Malaria Resistant Humans - Research Proposal Example

Malaria resistant humans Introduction Malaria, caused by the parasite Plasmodium spp and transmitted by the Anopheles mosquitoes, is viewed as the third deadly disease in the world. (Zimmerman et al. 1999). Many efforts are taken to control malaria but still it remains as a major problem over many years. The humans are exposed to the malaria parasite all over the world and only a few are not affected by the plasmodium spp. The variation in the immune system of the humans and the modification in the human blood cells is the major reason for the malaria resistance in humans. Our human body possesses a lot of adaptations against malaria. Polymorphisms are one of the reasons for the resistance to the infection of Plasmodium vivax. The variations conferring to the resistance have evolved from about 1000 – 5000 years. Many variants have been discussed for malaria. Some of them are globin, G6PD deficiency, Duffy, ovalocytosis, ABO and human leukocyte antigen variants. (Zimmerman et al. 1999). The most studied aspect of malaria resistance in humans is hemoglobin molecules. It was Haldane, who brought in 1949 into light about the link between genetic blood disorders and malaria protection. (Zimmerman et al. 1999). Mutation, natural selection and migration are the important mechanisms that play a major role in genetics. There is some interrelation between humans and malaria. Genetic factors and the higher levels of anti-malarial antibodies in the particular ethnic groups are the major reasons of malarial resistant to humans. The hemoglobin molecule is a protein that contains two alpha and two beta subunits. The genes encoding these sub units have many alleles. These alleles are largely affected by the point-mutations in the DNA sequence. Point mutations alter a single amino acid in the protein and reduce the red blood cells oxygen transport efficiency. (Sibinga and Klein 2000). In the Africa at the sub-Sahara region, an abnormality was studied at the beta sub unit of the hemoglobin molecule. The high frequency of the allele was the result of the point mutation. The glutamic acid in the beta sub unit was replaced by valine. This change created change in the shape of the hemoglobin molecule. (Walliker, Walliker and Chappell 1996). The mutation crystallized hemoglobin under low oxygen tension and the red blood cell shape changed from normal platelet shape to sickle- shaped. The patients are identified to have developed malaria resistance. Malaria resistance in humans might have occurred by modification in the immune system and the native genetic resistance. Genetic resistance to malaria is observed in some group of people in sub- Sahara and South East Asia. (Sibinga and Klein 2000). Malaria resistance is conferred in the genes of the people. It is a type of genetically controlled immunity present in some people to control the disease at the beginning stage itself. The lack of the expression of the gene Duffy antigen receptor for chemokines (DARC) in the blood cells of the sub-Saharan region has resulted in the high malaria resistance in those people. The DARC negative individuals contain a single point mutation at the 46th position. (Sibinga and Klein 2000). The DARC negative individuals are homozygous and the point mutation creates a variation in the structure of the erythrocyte. The toxin produced by the parasite Plasmodium vivax could not bind to the erythrocytes due to the structure variation. As a result, these individuals are resistant to malaria caused by the P. vivax. The hemoglobin disorders are classified into Hemoglobin S, Hemoglobin C, Hemoglobin E, alpha and beta thalassemia. Hemoglobin S (HbS) occurs due to the 6th codon point mutation in the beta globin gene. (Dronamraju and Arese 2006). The hemoglobin S homozygotes have sickle cell disease, which is the result of the red cell deformation and low oxygen tension. HbC is the structural variant caused by the replacement of lysine to glutamic acid. It is encoded at the HBB gene. It is present in the Africa but is less common than the HbS. If the lysine substitution occurs at the codon 26 for glutamic acid, it results in the HbE. (Dronamraju and Arese 2006). It is very common in South East Asia. For the isolation and identification of the DARC gene from the blood sample many techniques and technologies are used. The techniques that are used for the identification of the malaria parasite in the patients are DNA isolation, Agarose gel electrophoresis, Restriction digestion, Polymerase chain reaction ( PCR), Northern blotting, Restriction fragment length polymorphism ( RFLP) and Polyacrylamide gel electrophoresis (PAGE). (Dronamraju and Arese 2006). DNA is a double stranded molecule present in the all living organisms which encodes the instructions for the functioning and development of the organisms. DNA isolation is the process of extracting the DNA molecules from the living cell. (Scopes 1994). Restriction endonucleases are the bacterial enzymes that cleave the double stranded DNA at specific ends. Restriction endonucleases are of many types. Type II restriction endonucleases are mainly used for the DNA cleavage at specific sites. Each restriction enzyme has a specific code and the restriction enzyme uses these codes for cutting the DNA stands. Restriction enzymes cut the DNA to form sticky or blunt ends. These Restriction endonucleases require specific temperature, pH for maximum activity. The restriction enzymes are stored at 4°C to maintain their activity level. (Scopes 1994). The restriction enzymes sites act as markers on the DNA molecules and enable to locate the specific regions on the defined lengths of the DNA. These DNA fragments sizes are determined using the agarose gel electrophoresis. (Siwach and Singh 2007). Agarose gel electrophoresis is the standard method for the separation of the DNA segments based on their molecular weight. Agarose is the linear polymer containing alternating residues of D and L galactose joined by the glycosidic linkages. The electrical power is used for the separation of the DNA fragments in the gel. (Scopes 1994). The rate of migration of the DNA is inversely proportional to the size of the DNA. But this electrophoretic mobility of the DNA is affected by the shape of the DNA and the pore size of the Gel. (Scopes 1994). DNA molecules are negatively charged and so they move towards the Anode. The electrophoresis buffer acts as the medium for the charge transfer and enables the DNA molecules to move through the pores of the gel. When the molecules move down wards, the gel acts as a molecular sieve and hold the larger fragments at the top, leaving the smaller fragments down. (Scopes 1994). Thus the DNA fragments are separated based on their charge, amount of current applied and molecular weight, conformation of the DNA and concentration of the DNA. A DNA marker or DNA ladder is used to identify the size of the fragments. The force that separates the molecules through the gel matrix is the Electromotive force. (EMF). The molecular weight of the protein was estimated using the SDS –PAGE. (Scopes 1994). The polyacrylamide gel electrophoresis is the most widely used technique for the determination of the molecular weight of the protein. SDS Page method is the universally used method for analyzing the mixture of proteins according to their respective size. (Desai, Allen and Neuberger, 1981). Separation of the proteins does not occur with similar charge. Therefore the proteins are treated with ionic detergent called SDS before the start and during the course of the electrophoresis. On the basis of the mass but not the charge the molecules are separated. (Scopes 1994).The gel acts as a molecular sieve and hence separates the proteins on the basis of their size. Sty I is the restriction enzyme used for the analysis of the Fy antigen in the given gene. The recognition site of Sty I is CCWWGG. The primers for the Fy antigen can be designed by using the primer 3 software. (Saiki et al. 1985). The Restriction fragment length polymorphism is a technique used for the exploitation of the variations in the DNA sequences. It is an important tool for analyzing the genes and locating the genes for the genetic disorders. RFLP can be used as a tool for determining the risk factor for a disease. Before RFLP is performed the DNA sample is fragmented by using a specific restriction enzyme. The DNA fragments thus obtained using the restriction enzyme are separated based on their size by the Agarose gel electrophoresis. The gel is then transferred into the Nitrocellulose membrane using the southern blotting technique. Labeled DNA probe specific for the fragments are hybridized on the DNA. Each fragment is considered as an allele and the length of the detected fragment may vary between the individuals. (Saiki et al. 1985). DNA detection The malarial resistant genes are identified by the assessment of the association between the particular genetic markers and the disease. Similarly the mouse models are also used for the identification of the malarial resistant genes. (Walliker, Walliker and Chappell 1996). Malarial gene resistance can be detected b y the PCR and other nucleic acid amplification techniques. Many PCR assays are developed to detect the parasite Plasmodium falciparum and four other species. The association between the disease and the polymorphism are used for the analysis of the resistant gene in the human. The Fy gene that encodes the protein called Duffy antigen is present in all the red blood cells of the people in the world except the population from the sub-Saharan Africa. This can be used as the negative control for the detection of malaria in the individuals who are resistant to malaria. (Tournamille et al. 1995). A single point mutation present in the erythroid –specific transcription factor binding site of the GATA box promoter site of the FY gene results in the absence of the Duffy glycoprotein in the Sub Sahara African population. On analyzing the structure of the Duffy blood antigen, it was observed that the erythrocyte receptor site contains a widely expressed chemokine receptor. The Duffy antigen/chemokine receptor gene (DARC) contains a single exon. The resistant black people contain a silent FY*B allele with the single T to C substitution at the 46th position. This mutation causes the disruption at the GATA1 erythriod transcription factor. (Tournamille et al. 1995). To isolate the FY*B gene, allele specific PCR and PCR –RFLP are used. The FY*A allele is also used for the distinguision. The GATA box genotyping is then performed to identify the molecular basis. (Smit, Sibinga and Klein, 2000). Methodology The blood samples were collected from the cohort individuals and the DNA extraction was performed using the standard DNA extraction protocol from blood. The DNA is quantified and purified and the PCR amplification was carried out. Allele specific PCR genotyping was carried out to determine the allele FY*B. (Di Cristofaro et al. 2010). The primers P1 (CCT TTT TCC TGA GTG TAGT) and P2 (GCA GAG CTG CCA GCG GAA GA) are used for the isolation of the FY*B allele from the blood samples. These two regions are the regions with the polymorphisms in the erythroid specific transcription factor at the GATA box. PCR amplification was performed in the automated thermal cycler. The DNA was denatured at 94 ˚ C for 4 minutes, and re natured and amplified at 60˚ C and the final extension was carried out at 72 ˚ C for 10 minutes. (Zimmerman et al. 1999). Nested PCR amplification was practiced for the first reaction and the second set of primers are used for the amplification of the isolated amplified DNA. The primers used for this reaction are P38 (AGG CTT GTG CAG GCA GTG) and P39 (GGC ATA GGG ATA AGG GACT). (Zimmerman et al. 1999). These two primers are used to obtain a PCR product of 221 bp. These DNA are extracted from the gel and the Sty I enzymes digestion was carried out at 37˚ C for 4 hours and the digested products were separated by the Sodium dodecyl sulfate – polyacrylamide gel Electrophoresis ( SDS- PAGE). (Zimmerman et al. 1999). The PAGE is then stained in the silver nitrate solution and the bands were analyzed. (Arévalo-Herrera et al. 2011). The bands were analyzed. The bands 62, 77, and 82 bp were considered as the Fy antigen bands and the absence of FY antigen was confirmed by the presence of four bands 12, 62, 65, and 82 bp. (Parasol et al. 1998). Fig.1: fragments generated by the Sty I digestion of the DARC fragment. (Zimmerman et al. 1999). Fy positive individuals will have the wild type GATA box and will produce three bands on restriction digestion of sizes 62, 77, and 82 bp respectively. Whereas the mutant Fy negative individuals will produce four bands during the restriction digestion of sizes 12, 62, 65, and 82 bp respectively. PCR combined with RFLP can identify the single nucleotide change in the given samples. The difference between the Fy*A and Fy*B alleles is a single nucleotide change. Figure 2: RFLP patterns of the DNA for the samples. (Zimmerman et al. 1999). The first lane is the positive control for the Fy antigen. The 5 lanes (lane 2 – 6) indicate the malaria resistant humans sample containing the Fy antigen and the last lane (lane 7) is the negative lane indicating the mutant Fy antigen. The lanes 2-5 indicates that the patients have the Fy wild antigen and it is confirmed by the occurrence of the three bands in the gel 62, 77, and 82 bp respectively. (Zimmerman et al. 1999). The Fy mutant which is the negative control has four bands of size 12, 62, 65, and 82 bp respectively indicating that mutation has occurred in the DARC gene. Conclusion: By using the recombinant DNA technology, the DNA was isolated from the patient blood sample. The allele specific PCR was carried out to identifiy the FY*B gene. The gene was then amplified using the primers 38 and 39 and the restriction digestion was carried out to find out the presence of DARC allele in the FY*B gene. The Polyacrylamide gel electrophoresis was performed and stained with silver nitrate to visualize the bands. The presence of three bands in the patients sample indicates the presence of resistant gene in the patient. References: Arévalo-Herrera, M, Solarte, Y, Rocha, L, Alvarez, D, Beier, JC, Herrera, S., 2011. Characterization of Plasmodium vivax Transmission-Blocking Activity in Low to Moderate Malaria Transmission Settings of the Colombian Pacific Coast, The American journal of Tropical Medicine and hygiene, Vol.84, No.2, pp. 71 – 77. Desai, NN, Allen, A, and Neuberger, A., 1981. Some properties of the Lectin from Datura stramonium (thorn-apple) and the Nature of its Glycoprotein Linkages, Biochemistry Journal. Vol.197, pp. 345-353. Dronamraju, KR and Arese, P., 2006. Malaria: Genetic and Evolutionary aspects, Springer. 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