Amplifying GH1 Gene by IB3 PCR to Identify Genotype of IGHD Type II Patients with E3+5A>G Mutation

 

By: Eli Guttman, Cara Morrison, Katie Raber, and Nicole Rando

 

http://www.msu.edu/~guttman1


http://www.youtube.com/watch?v=2DYSRNqszWU&feature=youtu.be

 

Abstract

            The point mutation E3+5A>G occurs on the GH1 gene at the fifth nucleotide of exon three when guanine replaces adenine, changing amino acid 33 from glutamate to glycine. This causes autosomal dominant IGHD type II, resulting in diminished levels of growth hormone. The exon splice enhancer (ESE) is interrupted, disturbing pre-mRNA splicing of growth hormone (Moseley et al., 2002). Genomic DNA from peripheral mononuclear IGHD type II patients’ cells were extracted for allele specific PCR, where a 770 bp sequence containing the site of the mutation will be amplified to determine if the DNA is homozygous wild-type, heterozygous, or homozygous for the E3+5A>G mutation (Kamijo et al., 1999). Primers anneal at specific positions on the sequence to amplify target DNA through PCR. DNA is examined using agarose gel electrophoresis, determining the genotype of the 1,635 base-long gene (Lee et al., 2000). Lambda and E. coli DNA were amplified as a control, producing approximately 400 and 1500 bp products, respectively. Primers in a previously published paper were used as a control, producing a predicted 1,123 bp length DNA sequence from the GH1 gene (Moseley et al., 2002).We hypothesize the designed primers IGHDIIF, RP1, and WTF will yield a 770 bp product and correctly identify the presence of the E3+5A>G mutation due to the single base pair mismatch at the 3’ end of the primers. Psychological experiences of children with IGHD were simulated by walking in public with a mutation, expressed as a blue square on team members’ foreheads. The State Trait Anxiety Test was used to determine basic anxiety levels (Spielberger, 1983). Based on previous research, we hypothesized anxiety levels and glances will increase with the mutation due to feelings of self-consciousness (Stouthart et al. 2003). An ANOVA test was used to analyze anxiety level data, which was proven to be statistically significant, suggesting a correlation between anxiety levels and physical deformities. Correctly identifying the presence of the E3+5A>G mutation can result in early diagnosis and treatment, improving the quality of life for individuals with IGHD (Stouthart et al. 2003).


Discussion

Experiment Summary

Isolated Growth Hormone Deficiency (IGHD) type II is an autosomal dominant disease caused by mutations of the GH1 gene (Lee et al., 2000). These mutations result in a lack of production of the growth hormone protein (Millar et al., 2008). The E3+5A>G mutation is one variation of IGHD type II, involving a substitution of A to G at the fifth nucleotide of exon 3 on the GH1 gene (Moseley et al., 2002). This change in nucleotide results in a missense mutation, coding for the amino acid glycine instead of glutamate (Moseley et al., 2002). The mutation activates a cryptic 3’ splice site, altering the splicing pattern and resulting in the deletion of the first 15 nucleotides of E3 (Moseley et al., 2008).

Through the process of PCR, it is possible to analyze a DNA sequence and develop a clinical assay, thus diagnosing the presence of IGHD Type II (Moseley et al., 2002). By designing allele-specific primers, we are addressing the question of whether the E3+5A>G mutation can be identified by using PCR. We hypothesized designed primers IGHDIIF and RP1 will correctly identify the presence of the E3+5A>G mutation due to the single base pair mismatch at the third nucleotide from the 3’ end of the primer IGHDIIF, thereby allowing earlier diagnosis and treatment of IGHD  (Moseley et al., 2002).

To provide controls for the experiment, two separate experiments were conducted. In the first control experiment, a sequence on the 16S gene of E. Coli and a sequence on the Rz Gene of  lambda bacteriophage were amplified. It was predicted that successful amplification of the 16S gene using the primers 8F and 1512R would produce a 1500 bp product. It was also predicted that successful amplification of the Rz Gene using primers RzF and RzR would produce a 500 bp product. For the second control experiment, previously published primers that successfully amplified a 1123 bp product were used. These experiments were done in order to produce data showing the system works, as well as develop an experimental basis by which possible errors in other experiments can be pinpointed.

The question of how this disease would impact someone was also examined using sociological experiments designed to simulate experiences shared by those affected by the disease. The experiments were designed to address the issues of finances, treatment, and psychological effects of the disease. The psychological experiment involved simulating a situation which caused group members to experience feelings of low self-esteem and social anxiety that can accompany physical abnormalities (Nicholas et al., 1997). In order to simulate discomfort and social anxiety resulting from appearing differently from the social norm, research team members each bore a blue paper square on their forehead and walked individually around for a set period of time. An observer counted the number of people glancing or staring at the subject while the subject counted the total number of people passed. Each walk with a “mutation,” or blue square, was compared to a control walk where the subject walked without the blue square. Dr. Charles Spielberger’s “State-Trait Anxiety Inventory for Adults” was used to measure anxiety levels when performing this experiment (Spielberger, 1983). Two locations were used to conduct the experiment in order to collect and compare data from two environments which differ in their social makeup: MSU’s campus and Meridian Mall. The mall contains a large variety of age groups and people, which were predicted to be much less accepting of the “mutation” than a college campus made up of people of a similar age group predicted to be more accepting of the “mutation.”

In addition to walking individually, three team members walked in a group with one member with the “mutation” and two walking with no mutation. This experiment was conducted in order to provide data to address the question of whether or not the presence of peers lacking the mutation would alleviate anxiety of someone living with IGHD. The results shown in figure 5 support the prediction that the subject’s anxiety levels are significantly lower than when walking alone.

Results and Findings

            A 400 base pair product was amplified from the Rz gene of bacteriophage lambda using the primers RzR and RzF. A 1500 base pair product was amplified from the 16S gene of E Coli using the primers 8F and 1512R. The presence of bands signifies the annealing temperatures and the ratio of the components of the PCR cocktail are ideal for the primers used. The successful amplification of both sequences make it possible to use this combination of conditions as a control for future experiments. Using this as a control will help to pinpoint what may have gone wrong if no bands are present in other experiments, possibly eliminating variables such as errors in the PCR cocktails, the thermocycler, or the gels. In the second control experiment, published primers were used with the purpose of amplifying a 1,123 base pair product on the GH1 gene. Many different length sequences were amplified. However, there were two distinct bands present at the correct length. The presence of multiple non distinct bands is most likely caused by nonspecific binding. This nonspecific binding is predicted to have been caused by the primers annealing to multiple positions on the DNA strand due to annealing temperatures being too low.

To determine the statistical significance of the data collected by the glances received during the psychological experiment, a chi squared test of independence was done resulting in a p-value of .3754. An ANOVA test was used to statistically analyze the data from the anxiety test scores recorded from the psychological experiment, resulting in a p-value of .01. This data signifies the differences in number of glances were not statistically significant however differences in anxiety levels were statistically significant. This data contradicts the hypothesis that the glances would increase when group members had the mutation. This data is logical when taking into account that it is likely for people to look at other people when they pass each other, however different reactions occur when a person does not appear within the social norm. This was seen when rude and confused verbal comments were made both in reference of and directed towards the group member with the mutation. A previous study was conducted to analyze if there was any increase in anxiety or depression during a year-long discontinuation of growth hormone treatment where results showed that there was a significant increase in anxiety and depression during the growth hormone treatment discontinuation phase in comparison to the continued treatment following discontinuation (Stouthart et al., 2003).The data in our experiment supports the hypothesis that the anxiety levels of each group member would increase when the member was walking with the mutation because of feelings of self consciousness due to the physical abnormality, as well as supporting data from previously published papers.

Improvements in Experimental Design

One process that was implemented early on in the experimental process to improve experimental design was the use of a “mastermix”. This involved making sets of mixes of components in large quantities in order make multiple cocktails consistent. This helps to eliminate variables, decreasing the likelihood that a specific cocktail will have failed due to the lack of a certain component. Concentration of the PCR buffer was also altered to increase elements of the cocktail that could improve PCR.

Future Research

            The next steps in order to achieve amplification GH1 Gene by IB3 PCR to Identify Genotype of IGHD Type II Patients with E3+5A>G Mutation would be to continue running PCR experiments using the published primers found in the paper ‘An exon splice enhancer

mutation causes autosomal dominant GH deficiency’ (Moseley et al., 2002).  Though DNA amplification using primers CPF and CPR was successful, there was also non specific binding in our results.  In order to improve the quality of the gel, the annealing temperatures of the primers should be gradually increases until 72oC.  We predict that clearer bands at 1125 bp will result from an increase in temperature due to the trends in our data showing as temperatures increase so do the brightness of bands at 1125 bp.  After successful PCR amplification occurs with the published primers, primers WTF and RP1 can be used to amplify the 770 bp target DNA sequence of the wild type human epithelial cells. The concentration of buffer in PCR cocktails and temperatures may be increased, but annealing temperatures should not be increased above 51.8oC. After bands have successfully been amplified using these primers, DNA purification of IGHDII mutant human cells must be purified using Genomic Purification (See Methods section for instructions). After the mutant DNA has been purified, primers IGHDIIF and RP1 will be used to amplify the target DNA of 770 bp using PCR cocktails containing mutant human DNA containing the adenine to guanine point mutation on the fifth nucleotide of exon 3. Concentration of PCR buffer and temperatures may be increased in order to achieve results, although annealing temperatures should not be increased beyond 53.8oC.

 

Figure 1:

Well number:

       16    15    14   13    12    11    10     9      8     7     6      5      4     3      2      1

Annealing Temp (°C):

                       63.0   61.1  58.8   56.9  55.7  55.0  64.3   63.0   61.1  58.8   56.9  55.7             55.0

B
 

A
 
https://sphotos-a.xx.fbcdn.net/hphotos-ash3/11900_10151387438258144_257285322_n.jpghttps://sphotos-b.xx.fbcdn.net/hphotos-ash3/11716_10151387412758144_1008122089_n.jpg                                                                                                                                                                                                               

Figure 1A: Amplification of DNA strand from the GH1 gene by PCR and analyzed by gel electrophoresis. A target DNA sequence of 1,125 base pairs was amplified by PCR using primers CPF and CPR. The PCR cocktails were made up of 35µL of nuclease free water, 10 µL of 10X PCR buffer, 1 µL of Taq polymerase, 1 µL of dNTPs, 1 µL of DNA template, and 1 µL of each primer. The cocktails were ran in the thermocycler at 95°C for an initial three minutes and cycled between 30 seconds at 95°C for the denaturing phase, 30 seconds at the annealing temperature for the annealing phase, and one minute at 72°C for the elongation phase. The annealing temperatures were set on a gradient from 55.0°C to 65.0°C and 25 cycles were completed. Annealing temperatures are specified above for each well (wells 2 through 15). A 0.8% agarose gel made using TBE (Tris/Borate/EDTA) buffer, agarose, and Sybrsafe was ran at 135 V in which 7 µL of DNA and 3 µL of loading dye was pipetted into wells 2 through 15 and 5 µL of Fermentas’ 1kb Plus ladder was pipetted into well 1(labeled Ladder). The gel was then observed under an ultraviolet light in order to see the bands formed by the DNA product.

Figure 1B: Migration distance vs. molecular size of the 1 kb Plus DNA ladder was used to analyze PCR products from the GH1 gene. 5 µL of 1 kb Plus ladder was analyzed using a semi-log plot shown above in which the x-values represent the distance in centimeters that the bands of the ladder migrated away from the well and the y-values represent the size of the molecules in base pairs. A trend line was added in order to obtain an equation for further analysis. This equation was used to calculate the base pair length of the PCR product in which the distance migrated from the wells was input for the x-value of the equation, yielding a y-value representing the base pair length of the product. For the bands produced by the DNA, the base pair length produced was calculated to be 1,125 bp for wells 8 and 9 at annealing temperatures 63.0°C and 64.3°C. However, there is still non-specific binding present in wells 2 through 15. The R˛ value given in the figure represents the fit of the trend line where 1 represents a perfect fit.