Single Nucleotide Polymorphism in Prion Protein Alleles - Essay Example

? Sheep (Ovis aries) and goats (Capra aegagrus hircus) have been identified with a severe disease known as scrapie. Scrapie is one of the many types of diseases known as Transmissible spongiform encephalopathies (TSEs). These are a fatal and degenerative disease that affects the nervous system of sheep and goats. The disease appears to be caused by a polymorphism in the normal prion protein gene (PrP) that results in the formation of a mammalian prion precursor. More specifically there are three polymorphisms in sheep that are acutely linked to the occurrence of Scrapie: A136V, R154H and Q171R. Among these 3 codons there are 5 alleles generated that causes resistance and 3 alleles generated that causes susceptibility to the disease. Therefore, genotype of the breeds may dictate susceptibility or resistance to scrapie. Scrapie can be highly infectious and transmissible among similar species. But being a genetic disease, only individuals that are susceptible in the prion protein (PrP) gene could be infected, regardless of any environmental factors. The objective of this research is to analyze each species genotype as to their genetic make up for alleles. Single nucleotide polymorphism (SNP) in the genotype of the three codons mentioned earlier, renders sheep susceptible or resistant to the disease. The research will involve the genotypic comparison of two breeds of sheep: The Awassi flock and The North Country Cheviot flock, to see which genotype of the two breeds resembles the disease allele the most. DNA extraction and amplification will be done through PCR amplification. Furthermore, identification of the nucleotide sequences that closely resemble those of the disease allele will be determined using methods of restriction enzymes and specific primer binding sites. PROJECT SUMMARY The purpose of this study is to identify sheep susceptibility and resistance for scrapie from two different breeds based strictly on genotypic variations due to single nucleotide polymorphism (SNP) at codons 136, 154 and 171. The primary reason for carrying out this research is to increase awareness about the fatal neurological disease in order to encourage farmers and breeders to take proper measurements in identifying the correct symptoms and to use proper sterile techniques to minimize transmission of the disease from environmental factors. More drastically, with no treatment or cure for scrapie, breeders can anticipate selective breeding to eradicate scrapie by mating a resistant ram (ARR) with any ewe. Thus, it will assure a resistant genotype for scrapie in the offsprings. Although selective breeding showed to have a few successes in the past, the method is not guaranteed to work every time, since factors such as mutation and single nucleotide polymorphism (SNP) render the animal susceptible to acquiring the disease upon interaction. INTRODUCTION Scrapie is a dangerous, degenerative disease affecting the central nervous system (CNS) of sheep and goats. The disease is also referred to as la tremblante (French: trembling), Traberkrankheit (German: trotting disease), or rida (Icelandic: ataxia or tremor) (Detwiler 1992). The disease was first recognized as affecting sheep in Great Britain and other countries of Western Europe over 250 years ago. The earliest definite record of the incidence of scrapie was in Britain in 1732. Following this event, there were accounts of a scrapie-like disease occurring in the Dorset Horn, Wiltshire Horn and Norfolk Horn breeds in England between 1750 and the early 1800s. Scrapie progressed in becoming a major problem in the English Suffolk breed around 1950, leading to successive financial loss in flocks. In 1938, first report of the disease was identified in Canada and since then there have been over 167 flocks in 6 Canadian provinces with confirmed cases of classical scrapie (Plummer 1946). In general, there is a gradual development of clinical cases of scrapie that have been recorded in many regions around the world; notable exceptions are Australia and New Zealand. This is due to the stringent and rapid efforts to depopulate imported scrapie-infected sheep within these countries. This has led to the eradication of the disease and acknowledgment of being the only two scrapie-free countries (Detwiler 1992). As discussed, classical scrapie is a transmissible prion disease, and one of the many transmissible spongiform encephalopathies (TSEs). TSEs are slowly progressive, inevitably fatal, neurodegenerative disorders that are characterized by vacuolated brain neurons and the deposition of an abnormal form of a host protein (PrP). TSEs affect a range of mammalian species. Other prion diseases include chronic wasting disease (CWD) in deer, bovine spongiform encephalopathy (BSE) in cattle and Creutz-feldt-Jakob disease (CJD) in humans (Terry et al. 2011). Prion diseases are transmissible protein misfolding disorders associated with the accumulation of aggregates of misfolded conformations of a host-encoded prion protein (PrP). Prions are recognized as infectious pathogens that differ from viruses, bacteria, parasites and fungi, due to their structure and the disease that they cause. PrP may exist in two forms: a normal cellular prion protein labeled as PrPC and a pathogenic misfolded conformer labeled as PrPSc (Prusiner 1998). The PrP prion protein is found to consist of an estimated 250 amino acids (Hunter 1997). It is also found to be glycosylated at either one or two glycosylation sites and it appears to be attached to the outside of the neuronal cell membrane by a glycosylphosphatidylinositol (GPI) anchor (Prusiner 1998). The prion protein was first determined in an abnormal conformation (PrPSc) and is the essential component of scrapie-associated-fibrils (SAF), a general characteristic of TSEs (Hunter 1997). PrP­­Sc being the only validated biochemical marker for prion disease originates by the catalyzation of PrP­­C molecules into PrP­­Sc fibrils (Imran and Mahmood 2011). PrP­­Sc must come into direct or indirect contact with PrP­­C cell surfaces to convert them into more PrP­­Sc, which will help in proliferation of the disease and where PrPSc will act as a template for the deposition of more aggregates of PrP protein in a new host (Imran and Mahmood 2011). According to Prusiner (1998), prions are devoid of nucleic acid and upon conversion of PrPC to PrPSc, a portion of the ?-helix and coil structure of the prion will be refolded into a ?-sheet. Clinical and pathological phenotypes of scrapie may vary according to the prion strain and animals’ genetic background. Further, from a single isolate of scrapie multiple strains have been recovered and it has been seen that a PrPSc conformer can be dominantly expressed in one breed and under expressed in another (Imran & Mahmood 2011). A major concern in prion disease transmission is the spread of the disease agent by means of secretions and excretions (Maddison et al. 2010). This transmission is a major concern to the control and management of the disease, as transmission tends to occur within the protracted preclinical incubation period (Maddison et al. 2010). The period of incubation is when an infected animal shows no clinical symptoms, but is still capable of transmitting the disease to others. Based on their relative studies of scrapie within the ovine species, Imran and Mahmood (2011) were able to identify the incubation period of scrapie to be 2-5 years with death occurring within two weeks to six months after clinical onsets. With such a long incubation period it is difficult to recognize the infected animal and during the entire period they serve as vectors for transmission of disease to other animals. Scrapie being an infectious and contagious disease, it is found to have multiple means of transmission. Transmission can be caused through lateral/horizontal transmission and vertical transmission. Respectively, lateral or horizontal transmission is the propagation of infection between unrelated animals through direct and indirect contact. Whereas vertical transmission is the propagation of infection or genes that are accountable for the disease from parent to offspring via germ plasm at the time of fertilization or in the uterus during embryonic and fetal development (Detwiler 1992). Oral route of transmission is common in case of lambs and adult sheep that typically contract the agent through ingestion of infectious placentae or fetal fluids or during nibbling (Greenwood 2002). Means of scarification and the conjunctiva are other areas that are also found to be infectious. It is important to note that no infectivity has been found in ovine feces, saliva, urine, colostrum, or milk. Methods of transmission were studied using mouse inoculation test. The mouse inoculation test happens to be the most sensitive technique to demonstrate the presence of the scrapie agent in tissue samples of infected sheep. This gives the investigator a convenient laboratory model that provides information on the nature of the unusual infectious pathogens that caused scrapie (Prusiner 1991). Although it is fairly labor and time consuming, mice inoculated intracerebrally or intraperitoneally develop scrapie like symptoms after 400–700 days. This method allows for clinical symptoms to occur and be studied, due to a far smaller incubation period (Groschup et al 1996). During the course of infection, the infectious prion agent arises presumably from cells to cell infections from the alimentary tract via the lymphatic organs into the central nervous system (CNS). Accumulation of the PrPSc prion protein in the CNS causes formation of amyloid plaques in the brain (Imran and Mahmood 2011). This in turn leads to gradual degeneration of the CNS and the occurrence of numerous clinical symptoms. Since scrapie was identified as an infectious disease, numerous clinical symptoms have been associated with scrapie. The following symptoms appear to occur frequently in the infected animals. The majority of infected individuals portray signs of vigorous rubbing of the backsides, flanks and head, this “scrapping” or rubbing gives it the name scrapie (Detwiler 1992). In case of intense pruritus, this vigorous rubbing is one of the most common symptoms which cause wool loss (Detwiler 1992). Nibbling of the haired skin of the lower legs has been noted, whereas all infected animals appear to exhibit a pronounced nibbling reflex on deep palpation of the soft back tissue (Parry 1962). An acne form papular rash tends to appear on the haired portions if the skin, more specifically on the lower legs and nose (Parry 1962). The ears display signs of hematomata, which cause blood clotting and swelling in the majority of the cases (Parry 1962). Exercise intolerance develops in animals suffering from scrapie and fatigue develops even with moderate exercise (Parry 1962). In certain sheep, asthenia may rapidly progress to general weakness and then the animal shows complete incapability to rise (Parry 1962). There is progressive loss of body condition and emaciation despite measures of food intake to maintain body weight (Parry 1962). Nervous symptoms are evident through disturbances of gait, especially of the hind limbs, and a high stepping movement of the forelimbs may also occur (Parry 1962). In severe cases ataxia is seen, a condition where the animal has involuntary coordination of muscle movements that renders them incapable of walking or standing normally (Parry 1962). Other nervous symptoms like mental disorder, changes in behavior, personality changes, and dementia occur very frequently (Parry 1962). For instance, some become uncooperative and reject being driven; some become fearless of humans and predators while others become extremely impulsive and are distressed by any change in their daily routine (Parry 1962). In 5 % of the cases, blindness and tremor tends to occur (Plummer 1946). As a result of unstable cardiovascular system the body temperature and heart rate show uncommon fluctuations which remain within normal limits (Parry 1962). Autopsy results indicate that there is enlargement of adrenal glands and shrinking of thyroid glands in all infected sheep (Parry 1962). During the incubation period no symptoms are apparent, but the disease remains transmissible. The disease is slowly progressive with death in 2 weeks to 6 months, rarely a year after the occurrence of clinical symptoms (Parry 1962). It is important to note that no vaccination and treatment has been identified to treat scrapie and its relative symptoms (Babar et al. 2008). The genetic make up has been determined to be a significant factor that affects susceptibility to infection with scrapie in an animal (Greenwood 2002). The progression of scrapie is influenced by specific single nucleotide polymorphisms within the protein-coding sequence of the host prion protein gene (PRP), and the resulting amino acid changes (Hautaniemi et al. 2012). Further, it is the presence of specific amino acids at certain sites on the gene that confers scrapie susceptibility, whereas the presence of other amino acids at the same sites on the gene will confer resistance to the disease (Hautaniemi et al. 2012). Variation in three regions of the prion protein gene, namely codons 136, 154 and 171, are related to resistance to natural scrapie. For instance, changes due to SNPs of alanine (A) to valine (V) at codon 136, arginine (R) to histidine (H) at codon 154, and glutamine (Q) to arginine (R) at codon 171 will render a sheep resistant in acquiring the disease (Hunter 1997). A136R154Q171 haplotype is found to be the most susceptible to scrapie versus A136R154R171 haplotype, which is highly resistant (Hunter 1997). The resistant genotype can prevent the shedding of abnormal prion (PrPSc) at lambing, and there is no accumulation in the placenta and associated fluids (Greenwood 2002). Thus it renders the animal to be scrapie free. There are two other resistant alleles that are not very common and they are respectively V136R154Q171 and A136H154Q171 (Hautaniemi 2012). From the study it can be said that the specific amino acids and the sites that appear to confer susceptibility versus resistance vary among the breed of sheep and that not all breeds are resistant. The objective of the present study is to investigate the genetic makeup of different sheep breeds of the Awassi flock and the North Country Cheviot flock, at the prion gene in order to determine the degree of resistance of these breeds to natural scrapie. With this, we will be able to determine the probability of how close each breed genotype is to the disease allele of the PrP gene. DNA extraction and amplification will be done through PCR amplification. Furthermore, identification of the nucleotide sequences that closely resemble those of the disease allele will be determined using methods of restriction enzymes and specific primer binding sites. METHODOLOGY Sample collection To study the genotype of both breeds of sheep, we must collect a cell sample. Cells will be collected using a sterile swab technique. “Catch all” sample collection swabs are used, as they tend to contain a soft porous foam swab that allows a higher sample yield. Collection of cells will be from the oral cavity of 5 Awassi sheep and 5 North Country Cheviot sheep. Both flocks are from separate local farm in Edmonton, Alberta, in which no disease or sickness has been recorded in either flocks. No harm will be done to the animals as we are only collecting a cell sample from the inner cheek of the oral cavity by gently rubbing the swab on both inner cheek surfaces. DNA Extraction, PCR and DNA Sequencing This experiment will be conducted in a level 2 containment laboratory over a one-month period at Concordia University College of Alberta. For rapid preparation of DNA, for PCR asssays from buccal sample, a single-tube system of BuccalAmp™ DNA Extraction Kit can be used (BuccalAmp™…2012). DNA that could be used specifically with the kit can be from human or other mammalian buccal (cheek) swab samples. QuickExtract™ DNA Extraction Solution is present in the kit that allows the rapid processing of samples using a simplified protocol (BuccalAmp™…2012). The protocol consists of rotating the sample swab vigorously in the QuickExtractTM DNA extraction solution a minimum of 5 times, assuring to press the brush against the side of the tube and rotating it while removing it from the tube to ensure most of the liquid remains in the tube and no sample is lost. Then vortex the tube for 10 seconds followed with specific heating for 1 minute at 65 °C. Vortex once again for 15 seconds followed by heating at 98 °C for 2 minutes and vortex one last time for 15 seconds. The final product allows for a PCR-ready DNA to be obtained. No centrifugation step is needed and sample-handling times are even shorter and found to be more efficient (BuccalAmp™…2012). Through the kit it is much easier to process one to hundreds of samples in less than an hour without phenol, chloroform, or other toxic organic solvents (BuccalAmp™…2012). DNA yields obtained using the BuccalAmp Kit with Catch-All™ Swabs range from 1 to 7 µg of DNA per buccal sample which is enough to perform about 100 PCR assays (BuccalAmp™…2012). Polymerase Chain Reactions (PCR), a biochemical technology used to amplify a single or a few copies of a piece of DNA, is used to generate thousands to millions of copies of a particular DNA sequence. The concentration of reagents used in PCR reaction for a 25 ?L PCR reaction are: 2.5 ?L of 10X PCR buffer stock solution, .5?L of dNTPs (for the final concentration to be 200?M), 1 ?L of each PCR primer (at 3.2pmole/ ?L concentration), 1 ?L of 60ng/ ?L of DNA template, 0.5ul 25mM MgCl2, 0.125ul Taq DNA polymerase, and 18.375ul of sterile triple distilled water to equal a 25ul reaction (Gipson 2002). In addition, a negative control will be made using sterile water because it is more cost efficient. Be certain to store sample on ice until ready to amplify according to the following profile. A restriction enzyme, BspH1 will only be used to amplify the DNA sequence of codon 136. For codons 154 and 171, specific binding primers will be added to the PCR mix, to identify and amplify specific DNA sequences. These amplified DNA sequences of all three codons, will assist in determining resistance or susceptibility in the genotype. The PCR procedure consists of a denaturation step at 95?C for 1-5 minutes, followed by 30 cycles in the thermal cycler, where the cycles were of 95?C for 1 minute, 55?C for 1 minute, 72?C for 2 minutes and a last step of extension at 72?C for 10 minutes (Gipson 2002). PCR product was examined and visualized using 1.2 % agarose gel electrophoresis. Add 0.6 grams of agarose to 50 ?L of 1 x TAE buffer. Microwave the agarose to melt it completely, and upon cooling; add 3 ?L of Ethidium Bromide to the liquid agarose. Followed by gentle swirling to mix, pour solution into prepared gel tray and allow it to cool. To the PCR products we will then add 6 x loading dye (DAPI) and then load them, the DNA ladder and the negative control onto the gel. After running the gel at 80 V electrical current, we than examine the gel under a UV Transilluminator. We should be able to identify the DNA strands that have been cut with the restriction enzyme and those that have the primers annealed to them. DNA strands will be determined based on their size. Thus, we will be able to identify if both breeds of sheep contain a susceptible or resistant genotype for scrapie. REFERENCE 1. Babar ME, Hossain F, Benkel B, Khan, Q, Ahmad, J, Nadeem, A. 2006. Genetic variability at eight codons of the PRP gene in 9 sheep breeds of Pakistan. 8th World Congress on Genetics Applied to Livestock Production. 22-30. 2. Detwiler LA. 1992. Scrapie. Rev. sci. tech. Off. int. Epiz. 11(2): 491-537. 3. Epicentre: an illuminia company [Internet]. c2012. Epicentre [cited 2012 Dec 11]. Available from: http://www.epibio.com/item.asp?id=270. 4. Gipson J. 2002. PCR Protocol. [Internet] [cited 2012 Dec 11]. Available from: microgen.ouhsc.edu/manuals_protocols/pcr_protocol.doc. 5. Greenwood P. 2002. Federal disease control — Scrapie. Can Vet J. 43:625-629. 6. Groschup MH, Weiland F, Straub OC, Pfaff E. 1996. Detection of Scrapie Agent in the Peripheral Nervous System of a Diseased Sheep. 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