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NRAMP1 Gene Polymorphism in Patients with Rheumatoid Arthritis in Pakistani Population

Hafsah Muhammad Chishti 1,2 , Abdul Hameed 2 , Samina Jalali 1 , Muhammad Ajmal 2 , M. Nasir Malik 2. NRAMP1 Gene Polymorphism in Patients with Rheumatoid Arthritis in Pakistani Population. 1 Department of Animal Sciences, Quaid-i-Azam University, Islamabad

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NRAMP1 Gene Polymorphism in Patients with Rheumatoid Arthritis in Pakistani Population

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  1. Hafsah Muhammad Chishti1,2, Abdul Hameed2, Samina Jalali1, Muhammad Ajmal2, M. Nasir Malik2 NRAMP1 Gene Polymorphism in Patients with Rheumatoid Arthritis in Pakistani Population 1 Department of Animal Sciences, Quaid-i-Azam University, Islamabad 2 Institute of Biomedical and Genetic Engineering, KRL Hospital, Islamabad

  2. Rheumatoid Arthritis • Disease Characteristics • Prevalence • Symptoms • Diagnosis • Causes • Environmental • Genetic • Treatment

  3. Candidate Genes for RA • HLA-DRB1 • PTPN22 • TRAF1-C5 • CTLA • PADI4 • STAT4 • NRAMP1/ SLC11A1

  4. NRAMP1 / SLC11A1: The GENE • Natural Resistance Associated Macrophage Protein 1 • Located on chromosome 2q35 • Approximately 14 kb in length • Contains 15 exons • Has been linked to many infectious and autoimmune diseases • Regulates and also regulated by cellular iron levels

  5. NRAMP1: The Protein • Originally described in mice for its roles in regulating resistance and susceptibility to infectious agents. • Expressed in macrophages , neutrophils and in bone marrow derived dendritic cells • highly hydrophobic integral membrane protein • 12 transmembrane domains • transport activity and ion channel • Can resist several intracellular pathogenic microorganisms

  6. Functions of nramp1 Protein • Nrampl regulates in early pathway of • macrophage activation • activation for antimicrobial activity • These functions are of potential importance in the induction or maintenance, or both, of autoimmune disease. • An animal resistant to intracellular pathogens may be susceptible to autoimmune disease.

  7. NRAMP1 Gene Polymorphisms • Nine sequence variants reported in the NRAMP1 gene • one (GT)n in the 5’ region (promoter region) • three in introns, one 4-bp deletion in the 3’ UTR (1729+55 del4; 55 nt 3’ to the last codon in exon 15) • The remaining four variants are in the coding region: • two silent substitutions (274 C/T, 823 C/T) • two missense mutations (A318V, D543N)

  8. NRAMP1 Gene Polymorphisms continued… A318V At codon 318 in exon 9 C or T at nucleotide 1029 C coding for Alanine T coding for Valine 274C/T At codon 66 in exon 3 C or T at position 274 silent nucleotide substitution coding of Phenylalanine D543N • At codon 543 in exon 15 • G or A at nucleotide 1703 • G coding for Aspartic Acid • A coding for Asparagine

  9. Objectives • To investigate the NRAMP1 gene polymorphism(s) in Pakistani population. • To study the association of NRAMP1 gene polymorphisms (D543N, A318V, 274C/T) in susceptibility to RA.

  10. Materials and Methods

  11. Primers sequences, annealing temperature and PCR product sizes

  12. Master mix preparation for restriction enzyme digestion

  13. Statistical analysis • Allele frequency calculated on the basis of Hardy-Weinberg Equilibrium equation using formulae: p² + 2pq + q² = 1  & Allele frequency = No. of that allele in study group Total numbers of alleles • Comparisons of genotype frequencies done by applying chi-square test • Allele frequencies compared by fisher’s exact test, odds ratios and 95% CI (Confidence Interval), using statistical software InStat (version 3) • P<0.05 was considered significant.

  14. RESULTS

  15. Sizes of restriction products according to specific polymorphism

  16. Representative gel of PCR and restriction digestion results for D543N Lane 1 = Molecular ladder/marker; Lane 2 and Lane 11 = undigested PCR product; Lane 3–10 and 12–19 = Digested samples 1–8 and 9–16 respectively.

  17. PCR results for D543N after restriction digestion with Ava II Lane 1 = Molecular ladder/marker; Lane 2 = undigested PCR product; Lane 3–40 = samples 1–38 respectively.

  18. Number and percentage of total subjects, controls and patients with their genotyping for each polymorphism

  19. D543N genotypic percentages in total subjects, patients and controls

  20. A318V genotypic percentages in total subjects, controls and patients

  21. 274C/T genotypic percentages in total subjects, controls and patients

  22. Chi-Square Test Results D534N • None of the three genotypes G/G, G/A & A/A showed significant association with RA A318V • Genotype C/T showed significant associated with RA (P< 0.001) 274C/T • Significant association of T/T genotype with RA (P< 0.001) observed

  23. Number and frequency of G and A alleles of D543N

  24. Number and frequency of T and C alleles at A318V

  25. Number and frequency of T and C alleles at 274C/T

  26. Allele carriage frequencies in RA patients and controls

  27. Discussion

  28. Comparison of allelic frequencies at three loci of NRAMP1 in normal individuals of different populations

  29. Conclusion • The genotype C/T and the allele T of A318V are highly significantly associated (P<0.001) with susceptibility to RA in Pakistani population. • Similarly the genotype T/T of 274C/T showed significant association with the disease and related with susceptibility to RA. • None of the genotypes or alleles of D543N is associated with RA in Pakistani population.

  30. Thank you Any Questions?

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