              Both the SMN1 and SMN2 gene produce the survival of motor neuron (SMN) protein, which is used to maintain motor neurons (Akbari et al, 2011). SMN1 normally produces a full length functional protein, while SMN2 mainly produces a shorter, less functional protein in addition to a small amount of the full length protein. When the C to T mutation occurs in SMN1, the gene starts producing nonfunctional SMN proteins, and the amount of full length functional protein produced by the SMN2 gene is not enough to sustain the life of motor neurons within the body (Pedrotti et al, 2010).
              We originally predicted that Wprimer would anneal to the 6^th base pair of exon 7 only if the nucleotide base at that location was cytosine and produce a band of 757 base pairs when combined with wild-type DNA. We also predicted that Mprimer would anneal to the 6^th base pair of exon 7 only if the nucleotide base was thymine and produce a band of 757 base pairs when combined with mutant DNA. Rprimer would be used in both reactions and be able to discriminate between the SMN1 and SMN2 by annealing to a nucleotide sequence that is specific to SMN1. We hypothesized that by using the Yaku method to design primers, binding specificity would increase. This increased specificity is due to intentional mismatches on the 1^st and 3^rd bases in from the 3’ end of the forward primers. This reduces the chance of nonspecific binding by not allowing any of the three bases on the 3’end to anneal to the DNA, resulting in an extremely low chance that Taq polymerase can extend the primer (Yaku et al, 2008).
              All PCR reactions were run using wild-type DNA that was negative for SMA Type I. No reactions could be run using mutant DNA that was positive for SMA Type I because we were unable to obtain a genome containing the mutation. None of the reactions containing Wprimer and Rprimer yielded a band of any length. This could have been due to a number of different things: the DNA may not have been pure enough, improper times and temperatures could have been used in the PCR reactions, or there may have been incorrect amounts of each reactant in the PCR cocktail. However, these were ruled out through the use of controls.
              Several positive controls were used along with a negative control. One positive control was found in the manual PCR reactions, where a portion of the lambda virus RZ gene roughly 500 base pairs in length was successfully amplified and fluoresced in the gel. This was used to determine an appropriate volume of all reactants in a single PCR reaction cocktail as well as to determine an appropriate number of cycles to use in each PCR reaction. Another positive control was found in using primers from a previously published study on the SMN1 gene. Here, a portion of the wild-type SMN1 gene roughly 307 base pairs in length was successfully amplified and fluoresced in the gel. This was used to confirm that our purified DNA could be amplified using PCR and that our reaction cocktails were mixed properly. A negative control used in this experiment was running PCR reactions at various temperatures using Mprimer and Rprimer with wild-type DNA. As expected, no bands showed up in the gel at any of the temperatures because the sequence of Mprimer is not complementary to wild-type DNA.
              These results refute our hypothesis that the primers designed using the Yaku method would increase binding specificity because we were unable to successfully amplify wild-type DNA. Because all of the controls used were successful in supporting different aspects of our experimental design, this leads us to believe that either the Yaku method does not increase binding specificity. However, this does not necessarily mean that we hypothesized incorrectly. A number of things outside of the controls could have gone wrong and led to unsuccessful PCR reactions: the primers may have been designed with sequences that were not complementary to wild-type DNA, the primers ordered from IDT may have been faulty, or the proper annealing temperature may have not been found. Having mutant DNA would have enabled us to obtain much more data to interpret our hypothesis because it would have allowed us to test Mprimer with its complementary sequence.
              A sociological experiment was also conducted in which one member of the group simulated symptoms of SMA Type I in a wheelchair while the others observed the reactions of Michigan State University students coming from different academic backgrounds, science and non-science. The students with a science background were observed in Holmes Hall and the Natural Science Building while the students with a non-science background were observed in Case Hall and the Business College Complex. The reactions were classified as either adverse or normal. We hypothesized that students with a science background would have a lower rate of adverse reactions to a disabled person than students with a non-science background because they are exposed to and have more knowledge about disabilities (Corrigan et al, 2001). Our results refute this hypothesis because although students sampled in Holmes Hall and the Natural Science Building did in fact have fewer adverse reactions than the students sampled in Case Hall and the Business College Complex, the results were found to be statistically insignificant. One way in which this portion of the experiment could have been improved is with more trials. Each location was only visited once for 30 minutes and although nearly 700 people were observed, replications would be needed to further validate our findings. Another drawback to this experimental design is that we could not confirm whether the students being observed had an academic background in science or some other area. It was assumed that Holmes Hall and the Natural Science Building would have primarily science students because science classes are mainly held in those two locations. It was also assumed that Case Hall and the Business College Complex would have primarily non-science students because classes in subject areas other than science are mainly held in those two locations.

              The designed primers Wprimer and Rprimer have yet to anneal properly to DNA that is wild type for SMA. Unfortunately, not enough time was allotted to allow for as many replications as would need to be performed. Given six more weeks, different annealing temperatures would ideally be used as well as different volumes of the ingredients in the PCR reaction cocktail. For instance, the DNA template volume could be increased or the concentrations of MgCl₂ could be altered. Using a master mix to make each PCR tube did not work and neither did pipetting out each ingredient into each tube individually. This leads us to believe that the correct annealing temperature may have not been used yet or that human error caused the tube that was run at the correct temperature to not run properly. More replications at the previously tried annealing temperatures as well as new tests with new annealing temperatures should be conducted.
             Additionally, mutant DNA known to be positive for the SMA phenotype was never able to be acquired. If there was more time it would be ideal to obtain this DNA and try to amplify it using design primers Mprimer and Rprimer. Although the previously mentioned primers did not anneal to the wild type DNA as expected, this could be a false negative and does not imply that they are designed properly to detect the presence of the C to T mutation that causes the disease.
               The sociological experiment that was conducted was not found to be statistically significant. One thing that should be done to improve the validity of the collected data is to repeat the trials in each location multiple times. Each location was only visited once for 30 minutes and although many people were observed, it would be better to replicate the results in multiple trials.