Figure:

Figure 4: Amplification of exon 8 for the SMN1 gene.  Wells 1 and 6 contained 1kb plus ladder, well 2 contained wildtype DNA, well 3 contained type 1 SMA phenotype DNA, wells 4 and 5 contained type II SMA DNA. Bands are present after gel electrophoresis of regions amplified by the outer common primers (386 base pairs) and the sense forward primer (139 base pairs).  Optimal annealing temperature for the primers was 55°C, and the PCR cocktails were allowed to run for 35 cycles in the thermocycler.  Go-taq® green master mix provided the best results in PCR reactions.  Amplifications of SMN2 exon 8 regions were not present after gel electrophoresis, which is most likely due to the inability for the antisense primer to bind.  Excess primer is visible at the bottom of the figure.

Abstract:

Homozygous deletions of exon 7 of the telometric survival motor neuron (SMN1) gene are responsible for 94% of all cases of spinal muscular atrophy (Ogino & Wilson, 2002). This deletion leaves only the centromeric copy of the gene (SMN2) to produce truncated proteins as a result of alternative splicing (Lorson et al., 1999).  A single base pair difference C to T at the 840th base pair of exon 7 is the source of this alternative splicing. Exon 8 of SMN1 similarly contains a single nucleotide difference of G to A in SMN2 (Gambardella et al., 1998), and deletions of exon 8 in SMN1 have also been observed in SMA patients (Gambardella et al., 1998).  A tetra-primer ARMS PCR has been used to successfully distinguish both genes apart using the base pair differences for both exon 7 and 8 (Baris et al., 2010).

Deletions in exon 8 could in part be responsible for the vast range of phenotypic severity found in SMA (Gambardella et al., 1998). We hypothesized that deletions in exon 8 of SMN1 were one cause of the alternative splicing in the gene, causing insufficient SMN protein production (Gambardella et al., 1998). This was tested using PCR to identify deletions in exon 8 of SMN1 via DNA from SMA type I and II patients, where primers utilized the outer common band length (386 bp) as an indicator of missing nucleotide sequences.  Our tetra-primer PCR assay successfully amplified exon 8 of SMN1. The entire exon 8 sequence was amplified (386 bp) and SMN1 presence was distinguished in wild type and SMA type II DNA (161 bp). However, the expected varying band lengths of exon 8 outer common amplifications were not detected, indicating that there were no observable deletions in the exon. The tetra primer was also unsuccessful in amplifying SMN2 (287 bp) for wild type and all SMA DNA.

A psychological experiment was conducted wherein different symptoms of SMA were experimentally simulated and rated (Sheehan et at, 1996).  Results for all simulated symptoms upheld our predictions that severity of the treatments would decrease over time as individuals adjusted to their symptom (Dorner, 1975). These tests are important in understanding the severity, prevalence, and social impact of SMA.

Discussion:

Experiment Summary

        Spinal muscular atrophy (SMA) is one of the most common autosomal recessive diseases, occurring in approximately 1 in 10,000 live births and 1 in 50 people are carriers (Lorson et al., 1999).  The SMA disease is a result of insufficient survival motor neuron (SMN) protein being produced in the motor neurons.  The genes coding for this protein are named after the SMN protein, thus they are called, SMN1 and SMN2. The disease is due to a homozygous deletion or point mutation of exon 7 in SMN1 (Baris et al., 2010).  The genes are located on band 13.2 of chromosome 5; SMN1 is telomeric and SMN2 is centromeric and has 5 known base pair differences, two of which were studied (Baris et al., 2010). 

        The first difference occurs in exon 7 at position 840 on both the SMN1 and SMN2 genes, although the difference is in the same location, SMN1 is a cytosine (C) where it is a thymine (T) on SMN2 (Baris et al., 2010). The latter base change in particular has been well researched and is known to effect the splicing of exon 7. A healthy copy of SMN1 would have a C at the stated position on exon 7 and the protein would be produced optimally.  The SMN1 gene specifically constitutes for approximately 90% of the SMN protein. The last 10% comes from the SMN2 which has the T on exon 7, thus makes less efficient protein (Cartegni & Krainer, 2002).  The second difference studied occurs on exon 8 of SMN1 and SMN2 where the difference is a guanine (G) and adenine (A) respectively.  This exon is normally non-coding since the stop codon is located at the end of the exon 7.  When exon 7 is spliced out due to the single base pair difference observed in SMN2 exon 7, exon 8 is expressed (Sendtner, 2010).  If exon 8 is coded with the placement of the single mutation, the stop codon would come before the mutation. 

        PCR has been used to identify the C to T mutation or deletion of exon 7 in the SMN1 and SMN2 genes using a tetra primer assay (Baris et al., 2010), but the same technique has not been used on the known G to A mutation on exon 8 (Lorson et al., 1999).  Most SMA patients have deletions of both exon 7 and 8 of the SMN gene but it is possible to have an isolated deletion (Gambardella et al., 1998). We hypothesized that deletions of SMN1of exon 8, identified using a specifically designed tetra-primer PCR assay, were the cause of alternative splicing in the gene leading to SMA (Gambardella et al., 1998).  The deletions of exon 8 could lead to insufficient SMN protein production which causes the symptoms of SMA patients (Gambardella et al., 1998).

Original Predictions

        It was predicted that genomic purification of one swab of buccal cells would result in 0.5 – 3.0 μg of DNA because that was the expected yield using the MasterAmp Buccal Swap Kit.  Once DNA was purified, PCR trials could begin.  Based on a previous tetra-primer assay used to detect deletions of exon 7, we predicted that deletions of exon 8 could be detected with a similar design (Baris et al., 2010).  In addition, the exon 7 tetra primer assay served as a positive control for the designed exon 8 PCR assay. A tetra primer assay was designed for exon 8 in order to determine the presence of the SMN1 and SMN2 genes.  The primers contained an intentional base pair mismatch on the 3’ end in order to increase the chances of successfully amplifying the region on the SMN1 and 2 genes (Yaku et al, 2008).  The four primers were designed to pick up both versions of exon 8 where the G and A single nucleotide difference occurs (Figure 2).  A successful PCR cocktail was predicted to contain 2µl of 100µM for each primer (8 µl), 5µl 10X ThermoPol Reaction Buffer PCR buffer, 1µl 10mM dNTPs, 1µl MgCl2, 1µl Taq Polymerase, 32 µl of H2O, and 2 µl  of 70 ng purified human DNA cells (Baris et al., 2010). It was hypothesized that a positive test for wild-type DNA would result in three bands which included a common of 386 bp (two outer common primers, forward and  reverse), an SMN2 region of 287 bp (reverse inner with a forward common primer), and a SMN1region of 139 bp (reverse common with the forward inner primer) (Baris et al, 2010).   The SMN1 (139 bp) and the SMN2 (287 bp) allele-specific amplifications allowed for easy identification by gel electrophoresis due to the difference in base pair lengths. The use of the tetra-primer assay with mutant DNA was hypothesized to result in a missing band at 139 bp, 287 bp, or both due to deletions of exon 8 for SMN1 and 2.  Deletions to exon 8 would cause the primers to fail to anneal resulting in no amplification during gel electrophoresis. To avoid complications in reading the gels after electrophoresis, it was important for us to pinpoint reliable temperatures and primer locations for our PCR assay (Ye et al., 2001).  We predicted that the annealing temperature of the primers for exon 8 to be 58°C, based on temperature ranges calculated using the method for long primers (Jeong et al, 2011).

Results and Ultimate Findings

        Genomic purification yielded an average of 2400 ng (2.4 μg) of DNA for type II patients and 2200 ng of DNA for the type I patient.  Following the purification process, PCR trials were commenced. In order to obtain a successful assay, many experimental trials were attempted and controllable aspects were adjusted accordingly (annealing temperatures and cocktail concentrations).  It was very important to analyze and troubleshoot the cocktail ingredients as well as annealing temperatures and other PCR conditions.  The original predicted cocktail ingredients were adjusted numerous times and produced no amplification during PCR.  The exon 8 cocktail that resulted in successful PCR amplification contained 15 µl of nuclease-free H20, 2µl of 100µM for each primer (8µl), 2µl of 70 ng purified DNA, and 25µl of Go Taq® Green Master Mix.  This PCR cocktail was run at various annealing temperatures from 48̊C – 58̊C and an optimal annealing temperature of 55̊C was found.  This annealing temperature resulted in amplification of the common band (384 bp) and SMN1 band (139 bp) for both wild-type DNA and mutant DNA.  The SMN2 band (287 bp) didn’t show up for wild-type DNA, type I DNA, as well as type II DNA which suggests that the designed inner primer specific for SMN2 was unable to anneal.  Type I mutant DNA resulted in no band amplification for both the control and designed assay which could be a result massive exon 7 and 8 deletions. The exon 7 PCR assay served positive control for our designed assay (Baris et al., 2010).  This assay was replicated with an annealing temperature of 53̊C which gave successful amplification of specific band sizes.  Wild-type DNA resulted in three bands at 161 bp (SMN1), 239 bp (SMN2), 343 bp (common region).  Mutant type II DNA resulted in the SMN2 band (239 bp) and the common band (343) but lacked the SMN 1 band, suggesting a deletion (Baris et al., 2010). 

        We reject our primary hypothesis that deletions of exon 8 result in alternative splicing of the SMN protein due to the small sample size of mutant DNA. The isolated exon 8 deletion is rare which means to further test our hypothesis, a larger sample of mutant DNA, both type I and II, is necessary (Lorson et al., 1999). However, our tetra-primer assay served to successfully diagnose the presence of exon 8 deletions for SMN1 and can ultimately detect the existence of SMA.

Sociological Investigation.

         Our sociological investigation is designed to study the detrimental effects of SMA by scoring daily the effects of the disease on ones work/school (W/S), social life (SOC.), and family life/relationships (H/F) (Sheehan et al., 1996).  The experiment entails four treatments: the first, an individual uses a wheelchair in the setting of their daily lives; the second, an individual is handfed one meal every day; the third, an individual is not allowed to slurp, sip or a straw to eat their meals; and the fourth, an individual requires assistance whenever sitting up from a prone or supine position.   Over the 30 days, scores of 1 to 10 for of the three sections were recorded every day and added together for a total score out of 30 that were averaged every five days and graphically represented by a regression plot (Figures 8, 9, 10 & 11).  Analyzing the data revealed a sharp increasing trend followed by a slower, decreasing trend, in each category due to an adaptation to unchangeable circumstances (Dorner, 1975) (Figure 4).  Alongside our physical and psychological investigation, a still-photo documentary also demonstrates the effects of SMA in a visual format.

The first treatment had the highest categorical impact on the social life category on the SDS (Table 1).  Being bound to a wheelchair also affected H/F and W/S, but the ratings were much lower (Figures 9, 10 & 11).  The test subject noted that social life was most affected due to the lack of mobility, forcing him to remain in his residence. The individual was also distracted from school work due to the disability although to a much less extent.  The second treatment also provides evidence for the impact of the disease on social life. It was reported that the disruption was a result of dependence on others to feed her, and was further disrupted due to hesitating to ask for help from individuals who treated her indifferently.  This resulted in relatively moderate disruption rankings.  The third treatment resulted in the lowest average ratings in all categories across all treatments (Table 1).  It was reported that the individual with the impairment experienced very little distraction in daily life due to the nature of the simulation.  The individual also reported that she experienced the most disruption while in class, explaining the highest values in the W/S category.  Otherwise, the impairment did not hinder any specific aspect of her daily routine.  The fourth treatment shows mid-range to high ratings across all three categories with the W/S category being most affected (Table 1).  It is noted by the simulator that his privacy was violated and the dependence on others made him feel inferior, thus explaining the high SOC. scores.  The W/S category was the most disrupted aspect of this individual’s treatment, because the individual would not even be able to go to his classes if he did not have that assistance.

    The sociological experiment was conducted to give a more comprehensive understanding of the disruption of the daily lives for those who are living with SMA.  SMA is well-studied on the molecular level but the effects of genetic diseases are not experienced or understood colloquially by society.  Our goal was to promote the awareness and understanding of how the disease affects individuals on a more personal level.  Although our experiment was far from replicating the actual symptoms an SMA patient would experience, we feel that this experience gave us a much greater perception of the hardships associated with SMA.

4-Primer PCR assay amplifies exon 8 in SMN1 for SMA patients using human Buccal Cells.

By: “The Spartan Gene”

Nicole Patel, Josh Reside, Leah Creech, and Bo Parsons

Future Directions:

        Further research of the SMN exon 8 would involve redesigning the SMN2 exon 8 primer in the tetra primer. The exon 8 SMN2 primer used in this study most likely failed due to sensitivity of using tetra primer; there was an increased likelihood of complimentary sequences between the primers them selves. We would first create an assay with only two primers, the previously stated one and the anti-parallel common primer. If this did not give amplifications then it would be necessary to modify the primer. This would entail working with the length and the percent complimentary base pairs with in the primer and in comparison to the other primers.

        Supplemental to the redesigning of the primers we would want to adjust our PCR cocktail. We were not able to obtain amplification using the control cocktail that was taken from a previously successful assay (Baris et. al., 2010). This was most likely caused by our less accurate and sensitive instruments such as the mircopipettes and thermocyclers. We were able to successfully amplify using cocktail concentrations based on our E.coli control assay. This produced the desired amplifications but they were faint. Based off of this result we think that the concentration of magnesium ions would need to be adjusted, because they affect the ability of the polymerase to amplify.

        Enhancement of this study would involve the use of a large array of mutant DNA, because a larger sample size has a greater chance of obtaining an exon 8 deletion. In addition, larger sample sizes decreases variance. This would allow us to determine if our tetra primer can detect deletions of exon 8. These strategies for improving our diagnostic primers would allow for further inferences such as severity. Exon 8 deletions have been found, but little research has correlated the single base change to severity (Gambardella et al., 1998). Before we can link to severity the tetra primer method needs to be perfected.

    In order to refine the socio-psychological experiment modifications of the treatments would be necessary. Treatment three, Oral muscle weakness, was a poor reflection of SMA type I because it was the hardest to simulate due to the fact that it occurs in infants requiring a feeding tube. In replacement of this treatment we would simulate weakness of the neck muscles thus, one would have to rely on a neck brace for head support. This would better exemplify a common symptom of SMA.