Detection of the C282Y Mutation on the HFE Gene Using Allele Specific Primers on Wild Type and Mutated Human DNA

By: Josh Andaloro, Kate DeVries, Sam Pace, Jordon Van De Velde

LB 145 Cell and Molecular Biology

Tuesday and Thursday 4:10PM

Dr. Luckie

David Malakauskas, Kelsey DeLand and Natalie Palumbo

April 19, 2011

Abstract

Haemochromatosis is an autosomal recessive blood disorder that is promoted by the presence of the C282Y mutation. The mutation is the result of a base pair mismatch, later translating its corresponding codon into a defective HFE protein. This prevents the regulation of iron into cells, producing an overwhelming amount of iron in the body. (Neghina, 2010). We hypothesized that by using allele specific primers along with the proper annealing temperatures and salt concentration, we would accurately and efficiently be able to use PCR to detect the presence of the point mutation, C282Y, given any sample of human DNA. The PCR and gel electrophoresis assay were performed using both wild-type and mutant C282Y+ DNA. Five distinct primers were used, including two Yaku-Bonczyk to increase specificity. The results are not quite what we expected, being that there was unspecific binding and bands in locations they should not have been. In addition to this research, our research group volunteered to experience what it could be like living with haemochromatosis for a week. The experiment started by donating blood to represent the therapeutic phlebotomies that a typical patient would have to receive, and the following seven days consisted of consuming a diet low in iron to experience both physical and emotional challenges that may occur when living with the disease. We hypothesized that a low-iron diet will cause a decrease in one’s energy. The results of our experiments provide a way to diagnose patients with haemochromatosis faster and more accurately.

Discussion

Disease Background

Haemochromatosis involves improper regulation of iron within the body due to a defect of the HFE gene located on chromosome 6, position 21.3 (Smillie, 1997). Disease onset occurs typically after the age of 40 and is most commonly found among individuals of European descent (Thompson, 2009). Symptoms include chronic fatigue, abdominal pain, diabetes, cardio-myopathy, and liver disorders (Rochette et al, 2009). The disease is a result of a combination of H63D, S65C, and C282Y mutations occurring in the HFE genetic sequence, but the C282Y mutation is known to be the most frequently occurring among the three (Spinola et al, 2010). The C282Y mutation involves a point mutation of a nucleotide Guanine to Adenine on the 845th base pair of the HFE genetic sequence (Mura et al, 1991). This alteration codes for the amino acid Tyrosine instead of Cysteine on the 282^nd codon. The simple codon change alters the functionality of the HFE protein, causing it to degrade before making it to the cellular membrane. The absence of the regulatory protein inhibits iron intake and puts the patient at severe risk for iron overload. It was hypothesized that we would be able to successfully amplify the genetic sequence containing the C282Y mutation by altering annealing temperatures and salt concentrations (Markoulatos et al, 2002), using our allele specific primers.

Experimental Design and Predictions

We designed an interactive experiment in which we experienced what it would be like to live with Haemochromatosis for seven days. It was predicted that through our experiment, decreased iron levels and a low iron diet can lead to fatigue and a low level of awareness. Once a patient is diagnosed with the disease, phlebotomies are needed in order to reduce the high iron concentration in the bloodstream. It is essential that the patient follows a strict diet composed of foods both low in iron and vitamin C in order to keep iron levels to a minimum. The experiment began by donating blood as a substitution to the phlebotomy that a patient with the disease would normally receive. The following seven days consisted of consuming a diet low in iron. An overall level of awareness or well being was recorded for each day. It was found that throughout the week, the patients level of awarness decreased (Figure 3). This data is supported by other findings in which patients living with Haemochromatosis are more likely to be weary or fatigued. Clinical signs attributed to HFE-related Hereditary Haemochromatosis include liver failure, arthralgia, chronic fatigue, diabetes mellitus and congestive heart failure (Jacobs et al, 2007).

Polymerase chain reaction was performed in combination with gel electrophoresis to determine the presence or absence of the C282Y mutation in a sample of human DNA. To achieve the predicted bands of 436 base pairs in length a total of five primers were designed to amplify wild-type, mutant and heterozygous DNA. It was hypothesized that by altering the annealing temperature and salt concentrations, specific binding would occur and remove false positives, providing brighter and more profound bands. The forward primers that were used varied with the different PCR tests, but the reverse primer was selected to remain the same throughout the whole experiment. The reverse primer did not incorporate the mutation in its sequence, therefore, it could be used to amplify both the mutant and wild-type sequence. Two of the four forward primers were designed to anneal to either wild-type or mutated based on the differences between the last base pair on the 3’ end. The other two forward primers were Yaku-Bonczyk primers that were designed for specificity in the amplification of the DNA sequence. Our predictions were that when the wild-type primer was used, there would be successful amplification of a 436 base pair sequence for wild-type DNA and a band of this length would be seen in the gel. When the wild-type primer was used on the mutant DNA, it would not anneal because there was a mismatch on the direct 3’ end of the primer. When the mutant primer was used, successful amplification of the mutated DNA sequence occurred and a band length of 437 base pairs would be observed in the gel. The mutant primer would not anneal to the wild-type DNA for the same reason as before, a mismatch on the 3’ end of the sequence (Chavana, 1996). Two Yaku-Bonczyk primers were used as well so that the primers would be more specific due to a mismatch that occurs three base pairs in from the 3’ end of the sequence. With the mismatch, the wild-type primer would anneal to the wild-type DNA strand and skip the mutated base pair. When the forward wild-type Yaku primer was used with mutated DNA, the primer would not anneal to the DNA strand due to two base pair mismatches. No bands were expected to show up in the gel after such PCR trials.

Experimental Results and Findings

An optimal PCR condition was achieved for our allele specific primers. By testing different annealing temperatures while keeping a constant PCR cocktail an optimal annealing temperature was found to be 45ºC. Temperatures above/below the optimal annealing temperature produced dim or fewer expected bands (Figure 4). Once the annealing temperature was set, magnesium chloride concentrations were tested to obtain brighter, clearer bands. Excessive Mg²⁺ concentrations stabilize the DNA strand and prevents complete denaturation of DNA, which reduces yield (Markoulatos et al, 2002). Brighter and more distinct bands were achieved by adding 9.5 µg of MgCl₂ (Figures 1 & 2). Having the knowledge and capability to successfully detect a C282Y mutation can help with future research of the disease and allows for quick screening procedures for those thought to have the disease (Smillie, 1997). The significance of genetic testing for Hereditary Haemochromatosis is crucial for diagnosing the disease early and providing appropriate treatments for positively tested patients.

Looking Forward

Early on in our lab experiment, we failed to dilute our wild type and mutated DNA properly which resulted in a loss of valuable time and DNA. The MSU Diagnostics Lab was very generous and provided us with more DNA with which we diluted properly. We were able to obtain another teams wild-type DNA. Further PCR testing resulted in no bands when the new wild-type DNA was used with our allele-specific wild-type primers. The wild-type DNA may not have been purified properly which is why our primers would not anneal. With more time we could have performed DNA purification again or possibly have obtained wild-type DNA from the MSU Diagnostics Lab. Either way purer wild-type DNA would have produced bands with our wild-type primers. In several of our gels, non specific binding had occurred, creating undesired products of different sizes than expected. These products may have been primer dimers, where the primers annealed to each other. They could have also been amplified regions of the DNA template other than the desired target region. The cause of this may have been from too low of an annealing temperature where the primers anneal non specifically to different parts of the DNA template. An analysis of a more specific annealing temperature and the time spent at that annealing temperature is suggested for further research. In order to gain a greater product yield and higher specificity, cycle numbers should be studied as well. With more time and supplies, an efficient annealing temperature could be found.