Genotypic Identification of Diabetic Patients with the

B24 INS Gene Mutation using IB3-1 Cells and Allele Specific PCR

Team Naples:

Elizabeth DeCesare, Shannen Serylo, Max Suris, and Anthony Kazyaka

Abstract

Allele-specific polymerase chain reaction (ASPCR) is used to diagnose type II diabetes. The experiment’s purpose is to detect the presence of a mutation known as B24 in genomic DNA. This provides additional confidence when determining if individuals carry the mutation. The mutation is a single base pair substitution from phenylalanine (UUC) to serine (UCC) (Tager, 1964). ASPCR was utilized to distinguish whether samples of human epithelial cells had the B24 mutation within the INS gene. The DNA tested was extracted from the epithelial cells (IB3-1) of cystic fibrosis patients. We hypothesized that by creating a PCR cocktail with proper annealing temperatures, we could determine the presence of the B24 mutation. The product would then be used to identify if the DNA contained the mutation by the appearance of bands 444bp long. If it didn’t, then bands would not appear. This would be measured using gel electrophoresis. Due to difficulties in obtaining DNA containing the B24 mutation, this hypothesis could not be fully tested. In addition to genotypic studies, we studied personal blood glucose levels and happiness levels on a non-diabetic diet, and also following an 1800-calorie diet for diabetics. Online aliases were created personifying different types of individuals, including obese males or healthy-weight females, in order to quantify public prejudices of people with diabetes. We hypothesized that caloric intake would affect both blood sugar levels and happiness levels, due to lower sugar consumption, and that there is a negative stigma associated with a person diagnosed with type II diabetes. The predictions made for this experiment were that blood glucose levels and happiness levels would drop while following the diet, and that members of the general public would respond negatively when presented with an alias of an overweight body type, and more positively when the alias is of a normal body weight. The blood sugar levels, however, did not change with statistical significance, yielding an R² value of 0.004. The changes in happiness were also not significant, with R²=0.171 and m=0.037, signifying a very loose correlation. Additionally, we found that the general public responded with positive feedback, regardless of body type.

Figures

Figure 4: PCR amplification of lambda virus versus control primers at 55°C. Lambda virus and control primer cocktails were run in the same PCR machine at an annealing temperature of 55°C. The targeted region of DNA was a length of 500 base pairs for lambda virus. Lanes 2, 3 and 4, show non-specific binding at a base pair length of 300 and 400 base pairs. Since the control primer cocktail did not have mutant DNA to initiate annealing, the lack of bands in lanes 5-7 were expected. Testing the control primers against lambda in the same apparatus and same gel, ruled out the error of an incorrect procedure.

Discussion

Experiment Summary and Original Predictions

An individual with type II diabetes produces inadequate insulin for glucose oxidation. The presence of the B24 point mutation in the INS gene results in mutated preproinsulin production (Steiner et al, 1990). The B24 mutation creates a form of insulin that the body cannot identify or use properly (Hua et al, 1993). The insulin produced is mutated by a small, hydrophilic side chain that obstructs insulin receptor-binding sites (Tager et al, 1964). We hypothesized that by creating allele specific primes to match a single base pair mutation, we could develop an accurate diagnostic test for patients with the B24 mutation using the correctly calculated annealing temperature, primer concentration and cocktail ratios in a PCR experiment (Yano et al, 1992).

The B24 mutation can be identified using PCR through denaturing, annealing, and elongating the amplified target DNA to be examined for success using gel electrophoresis. We intend that our wild type primers, in the presence of wild type DNA, and mutant type primers, in the presence of mutant type DNA, will both individually yield a PCR product of 444 base pairs. Although when wild type primer is in the presence of mutant DNA, no PCR products will be produced. Likewise, when mutant type primer is in the presence of wild type DNA, PCR products will not be produced. Precisely, this means that primers will be specifically designed for a genetic sequence that should provide bands 444 base pairs long.

In conjunction to our molecular study of the insulin gene mutation, we underwent a sociological and psychological study of diabetes. To experience how the general public views individuals with type II diabetes, aliases with various body types and profiles were created and posted to forums requesting advice on how to manage their diabetes. These profiles ranged from in-shape individuals in their late teens, to obese individuals in their mid-twenties, with variations such as being a vegetarian also given in the description. In addition to this, daily blood glucose readings were taken and analyzed for trends in the data. Our final test during the 30-day sociological experiment consisted of a survey determining one’s mood and happiness. These were taken to examine the affects diabetes would inflict on individuals, at the end of each week. Predictions for the 30-days experiment projected responses to the alias’s that were entirely based around weight, and also that these responses would provide undesirable feedback. The blood glucose levels were not expected to change, or drop an insignificant amount over the 30 days. Lastly, the anticipated happiness of each member was proposed to decline, due to new changes in diet that would cause a small bodily unbalance.

Results and Findings

In order to create an additional experimental control that was used in future gels for a reference, we began by creating a PCR experiment with solely the Lambda virus. This provided us with the experience we needed to calculate correct annealing temperatures (50-55°C, see Methods section), and proper concentrations in the cocktail. This approach ensured that that our ladder was working correctly, the gel electrophoresis apparatus was functioning properly and the gel was made correctly (see Methods section). After running this gel, we were able to gauge the precise base pair length of the bands based off of the 1Kb+ ladder (Figure 3). This was then used in future gels for as a base of length in addition to the ladder.

In order to further our research we found a published paper in an online database that performed similar studies on the INS gene, with mutations that are linked to type II diabetes (Hoban et al, 1991). We then ordered the exact same primers from a company that synthesizes primers. When we ran these primers against the DNA derived from the IB3-1 cells, we did not expect to get bands. The cells we received did not contain any INS mutations therefore there are no places for the primers to bind. We ran a PCR with the Lambda virus and the control primers under the same conditions. This supports our predictions because the only thing that appeared was the lambda bands (Figure 4). We can conclude that our results did support our hypothesis because of the lack of bands present.

The 30-day experiment yielded impressive data. The alias’s created all received substantial feedback on the World Wide Web. The responses were particularly constructive in the direction they posed for each alias to now live with type II diabetes. Each prediction we made for this aspect of the 30 days was incorrect. The outstanding topic of the responses was what diet should be followed. Also the responses were entirely supportive, with little to no negativity. As predicted, after 30 days monitoring blood glucose levels, before each meal, there were no substantial changes in our numbers. This would best be accounted for by the properly functioning insulin in each non-diabetic group member. The happiness data during the 30-days showed a very loose positive correlation; again we can attribute this small relation to each member having working insulin. This means that the body can account for the changes in diet, so there was not a strong mental effect.

Weaknesses in the Experiments

During our research, some technical difficulties were encountered. After using gel electrophoresis, our bands did not appear when testing the lambda virus. This specifically affected the outcome of our research because PCR had the potential to be successful but there is no way of knowing until a proper gel was made using the same PCR cocktail previously used. We speculated to fix this complication; 2.5 μL of SYBR Safe should have been added when making the gel originally. In addition, after another PCR attempt was made using the same PCR cocktail, only the 1Kb+ ladder appeared again, with no evidence of lambda bands. Therefore we can assume the error was in our PCR cocktail, we moved forward and fixed the problem by correctly making a new PCR cocktail.

This was not the only difficulty we faced with PCR experiments, during our trial with the control primers taken from (Hoban et al, 1991) we saw faint bands in our first gel, no bands at all in our second, then in our final we saw specific bands with faint non-specific bands below. We know that faintness in bands or no bands at all comes from too few cycles, or extension and/or annealing times being too short. With that information we adjusted our annealing times, which provided us with bands (Figure 3).

There were also many opportunities for error in our psychological and sociological experiments, since there were many variables that could affect the recorded data. Each alias provided an altered background description from the next, and each responder only commented on one. This creates for a situation where each alias is interpreted and analyzed differently. Also, because a “low glycemic diet” was supposed to be kept during the second portion of the 30-days experiment, any food/drink that was not part of the diet skewed our data. In addition, another error could come from false readings of blood glucose levels. If one eats at any time less than two hours prior to testing their blood glucose level, a higher glucose level would be yielded due to food consumption. This would ultimately give a false reading since it is not a reliable measure. In order to avoid this error, we did not eat meals three hours prior to testing and kept snacking to the absolute minimum. There was also room for a lot of inaccurate data with regards to the happiness test. Our data for each test was coming from the basis of our diet, but in reality there are so many more variables that could affect ones happiness at any given time. Which means, our perceived happiness at the beginning of the week was most likely very different at the end of the week when the survey was completed.

Future Directions

In the future, multiple forward primers could be created to match the multiple variations in the mutation presence on the INS gene in order to continue our research. This experiment would further support our hypothesis, that by creating a conducive PCR reaction with annealing temperatures that corresponded to our primers could determine the presence of the B24 mutation, a single base pair mismatch. Patients with abnormal insulin can be associated with human diabetes (Shoelson et al, 1983). When phenylalanine is replaced with serine, people have reduced insulin receptor-binding capability (Steiner et al, 1990). Correspondingly, substitution in the B24 mutation creates altered structures and less possibility for receptor competence (Tager, 1984). Which adds another direction we could take our research. If we were able to study the structure that the mutation creates then our research could be applied to many similar mutations.

In addition if we could obtain cells that contained the B24 mutation within their genetic sequence we could then use our mutant primers to get results in our gels. To further this research, we could find a type II diabetic who also has the B24 mutation, and has kin that are available for testing. Next we would obtain cells from the individual’s offspring and perform genomic purification. The extracted DNA of the kin would be tested for the B24 mutation. If this individual shows the B24 mutation and also has type II diabetes we would be able to associate the two more closely. As seen in a similar experiment, where the individual’s offspring had the mutation without type II diabetes, then we could rule out the B24 mutation as the ultimate cause of the diabetes (Warren-Perry et al, 1997).

In order to make the 30 days) experiment more accurate, at the beginning and the end of the trial period we would submit blood for an hemoglobin A1c (HbA1C) test, which is the most accurate measurement for glycohemoglobin in the bloodstream. This would show even the slightest differences in our glycohemoglobin levels over the course of the experiment, thus providing us with more accurate data.

Psychological and Sociological Experiments

Video - https://www.youtube.com/watch?feature=player_embedded&v=jqH0Nw89cZE