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Bioinformatics Analysis of COPD-Associated IL-6 and CHRNA3 Gene Variations

This study delves into the genetic variations of IL-6 and CHRNA3 in COPD and employs bioinformatics tools to assess their functional effects. Results show significant findings that may lead to phenotypic diversity in COPD. Future work involves experimental validation of the predicted impacts.

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Bioinformatics Analysis of COPD-Associated IL-6 and CHRNA3 Gene Variations

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  1. 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

  2. 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

  3. 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

  4. 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

  5. Previous studies CHRNA3 IL-6 rs1818879 IL-6 gene rs1051730 CHRNA3 gene COPD COPD

  6. Genetic variants in question Chromosome 7 • Inflammatory diseases • Endogenous pyrogen IL-6 Pro-inflammatory cytokines

  7. Genetic variants in question Chromosome 15 • Nicotinic addiction • Up-regulated with chronic tobacco smoking CHRNA3 Nicotinic Acetylcholine Receptor alpha subunit (nAChR)

  8. 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

  9. 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

  10. 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

  11. Methodology of mfold Predicts RNA secondary structures

  12. 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

  13. 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

  14. Results CHRNA3

  15. Results First transcript consisted of 3202 bp Wild-Type Risk structure

  16. Results Second transcript consisted of 2030 bp Wild-Type Risk structure

  17. Results Third transcript consisted of 2030 bp Wild-Type Risk structure

  18. 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

  19. 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)

  20. 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

  21. Thank you

  22. 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

  23. 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

  24. 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

  25. 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

  26. RNA 2ndry structure SNP experimental design… C)Exon 21 SNP (rs13180) – Common primer Risk WT RS Common primer generate 165bp 75bp 55bp RNase T1

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