Diagnosis of Fibrodysplasia Ossificans Progressiva (R206H) on gene ACVR1 via Tetra Primer ARMS PCR

        An allele-specific diagnosis of Fibrodysplasia Ossificans Progressiva (FOP), a rare genetic disorder that causes the ossification of muscle, tendons, ligaments and other connective tissues, was done via Tetra-primer ARMS-PCR. Tetra-primer ARMS-PCR, which utilizes four primers rather than two, is a method specifically designed to detect single nucleotide polymorphisms (SNPs). The genetic mutation (R206H) is caused by an SNP in which nucleotide 617 of the ACVR1 gene, which is located in the 206th codon, mutates from a guanine to an adenine (Shore et al, 2006). Our hypothesized PCR results were based on information obtained from an online primer generating software found in a paper in which a similar assay was used. The software, which produced primers specifically for detecting the R206H mutation on the ACVR1 gene also projected expected band lengths for the PCR products of lengths 210 base pairs, 259 base pairs and 422 base pairs, corresponding to products of the R206H mutation, the wild type allele, and the non-specific product produced by the outer primers, respectively (Ye et al, 2001). A sociological experiment was also carried out to emulate the progressive physical restraints of FOP by successively adding more restriction to our joints through the use of various braces on our bodies. It was hypothesized that by the third stage (leg, neck, and hand braces) of our simulated FOP, the symptoms imposed would have become so physically challenging that the majority of everyday tasks would take a significantly longer time to accomplish (Pignolo et al, 2011) Time trials collected for daily activities over the four stages of the emulated disease were analyzed with a two-tailed T-test. The statistical test first yielded p-values that demonstrated significantly longer times to carry out all tested daily tasks in the third stage of the disease. Beyond that, a test of mobility and accessibility in the mall was carried out to see how accommodating a public setting is of a person with such a debilitating disease.

Research Summary

        Fibrodysplasia Ossificans Progressiva (FOP) historically referred to as “Stone Man Syndrome” is a rare congenital disease that affects nearly 1 in 2,000,000 people (Connor and Evans,1982). The disease causes bone formation to occur around the patient’s muscles, tendons, ligaments and other connective tissue (Hair, 2002). The ossification of the victim’s joints becomes so severe that it eventually immobilizes the patient completely causing their body to be locked in place. FOP is caused by the R206H single point mutation on the ACVR1 gene located on chromosome 2q23-24. This mutation specifically alters codon 206 from CGC to CAC (Shore et al, 2006). This results in the amino acid histidine to be translated instead of arginine. A PCR based diagnostic test was designed in order to successfully detect the mutation in human DNA. Specifically, the Tetra-Primer ARMS-PCR method used by Ye and his colleagues in their studies determining an efficient procedure for genotyping single nucleotide polymorphisms, was used utilized in our experiment (Ye et al, 2001). A useful online software exists for this specific form of PCR where we were able to input our DNA sequence, the desired location for amplification, and other required criteria to produce optimal primer sets for our use. Not only did the software produce primers for amplification of the specific region of DNA, it also produced expected band lengths for the PCR products that would result from the given primers. Having based our hypothesis on the results of the primer designing computer software, we expected to observe three different PCR products in our gel with the band lengths 195 base pairs, 253 base pairs and 394 base pairs, corresponding to the PCR products of the R206H mutation, the wild type allele, and the non-specific product produced by the outer primers respectively. In addition to the assay we developed that enabled us to better understand the genetic portion of the disease, we also constructed an experiment to observe the sociological implications of having FOP. With no known treatment for the disease, we desired to learn how people with FOP cope with the everyday difficulties they face. Each day, in order to both mentally and physically challenge ourselves in a similar manner to an FOP patient, we added different “growths” and restrictions to simulate the progression and symptoms of FOP. For all intents and purposes we treated each new day as the next year in the life of an FOP patient. We started these trials at age 10 and ended them at age 40 because the median life expectancy of a person with FOP is 40 years and starting at age 10 allows us to start the simulation with mild symptoms and conclude it with extremely severe symptoms (Pignolo et al, 2011).

Original Predictions and Results

        Prior to the actual PCR test, we first had to extract and purify DNA from human bronchial epithelial cells that had Cystic Fibrosis, which were given to us by Dr. Luckie at the Lyman Briggs College at Michigan State University. To get rid of any unwanted impurities in the DNA sample, we followed a protocol from the generation capture column purification kit for human cells (Qiagen, 2010). Following the protocol, we purified two different samples of provided DNA. The purity of the extracted DNA was calculated by an Epoch Spectrophotometer, which yielded 260/280 ratios of 1.781 and 1.363. Two different PCR experiments were done regarding the R206H mutation on the ACVR1 gene. The first experiment used primers that were reported to have successfully amplified the region of the ACVR1 gene where the R206H mutation occurs (Shore et al, 2006). The forward primer 5’-CCAGTCCTTCTTCCTTCTTCC-3’ and reverse primer 5’-AGCAGATTTTCCAAGTTCCATC-3’ were used as control primers in our research. The control primers successfully produced band lengths of 350 base pairs as predicted when an annealing temperature of 55°C was used (Figure 8). For our own designed primers, we used four primers, two inner primers and two outer primers, generated by an online computer software specifically for the Tetra-Primers ARMS-PCR method (Figure 1). Annealing temperatures of in the range of 59°C through 66°C were used for all 5 trials conducted for the design primers because the primer generating software displayed a melting temperature of 69°C for all of the primers. The FIprimer is the primer designed to specifically anneal to the mutant A-allele in DNA, and will produce a band length of 195 base pairs in successful PCR. Conversely, the RIprimer is the primer designed to specifically anneal to the wild-type G-allele in DNA, and will produce a band length of 253 base pairs in successful PCR (Figure 2). Regardless of the presence or absence of the mutation, the two designed outer primers will anneal to the DNA and produce a length of 394 base pairs in successful PCR. Therefore, wild-type DNA should produce band lengths of 394 base pairs corresponding to the outer primers, and 253 base pairs corresponding to the RIprimer in successful PCR. Since the mutation is dominant, a PCR reaction with DNA that is heterozygous for the A allele will produce the same band lengths as the wild-type DNA, but with an additional 195 base pair product corresponding to the FIprimer. After allowing a PCR reaction to be carried out with our designed primers and wild-type DNA, we obtained products with band lengths of around 20 base pairs (Figure 7). These unpredicted products are likely a our primers, which did not anneal to the DNA in the PCR. For the second half of this project we have attempted to simulate 30 years of an FOP patient’s life in 30 days. In a previous study, the researchers determined that the median lifespan for an FOP patient is 40 years, and of those people most are in a wheelchair by the age of thirty (Pignolo et al, 2011). Each day restrictions were added to our bodies to simulate the progressive nature of the disease through the use of various medical braces and a wheelchair (Figure 4). We tested how these restrictions affected how long it took us to complete basic everyday activities such as climbing up to a lofted bed, getting in the car, walking to class, and simply folding laundry. It was predicted that by day eleven when “ossification” of our bones had taken considerable effect, the majority of normal everyday activities would take a significantly longer time to accomplish due to our physical restrictions compared to an ordinary person without FOP. After taking note of how long it took to complete these activities, it is clear that in the later stages of the disease it takes a lot longer (Figure 9). After running our time trial values through a two-tailed T-test, we found that all four of the daily tasks that were tested took significantly longer to complete in our simulated stage 3 of the disease that had the wrists, knee, and neck restricted (Table 3). Based on previous research that concluded women with a physical disability over time have a significantly lower self esteem, we anticipated to experience a decrease in self esteem from the start of the experiment and progressively worsening as the experiment went on as a result of negative social effects such as receiving negative glares and whispers from strangers (Nosek et al, 2003). Such negative reactions were experienced when we subjected ourselves to a mall setting and walked around observing passer-by’s reactions. Beyond that, we also came to find that many of the stores were set up in a way that was completely inaccessible for a wheelchair. In one case, the store we wanted to enter had stairs in the front and no accessible entrance was located. To analyze and determine the significance of the progressive stages, a two-tailed T-test was generated from the time trials. The hypothesis that the restrictions of the disease would lead to daily tasks taking a significantly longer amount of time in our third stage of the disease was proved accurate with an average p-value among the four activities tested of .0174 from the two-tailed T-test (Table 3).

Future Research

        In the future we plan to modify the PCR mixture used for the designed primers in order for our primers to successfully anneal to the DNA. In addition to the PCR mixture being changed, we plan on altering the temperatures and lengthening the times utilized for the PCR. Once successful bands are produced with the wild-type DNA and the designed primers, our plan is to run a PCR with the designed primers and DNA containing the R206H mutation. in the absence of obtaining mutant DNA for another researcher we plan to carry out mutagenesis, which is a technique where by primers and wild-type DNA are run through a special PCR process that that induces the necessary point mutation in our DNA, thereby creating the mutant DNA need to carry out our following research.

        With no known cure, there is a great need for continued research on Fibrodysplasia Ossificans Progressiva. In a 2006 interview with Penn Medicine, Dr. Kaplan, one of the world’s leading investigators of FOP, stated that he believes that understanding the molecular mechanisms behind the R206H mutation will allow scientists to use that knowledge to not only to help FOP patients, but also develop procedures to treat many other skeletal diseases. In addition to a lack of current treatments, FOP far too often is misdiagnosed as fibrosis or cancer. The biopsies and surgeries that come with the misdiagnoses end up causing even more damage to the patient (Kaplan et al, 2008). Future funding and research should also consider the social aspect aspects of living with a disability. A better public understanding perhaps will improve the lives of those with FOP and other debilitating diseases by making those affected feel more supported and comfortable rather than ostracized (Nosek et al, 2003).

View our 30 day still video on the sociological implications of FOP View video here!

View our blog that recorded our personal struggles and experiences while we simulated the life of an FOP patient. Click here to view it!.