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Bioinformatics Analysis of Chronic Obstructive Pulmonary Disease (COPD) – Associated Interleukin-6 and CHRNA3 Genetic Variations. Arwa Kheder Al- Zahrani Dareen Sami Ashgar Hanadi Mohammed Zbrmawi Tasneem Abdella Al- Sharief Supervised by: Dr. Aiman Al-Saegh
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Bioinformatics Analysis of Chronic Obstructive Pulmonary Disease (COPD) – Associated Interleukin-6 and CHRNA3 Genetic Variations Arwa Kheder Al-Zahrani Dareen Sami Ashgar Hanadi Mohammed Zbrmawi Tasneem Abdella Al-Sharief Supervised by: Dr. Aiman Al-Saegh Assistant professor of Molecular Diagnostics
Chronic Obstructive Pulmonary Disease (COPD) • COPD is characterized by airflow limitation • COPD has three phenotypes: • According to World Health Organization (WHO), it is the fourth leading cause of death in the world Healthy Bronchitis Healthy Emphysema
Risk factors Tobacco smoke • Primary risk factor of COPD • Tobacco smoke accounts for 50-80% of COPD cases • Risk of COPD falls to half with smoking cessation Pollutants Viral infections Age Gender
Continued.. Genetics Does genetics play a role in COPD? Alpha 1 anti-trypsin only 1% of COPD cases How? Familial studies What is a SNP? Single base-pair change in DNA sequence
Previous studies CHRNA3 IL-6 rs1818879 IL-6 gene rs1051730 CHRNA3 gene COPD COPD
Genetic variants in question Chromosome 7 • Inflammatory diseases • Endogenous pyrogen IL-6 Pro-inflammatory cytokines
Genetic variants in question Chromosome 15 • Nicotinic addiction • Up-regulated with chronic tobacco smoking CHRNA3 Nicotinic Acetylcholine Receptor alpha subunit (nAChR)
Aim To use the available bioinformatics tools to evaluate: • The functional effect of SNP located on 3’UTR of IL-6 rs1818879 mRNA 5’UTR 3’UTR • Possible effect of synonymous SNP located on coding region of CHRNA3
Bioinformatics A computational approach to resemble what happens inside our bodies using computational algorithms and thermodynamics stability • CHRNA3 IL-6 Using miRBase software to predict the effect of SNP (rs1818879) binding probability of micro-RNA (miRNA) • Using mfold software to test the possible impact of synonymous SNP (rs1051730) • on RNA secondary structure
Methodology of MirBase Evaluation of the similarity between miRNA and mRNA sequence G G C U C A C G C C U A U A A U C C C A G C U G A G C U C A U G C C U G A U C C C A G C
Methodology of mfold Predicts RNA secondary structures
Results IL-6 mRNA G G C U C A C G C C U A U A A U C C C A G C Mir-619-5p p=0.025 U G A G C U C A U G C C U G A U C C C A G C rs1818879 mRNA 5’UTR 3’UTR
Results IL-6 mRNA G G C U C A C G C C U A U A A U C C C A G C Mir-619-5p p=0.13 U G A G C U C A U A C C U G A U C C C A G C rs1818879 mRNA 5’UTR 3’UTR
Results CHRNA3
Results First transcript consisted of 3202 bp Wild-Type Risk structure
Results Second transcript consisted of 2030 bp Wild-Type Risk structure
Results Third transcript consisted of 2030 bp Wild-Type Risk structure
Conclusion • Demonstration of the binding probability between variant in IL-6 and hsa-mir-619-5p • Prediction of RNA secondary structure of CHRNA3 wild and risk allele
Bartoszewki and colleagues(2017) • Significant ? • Reliable ? • Can these findings lead to phenotypic diversity ? “The results presented herein offer significant experimental proof regarding this important prediction.”ــBartoszewski and colleagues(2017)
Future work • Experimentally validate the predicted impact of both variants on miRNA and mRNA • To investigate other COPD associated variants in IL-6 and CHRNA3 • Use these biomarkers for predictive and meditational purposes
Experimental design miRNA Wild-type allele Cells Mutated allele Cells Pulmonary cells IL-6(+) miRNAs (mir-619-5p) not expressed Transfect cells with mir-619-5p Transfect cells with mir-619-5p Extract RNA/Protein Extract RNA/Protein Measure IL-6expression using RT-PCR/Western blotting
Synonymous SNP… CHRNA3 Exon 7 SNP (rs1051730) • Experimental design: cDNA expression clone (CHRNA3 coding sequence) Introduce the minor allele of rs1051730 by mutagenesis In vitro transcription (T7 Polymerase) and purification Folding buffer RNase digestion (T1 cleaves at 3’ single-stranded CU) Reverse-Transcription PCR
RNA 2ndry structure SNP experimental design… Exon 7 SNP (rs1051730) – Wild-Type primer Wild-type Risk WT primer RNase T1 Wild-type primer generate 150bp RNase T1
RNA 2ndry structure SNP experimental design… C)- Exon 21 SNP (rs13180) – Risk allele primer RNase T1 RNase T1 Risk Wild-type Risk allele primer Risk allele primers generate 75bp Risk allele primer
RNA 2ndry structure SNP experimental design… C)Exon 21 SNP (rs13180) – Common primer Risk WT RS Common primer generate 165bp 75bp 55bp RNase T1