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Title: Molecular Characterization of Toll Like Receptor-2 Gene for Single Nucleotide Polymorphism Among Complicated Measle Cases in Peshawar, Pakistan
Authors: Ilyas, Muhammad
Keywords: Biological & Medical Sciences
Issue Date: 2021
Publisher: University of Peshawar, Peshawar.
Abstract: Measles is highly contagious viral disease and transmitted form one person to another through respiratory tract by coughing, sneezing or direct contact with respiratory droplets. Measles infection play clever role in targeting IL-12 suppression, providing an opportunity for replication of virus inside the host. Host immune responses decreased during the course of measles infection. It provides a platform for secondary bacterial infections which increase the risk for early childhood mortality. Malnutrition and immune suppression are the leading risk factors responsible for several measles complications like Encephalitis, Blindness, Severe diarrhea, Otitis media, Hepatitis and Myocarditis. Recent studies suggested that signaling through TLRs play important role in measles pathogenicity and immunogenicity. For the last few years extensive research profile augment the role of TLR2 in immunity of vertebrate animals. Important functional feature of TLR2 is to form heterodimers with other TLRs necessary for its activation. The presence of single nucleotide polymorphism in the human genome makes individual differences like difference in phenotype, susceptibility of the host to various diseases, drugs, chemicals and pathogens. The current study is designed to identify genetic polymorphism in TLR2 gene among complicated measles cases. In the present study a total of 100 measles infected children associated with various complications were included after fulfilling the inclusion and exclusion criteria. All demographic data and clinical information were collected on separate performa. The blood sample was collected and used for determination of anti-measles IgM antibody through ELISA technique. Out 100 patients, 50 whose serum IgM titers were ≥180 I.U were selected for further molecular characterization of TLR2 gene. Genomic DNA was extracted and visualized through gel electrophoresis. Primers were designed against all the exonic xvii regions of the TLR2 gene and amplified through Polymerase Chain Reaction (PCR). All the PCR products were also visualized through gel electrophoresis. After purification, the PCR products were sent for sequencing. Identification of single nucleotide polymorphism was done through BioEdit ClustalW. After the identification of SNPs, in-silico verification tools such as Polyphen-2, Mupro, SIFT, FATHM, and Provean were used to examine the effect of polymorphism on the structure and function of the TLR2 protein. Protein structure of the reference and mutant were made through Swiss Model Homology and both structures were compared and analyzed. In this study out of 100 patients, female (52%) slightly exceeded the males’ patients (48%). In terms of measles vaccination status, 76% of children were found to be unvaccinated, while 24% were vaccinated. Among the infected children, 77% had elevated IgM level, 8% had intermediate and 15% had found negative. Major complications were Pneumonia, Lower Respiratory Tract Infection, Diarrhea, Otitis media, Myocarditis and SSPE, more than one complication were also observed among unvaccinated children. Majority of the infected children (n=63) were under the age of 12 months and were experiencing measles complications. In terms of molecular characterization of the TLR2 gene, the single nucleotide polymorphism rs1816702 was observed in the regulatory region of the TLR2 gene, while rs3804099 and rs3804100 were observed in the coding region. In patients, overall percentage of rs1816702, rs3804099 and rs3804100 was 60%, 44%, and 40% respectively, while in the control group it was 44%, 26%, and 24%. Novel single nucleotide polymorphisms (G>A) (4%), (A>T, A>G) (8%) and (T>C, A>C) (2%) were only seen in the patient group. Significant association of Pneumonia (p 0.002) and Lower Respiratory Tract Infection (p-0.003) was established with rs1816702. Similarly positive association (p-0.003) was seen between rs380410 and xviii Lower Respiratory Tract Infection. Protein structure models were developed from the Reference protein (TLR2) and mutant T513A by using 2Z7X (PDB) as a template. Both structures were superimposed and RMSD (0.66) was calculated. Both structures showed 99.81% similarities. According to the Ramachandran plot, >90% of the residues were found in the favorable region. The PDB files for each target uploaded in Verify 3D sever to validate the 3D structures of the target model. It has been observed that 3D model of the mutant (T513A) showed 93.79% of the residues have average 3D-ID score ≥=0.2. It has been observed that 3D model of reference protein showed 93.74% of the residues have average 3D-ID score ≥=0.2. Protein structure analysis revealed that hydrogen bond is present between the two threonines at position 513 and 512 with distance of 2.52Å in the reference protein. But in mutant (T513A) substitution of threonine by alanine at position 513 abolish hydrogen bond between alanine and threonine, but almost the effect of non-synonymous polymorphism was neutral on protein structure. It was concluded that major complications observed in the present study were Pneumonia, Lower Respiratory Tract Infection, Diarrhea, Otitis media, Myocarditis and SSPE, while some patients suffered from more than one complication. Synonymous polymorphism rs1816702 was found in the regulatory region and rs3804099 and rs3804100 was in the coding region of TLR2. Non synonymous novel polymorphism was found in the coding region of TLR2 gene. The effect of the novel non-synonymous polymorphism on protein structure and function of TLR2 was neutral.
Gov't Doc #: 23845
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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